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1.  A Novel Role for Ecdysone in Drosophila Conditioned Behavior: Linking GPCR-Mediated Non-canonical Steroid Action to cAMP Signaling in the Adult Brain 
PLoS Genetics  2013;9(10):e1003843.
The biological actions of steroid hormones are mediated primarily by their cognate nuclear receptors, which serve as steroid-dependent transcription factors. However, steroids can also execute their functions by modulating intracellular signaling cascades rapidly and independently of transcriptional regulation. Despite the potential significance of such “non-genomic” steroid actions, their biological roles and the underlying molecular mechanisms are not well understood, particularly with regard to their effects on behavioral regulation. The major steroid hormone in the fruit fly Drosophila is 20-hydroxy-ecdysone (20E), which plays a variety of pivotal roles during development via the nuclear ecdysone receptors. Here we report that DopEcR, a G-protein coupled receptor for ecdysteroids, is involved in activity- and experience-dependent plasticity of the adult central nervous system. Remarkably, a courtship memory defect in rutabaga (Ca2+/calmodulin-responsive adenylate cyclase) mutants was rescued by DopEcR overexpression or acute 20E feeding, whereas a memory defect in dunce (cAMP-specific phosphodiestrase) mutants was counteracted when a loss-of-function DopEcR mutation was introduced. A memory defect caused by suppressing dopamine synthesis was also restored through enhanced DopEcR-mediated ecdysone signaling, and rescue and phenocopy experiments revealed that the mushroom body (MB)—a brain region central to learning and memory in Drosophila—is critical for the DopEcR-dependent processing of courtship memory. Consistent with this finding, acute 20E feeding induced a rapid, DopEcR-dependent increase in cAMP levels in the MB. Our multidisciplinary approach demonstrates that DopEcR mediates the non-canonical actions of 20E and rapidly modulates adult conditioned behavior through cAMP signaling, which is universally important for neural plasticity. This study provides novel insights into non-genomic actions of steroids, and opens a new avenue for genetic investigation into an underappreciated mechanism critical to behavioral control by steroids.
Author Summary
The brain is a prominent target of steroid hormones, which control a variety of neurobiological processes and are critical to the regulation of behavior. Some effects of these hormones involve changes in gene expression and thus emerge slowly, over the course of hours or even days. Other responses to steroids occur rapidly and are independent of transcriptional regulation. Their functions and mechanisms of action are poorly understood, particularly in the context of steroid-mediated control of behavior. Here we show, using the genetic model organism Drosophila melanogaster (the fruit fly), that an unconventional, membrane-bound receptor for the molting hormone ecdysone transmits a novel form of steroid signaling in the adult brain. Our study shows that this novel form of steroid signaling has a robust interface with the classical “memory genes” that encode central components of the so-called cAMP signaling pathway, which is universally important for neuronal and behavioral plasticity. These findings underscore the significance of steroid signaling in memory processing, and provide a foundation for the genetic analysis of rapid, unconventional steroid signaling in behavioral regulation.
PMCID: PMC3794910  PMID: 24130506
2.  Distinct, genome-wide, gene-specific selectivity patterns of four glucocorticoid receptor coregulators 
Glucocorticoids are a class of steroid hormones that bind to and activate the glucocorticoid receptor (GR), which then positively or negatively regulates transcription of many genes that govern multiple important physiological pathways such as inflammation and metabolism of glucose, fat and bone. The remodeling of chromatin and regulated assembly or disassembly of active transcription complexes by GR and other DNA-binding transcription factors is mediated and modulated by several hundred transcriptional coregulator proteins. Previous studies focusing on single coregulators demonstrated that each coregulator is required for regulation of only a subset of all the genes regulated by a steroid hormone. We hypothesized that the gene-specific patterns of coregulators may correspond to specific physiological pathways such that different coregulators modulate the pathway-specificity of hormone action, thereby providing a mechanism for fine tuning of the hormone response. We tested this by direct comparison of multiple coregulators, using siRNA to deplete the products of four steroid hormone receptor coregulator genes (CCAR1, CCAR2, CALCOCO1 and ZNF282). Global analysis of glucocorticoid-regulated gene expression after siRNA mediated depletion of coregulators confirmed that each coregulator acted in a selective and gene-specific manner and demonstrated both positive and negative effects on glucocorticoid-regulated expression of different genes. We identified several classes of hormone-regulated genes based on the effects of coregulator depletion. Each coregulator supported hormonal regulation of some genes and opposed hormonal regulation of other genes (coregulator-modulated genes), blocked hormonal regulation of a second class of genes (coregulator-blocked genes), and had no effect on hormonal regulation of a third gene class (coregulator-independent genes). In spite of previously demonstrated physical and functional interactions among these four coregulators, the majority of the several hundred modulated and blocked genes for each of the four coregulators tested were unique to that coregulator. Finally, pathway analysis on coregulator-modulated genes supported the hypothesis that individual coregulators may regulate only a subset of the many physiological pathways controlled by glucocorticoids. We conclude that gene-specific actions of coregulators correspond to specific physiological pathways, suggesting that coregulators provide a potential mechanism for physiological fine tuning in vivo and may thus represent attractive targets for therapeutic intervention.
PMCID: PMC4242289  PMID: 25422592
coregulators; gene expression; microarray; glucocorticoid receptor; gene regulation
3.  Transcriptional Control of Steroid Biosynthesis Genes in the Drosophila Prothoracic Gland by Ventral Veins Lacking and Knirps 
PLoS Genetics  2014;10(6):e1004343.
Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni), have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH) signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld), controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development.
Author Summary
Steroid hormones play important roles in physiology and disease. These hormones are molecules produced and secreted by endocrine cells in the body and control sexual maturation, metabolism and reproduction. We found transcriptional regulators that underlie the specialized function of endocrine steroid-producing cells. In the steroid-producing cells of the fruit fly Drosophila, Ventral veins lacking (Vvl) and Knirps (Kni) turn on all the genes required for steroid production. When Vvl or Kni were inactivated in the cells where the hormone is made, the genes involved in steroid production were not activated. Because of the reduced steroid production, the juvenile larvae failed to develop and undergo maturation to adulthood. Inactivation of Vvl and Kni also reduces endocrine cell growth by disturbing their response to growth promoting signals. Genetic variations in humans with the loss of a homolog of Vvl have been associated with disorders caused by insufficient steroid production. Together with the fact that Vvl is highly expressed in the steroid-producing cells of Drosophila, this suggests that Vvl may be a conserved master regulator of steroid production. Our findings provide insight into the network of factors that control endocrine cell function and steroid hormone levels that could have implication for human diseases.
PMCID: PMC4063667  PMID: 24945799
4.  Kinases and protein phosphorylation as regulators of steroid hormone action  
Although the primary signal for the activation of steroid hormone receptors is binding of hormone, there is increasing evidence that the activities of cell signaling pathways and the phosphorylation status of these transcription factors and their coregulators determine the overall response to the hormone. In some cases, enhanced cell signaling is sufficient to cause activation of receptors in medium depleted of steroids. Steroid receptors are targets for multiple kinases. Many of the phosphorylation sites contain Ser/Thr-Pro motifs implicating proline-directed kinases such as the cyclin-dependent kinases and the mitogen-activated kinases (MAPK) in receptor phosphorylation. Although some sites are constitutively phosphorylated, others are phosphorylated in response to hormone. Still others are only phosphorylated in response to specific cell signaling pathways. Phosphorylation of specific sites has been implicated not only in overall transcriptional activity, but also in nuclear localization, protein stability, and DNA binding. The studies of the roles of phosphorylation in coregulator function are more limited, but it is now well established that many of them are highly phosphorylated and that phosphorylation regulates their function. There is good evidence that some of the phosphorylation sites in the receptors and coregulators are targets of multiple signaling pathways. Individual sites have been associated both with functions that enhance the activity of the receptor, as well as with functions that inhibit activity. Thus, the specific combinations of phosphorylations of the steroid receptor combined with the expression levels and phosphorylation status of coregulators will determine the genes regulated and the biological response.
PMCID: PMC1876600  PMID: 17525795
5.  E1A-Mediated Repression of Progesterone Receptor-Dependent Transactivation Involves Inhibition of the Assembly of a Multisubunit Coactivation Complex 
Molecular and Cellular Biology  2000;20(6):2138-2146.
The steroid hormone progesterone acts via high-affinity nuclear receptors that interact with specific DNA sequences located near the promoter of the hormone-responsive gene. Recent studies suggested that the hormone-occupied progesterone receptor (PR) mediates gene activation by recruiting a cellular coregulatory factor, termed coactivator, to the target promoter. The identity and mechanism of action of the coactivator(s) that regulates transcriptional activity of PR are currently under investigation. Here we provide evidence that the hormone-occupied PR forms a multisubunit receptor-coactivator complex containing two previously described coactivators, CREB-binding protein (CBP) and steroid receptor coactivator 1 (SRC-1, a member of the p160 family of coactivators), in nuclear extracts of human breast tumor T47D cells. The association of CBP and SRC-1/p160 with the receptor complex is entirely hormone dependent. Both CBP and SRC-1/p160 possess intrinsic histone acetyltransferase (HAT) activity, and it has been recently proposed that these coactivators function by modulating chromatin structure at the promoter of the target gene. Interestingly, addition of purified CBP to the nuclear extracts of T47D cells markedly stimulated progesterone- and PR-dependent transcription from a nucleosome-free, progesterone response element (PRE)-linked reporter DNA template. Furthermore, depletion of SRC-1/p160 by immunoprecipitation from these transcriptional extracts also significantly impaired PR-mediated RNA synthesis from a naked PRE-linked DNA template. These results strongly implied that CBP and SRC-1/p160 facilitate receptor-mediated transcription in these cell extracts through mechanisms other than chromatin remodeling. We also observed that the adenoviral oncoprotein E1A, which interacts directly with CBP, repressed PR-mediated transactivation when added to the nuclear extracts of T47D cells. Supplementation with purified CBP overcame this inhibition, indicating that the inhibitory effect of E1A is indeed due to a blockade of CBP function. Most importantly, we noted that binding of E1A to CBP prevented the assembly of a coactivation complex containing PR, CBP, and SRC-1/p160, presumably by disrupting the interaction between CBP and SRC-1/p160. These results strongly suggested that E1A repressed receptor-mediated transcription by blocking the formation or recruitment of coactivation complexes. Collectively, our results support the hypothesis that the assembly of a multisubunit coactivation complex containing PR, CBP, and SRC-1/p160 is a critical regulatory step during hormone-dependent gene activation by PR and that the fully assembled complex has the ability to control transcription through mechanisms that are independent of the histone-modifying activities of its component coactivators.
PMCID: PMC110830  PMID: 10688660
6.  Boolean modeling of transcriptome data reveals novel modes of heterotrimeric G-protein action 
Classical mechanisms of heterotrimeric G-protein signaling are observed to function in regulation of the transcriptome. Conversely, many theoretical regulatory modes of the G-protein are not manifested in the transcriptomes we investigate.A new mechanism of G-protein signaling is revealed, in which the β subunit regulates gene expression identically in the presence or absence of the α subunit.We find evidence of cross-talk between G-protein-mediated and hormone-mediated transcriptional regulation.We find evidence of system specificity in G-protein signaling.
Heterotrimeric G-proteins, composed of α, β, and γ subunits, participate in a wide range of signaling pathways in eukaryotes (Morris and Malbon, 1999). According to the typical, mammalian paradigm, in its inactive state, the G-protein exists as an associated heterotrimer. G-protein signaling begins with ligand binding that results in a conformational change in a G-protein-coupled receptor (GPCR). Once activated by the GPCR, the Gα separates from the associated Gβγ dimer and the freed Gα and Gβγ proteins can then interact with downstream effector molecules, alone or in combination, to transduce the signal. Subsequent to signal propagation, Gα re-associates with the Gβγ dimer to reform the G-protein complex.
There are several classical routes for signal propagation through heterotrimeric G-proteins that have been categorized in mammalian systems (Marrari et al, 2007; Dupre et al, 2009). One route, which we designate classical I, requires the presence of both subunits, and can invoke one of two distinct mechanisms. In one mechanism, on GPCR activation, freed Gα and Gβγ each interact with downstream effectors to elicit the downstream response. In a related mechanism, Gα but not Gβγ interacts with downstream effectors, but the Gβγ dimer is nevertheless required to facilitate coupling of Gα with the relevant GPCR (Marrari et al, 2007). In a second route, which we designate classical II, it is solely the Gβγ dimer that interacts with downstream effectors; in this case, sequestration of Gβγ within the heterotrimer prevents signal propagation. In addition, a few non-classical G-protein regulatory modes have also been implicated in some systems, for example signaling by the intact heterotrimer in yeast (Klein et al, 2000; Frank et al, 2005). Observations such as these lead to a fundamental question, namely, which of all the theoretical regulatory modes of G-protein signaling are realized biologically. Our study answers this question in the context of the model plant Arabidopsis thaliana, and in addition analyzes the manner in which G-protein signaling couples with signaling by the plant hormone abscisic acid. The Arabidopsis genome encodes only one canonical Gα subunit, GPA1, and one canonical Gβ subunit, AGB1, and knockout mutants are available for each of these, allowing clear dissection of Gα- and Gβ-related phenotypes.
Abscisic acid (ABA) is a major plant hormone, which inhibits growth and promotes tolerance of abiotic stresses such as drought, salinity, and cold. ABA signaling is known to interact with heterotrimeric G-protein signaling in both developmental and stress responses in a complex manner, causing, for example, ABA hyposensitivity of guard cell stomatal opening in gpa1 and agb1 single mutants as well as agb1 gpa1 double mutants (Fan et al, 2008), but ABA hypersensitivity of the inhibition of seed germination and post-germination seedling development in the same mutants (Pandey et al, 2006). These experimental observations implicate G-proteins as one of the components of ABA signaling, but to date no systematic study has been conducted in either plant or metazoan systems to define the co-regulatory modes of a G-protein and a hormone.
In this study, we conduct genome-wide gene expression profiling in G-protein subunit mutants of A. thaliana guard cells and leaves, with or without treatment with ABA. By introducing one or more mediators acting downstream of the G-protein and ABA to control transcript levels, we propose nine G-protein/ABA signaling pathways including ABA-independent G-protein signaling pathways, G-protein-independent ABA signaling pathways, and seven distinct ABA–G-protein-coupled signaling pathways (Figure 1). We develop a Boolean modeling framework to systematically enumerate 14 possible theoretical regulatory modes of the G-protein and 142 co-regulatory modes of the G-protein and ABA, and then use a pattern matching approach to associate target genes with theoretical regulatory modes.
Our analysis shows that the G-protein regulatory mode that requires the presence of both Gα and Gβγ subunits (consistent with classical I mechanisms), is well represented in both guard cells and leaves. The G-protein regulatory mode that requires a freed Gβγ subunit (classical II G-protein regulatory mechanism) is well supported in guard cells and somewhat less so in leaves. In addition, a G-protein regulatory mode representing a non-classical regulatory mechanism is prevalent in guard cells but less so in leaves (Figure 5). In this regulatory mode, signaling by Gβ(γ) occurs, and this signaling is not regulated in any way by Gα.
By relating the target genes with the nine proposed G-protein/ABA signaling pathways, we are able to gauge the plausibility of regulatory modes of the G-protein and ABA at the pathway level. We find that G-protein-independent ABA signaling pathways are prevalent in both guard cells and leaves. The existence of an ABA-independent regulatory activity of the G-protein is well supported in guard cells, but not supported in leaves. Additive regulation by G-protein signaling plus G-protein-independent ABA signaling is rare in both guard cells and leaves. In addition, combinatorial cross-talk between G-protein signaling and ABA signaling and additive cross-talk between ABA–G-protein signaling and G-protein-independent ABA signaling are observed in both guard cells and leaves. Our transcriptome analysis indicates that in some cases, ABA definitely does not influence G-protein signaling, though it may do so in some other cases.
To investigate whether previously observed hypersensitivity or hyposensitivity of developmental and dynamic transient responses to ABA in G-protein mutants is recapitulated at the level of transcriptional regulation, we compare gene regulation by ABA in guard cells and leaves of the G-protein mutants versus wild type. We find that in guard cells, equal ABA hyposensitivity of all mutants combined is significant, although hyposensitivity in individual mutants is not. There is also a separate group of genes in guard cells that show ABA hypersensitivity in the gpa1 mutant, suggesting complex interactions between ABA and G-protein signaling in gene regulation in this cell type. In leaves, ABA hyposensitivity of gene expression in the three individual mutants and equal hyposensitivity in all mutants are strongly supported. In addition, several of the functional categories identified by our analysis of G-protein regulatory modes have been implicated in previous physiological analyses of G-protein mutants, providing validation to the biological interpretation of our results.
In summary, by conducting a genome-wide gene expression profiling study in G-protein subunit mutants of A. thaliana guard cells and leaves and developing a Boolean modeling framework, we systematically evaluate the biological utilization of mechanisms of G-protein regulatory action and reveal novel regulatory modes of the G-protein. The results generate empirical evidence and insights regarding molecular events of G-protein signaling and response at the physiological level in both plants and mammals.
Heterotrimeric G-proteins mediate crucial and diverse signaling pathways in eukaryotes. Here, we generate and analyze microarray data from guard cells and leaves of G-protein subunit mutants of the model plant Arabidopsis thaliana, with or without treatment with the stress hormone, abscisic acid. Although G-protein control of the transcriptome has received little attention to date in any system, transcriptome analysis allows us to search for potentially uncommon yet significant signaling mechanisms. We describe the theoretical Boolean mechanisms of G-protein × hormone regulation, and then apply a pattern matching approach to associate gene expression profiles with Boolean models. We find that (1) classical mechanisms of G-protein signaling are well represented. Conversely, some theoretical regulatory modes of the G-protein are not supported; (2) a new mechanism of G-protein signaling is revealed, in which Gβ regulates gene expression identically in the presence or absence of Gα; (3) guard cells and leaves favor different G-protein modes in transcriptome regulation, supporting system specificity of G-protein signaling. Our method holds significant promise for analyzing analogous ‘switch-like' signal transduction events in any organism.
PMCID: PMC2913393  PMID: 20531402
abscisic acid; Arabidopsis thaliana; Boolean modeling; heterotrimeric G-protein; transcriptome
7.  Signaling by Tyrosine Kinases Negatively Regulates the Interaction between Transcription Factors and SMRT (Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor) Corepressor 
Nuclear hormone receptors are hormone-regulated transcription factors that bind to specific sites on DNA and modulate the expression of adjacent target genes. Many nuclear hormone receptors display bimodal transcriptional properties; thyroid hormone receptors, for example, typically repress target gene expression in the absence of hormone, but activate target gene expression in the presence of hormone. The ability to repress is closely linked to the ability of the apo-receptor to physically bind to auxiliary corepressor proteins denoted SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor), which, in turn, help mediate the actual molecular events involved in transcriptional silencing. We report here that repression by thyroid hormone receptors can be regulated not only by cognate hormone, but also by certain tyrosine kinase signal transduction pathways, such as that represented by the epidermal growth factor-receptor. Activation of tyrosine kinase signaling leads to inhibition of T3R-mediated repression with relatively little effect on activation. These effects appear to be mediated by a kinase-initiated disruption of the ability of T3R to interact with SMRT corepressor. Intriguingly, tyrosine kinase signaling similarly disrupted the interactions of SMRT with v-Erb A, with retinoic acid receptors, and with PLZF, a nonreceptor transcriptional repressor. We conclude that tyrosine kinase signaling exerts potentially important regulatory effects on transcriptional silencing mediated by a variety of transcription factors that operate through the SMRT corepressor complex.
PMCID: PMC2653426  PMID: 9717842
8.  Specific DNA binding of Stat5, but not of glucocorticoid receptor, is required for their functional cooperation in the regulation of gene transcription. 
Molecular and Cellular Biology  1997;17(11):6708-6716.
Prolactin and glucocorticoid hormone are signals which regulate the transcription of milk protein genes in mammary epithelial cells. We have investigated the molecular mechanisms by which these hormones cooperate in the induction of transcription. Both hormones activate latent transcription factors in the cytoplasm of mammary epithelial cells. Prolactin exerts its effect through binding to the extracellular domain of the prolactin receptor and through receptor dimerization. This leads to the activation of a protein tyrosine kinase (Jak2), which is noncovalently associated with the cytoplasmic domain of the prolactin receptor. Jak2 phosphorylates the signal transducer and transcription activator (Stat5) which causes its dimerization and nuclear translocation where Stat5 specifically binds to sequence elements in the promoter regions of milk protein genes. In comparison, the glucocorticoid receptor is activated by a lipophilic steroid ligand in the cytoplasm which causes allosteric changes in the molecule, dimerization, and nuclear localization. It has been demonstrated that Stat5 and the glucocorticoid receptor form a molecular complex which cooperates in the induction of transcription of the beta-casein gene. We have defined the DNA sequence requirements for this cooperative mechanism and have delimited the functional domains in Stat5 and the glucocorticoid receptor that are necessary for the functional interaction. We find that the Stat5 response element (Stat5RE) within the beta-casein gene promoter is sufficient to elicit the cooperative action of Stat5 and the glucocorticoid receptor on transcription. Activation of Stat5 through phosphorylation of tyrosine 694 is an absolute prerequisite for transcription. Deletion of the transactivation domain of Stat5 results in a molecule which cannot mediate transactivation by itself but can still cooperate with the glucocorticoid receptor. Mutated variants of the glucocorticoid receptor with a nonfunctional DNA binding domain or a DNA binding domain contributed by the estrogen receptor are still able to cooperate with Stat5 in transcriptional induction. Deletion of the ligand binding domain of the glucocorticoid receptor does not impede cooperation with Stat5, whereas deletion of the AF-1 transactivation domain does prevent cooperation. Our results indicate that the glucocorticoid receptor acts as a ligand-dependent coactivator of Stat5 independently of its DNA binding function.
PMCID: PMC232525  PMID: 9343435
9.  Constitutive activation of gene expression by thyroid hormone receptor results from reversal of p53-mediated repression. 
Molecular and Cellular Biology  1997;17(12):7195-7207.
Thyroid hormone receptor (T3R) is a member of the steroid hormone receptor gene family of nuclear hormone receptors. In most cells T3R activates gene expression only in the presence of its ligand, L-triiodothyronine (T3). However, in certain cell types (e.g., GH4C1 cells) expression of T3R leads to hormone-independent constitutive activation. This activation by unliganded T3R occurs with a variety of gene promoters and appears to be independent of the binding of T3R to specific thyroid hormone response elements (TREs). Previous studies indicate that this constitutive activation results from the titration of an inhibitor of transcription. Since the tumor suppresser p53 is capable of repressing a wide variety of gene promoters, we considered the possibility that the inhibitor is p53. Evidence to support this comes from studies indicating that expression of p53 blocks T3R-mediated constitutive activation in GH4C1 cells. In contrast with hormone-independent activation by T3R, p53 had little or no effect on T3-dependent stimulation which requires TREs. In addition, p53 mutants which oligomerize with wild-type p53 and interfere with its function also increase promoter activity. This enhancement is of similar magnitude to but is not additive with the stimulation mediated by unliganded T3R, suggesting that they target the same factor. Since p53 mutants are known to target wild-type p53 in the cell, this suggests that T3R also interacts with p53 in vivo and that endogenous levels of p53 act to suppress promoter activity. Evidence supporting both functional and physical interactions of T3R and p53 in the cell is presented. The DNA binding domain (DBD) of T3R is important in mediating constitutive activation, and the receptor DBD appears to functionally interact with the N terminus of p53 in the cell. In vitro binding studies indicate that the T3R DBD is important for interaction of T3R with p53 and that this interaction is reduced by T3. These findings are consistent with the in vivo studies indicating that p53 blocks constitutive activation but not ligand-dependent stimulation. These studies provide insight into mechanisms by which unliganded nuclear hormone receptors can modulate gene expression and may provide an explanation for the mechanism of action of the v-erbA oncoprotein, a retroviral homolog of chicken T3R alpha.
PMCID: PMC232577  PMID: 9372952
10.  Non-genomic mechanisms of progesterone action in the brain 
Progesterone is a gonadal steroid hormone whose physiological effects extend well beyond the strict confines of reproductive function. In fact, progesterone can have important effects on a variety of tissues, including the bone, the heart and the brain. Mechanistically, progesterone has been thought to exert its effects through the progesterone receptor (PR), a member of the nuclear steroid hormone superfamily, and as such, acts through specific progesterone response elements (PRE) within the promoter region of target genes to regulate transcription of such genes. This has been often described as the “genomic” mechanism of progesterone action. However, just as progesterone has a diverse range of tissue targets, the mechanisms through which progesterone elicits its effects are equally diverse. For example, progesterone can activate alternative receptors, such as membrane-associated PRs (distinct from the classical PR), to elicit the activation of several signaling pathways that in turn, can influence cell function. Here, we review various non-nuclear (i.e., non-genomic) signaling mechanisms that progesterone can recruit to elicit its effects, focusing our discussion primarily on those signaling mechanisms by which progesterone influences cell viability in the brain.
PMCID: PMC3776940  PMID: 24065876
progesterone; non-genomic; progesterone receptor; signaling; brain
11.  The phosphoproteome of toll-like receptor-activated macrophages 
First global and quantitative analysis of phosphorylation cascades induced by toll-like receptor (TLR) stimulation in macrophages identifies nearly 7000 phosphorylation sites and shows extensive and dynamic up-regulation and down-regulation after lipopolysaccharide (LPS).In addition to the canonical TLR-associated pathways, mining of the phosphorylation data suggests an involvement of ATM/ATR kinases in signalling and shows that the cytoskeleton is a hotspot of TLR-induced phosphorylation.Intersecting transcription factor phosphorylation with bioinformatic promoter analysis of genes induced by LPS identified several candidate transcriptional regulators that were previously not implicated in TLR-induced transcriptional control.
Toll-like receptors (TLR) are a family of pattern recognition receptors that enable innate immune cells to sense infectious danger. Recognition of microbial structures, like lipopolysaccharide (LPS) of Gram-negative bacteria by TLR4, causes within hours substantial re-programming of macrophage gene expression, including up-regulation of chemokines driving inflammation, anti-microbial effector molecules and cytokines directing adaptive immune responses. TLR signalling is initiated by the adapter protein Myd88 and leads to the activation of kinase cascades that result in activation of the MAPK and NFkB pathways. Phosphorylation has an essential role in these early steps of TLR signalling, and in addition regulates critical transcription factors (TFs). Although TLR signalling has been extensively studied, a comprehensive analysis of phosphorylation events in TLR-activated macrophages is lacking. It is therefore unknown whether the canonical MAPK and NFkB pathways comprise the main phosphorylation events and which other molecular functions and processes are regulated by phosphorylation after stimulation with LPS.
Recent progress in mass spectrometry-based proteomics has opened the possibility to quantitatively investigate global changes in protein abundance and post-translational modifications. Stable isotope labelling with amino acids in cell culture (SILAC) allows highly accurate quantification, and has proved especially useful for direct comparison of phosphopeptide abundance in time-course or treatment analyses.
Here, we adapted SILAC to primary mouse macrophages, and performed a global, quantitative and kinetic analysis of the macrophage phosphoproteome after LPS stimulation. Bioinformatic analyses were used to identify kinases, pathways and biological processes enriched in the LPS-regulated phosphoproteome. To connect TF phosphorylation with transcription, we generated a parallel dataset of nascent RNA and used in silico promoter analysis to identify transcriptional regulators with binding site enrichment among the LPS-regulated gene set.
After establishing SILAC conditions for efficient labelling of primary bone marrow-derived macrophages in two independent experiments 1850 phosphoproteins with a total of 6956 phosphorylation sites were reproducibly identified. Phosphoproteins were detected from all cellular compartments, with a clear enrichment for nuclear and cytoskeleton-associated proteins. LPS caused major regulation of a large fraction of phosphopeptides, with 24% of all sites up-regulated and 9% down-regulated after stimulation (Figure 3A and B). These changes were highly dynamic, as the majority of the regulated phosphopeptides were up-regulated or down-regulated transiently or in a delayed manner (Figure 3C). Overall, the extent of changes in the phosphoproteome was comparable to the transcriptional re-programming, underscoring the importance of phosphorylation cascades in TLR signalling. Our parallel transcriptome data also showed that widespread phosphorylation precedes massive transcriptional changes.
To obtain footprints of kinase activation in response to TLR ligation, we searched phosphopeptide sequences for known linear sequence motifs of 33 kinases and identified kinase motifs enriched among LPS-regulated phosphorylation sites (compared to non-regulated phosphorylation sites) (Table I). Motif ERK/MAPK was highly enriched, in accordance with the essential role of the MAPK module in TLR signalling. Other kinases with motif enrichment have also recently been linked to TLR signalling (e.g. PKD; AKT and its targets GSK3 and mTOR). However, the DNA damage-actviated kinases ATM/ATR and the cell cycle-associated kinases AURORA and CHK1/2 have not been associated with the macrophage response to TLR activation yet. These finding shed new light on older data on the effect of TLR on macrophage proliferation in response to macrophage colony stimulating factor. Of interest, in follow-up experiments using pharmacological inhibitors of the kinases with motif enrichment, we observed that inhibition of ATM kinase activity caused increased LPS-induced expression of several cytokines and chemokines, suggesting that this pathway regulates inflammatory responses.
In further bioinformatic analyses, the Gene Ontology and signalling pathway annotations of phosphoproteins were used to identify signalling pathways and cellular processes targeted by TLR4-controlled phosphorylation (Table II). Among the expected hits, based on the known TLR pathways, were TLR signalling, MAPK and AKT as well as mTOR signalling. Of interest, the annotation terms ‘Rho GTPase cycle' and ‘cytoskeleton' were significantly enriched among LPS-regulated phosphoproteins, indicating a more prominent role for cytoskeletal proteins in the transduction of TLR signals or in the biological response to it.
We were especially interested in the phosphorylation of TFs and its regulation by LPS (Figure 6A). We hypothesised that functionally important TFs should have an increased frequency of binding sites in the promoters of LPS-regulated genes (Figure 6B). To identify transcriptionally regulated genes with high sensitivity, we isolated nascent RNA after metabolic labelling (Figure 6C–E). In silico promoter scanning using Genomatix software for binding sites for all 50 TF families with phosphorylated members was used to test for enrichment in transciptionally induced genes (Figure 6F). At the early time point, binding site enrichment for the canonical TLR-associated TF NFkB was detected, and in addition we found that several other TF families with an established role in the transcription of individual LPS-target genes showed binding site enrichment (CEBP, MEF2, NFAT and HEAT). In addition, enrichment for OCT and HOXC binding sites at the early time point and SORY matrices later after stimulation indicated an involvement of the phosphorylated members of the respective TF families in the execution of TLR-induced transcriptional responses. An initial test of the function for a few of these candidate transcriptional regulators was performed using siRNA knockdown in primary macrophages. These experiments suggested that knock down of the SORY binding phosphoprotein Capicua homolog (Cic) and to a lesser extent of the CREB family member Atf7 selectively attenuates LPS-induced expression of Il1a and Il1b.
In summary, this study provides a novel and global perspective on innate immune activation by TLR signalling (Figure 5). We quantitatively detected a large number of previously unknown site-specific phosphorylation events, which are now publicly available through the Phosida database. By combining different data mining approaches, we consistently identified canonical and newly implicated TLR-activated signalling modules. In particular, the PI3K/AKT and the related mTOR pathway were highlighted; furthermore, DNA damage–response associated ATM/ATR kinases and the cytoskeleton emerged as unexpected hotspots for phosphorylation. Finally, weaving together corresponding phophoproteome and nascent transcriptome datasets through the loom of in silico promoter analysis we identified TFs with a likely role in mediating TLR-induced gene expression programmes.
Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome of primary macrophages using stable isotope labelling with amino acids in cell culture, phosphopeptide enrichment and high-resolution mass spectrometry. In parallel, nascent RNA was profiled to link transcription factor (TF) phosphorylation to TLR4-induced transcriptional activation. We reproducibly identified 1850 phosphoproteins with 6956 phosphorylation sites, two thirds of which were not reported earlier. LPS caused major dynamic changes in the phosphoproteome (24% up-regulation and 9% down-regulation). Functional bioinformatic analyses confirmed canonical players of the TLR pathway and highlighted other signalling modules (e.g. mTOR, ATM/ATR kinases) and the cytoskeleton as hotspots of LPS-regulated phosphorylation. Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene expression.
PMCID: PMC2913394  PMID: 20531401
macrophage; nascent RNA; phosphoproteome; SILAC; toll-like receptors
12.  Synthesis and Functional Analysis of Novel Bivalent Estrogens 
Steroids  2010;75(12):825-833.
The steroid hormone estrogen plays a critical role in female development and homeostasis. Estrogen mediates its effects through binding and activation of specific estrogen receptors alpha (ERα) and beta (ERβ), members of the steroid/nuclear receptor family of ligand-induced transcription factors. Due to their intimate roles in genomic and nongenomic signaling pathways, these hormones and their receptors have been also implicated in the pathologies of a variety of cancers and metabolic disorders, and have been the target of large therapeutic development efforts. The binding of estrogen to its respective receptors initiates a cascade of events that include receptor dimerization, nuclear localization, DNA binding and recruitment of co-regulatory protein complexes. In this manuscript, we investigate the potential for manipulating steroid receptor gene expression activity through the development of bivalent steroid hormones that are predicted to facilitate hormone receptor dimerization events. Data are presented for the development and testing of novel estrogen dimers, linked through their C-17 moiety, that can activate estrogen receptor alpha (ERα)-mediated transcription events with efficacy and potency equal to or greater than that of ERα’s cognate ligand, 17β-estradiol. These bivalent estrogen structures open the door to the development of a variety of steroid therapeutics that could dramatically impact future drug development in this area.
PMCID: PMC2948962  PMID: 20685325
ERα; steroid hormone; Girard reagent; dimerization
13.  Expression of Membrane Progesterone Receptors (mPR/PAQR) in Ovarian Cancer Cells: Implications for Progesterone-Induced Signaling Events 
Hormones & cancer  2010;1(4):167-176.
The high mortality rates associated with ovarian cancer are largely due to a lack of highly effective treatment options for advanced stage disease; a time when initial diagnosis most commonly occurs. Recent evidence suggests that the steroid hormone, progesterone, may possess anti-tumorigenic properties. With the discovery of a new class of membrane-bound progesterone receptors (mPRs) belonging to the progestin and adipoQ receptor (PAQR) gene family in the ovary, there are undefined mechanisms by which progesterone may inhibit tumor progression. Therefore, our goal was to define potential mPR-dependent signaling mechanisms operative in ovarian cancer cells. We detected abundant mPRα (PAQR7), mPRβ (PAQR8), and mPRγ (PAQR5), but not classical nuclear PR (A or B isoforms) mRNA expression and mPRα protein expression in a panel of commonly used ovarian cancer cell lines. In contrast to mPR action in breast cancer cells, progesterone alone failed to induce changes in cyclic adenosine monophosphate (cAMP) levels in ovarian cancer cells. However, progesterone enhanced cAMP production by β1,2-adrenergic receptors and increased isoproterenol-induced transcription from a cAMP response element (CRE)-driven reporter gene. Independently of β-adrenergic signaling, we additionally observed activation of both JNK1/2 and p38 MAPK in response to progesterone alone. This finding was supported by the results of a screen for potential mPR gene targets. Progesterone induced a significant increase in transcription of the pro-apoptotic marker BAX, whose activity and expression has been linked to JNK1/2 and p38 signaling. Inhibitors of JNK, but not p38, blocked progesterone-induced BAX expression. Taken together, these observations implicate at least two distinct signaling pathways that may be utilized by mPRs in ovarian cancer cells that exhibit regulatory genomic changes. These studies on mPR signaling in ovarian cancer lay the foundation for future work aimed at understanding how progesterone exerts its anti-tumorigenic effects in the ovary and suggest that pharmacologic activation of mPRs, abundantly expressed in ovarian cancers, may provide a new treatment option for patients with advanced stage disease.
PMCID: PMC3926102  PMID: 21761364
Ovarian cancer; Progesterone; Membrane progesterone receptor (mPR); PAQR; cAMP; Jun kinase (JNK); p38 Mitogen-activated protein kinase; Apoptosis
14.  Negative regulation of expression of the pituitary-specific transcription factor GHF-1/Pit-1 by thyroid hormones through interference with promoter enhancer elements. 
Molecular and Cellular Biology  1995;15(11):6322-6330.
Expression of the growth hormone gene is due to the presence of the pituitary-specific transcription factor GHF-1/Pit-1. The action of the thyroid hormone T3 is mediated by nuclear receptors that regulate transcription by interaction with DNA elements located near promoters of the regulated genes. In this study, we show that T3 inhibits expression of the GHF-1/Pit-1 gene in rat pituitary GH4C1 cells by a novel mechanism that involves transcriptional interference with other regulatory elements of the promoter. Sequences between bp -90 and -200 of the rat GHF-1/Pit-1 gene which do not contain a hormone response element but contain two cyclic AMP-responsive elements mediate most of the repressive effect of T3. The hormone reduces basal levels of GHF-1/Pit-1 promoter activity and antagonizes its response to cyclic AMP and the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate). A similar repression is found with a heterologous promoter that contains four copies of the cyclic AMP-responsive element motif. This regulation provides a novel example of the cross-talk between the thyroid hormone receptor and the signal transduction pathways used by different hormones and growth factors. Additionally, T3 interferes with in vitro binding of GHF-1/Pit-1 to a positive autoregulatory element located at bp -45 to -63 and has a detectable inhibitory effect on the activity of a promoter construct which extends to bp -90 of 5'-flanking DNA. The regulation of the transcription factor provides a novel example of negative transcriptional regulation by thyroid hormones.
PMCID: PMC230884  PMID: 7565785
15.  Dissection of Estrogen Receptor Alpha Signaling Pathways in Osteoblasts Using RNA-Sequencing 
PLoS ONE  2014;9(4):e95987.
The effects of 17-β-estradiol in osteoblasts are primarily mediated by the nuclear transcription factors, estrogen receptor (ER)α and ERβ. ERs function through three general modes of action: DNA-binding dependent through estrogen response elements (EREs; designated nuclear ERE signaling); nuclear signaling via protein-protein interactions to other transcription factors (nuclear non-ERE signaling); and extra-nuclear signaling (membrane-bound functions of ERs). Identification of the specific transcriptional signatures regulated by each of these modes of action should contribute to an enhanced understanding of estrogen signaling in osteoblasts. To achieve this goal, we utilized specific mutations of ERα that eliminate the ability of the receptor to signal through a specific mode of action. The non-classical ERα knock-in (NERKI) mutation is incapable of signaling through direct DNA binding to EREs and the nuclear only ERα (NOER) mutation eliminates all membrane-localized signaling. Comparison of the gene expression patterns elicited by these mutations with the wild-type ERα (WT) pattern provides mode-specific data concerning transcriptional regulation by ERα. We expressed these constructs in the ER-negative osteoblastic cell line hFOB (−/+ estrogen) and performed global RNA-sequencing. Using a series of pair-wise comparisons, we generated three lists of genes that were regulated either by the nuclear ERE-dependent, nuclear ERE-independent, or extra-nuclear actions of ERα. Pathway and gene ontology analyses revealed that genes regulated through the nuclear ERE and nuclear non-ERE pathways were largely involved in transcriptional regulation, whereas genes regulated through extra-nuclear mechanisms are involved in cytoplasmic signaling transduction pathways. We also intersected our data with genes linked to bone density and fractures from a recent genome-wide association study and found 25 of 72 genes (35%) regulated by estrogen. These data provide a comprehensive list of genes and pathways targeted by these specific modes of ERα action and suggest that “mode-specific” ligands could be developed to modulate specific ERα functionality in bone.
PMCID: PMC4002480  PMID: 24776842
16.  Riboswitches as hormone receptors: hypothetical cytokinin-binding riboswitches in Arabidopsis thaliana 
Biology Direct  2010;5:60.
Riboswitches are mRNA elements that change conformation when bound to small molecules. They are known to be key regulators of biosynthetic pathways in both prokaryotes and eukaryotes.
Presentation of the Hypothesis
The hypothesis presented here is that riboswitches function as receptors in hormone perception. We propose that riboswitches initiate or integrate signaling cascades upon binding to classic signaling molecules. The molecular interactions for ligand binding and gene expression control would be the same as for biosynthetic pathways, but the context and the cadre of ligands to consider is dramatically different. The hypothesis arose from the observation that a compound used to identify adenine binding RNA sequences is chemically similar to the classic plant hormone, or growth regulator, cytokinin. A general tenet of the hypothesis is that riboswitch-binding metabolites can be used to make predictions about chemically related signaling molecules. In fact, all cell permeable signaling compounds can be considered as potential riboswitch ligands. The hypothesis is plausible, as demonstrated by a cursory review of the transcriptome and genome of the model plant Arabidopsis thaliana for transcripts that i) contain an adenine aptamer motif, and ii) are also predicted to be cytokinin-regulated. Here, one gene, CRK10 (for Cysteine-rich Receptor-like Kinase 10, At4g23180), contains an adenine aptamer-related sequence and is down-regulated by cytokinin approximately three-fold in public gene expression data. To illustrate the hypothesis, implications of cytokinin-binding to the CRK10 mRNA are discussed.
Testing the hypothesis
At the broadest level, screening various cell permeable signaling molecules against random RNA libraries and comparing hits to sequence and gene expression data bases could determine how broadly the hypothesis applies. Specific cases, such as CRK10 presented here, will require experimental validation of direct ligand binding, altered RNA conformation, and effect on gene expression. Each case will be different depending on the signaling pathway and the physiology involved.
Implications of the hypothesis
This would be a very direct signal perception mechanism for regulating gene expression; rivaling animal steroid hormone receptors, which are frequently ligand dependent transcription initiation factors. Riboswitch-regulated responses could occur by modulating target RNA stability, translatability, and alternative splicing - all known expression platforms used in riboswitches. The specific illustration presented, CRK10, implies a new mechanism for the perception of cytokinin, a classic plant hormone. Experimental support for the hypothesis would add breadth to the growing list of important functions attributed to riboswitches.
This article was reviewed by Anthony Poole, Rob Knight, Mikhail Gelfand.
PMCID: PMC2974657  PMID: 20961447
17.  Cellular reprogramming by the conjoint action of ERα, FOXA1, and GATA3 to a ligand-inducible growth state 
Estrogen receptor α (ERα), FOXA1, and GATA3 form a functional enhanceosome in MCF-7 breast carcinoma cell that is significantly associated with active transcriptional features such as enhanced p300 co-activator and RNA Pol II recruitment as well as chromatin opening.The enhanceosome exerts significant impact and optimal transcriptional control in the regulation of E2-responsive genes.The presence of FOXA1 and GATA3 is indispensable in restoring the ERα growth-response machinery in the ERα-negative cells and recapitulating the appropriate expression cassette.
Estrogen receptor α (ERα) is a ligand-inducible hormone nuclear receptor that has important physiology and pathology roles in reproduction, cancer, and cardiovascular biology. The regulation of ERα involves its binding to the DNA recognition sequence also known as estrogen-response elements (EREs) and recruits a variety of co-activators, corepressors, and chromatin remodeling enzymes to initiate transcription machinery. In our previous (Lin et al, 2007) and recent (Joseph et al, 2010) studies, we have identified high confidence ERα binding sites in MCF-7 human mammary carcinoma cells. With known motif scanning and de novo motif detection, we identified that FOXA1 and GATA3 motifs were commonly enriched around ERα binding sites. Moreover, numerous microarray studies have documented the co-expression of ERα, FOXA1, and GATA3 in primary breast tumors (Badve et al, 2007; Wilson and Giguere, 2008). This evidence suggests that these three transcription factors (TFs) may cluster on DNA binding sites and contribute to the breast cancer phenotype. However, there is little understanding as to the nature of their coordinated interaction at the genome level or the biological consequences of their detailed interaction.
We mapped the genome-wide binding profiles of ERα, FOXA1, and GATA3 using the massive parallel chromatin immunoprecipitation-sequencing (ChIP-seq) approach. We observed that ERα, FOXA1, and GATA3 colocalized in a coordinated manner where ∼30% of all ERα binding sites were overlapped with FOXA1 and GATA3 bindings upon estrogen (E2) stimulation. Moreover, we found that the ERα+FOXA1+GATA3 conjoint sites were associated with highest p300 co-activator recruitment, RNA Pol II occupancy, and chromatin opening. Such results indicate that these three TFs form a functional enhanceosome and cooperatively modulate the transcriptional networks previously ascribed to ERα alone. And such enhanceosome binding sites appear to regulate the genes driving core ERα function.
To further validate that ERα+FOXA1+GATA3 co-binding represents an optimal configuration for E2-mediated transcriptional activation, we have performed luciferase reporter assays on GREB1 locus that actively engages ERα enhanceosome sites in gene regulation (Figure 5C). The presence of ERα induced the GREB1 luciferase activity to ∼246% (as compared with the control construct). The individual presence of FOXA1 and GATA3 or combination of both only produced subtle changes to the GREB1 luciferase activity. The combination of ERα+FOXA1 and ERα+GATA3 has increased the luciferase activity to ∼330%. Interestingly, the assemblage of ERα+FOXA1+GATA3 provided the optimal ER responsiveness to 370%. This suggests that ERα provides the fundamental gene regulatory module but that FOXA1 and GATA3 incrementally improve ERα-regulated transcriptional induction.
It is known that ERα is a ligand-activated TF that mediates the proliferative effects of E2 in breast cancer cells. Garcia et al (1992) showed inhibited growth in MDA-MB-231 cells with forced expression of ERα upon E2 treatment. The rationale for these different outcomes has remained elusive. We posited that these higher order regulatory mechanisms of ERα function such as the formation and composition of enhanceosomes may explain the establishment of transcriptional regulatory cassettes favoring either growth enhancement or growth repression.
To test this hypothesis, we stably transfected the MDA-MB-231 cells with individual ERα, FOXA1, GATA3, or in combinations (Figure 6A). We observed inhibited growth in cells with enforced expression of ERα or FOXA1. There was unaltered growth in cells with expression of GATA3. Co-expression of ERα+FOXA1 or ERα+GATA3 exhibited inhibition of cell proliferation as compared with control cells. However, the co-expression of ERα together with FOXA1 and GATA3 resulted in marked induction of cell proliferation under E2 stimulation. We have recapitulated this cellular reprogramming in another ERα-negative breast cancer cell line, BT-549 and observed similar E2-responsive growth induction in the ERα+FOXA1+GATA3-expressing BT-549 cells. This suggests that only with the full activation of conjoint binding sites by the three TFs will the proliferative phenotype associated with ligand induced ERα be manifest.
To assess the nature of this transcriptional reprogramming, we asked the question if the reprogrammed MDA-MB-231 cells display any similarity in the expression profile of the ERα-positive breast cancer cell line, MCF-7 (Figure 6C). We combined the E2-regulated genes from these differently transfected MDA-MB-231 cells, and compared their expressions in these MDA-MB-231-transfected cells and MCF-7 cells. Strikingly, we found that the expression profiles of ERα+FOXA1+GATA3-expressing MDA-MB-231 cells display a good correlation (R=0.42) with the E2-induced expression profile of MCF-7. We did not observe such correlation between the expression profiles of MDA-MB-231 transfected with ERα only (R=−0.21). Furthermore, we observed that there is marginal induced expression of luminal marker genes and reduced expression of basal genes in the ERα+FOXA1+GATA3-expressing MDA-MB-231 as compared with the vector control cells. This suggests that the enhanceosome component is competent to partially reprogramme the basal cells to resemble the luminal cells.
Taken together, we have uncovered the genomics impact as well as the functional importance of an enhanceosome comprising ERα, FOXA1, and GATA3 in the estrogen responsiveness of ERα-positive breast cancer cells. This enhanceosome exerts significant combinatorial control of the transcriptional network regulating growth and proliferation of ERα-positive breast cancer cells. Most importantly, we show that the transfection of the enhanceosome component was necessary to reprogramme the ERα-negative cells to restore the estrogen-responsive growth and to transcriptionally induce a basal to luminal transition.
Despite the role of the estrogen receptor α (ERα) pathway as a key growth driver for breast cells, the phenotypic consequence of exogenous introduction of ERα into ERα-negative cells paradoxically has been growth inhibition. We mapped the binding profiles of ERα and its interacting transcription factors (TFs), FOXA1 and GATA3 in MCF-7 breast carcinoma cells, and observed that these three TFs form a functional enhanceosome that regulates the genes driving core ERα function and cooperatively modulate the transcriptional networks previously ascribed to ERα alone. We demonstrate that these enhanceosome occupied sites are associated with optimal enhancer characteristics with highest p300 co-activator recruitment, RNA Pol II occupancy, and chromatin opening. Most importantly, we show that the transfection of all three TFs was necessary to reprogramme the ERα-negative MDA-MB-231 and BT-549 cells to restore the estrogen-responsive growth resembling estrogen-treated ERα-positive MCF-7 cells. Cumulatively, these results suggest that all the enhanceosome components comprising ERα, FOXA1, and GATA3 are necessary for the full repertoire of cancer-associated effects of the ERα.
PMCID: PMC3202798  PMID: 21878914
enhanceosome; estrogen receptor α; FOXA1; GATA3; synthetic phenotypes
18.  Target Genes of the MADS Transcription Factor SEPALLATA3: Integration of Developmental and Hormonal Pathways in the Arabidopsis Flower 
PLoS Biology  2009;7(4):e1000090.
The molecular mechanisms by which floral homeotic genes act as major developmental switches to specify the identity of floral organs are still largely unknown. Floral homeotic genes encode transcription factors of the MADS-box family, which are supposed to assemble in a combinatorial fashion into organ-specific multimeric protein complexes. Major mediators of protein interactions are MADS-domain proteins of the SEPALLATA subfamily, which play a crucial role in the development of all types of floral organs. In order to characterize the roles of the SEPALLATA3 transcription factor complexes at the molecular level, we analyzed genome-wide the direct targets of SEPALLATA3. We used chromatin immunoprecipitation followed by ultrahigh-throughput sequencing or hybridization to whole-genome tiling arrays to obtain genome-wide DNA-binding patterns of SEPALLATA3. The results demonstrate that SEPALLATA3 binds to thousands of sites in the genome. Most potential target sites that were strongly bound in wild-type inflorescences are also bound in the floral homeotic agamous mutant, which displays only the perianth organs, sepals, and petals. Characterization of the target genes shows that SEPALLATA3 integrates and modulates different growth-related and hormonal pathways in a combinatorial fashion with other MADS-box proteins and possibly with non-MADS transcription factors. In particular, the results suggest multiple links between SEPALLATA3 and auxin signaling pathways. Our gene expression analyses link the genomic binding site data with the phenotype of plants expressing a dominant repressor version of SEPALLATA3, suggesting that it modulates auxin response to facilitate floral organ outgrowth and morphogenesis. Furthermore, the binding of the SEPALLATA3 protein to cis-regulatory elements of other MADS-box genes and expression analyses reveal that this protein is a key component in the regulatory transcriptional network underlying the formation of floral organs.
Author Summary
Most regulatory genes encode transcription factors, which modulate gene expression by binding to regulatory sequences of their target genes. In plants in particular, which genes are directly controlled by these transcription factors, and the molecular mechanisms of target gene recognition in vivo, are still largely unexplored. One of the best-understood developmental processes in plants is flower development. In different combinations, transcription factors of the MADS-box family control the identities of the different types of floral organs: sepals, petals, stamens, and carpels. Here, we present the first genome-wide analysis of binding sites of a MADS-box transcription factor in plants. We show that the MADS-domain protein SEPALLATA3 (SEP3) binds to the regulatory regions of thousands of potential target genes, many of which are also transcription factors. We provide insight into mechanisms of DNA recognition by SEP3, and suggest roles for other transcription factor families in SEP3 target gene regulation. In addition to effects on genes involved in floral organ identity, our data suggest that SEP3 binds to, and modulates, the transcription of target genes involved in hormonal signaling pathways.
The key floral regulator SEPALLATA3 binds to the promoters of a large number of potential direct target genes to integrate different growth-related and hormonal pathways in flower development.
PMCID: PMC2671559  PMID: 19385720
19.  High-Mobility Group Chromatin Proteins 1 and 2 Functionally Interact with Steroid Hormone Receptors To Enhance Their DNA Binding In Vitro and Transcriptional Activity in Mammalian Cells 
Molecular and Cellular Biology  1998;18(8):4471-4487.
We previously reported that the chromatin high-mobility group protein 1 (HMG-1) enhances the sequence-specific DNA binding activity of progesterone receptor (PR) in vitro, thus providing the first evidence that HMG-1 may have a coregulatory role in steroid receptor-mediated gene transcription. Here we show that HMG-1 and the highly related HMG-2 stimulate DNA binding by other steroid receptors, including estrogen, androgen, and glucocorticoid receptors, but have no effect on DNA binding by several nonsteroid nuclear receptors, including retinoid acid receptor (RAR), retinoic X receptor (RXR), and vitamin D receptor (VDR). As highly purified recombinant full-length proteins, all steroid receptors tested exhibited weak binding affinity for their optimal palindromic hormone response elements (HREs), and the addition of purified HMG-1 or -2 substantially increased their affinity for HREs. Purified RAR, RXR, and VDR also exhibited little to no detectable binding to their cognate direct repeat HREs but, in contrast to results with steroid receptors, the addition of HMG-1 or HMG-2 had no stimulatory effect. Instead, the addition of purified RXR enhanced RAR and VDR DNA binding through a heterodimerization mechanism and HMG-1 or HMG-2 had no further effect on DNA binding by RXR-RAR or RXR-VDR heterodimers. HMG-1 and HMG-2 (HMG-1/-2) themselves do not bind to progesterone response elements, but in the presence of PR they were detected as part of an HMG-PR-DNA ternary complex. HMG-1/-2 can also interact transiently in vitro with PR in the absence of DNA; however, no direct protein interaction was detected with VDR. These results, taken together with the fact that PR can bend its target DNA and that HMG-1/-2 are non-sequence-specific DNA binding proteins that recognize DNA structure, suggest that HMG-1/-2 are recruited to the PR-DNA complex by the combined effect of transient protein interaction and DNA bending. In transient-transfection assays, coexpression of HMG-1 or HMG-2 increased PR-mediated transcription in mammalian cells by as much as 7- to 10-fold without altering the basal promoter activity of target reporter genes. This increase in PR-mediated gene activation by coexpression of HMG-1/-2 was observed in different cell types and with different target promoters, suggesting a generality to the functional interaction between HMG-1/-2 and PR in vivo. Cotransfection of HMG-1 also increased reporter gene activation mediated by other steroid receptors, including glucocorticoid and androgen receptors, but it had a minimal influence on VDR-dependent transcription in vivo. These results support the conclusion that HMG-1/-2 are coregulatory proteins that increase the DNA binding and transcriptional activity of the steroid hormone class of receptors but that do not functionally interact with certain nonsteroid classes of nuclear receptors.
PMCID: PMC109033  PMID: 9671457
20.  The auxin signalling network translates dynamic input into robust patterning at the shoot apex 
We provide a comprehensive expression map of the different genes (TIR1/AFBs, ARFs and Aux/IAAs) involved in the signalling pathway regulating gene transcription in response to auxin in the shoot apical meristem (SAM).We demonstrate a relatively simple structure of this pathway using a high-throughput yeast two-hybrid approach to obtain the Aux/IAA-ARF full interactome.The topology of the signalling network was used to construct a model for auxin signalling and to predict a role for the spatial regulation of auxin signalling in patterning of the SAM.We used a new sensor to monitor the input in the auxin signalling pathway and to confirm the model prediction, thus demonstrating that auxin signalling is essential to create robust patterns at the SAM.
The plant hormone auxin is a key morphogenetic signal involved in the control of cell identity throughout development. A striking example of auxin action is at the shoot apical meristem (SAM), a population of stem cells generating the aerial parts of the plant. Organ positioning and patterning depends on local accumulations of auxin in the SAM, generated by polar transport of auxin (Vernoux et al, 2010). However, it is still unclear how auxin is distributed at cell resolution in tissues and how the hormone is sensed in space and time during development. A complex ensemble of 29 Aux/IAAs and 23 ARFs is central to the regulation of gene transcription in response to auxin (for review, see Leyser, 2006; Guilfoyle and Hagen, 2007; Chapman and Estelle, 2009). Protein–protein interactions govern the properties of this transduction pathway (Del Bianco and Kepinski, 2011). Limited interaction studies suggest that, in the absence of auxin, the Aux/IAA repressors form heterodimers with the ARF transcription factors, preventing them from regulating target genes. In the presence of auxin, the Aux/IAA proteins are targeted to the proteasome by an SCF E3 ubiquitin ligase complex (Chapman and Estelle, 2009; Leyser, 2006). In this process, auxin promotes the interaction between Aux/IAA proteins and the TIR1 F-box of the SCF complex (or its AFB homologues) that acts as an auxin co-receptor (Dharmasiri et al, 2005a, 2005b; Kepinski and Leyser, 2005; Tan et al, 2007). The auxin-induced degradation of Aux/IAAs would then release ARFs to regulate transcription of their target genes. This includes activation of most of the Aux/IAA genes themselves, thus establishing a negative feedback loop (Guilfoyle and Hagen, 2007). Although this general scenario provides a framework for understanding gene regulation by auxin, the underlying protein–protein network remains to be fully characterized.
In this paper, we combined experimental and theoretical analyses to understand how this pathway contributes to sensing auxin in space and time (Figure 1). We first analysed the expression patterns of the ARFs, Aux/IAAs and TIR1/AFBs genes in the SAM. Our results demonstrate a general tendency for most of the 25 ARFs and Aux/IAAs detected in the SAM: a differential expression with low levels at the centre of the meristem (where the stem cells are located) and high levels at the periphery of the meristem (where organ initiation takes place). We also observed a similar differential expression for TIR1/AFB co-receptors. To understand the functional significance of the distribution of ARFs and Aux/IAAs in the SAM, we next investigated the global structure of the Aux/IAA-ARF network using a high-throughput yeast two-hybrid approach and uncover a rather simple topology that relies on three basic generic features: (i) Aux/IAA proteins interact with themselves, (ii) Aux/IAA proteins interact with ARF activators and (iii) ARF repressors have no or very limited interactions with other proteins in the network.
The results of our interaction analysis suggest a model for the Aux/IAA-ARF signalling pathway in the SAM, where transcriptional activation by ARF activators would be negatively regulated by two independent systems, one involving the ARF repressors, the other the Aux/IAAs. The presence of auxin would remove the inhibitory action of Aux/IAAs, but leave the ARF repressors to compete with ARF activators for promoter-binding sites. To explore the regulatory properties of this signalling network, we developed a mathematical model to describe the transcriptional output as a function of the signalling input that is the combinatorial effect of auxin concentration and of its perception. We then used the model and a simplified view of the meristem (where the same population of Aux/IAAs and ARFs exhibit a low expression at the centre and a high expression in the peripheral zone) for investigating the role of auxin signalling in SAM function. We show that in the model, for a given ARF activator-to-repressor ratio, the gene induction capacity increases with the absolute levels of ARF proteins. We thus predict that the differential expression of the ARFs generates differences in auxin sensitivities between the centre (low sensitivity) and the periphery (high sensitivity), and that the expression of TIR1/AFB participates to this regulation (prediction 1). We also use the model to analyse the transcriptional response to rapidly changing auxin concentrations. By simulating situations equivalent either to the centre or the periphery of our simplified representation of the SAM, we predict that the signalling pathway buffers its response to the auxin input via the balance between ARF activators and repressors, in turn generated by their differential spatial distributions (prediction 2).
To test the predictions from the model experimentally, we needed to assess both the input (auxin level and/or perception) and the output (target gene induction) of the signalling cascade. For measuring the transcriptional output, the widely used DR5 reporter is perfectly adapted (Figure 5) (Ulmasov et al, 1997; Sabatini et al, 1999; Benkova et al, 2003; Heisler et al, 2005). For assaying pathway input, we designed DII-VENUS, a novel auxin signalling sensor that comprises a constitutively expressed fusion of the auxin-binding domain (termed domain II or DII) (Dreher et al, 2006; Tan et al, 2007) of an IAA to a fast-maturating variant of YFP, VENUS (Figure 5). The degradation patterns from DII-VENUS indicate a high auxin signalling input both in flower primordia and at the centre of the SAM. This is in contrast to the organ-specific expression pattern of DR5::VENUS (Figure 5). These results indicate that the signalling pathway limits gene activation in response to auxin at the meristem centre and confirm the differential sensitivity to auxin between the centre and the periphery (prediction 1). We further confirmed the buffering capacities of the signalling pathway (prediction 2) by carrying out live imaging experiments to monitor DII-VENUS and DR5::VENUS expression in real time (Figure 5). This analysis reveals the presence of important temporal variations of DII-VENUS fluorescence, while DR5::VENUS does not show such global variations. Our approach thus provides evidence that the Aux/IAA-ARF pathway has a key role in patterning in the SAM, alongside the auxin transport system. Our results illustrate how the tight spatio-temporal regulation of both the distribution of a morphogenetic signal and the activity of the downstream signalling pathway provides robustness to a dynamic developmental process.
A comprehensive expression and interaction map of auxin signalling factors in the Arabidopsis shoot apical meristem is constructed and used to derive a mathematical model of auxin signalling, from which key predictions are experimentally confirmed.
The plant hormone auxin is thought to provide positional information for patterning during development. It is still unclear, however, precisely how auxin is distributed across tissues and how the hormone is sensed in space and time. The control of gene expression in response to auxin involves a complex network of over 50 potentially interacting transcriptional activators and repressors, the auxin response factors (ARFs) and Aux/IAAs. Here, we perform a large-scale analysis of the Aux/IAA-ARF pathway in the shoot apex of Arabidopsis, where dynamic auxin-based patterning controls organogenesis. A comprehensive expression map and full interactome uncovered an unexpectedly simple distribution and structure of this pathway in the shoot apex. A mathematical model of the Aux/IAA-ARF network predicted a strong buffering capacity along with spatial differences in auxin sensitivity. We then tested and confirmed these predictions using a novel auxin signalling sensor that reports input into the signalling pathway, in conjunction with the published DR5 transcriptional output reporter. Our results provide evidence that the auxin signalling network is essential to create robust patterns at the shoot apex.
PMCID: PMC3167386  PMID: 21734647
auxin; biosensor; live imaging; ODE; signalling
21.  Cryptocephal, the Drosophila melanogaster ATF4, Is a Specific Coactivator for Ecdysone Receptor Isoform B2 
PLoS Genetics  2012;8(8):e1002883.
The ecdysone receptor is a heterodimer of two nuclear receptors, the Ecdysone receptor (EcR) and Ultraspiracle (USP). In Drosophila melanogaster, three EcR isoforms share common DNA and ligand-binding domains, but these proteins differ in their most N-terminal regions and, consequently, in the activation domains (AF1s) contained therein. The transcriptional coactivators for these domains, which impart unique transcriptional regulatory properties to the EcR isoforms, are unknown. Activating transcription factor 4 (ATF4) is a basic-leucine zipper transcription factor that plays a central role in the stress response of mammals. Here we show that Cryptocephal (CRC), the Drosophila homolog of ATF4, is an ecdysone receptor coactivator that is specific for isoform B2. CRC interacts with EcR-B2 to promote ecdysone-dependent expression of ecdysis-triggering hormone (ETH), an essential regulator of insect molting behavior. We propose that this interaction explains some of the differences in transcriptional properties that are displayed by the EcR isoforms, and similar interactions may underlie the differential activities of other nuclear receptors with distinct AF1-coactivators.
Author Summary
Nuclear receptors are proteins that regulate gene expression in response to steroid and thyroid hormones and other small lipid-soluble signaling molecules. In many cases, nuclear receptor genes encode multiple variants (isoforms) that direct tissue- and stage-specific hormonal responses. The sequence differences among isoforms are often found at the protein N-terminus, which mediates hormone-independent interactions with unknown regulatory partners to control target gene expression. Here, we show that the fruit fly Cryptocephal (CRC) protein is a specific coactivator for one of three isoforms of the receptor for the insect molting steroid, ecdysone. Our findings reveal a mechanism for differential activation of gene expression in response to ecdysone during insect molting and metamorphosis, and contribute to our understanding of isoform-specific functions of nuclear hormone receptors.
PMCID: PMC3415445  PMID: 22912598
22.  Unraveling condition-dependent networks of transcription factors that control metabolic pathway activity in yeast 
While typically many expression levels change in transcription factor mutants, only few of these changes lead to functional changes. The predictive capability of expression and DNA binding data for such functional changes in metabolism is very limited.Large-scale 13C-flux data reveal the condition specificity of transcriptional control of metabolic function.Transcription control in yeast focuses on the switch between respiration and fermentation.Follow-up modeling on the basis of transcriptomics and proteomics data suggest the newly discovered Gcn4 control of respiration to be mediated via PKA and Snf1.
Effective control and modulation of cellular behavior is of paramount importance in medicine (Kreeger and Lauffenburger, 2010) and biotechnology (Haynes and Silver, 2009), and requires profound understanding of control mechanisms. In this study, we aim to elucidate the extent to which transcription factors control the operation of yeast metabolism. As a quantitative readout of metabolic function, we monitored the traffic of small molecules through various pathways of central metabolism by 13C-flux analysis (Sauer, 2006). The choosen growth conditions represent two different regulatory states of reduced (galactose) and maximal carbon source repression (glucose), as well as a different nitrogen metabolism and two common, permanent stress conditions.
Depending on the growth condition, between 7 and 13% of the deleted transcription factors altered the determined flux ratios (Figure 3). Of the six quantified flux ratios, only the glycolysis/pentose phosphate pathway split, and the convergent ratio of anaplerosis and TCA cycle were controlled by the deleted transcription factors. Thus, we concluded that 23 transcription factors control flux distributions under at least one of the tested growth conditions, leading to 42 condition-dependent interactions of transcription factors with metabolic pathway activity (Figure 4). With two exceptions, all other identified transcription factors interactions controlled the TCA cycle flux. This condition-specific active control of metabolic function could not have been predicted from DNA binding and expression data; that is, 26.1% false negatives, 48.6% true positives.
Of the 23 transcription factors that controlled TCA cycle flux distributions under the tested conditions, only Bas1, Gcn4, Gcr2 and Pho2 exerted control under more than one condition. We identified Cit1, Mdh1 and Idh1/2 with a proteomics approach as the relevant target enzyme that increase the TCA cycle flux. Next, we asked whether Bas1, Gcr2, Gcn4 and Pho2 act directly on the TCA cycle or mediate their effect indirectly. Based on the transcriptomics data, the pattern of differentially activated transcription factors inferred by the differential expression of their target genes suggested reduced glucose repression in all four mutants as the common mechanism.
Starting from the currently largest set of 13C-based flux distributions, we identified networks of individual transcription factors that control metabolic pathway activity. These networks of active metabolic control have the following properties. First, they are highly condition dependent, as at most four transcription factors control the same metabolic flux distribution under more than one growth conditions. Second, they focus almost exclusively on the TCA cycle, thereby controlling the switch between respiratory and fermentative metabolism. Third, with four to 14 active transcription factors, they are small compared with gene regulation networks that were obtained from expression and DNA binding data. For the metabolic network studied here, robustness is also apparent from the fact that upregulated TCA cycle fluxes were not sufficient to achieve full respiratory metabolism; that is, absent or low ethanol formation. Several explanations could potentially explain the observed robustness. The most likely explanation is that environmental signals might be transmitted by different signaling pathways to several transcription factors, whose orchestrated action on multiple target genes is necessary to achieve a functional flux response. This hypothesis would explain why several transcription factors exert flux effects on the same pathway, but each flux effect is relatively small, as further, coordinated manipulations would be necessary to further improve the respiratory flux. Our findings demonstrate the importance of identifying and quantifying the extent to which regulatory effectors alter cellular function.
Which transcription factors control the distribution of metabolic fluxes under a given condition? We address this question by systematically quantifying metabolic fluxes in 119 transcription factor deletion mutants of Saccharomyces cerevisiae under five growth conditions. While most knockouts did not affect fluxes, we identified 42 condition-dependent interactions that were mediated by a total of 23 transcription factors that control almost exclusively the cellular decision between respiration and fermentation. This relatively sparse, condition-specific network of active metabolic control contrasts with the much larger gene regulation network inferred from expression and DNA binding data. Based on protein and transcript analyses in key mutants, we identified three enzymes in the tricarboxylic acid cycle as the key targets of this transcriptional control. For the transcription factor Gcn4, we demonstrate that this control is mediated through the PKA and Snf1 signaling cascade. The discrepancy between flux response predictions, based on the known regulatory network architecture and our functional 13C-data, demonstrates the importance of identifying and quantifying the extent to which regulatory effectors alter cellular functions.
PMCID: PMC3010106  PMID: 21119627
metabolic flux; omics data; regulatory network; transcription factor; transcriptional regulation
23.  Dual Requirement for the EcR/USP Nuclear Receptor and the dGATAb Factor in an Ecdysone Response in Drosophila melanogaster 
Molecular and Cellular Biology  1999;19(8):5732-5742.
The EcR/USP nuclear receptor controls Drosophila metamorphosis by activating complex cascades of gene transcription in response to pulses of the steroid hormone ecdysone at the end of larval development. Ecdysone release provides a ubiquitous signal for the activation of the receptor, but a number of its target genes are induced in a tissue- and stage-specific manner. Little is known about the molecular mechanisms involved in this developmental modulation of the EcR/USP-mediated pathway. Fbp1 is a good model of primary ecdysone response gene expressed in the fat body for addressing this question. We show here that the dGATAb factor binds to three target sites flanking an EcR/USP binding site in a 70-bp enhancer that controls the tissue and stage specificity of Fbp1 transcription. We demonstrate that one of these sites and proper expression of dGATAb are required for specific activation of the enhancer in the fat body. In addition, we provide further evidence that EcR/USP plays an essential role as a hormonal timer. Our study provides a striking example of the integration of molecular pathways at the level of a tissue-specific hormone response unit.
PMCID: PMC84424  PMID: 10409761
24.  Target Gene Analysis by Microarrays and Chromatin Immunoprecipitation Identifies HEY Proteins as Highly Redundant bHLH Repressors 
PLoS Genetics  2012;8(5):e1002728.
HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.
Author Summary
NOTCH signaling is a central developmental pathway that influences a multitude of cell fate decisions and differentiation steps as well as later tissue homeostasis and regeneration. The three HEY genes encode basic helix-loop-helix transcription factors that are critical effectors to convey signaling by NOTCH receptors and similar signaling systems. This is underscored by the multitude of developmental defects observed in HEY single- and double-mutant mice. The mode of action of HEY proteins remained largely unexplored, however. By gene expression analysis and chromatin immunoprecipitation we have now identified a large set of HEY target genes. While only 500–2,000 mRNAs are regulated by HEY1 or HEY2, there are around 10,000 binding sites in the genome. HEY proteins act as transcriptional repressors that bind close to transcriptional start sites in all cases tested. In contrast, gene activation seems to be mediated by indirect/secondary mechanisms. The extent of regulation is rather limited, implicating HEY genes in modulating rather than switching on or off target gene expression. All our in vitro and in vivo data point to a high degree of redundancy between the three HEY genes, suggesting that tissue specific patterns and expression levels determine the final outcome of HEY induced cellular responses.
PMCID: PMC3355086  PMID: 22615585
25.  Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis 
eLife  2013;2:e00675.
The gaseous plant hormone ethylene regulates a multitude of growth and developmental processes. How the numerous growth control pathways are coordinated by the ethylene transcriptional response remains elusive. We characterized the dynamic ethylene transcriptional response by identifying targets of the master regulator of the ethylene signaling pathway, ETHYLENE INSENSITIVE3 (EIN3), using chromatin immunoprecipitation sequencing and transcript sequencing during a timecourse of ethylene treatment. Ethylene-induced transcription occurs in temporal waves regulated by EIN3, suggesting distinct layers of transcriptional control. EIN3 binding was found to modulate a multitude of downstream transcriptional cascades, including a major feedback regulatory circuitry of the ethylene signaling pathway, as well as integrating numerous connections between most of the hormone mediated growth response pathways. These findings provide direct evidence linking each of the major plant growth and development networks in novel ways.
eLife digest
All multicellular organisms, including plants, produce hormones—chemical messengers that are released in one part of an organism but act in another. The binding of hormones to receptor proteins on the surface of target cells activates signal transduction cascades, leading ultimately to changes in the transcription and translation of genes.
Ethylene is a gaseous plant hormone that acts at trace levels to stimulate or regulate a variety of processes, including the regulation of plant growth, the ripening of fruit and the shedding of leaves. Plants also produce ethylene in response to wounding, pathogen attack or exposure to environmental stresses, such as extreme temperatures or drought. Although the effects of ethylene on plants are well documented, much less is known about how its functions are controlled and coordinated at the molecular level.
Here, Chang et al. reveal how ethylene alters the transcription of DNA into messenger DNA (mRNA) in the plant model organism, Arabidopsis thaliana. Ethylene is known to exert some of its effects via a protein called EIN3, which is a transcription factor that acts as the master regulator of the ethylene signaling pathway. To identify the targets of EIN3, Chang et al. exposed plants to ethylene and then used a technique called ChIP-Seq to identify those regions of the DNA that EIN3 binds to. At the same time, they used genome-wide mRNA sequencing to determine which genes showed altered transcription.
Over the course of 24 hr, ethylene induced four distinct waves of transcription, suggesting that discrete layers of transcriptional control are present. EIN3 binding also controlled a multitude of downstream transcriptional cascades, including a major negative feedback loop. Surprisingly, many of the genes that showed altered expression in response to EIN3 binding were also influenced by hormones other than ethylene.
In addition to extending our knowledge of the role of EIN3 in coordinating the effects of ethylene, the work of Chang et al. reveals the extensive connectivity between pathways regulated by distinct hormones in plants. The results may also make it easier to identify key players involved in hormone signaling pathways in other plant species.
PMCID: PMC3679525  PMID: 23795294
transcriptional regulation; temporal modulation; network; ethylene; hormone; Physcomitrella patens; Arabidopsis

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