Central regulators of cell fate, or selector genes, establish the identity of cells by direct regulation of large cohorts of genes. In Caenorhabditis elegans, foregut (or pharynx) identity relies on the FoxA transcription factor PHA-4, which activates different sets of target genes at various times and in diverse cellular environments. An outstanding question is how PHA-4 distinguishes between target genes for appropriate transcriptional control. We have used the Nuclear Spot Assay and GFP reporters to examine PHA-4 interactions with target promoters in living embryos and with single cell resolution. While PHA-4 was found throughout the digestive tract, binding and activation of pharyngeally expressed promoters was restricted to a subset of pharyngeal cells and excluded from the intestine. An RNAi screen of candidate nuclear factors identified emerin (emr-1) as a negative regulator of PHA-4 binding within the pharynx, but emr-1 did not modulate PHA-4 binding in the intestine. Upon promoter association, PHA-4 induced large-scale chromatin de-compaction, which, we hypothesize, may facilitate promoter access and productive transcription. Our results reveal two tiers of PHA-4 regulation. PHA-4 binding is prohibited in intestinal cells, preventing target gene expression in that organ. PHA-4 binding within the pharynx is limited by the nuclear lamina component EMR-1/emerin. The data suggest that association of PHA-4 with its targets is a regulated step that contributes to promoter selectivity during organ formation. We speculate that global re-organization of chromatin architecture upon PHA-4 binding promotes competence of pharyngeal gene transcription and, by extension, foregut development.
Central regulators of cell fate establish the identity of cells by direct regulation of large cohorts of genes. In Caenorhabditis elegans, foregut (or pharynx) identity relies on the FoxA transcription factor PHA-4, which activates different target genes in different cellular environments. An outstanding question is how PHA-4 distinguishes between target genes for appropriate transcriptional control. Here we examine PHA-4 interactions with target promoters in living embryos and with single-cell resolution. While PHA-4 was found throughout the digestive tract, binding and activation of pharyngeally expressed promoters was restricted to a subset of pharyngeal cells and excluded from the intestine. An RNAi screen identified emerin (emr-1) as a negative regulator of PHA-4 binding within the pharynx. Upon promoter association, PHA-4 induced large-scale chromatin de-compaction, which, we hypothesize, facilitates promoter access. Our results reveal two tiers of PHA-4 regulation. PHA-4 binding is prohibited in intestinal cells and is limited in the pharynx by the nuclear lamina component EMR-1/emerin. The data suggest that association of PHA-4 with its targets is a regulated step that contributes to promoter selectivity during organ formation. We speculate that global re-organization of chromatin architecture upon PHA-4 binding promotes competence of pharyngeal gene transcription and, by extension, foregut development.
We have investigated the cis-regulatory network that mediates temporal gene expression during organogenesis. Previous studies demonstrated that the organ selector gene pha-4/FoxA is critical to establish the onset of transcription of Caenorhabditis elegans foregut (pharynx) genes. Here, we discover additional cis-regulatory elements that function in combination with PHA-4. We use a computational approach to identify candidate cis-regulatory sites for genes activated either early or late during pharyngeal development. Analysis of natural or synthetic promoters reveals that six of these sites function in vivo. The newly discovered temporal elements, together with predicted PHA-4 sites, account for the onset of expression of roughly half of the pharyngeal genes examined. Moreover, combinations of temporal elements and PHA-4 sites can be used in genome-wide searches to predict pharyngeal genes, with more than 85% accuracy for their onset of expression. These findings suggest a regulatory code for temporal gene expression during foregut development and provide a means to predict gene expression patterns based solely on genomic sequence.
Computational analysis combined with validation by reporter gene studies is uncovering the code for temporal gene regulation in the C. elegans foregut - a model for organogenesis
The Caenorhabditis elegans pharynx (or foregut) functions as a pump that draws in food (bacteria) from the environment. While the “organ identity factor” PHA-4 is critical for formation of the C. elegans pharynx as a whole, little is known about the specification of distinct cell types within the pharynx. Here, we use a combination of bioinformatics, molecular biology, and genetics to identify a helix-loop-helix transcription factor (HLH-6) as a critical regulator of pharyngeal gland development. HLH-6 is required for expression of a number of gland-specific genes, acting through a discrete cis-regulatory element named PGM1 (Pharyngeal Gland Motif 1). hlh-6 mutants exhibit a frequent loss of a subset of glands, while the remaining glands have impaired activity, indicating a role for hlh-6 in both gland development and function. Interestingly, hlh-6 mutants are also feeding defective, ascribing a biological function for the glands. Pharyngeal pumping in hlh-6 mutants is normal, but hlh-6 mutants lack expression of a class of mucin-related proteins that are normally secreted by pharyngeal glands and line the pharyngeal cuticle. An interesting possibility is that one function of pharyngeal glands is to secrete a pharyngeal lining that ensures efficient transport of food along the pharyngeal lumen.
To make an organ, cells must be instructed to be part of a common structure yet must also be assigned specific roles or identities within that structure. For example, the stomach contains a variety of different kinds of cells, including muscles, nerves, and glands. This same complexity is seen even in relatively simple organs, like the pharynx (foregut) of the nematode C. elegans. The pharynx is a neuromuscular organ that pumps in food (bacteria) from the environment. This organ is relatively simple (containing only 80 cells) yet contains five distinct kinds of cells. How these different cells are specified is unclear but likely involves combinations of developmental regulators known as transcription factors. Here, we examine one cell type, the pharyngeal glands, and identify a key regulator of their development, the transcription factor HLH-6. Interestingly, HLH-6 is closely related to a mammalian transcription factor, Sgn1, which is involved in development of mammalian salivary glands, suggesting that C. elegans pharyngeal glands are evolutionarily related to mammalian salivary glands. A further connection is that the pharyngeal glands of C. elegans appear to be required for efficient feeding, possibly by secreting mucin-like proteins that ensure the smooth passage of food along the digestive tract.
pha-4 encodes a forkhead box (FOX) A transcription factor serving as the C. elegans pharynx organ identity factor during embryogenesis. Using Serial Analysis of Gene Expression (SAGE), comparison of gene expression profiles between growing stages animals and long-lived, developmentally diapaused dauer larvae revealed that pha-4 transcription is increased in the dauer stage.
Knocking down pha-4 expression by RNAi during post-embryonic development showed that PHA-4 is essential for dauer recovery, gonad and vulva development. daf-16, which encodes a FOXO transcription factor regulated by insulin/IGF-1 signaling, shows overlapping expression patterns and a loss-of-function post-embryonic phenotype similar to that of pha-4 during dauer recovery. pha-4 RNAi and daf-16 mutations have additive effects on dauer recovery, suggesting these two regulators may function in parallel pathways. Gene expression studies using RT-PCR and GFP reporters showed that pha-4 transcription is elevated under starvation, and a conserved forkhead transcription factor binding site in the second intron of pha-4 is important for the neuronal expression. The vulval transcription of lag-2, which encodes a ligand for the LIN-12/Notch lateral signaling pathway, is inhibited by pha-4 RNAi, indicating that LAG-2 functions downstream of PHA-4 in vulva development.
Analysis of PHA-4 during post-embryonic development revealed previously unsuspected functions for this important transcriptional regulator in dauer recovery, and may help explain the network of transcriptional control integrating organogenesis with the decision between growth and developmental arrest at the dauer entry and exit stages.
Dietary restriction (DR) is a robust means of extending adult lifespan and postponing age-related disease in many species, including yeast, worms, flies and rodents1,2. Studies of the genetic requirements for lifespan extension by DR in the nematode Caenorhabditis elegans (C. elegans) have implicated a number of key players in this process3–5, including the nutrient-sensing target of rapamycin (TOR) pathway6 and the Foxa transcription factor PHA-47. However, little is known about the metabolic signals that coordinate the organismal response to DR and maintain homeostasis when nutrients are limited. The endocannabinoid (EC) system is an excellent candidate to play such a role given its involvement in regulating nutrient intake and energy balance8. Despite this, a direct role for EC signaling in DR or lifespan determination has yet to be demonstrated, in part due to the apparent absence of EC signaling pathways in model organisms that are amenable to lifespan analysis9. N-acylethanolamines (NAEs) are lipid-derived signaling molecules, which include the mammalian EC arachidonoyl ethanolamide (AEA). Here we identify NAEs in C. elegans, show that NAE abundance is reduced under DR and that NAE deficiency is sufficient to extend lifespan through a DR mechanism requiring PHA-4. Conversely, dietary supplementation with the nematode NAE eicosapentaenoyl ethanolamide (EPEA) not only inhibits DR-induced lifespan extension in wild type animals, but also suppresses lifespan extension in a TOR pathway mutant. This demonstrates a role for NAE signaling in aging and suggests that NAEs represent a signal that coordinates nutrient status with metabolic changes that ultimately determine lifespan.
The histone variant H2A.Z is evolutionarily conserved and plays an essential role in mice, Drosophila, and Tetrahymena. The essential function of H2A.Z is unknown, with some studies suggesting a role in transcriptional repression and others in activation. Here we show that Caenorhabditis elegans HTZ-1/H2A.Z and the remodeling complex MYS-1/ESA1–SSL-1/SWR1 synergize with the FoxA transcription factor PHA-4 to coordinate temporal gene expression during foregut development. We observe dramatic genetic interactions between pha-4 and htz-1, mys-1, and ssl-1. A survey of transcription factors reveals that this interaction is specific, and thus pha-4 is acutely sensitive to reductions in these three proteins. Using a nuclear spot assay to visualize HTZ-1 in living embryos as organogenesis proceeds, we show that HTZ-1 is recruited to foregut promoters at the time of transcriptional onset, and this recruitment requires PHA-4. Loss of htz-1 by RNAi is lethal and leads to delayed expression of a subset of foregut genes. Thus, the effects of PHA-4 on temporal regulation can be explained in part by recruitment of HTZ-1 to target promoters. We suggest PHA-4 and HTZ-1 coordinate temporal gene expression by modulating the chromatin environment.
During development, a single fertilized egg gives rise to the different cell types within an embryo. These different cell types are characterized by the different genes that they express. A critical question in biology is how embryonic cells activate genes at the appropriate time and place to generate the different cell types. In this paper, the authors explore temporal regulation of gene expression during formation of an organ, namely the Caenorhabditis elegans foregut. They have discovered that foregut genes require a variant of the canonical H2A histone for the onset of transcription. This variant, called H2A.Z, or htz-1 in C. elegans, promotes transcription by modifying how DNA is packaged within cells. Their data suggest that a key regulator of foregut development, the transcription factor PHA-4, recruits HTZ-1 to pharyngeal promoters, and this association contributes to their timely activation.
Foxa is a forkhead transcription factor that is expressed in the endoderm lineage across metazoans. Orthologs of foxa are expressed in cells that intercalate, polarize and form tight junctions in the digestive tracts of the mouse, the sea urchin and the nematode and in the chordate notochord. The loss of foxa expression eliminates these morphogenetic processes. The remarkable similarity in foxa phenotypes in these diverse organisms raises the following questions – why is the developmental role of Foxa so highly conserved? Is foxa transcriptional regulation as conserved as its developmental role? Comparison of the regulation of foxa orthologs in sea urchin and in C. elegans shows that foxa transcriptional regulation has diverged significantly between these two organisms, particularly in the cells that contribute to the C. elegans pharynx formation. We suggest that the similarity of foxa phenotype is due to its role in an ancestral gene regulatory network that controlled intercalation followed by mesenchymal to epithelial transition. foxa transcriptional regulation had evolved to support the developmental program in each species so foxa would play its role controlling morphogenesis at the necessary embryonic address.
gene regulatory networks; cis-regulatory analysis; evolution; mesenchymal to epithelial transition
FoxA factors are critical regulators of embryonic development and post-embryonic life, but little is know about the upstream pathways that modulate their activity . C. elegans pha-4 encodes a FoxA transcription factor that is required to establish the foregut in embryos, and to control growth and longevity after birth [2–5]. We previously identified the AAA+ ATPase homologue ruvb-1 as a potent suppressor of pha-4 mutations .
Here we show that ruvb-1 is a component of the TOR pathway in C. elegans (CeTOR). Both ruvb-1 and let-363/TOR control nucleolar size and promote localization of box C/D snoRNPs to nucleoli, suggesting a role in rRNA maturation. Inactivation of let-363/TOR or ruvb-1 suppresses the lethality associated with reduced pha-4 activity. The CeTOR pathway controls protein homeostasis and also contributes to adult longevity [7, 8]. We find that pha-4 is required to extend adult lifespan in response to reduced CeTOR signaling. Mutations in the predicted CeTOR target rsks-1/S6 kinase or in ife-2/eIF4E also reduce protein biosynthesis and extend lifespan [9–11], but only rsks-1 mutations require pha-4 for adult longevity. In addition, rsks-1, but not ife-2, can suppress the larval lethality associated with pha-4 loss-of-function mutations.
The data suggest that pha-4 and the CeTOR pathway antagonize one another to regulate post-embryonic development and adult longevity. We suggest a model in which nutrients promote TOR and S6 kinase signaling, which represses pha-4/FoxA, leading to a shorter lifespan. A similar regulatory hierarchy may function in other animals to modulate metabolism, longevity or disease.
A key question in development is how pluripotent progenitors are progressively restricted to acquire specific cell fates. Here we investigate how embryonic blastomeres in C. elegans develop into foregut (pharynx) cells in response to the selector gene PHA-4/FoxA. When pha-4 is removed from pharyngeal precursors, they exhibit two alternative responses. Before late-gastrulation (8E stage), these cells lose their pharyngeal identity and acquire an alternative fate such as ectoderm (Specification stage). After the Specification stage, mutant cells develop into aberrant pharyngeal cells (Morphogenesis/Differentiation stage). Two lines of evidence suggest that the Specification stage depends on transcriptional repression of ectodermal genes by pha-4. First, pha-4 exhibits strong synthetic phenotypes with the B class synMuv gene tam-1 (Tandam Array expression Modifier 1) and with a mediator of transcriptional repression, the NuRD complex (NUcleosome Remodeling and histone Deacetylase). Second, pha-4 associates with the promoter of the ectodermal regulator lin-26 and is required to repress lin-26 expression. We propose that restriction of early blastomeres to the pharyngeal fate depends on both repression of ectodermal genes and activation of pharyngeal genes by PHA-4.
embryogenesis; Mi-2; organogenesis; pharynx; synMuv; selector gene
Bacillus megaterium can produce poly-β-hydroxybutyrate (PHB) as carbon and energy storage materials. We now report that the phaQ gene, which is located upstream of the phasin-encoding phaP gene, codes for a new class of transcriptional regulator that negatively controls expression of both phaQ and phaP. A PhaQ binding site that plays a role in this control has been identified by gel mobility shift assays and DNase I footprinting analysis. We have also provided evidence that PhaQ could sense the presence of PHB in vivo and that artificial PHB granules could inhibit the formation of PhaQ-DNA complex in vitro by binding to PhaQ directly. These suggest that PhaQ is a PHB-responsive repressor.
Based on metabolic and morphological similarities between infective third-stage larvae of parasitic nematodes and dauer larvae of Caenorhabditis elegans, it is hypothesized that similar genetic mechanisms control the development of these forms. In the parasite Strongyloides stercoralis, FKTF-1 is an ortholog of DAF-16, a forkhead transcription factor that regulates dauer larval development in C. elegans. Using transgenesis, we investigated the role of FKTF-1 in S. stercoralis' infective larval development. In first-stage larvae, GFP-tagged recombinant FKTF-1b localizes to the pharynx and hypodermis, tissues remodeled in infective larvae. Activating and inactivating mutations at predicted AKT phosphorylation sites on FKTF-1b give constitutive cytoplasmic and nuclear localization of the protein, respectively, indicating that its post-translational regulation is similar to other FOXO-class transcription factors. Mutant constructs designed to interfere with endogenous FKTF-1b function altered the intestinal and pharyngeal development of the larvae and resulted in some transgenic larvae failing to arrest in the infective stage. Our findings indicate that FKTF-1b is required for proper morphogenesis of S. stercoralis infective larvae and support the overall hypothesis of similar regulation of dauer development in C. elegans and the formation of infective larvae in parasitic nematodes.
Parasitic nematodes are an important threat to public health in much of the world. Understanding how these worms find and invade their hosts may lead to improved therapies. The infectious forms of many parasitic nematodes developmentally arrest as infective third-stage larvae that require hosts to reactivate. Development of these larvae has been compared to that of the diapausing dauer larvae of Caenorhabditis elegans. Our lab studies the development of the human nematode parasite Strongyloides stercoralis. We identified S. stercoralis' FKTF-1 as an ortholog of DAF-16, a forkhead transcription factor controlling dauer larval development in C. elegans. Transgenes were introduced into S. stercoralis to investigate the possibility that FKTF-1 regulates development of its infective larvae. We discovered that recombinant FKTF-1b tagged with GFP localizes to specific tissues remodeled in infective larvae. Furthermore, mutant forms of FKTF-1b designed to interfere with endogenous FKTF-1b function resulted in incomplete development of the infective larval structures and prevented some transgenic larvae from arresting in the infective stage. Indicating that FKTF-1b is required for the proper development of Strongyloides stercoralis infective larvae, our findings support the hypothesis of similar controls over parasitic and free-living nematode development and pave the way for further comparative studies.
Foxa2 is a critical transcription factor that controls liver development and plays an important role in hepatic gluconeogensis in adult mice. Here, we use genome-wide location analysis for Foxa2 to identify its targets in the adult liver. We then show by computational analyses that Foxa2 containing cis-regulatory modules are not constructed from a random assortment of binding sites for other transcription factors expressed in the liver, but rather that their composition depends on the strength of the Foxa2 consensus site present. Genes containing a cis-regulatory module with a medium or weak Foxa2 consensus site are much more liver-specific than the genes with a strong consensus site. We not only provide a better understanding of the mechanisms of Foxa2 regulation but also introduce a novel method for identification of different cis-regulatory modules involving a single factor.
All known genomes code for a large number of transcription factors. It is important to develop methods that will reveal how these transcription factors act on a genome wide level, that is, through what target genes they exert their function.
We describe here a program pipeline aimed at identifying transcription factor target genes in whole genomes. Starting from a consensus binding site, represented as a weight matrix, potential sites in a pre-filtered genome are identified and then further filtered by assessing conservation of the putative site in the genome of a related species, a process called phylogenetic footprinting. CisOrtho has been successfully used to identify targets for two homeodomain transcription factors in the genomes of the nematodes Caenorhabditis elegans and Caenorhabditis briggsae.
CisOrtho will identify targets of other nematode transcription factors whose DNA binding specificity is known and can be easily adapted to search other genomes for transcription factor targets.
Dietary Restriction (DR) extends longevity in diverse species suggesting that there is a conserved mechanism for nutrient regulation and prosurvival responses1. We have discovered a role for the HECT E3 ubiquitin ligase WWP-1 as a positive regulator of lifespan in C. elegans in response to diet restriction. We find that overexpression of wwp-1 in worms extends lifespan up to 20% under conditions of ad libitum feeding. This extension is dependent upon the FoxA transcription factor pha-4, and independent of the FoxO transcription factor, daf-16. Reduction of wwp-1 completely suppresses the extended longevity of diet-restricted animals. However, loss of wwp-1 does not affect the long lifespan of animals with compromised mitochondrial function or reduced insulin/IGF-1 signaling. Overexpression of a mutant form of WWP-1 lacking catalytic activity suppresses the increased lifespan of diet-restricted animals, indicating that WWP-1 ubiquitin ligase activity is essential for longevity. Additionally, we find that the E2 ubiquitin conjugating enzyme, UBC-18, is essential and specific for DR induced longevity. UBC-18 interacts with WWP-1 and is required for the ubiquitin ligase activity of WWP-1 and the extended longevity of worms overexpressing wwp-1. Taken together, our results indicate that WWP-1 and UBC-18 function to ubiquitinate substrates that regulate DR induced longevity.
The provision of stress resistance diverts resources from development and reproduction and must therefore be tightly regulated. In Caenorhabditis elegans, the switch to increased stress resistance to promote survival through periods of starvation is regulated by the DAF-16/FOXO transcription factor. Reduction-of-function mutations in AGE-1, the C. elegans Class IA phosphoinositide 3-kinase (PI3K), increase lifespan and stress resistance in a daf-16 dependent manner. Class IA PI3Ks downregulate FOXOs by inducing their translocation to the cytoplasm. However, the circumstances under which AGE-1 is normally activated are unclear. To address this question we used C. elegans first stage larvae (L1s), which when starved enter a developmentally-arrested diapause stage until food is encountered.
We find that in L1s both starvation and daf-16 are necessary to confer resistance to oxidative stress in the form of hydrogen peroxide. Accordingly, DAF-16 is localised to cell nuclei after short-term starvation. However, after long-term starvation, DAF-16 unexpectedly translocates to the cytoplasm. This translocation requires functional age-1. H2O2 treatment can replicate the translocation and induce generation of the AGE-1 product PIP3. Because feeding reduces to zero in ageing adult C. elegans, these animals may also undergo long-term starvation. Consistent with our observation in L1s, DAF-16 also translocates to the cytoplasm in old adult worms in an age-1-dependent manner.
DAF-16 is activated in the starved L1 diapause. The translocation of DAF-16 to the cytoplasm after long-term starvation may be a feedback mechanism that prevents excessive expenditure on stress resistance. H2O2 is a candidate second messenger in this feedback mechanism. The lack of this response in age-1(hx546) mutants suggests a novel mechanism by which this mutation increases longevity.
Transcriptional regulation, a primary mechanism for controlling the development of multicellular organisms, is carried out by transcription factors (TFs) that recognize and bind to their cognate binding sites. In Caenorhabditis elegans, our knowledge of which genes are regulated by which TFs, through binding to specific sites, is still very limited. To expand our knowledge about the C. elegans regulatory network, we performed a comprehensive analysis of the C. elegans, Caenorhabditis briggsae, and Caenorhabditis remanei genomes to identify regulatory elements that are conserved in all genomes. Our analysis identified 4959 elements that are significantly conserved across the genomes and that each occur multiple times within each genome, both hallmarks of functional regulatory sites. Our motifs show significant matches to known core promoter elements, TF binding sites, splice sites, and poly-A signals as well as many putative regulatory sites. Many of the motifs are significantly correlated with various types of experimental data, including gene expression patterns, tissue-specific expression patterns, and binding site location analysis as well as enrichment in specific functional classes of genes. Many can also be significantly associated with specific TFs. Combinations of motif occurrences allow us to predict the location of cis-regulatory modules and we show that many of them significantly overlap experimentally determined enhancers. We provide access to the predicted binding sites, their associated motifs, and the predicted cis-regulatory modules across the whole genome through a web-accessible database and as tracks for genome browsers.
cis-regulatory element; cis-regulatory module; transcription factor; transcriptional regulation; Caenorhabditis elegans
Availability of food is often a limiting factor in nature. Periods of food abundance are followed by times of famine, often in unpredictable patterns. Reliable information about the environment is a critical ingredient of successful survival strategy. One way to improve accuracy is to integrate information communicated by other organisms. To test whether such exchange of information may play a role in determining starvation survival strategies, we studied starvation of L1 larvae in C. elegans and other Caenorhabditis species. We found that some species in genus Caenorhabditis, including C. elegans, survive longer when starved at higher densities, while for others survival is independent of the density. The density effect is mediated by chemical signal(s) that worms release during starvation. This starvation survival signal is independent of ascarosides, a class of small molecules widely used in chemical communication of C. elegans and other nematodes.
Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.
Gene duplication is a powerful driver of evolution. Newly duplicated genes acquire new roles that are relevant to fitness, or they will be lost over time. A potential path to functional relevance is mutation of the coding sequence leading to the acquisition of novel biochemical properties, as analyzed here for the highly homologous paralogs Foxa1 and Foxa2 transcriptional regulators. We determine by genome-wide location analysis (ChIP-Seq) that, although Foxa1 and Foxa2 share a large fraction of binding sites in the liver, each protein also occupies distinct regulatory elements in vivo. Foxa1-only sites are enriched for p53 binding sites and are frequently found near genes important to cell cycle regulation, while Foxa2-restricted sites show only a limited match to the forkhead consensus and are found in genes involved in steroid and lipid metabolism. Thus, Foxa1 and Foxa2, while redundant during development, have evolved divergent roles in the adult liver, ensuring the maintenance of both genes during evolution.
The duplication of a gene from a common ancestor, resulting in two copies known as paralogs, plays an important role in evolution. Newly duplicated genes must acquire new functions in order to remain relevant, otherwise they are lost via mutation over time. We have performed genome-wide location analysis (ChIP–Seq) in adult liver to examine the differences between two paralogous DNA binding proteins, Foxa1 and Foxa2. While Foxa1 and Foxa2 bind a number of common genomic locations, each protein also localizes to distinct regulatory regions. Sites specific for Foxa1 also contain a DNA motif bound by tumor suppressor p53 and are found near genes important to cell cycle regulation, while Foxa2-only sites are found near genes essential to steroid and lipid metabolism. Hence, Foxa1 and Foxa2 have developed unique functions in adult liver, contributing to the maintenance of both genes during evolution.
DRM is a conserved transcription factor complex that includes E2F/DP and pRB
family proteins and plays important roles in development and cancer. Here we
describe new aspects of DRM binding and function revealed through genome-wide
analyses of the Caenorhabditis elegans DRM subunit LIN-54. We
show that LIN-54 DNA-binding activity recruits DRM to promoters enriched for
adjacent putative E2F/DP and LIN-54 binding sites, suggesting that these two
DNA–binding moieties together direct DRM to its target genes. Chromatin
immunoprecipitation and gene expression profiling reveals conserved roles for
DRM in regulating genes involved in cell division, development, and
reproduction. We find that LIN-54 promotes expression of reproduction genes in
the germline, but prevents ectopic activation of germline-specific genes in
embryonic soma. Strikingly, C. elegans DRM does not act
uniformly throughout the genome: the DRM recruitment motif, DRM binding, and
DRM-regulated embryonic genes are all under-represented on the X chromosome.
However, germline genes down-regulated in lin-54 mutants are
over-represented on the X chromosome. We discuss models for how loss of
autosome-bound DRM may enhance germline X chromosome silencing. We propose that
autosome-enriched binding of DRM arose in C. elegans as a
consequence of germline X chromosome silencing and the evolutionary
redistribution of germline-expressed and essential target genes to autosomes.
Sex chromosome gene regulation may thus have profound evolutionary effects on
genome organization and transcriptional regulatory networks.
X chromosomes differ in number between the sexes and differ from autosomes in
their associated proteins and gene regulatory properties. In C.
elegans both X chromosomes are partially silenced in hermaphrodite
germlines. Germline-expressed and essential genes are autosome-enriched and are
thought to have fled the X chromosome during evolution because silencing these
genes would result in sterility or lethality. We discovered that the C.
elegans DRM complex, which controls transcription of genes
implicated in development and cancer, avoids the X chromosome. We first describe
how DNA–binding components of the DRM complex together recognize DNA
sequences upstream of its target genes, and we describe that DRM controls
different target genes in the germline versus the soma. We show that the DRM
binding motif, the genes bound by DRM, and the embryonic genes regulated by DRM
are all under-represented on the X chromosome. Interestingly, compromising DRM
function in the germline enhances X chromosome silencing, and we discuss how
autosome-bound DRM might regulate X-linked genes in trans. We
propose that autosome-enriched binding of DRM co-evolved with the redistribution
of its germline-expressed and essential target genes to autosomes. Our data
highlight how X chromosome gene regulation may impact both the genomic
distribution of gene sets and their transcriptional regulators.
The cellular recycling process of autophagy is emerging as a key player in several longevity pathways in C. elegans; however, the underlying mechanism by which autophagy modulates aging is currently unknown. Here, we identify a role for autophagy in the extended lifespan induced by germline removal in C. elegans, and show that autophagy and lipid metabolism work interdependently to modulate aging in this longevity model.
Germline ablation extends lifespan in C. elegans via genes such as the lipase LIPL-4, however less is known of the cellular basis by which longevity is achieved in these animals. Here, we show that germline loss induces autophagy gene expression via the FOXA transcription factor PHA-4, and that autophagy is required to extend longevity. We identify a novel link between autophagy and LIPL-4, as autophagy is required to maintain high lipase activity in germline-deficient animals. Reciprocally, lipl-4 is primarily expressed in autophagy-positive tissues and is required for autophagy induction. Coordination between autophagy and lipolysis is further supported by the finding that inhibition of TOR, a major negative regulator of autophagy, induces lipl-4 expression, and TOR levels are reduced in germline-less animals. TOR may therefore function as a common upstream regulator of both autophagy and of lipl-4 expression in germline-less animals. Importantly, we find that the link between autophagy and LIPL-4 is relevant to longevity, as autophagy is induced in animals overexpressing LIPL-4 and autophagy is required for the reported lifespan extension observed in these animals, recapitulating observations in germline-less animals.
Collectively, our data offer a novel mechanism by which autophagy and the lipase LIPL-4 interdependently modulates aging in germline-deficient C. elegans by maintaining lipid homeostasis to prolong lifespan.
Autophagy; lipase; lipolysis; lipophagy; TOR; PHA-4; DAF-16; aging; C. elegans
Cholesterol transport is an essential process in all multicellular
organisms. In this study we applied two recently developed approaches
to investigate the distribution and molecular mechanisms of cholesterol
transport in Caenorhabditis elegans. The distribution of
cholesterol in living worms was studied by imaging its fluorescent
analog, dehydroergosterol, which we applied to the animals by feeding.
Dehydroergosterol accumulates primarily in the pharynx, nerve ring,
excretory gland cell, and gut of L1–L3 larvae. Later, the bulk of
dehydroergosterol accumulates in oocytes and spermatozoa. Males display
exceptionally strong labeling of spermatids, which suggests a possible
role for cholesterol in sperm development. In a complementary approach,
we used a photoactivatable cholesterol analog to identify
cholesterol-binding proteins in C. elegans. Three major
and several minor proteins were found specifically cross-linked to
photocholesterol after UV irradiation. The major proteins were
identified as vitellogenins. rme-2 mutants, which lack
the vitellogenin receptor, fail to accumulate dehydroergosterol in
oocytes and embryos and instead accumulate dehydroergosterol in the
body cavity along with vitellogenin. Thus, uptake of cholesterol by
C. elegans oocytes occurs via an endocytotic pathway
involving yolk proteins. The pathway is a likely evolutionary ancestor
of mammalian cholesterol transport.
FOXA1 and FOXA3 binding patterns in HepG2 cells, together with their possible molecular interactions with FOXA2 and each other, are revealed by ChIP-seq.
The forkhead box/winged helix family members FOXA1, FOXA2, and FOXA3 are of high importance in development and specification of the hepatic linage and the continued expression of liver-specific genes.
Here, we present a genome-wide location analysis of FOXA1 and FOXA3 binding sites in HepG2 cells through chromatin immunoprecipitation with detection by sequencing (ChIP-seq) studies and compare these with our previous results on FOXA2. We found that these factors often bind close to each other in different combinations and consecutive immunoprecipitation of chromatin for one and then a second factor (ChIP-reChIP) shows that this occurs in the same cell and on the same DNA molecule, suggestive of molecular interactions. Using co-immunoprecipitation, we further show that FOXA2 interacts with both FOXA1 and FOXA3 in vivo, while FOXA1 and FOXA3 do not appear to interact. Additionally, we detected diverse patterns of trimethylation of lysine 4 on histone H3 (H3K4me3) at transcriptional start sites and directionality of this modification at FOXA binding sites. Using the sequence reads at polymorphic positions, we were able to predict allele specific binding for FOXA1, FOXA3, and H3K4me3. Finally, several SNPs associated with diseases and quantitative traits were located in the enriched regions.
We find that ChIP-seq can be used not only to create gene regulatory maps but also to predict molecular interactions and to inform on the mechanisms for common quantitative variation.
Oxidative stress has been hypothesized to play a role in normal aging. The SKN-1 transcription factor regulates the response to oxidative stress and also is necessary for intestinal development in Caenorhabditis elegans. Using transcriptome analysis, we found that oxidative stress induces almost a thousand genes, including the antioxidant and heat-shock responses, as well as genes responsible for xenobiotic detoxification. There were also 392 down-regulated genes including many involved in metabolic homeostasis, organismal development, and reproduction. Many of these oxidative-stress-induced transcriptional changes are dependent on SKN-1 action; the induction of the heat-shock response is not. When we used RNAi to inhibit genes, we found that most had no effect on either resistance to oxidative stress or longevity; however two SKN-1-dependent genes, nlp-7 and cup-4, that were up-regulated by oxidative stress were found to be required for resistance to oxidative stress and for normal life span. nlp-7 encodes a neuropeptide-like protein, expressed in neurons, while cup-4 encodes a coelomocyte-specific, ligand-gated ion channel. RNAi of nlp-7 or cup-4 increased sensitivity to oxidative stress and reduced lifespan. Among down-regulated genes, only inhibition of ent-1, a nucleoside transporter, led to increased resistance to oxidative stress; inhibition had no effect on lifespan. In contrast, RNAi of nhx-2, a Na+/H+ exchanger, extended lifespan significantly without affecting sensitivity to oxidative stress. These findings show that oxidative stress causes a transcriptional shift from growth and maintenance towards the activation of cellular defense mechanisms; many of these transcriptional alterations are SKN-1-dependent.
oxidative stress; C. elegans; microarray; SKN-1; aging; longevity
Third-stage infective larvae (L3) of hookworms are in an obligatory state of developmental arrest that ends upon entering the definitive host, where they receive a signal that re-activates development. Recovery from the developmentally arrested dauer stage of Caenorhabditis elegans is analogous to the resumption of development during hookworm infection. Insulin-like signaling (ILS) mediates recovery from arrest in C. elegans and activation of hookworm dauer L3. In C. elegans, phosphorylation of the forkhead transcription factor DAF-16 in response to ILS creates binding cites for the 14-3-3 protein Ce-FTT-2, which translocates DAF-16 out of the nucleus, resulting in resumption of reproductive development.
To determine if hookworm 14-3-3 proteins play a similar role in L3 activation, hookworm FTT-2 was identified and tested for its ability to interact with A. caninum DAF-16 in vitro. The Ac-FTT-2 amino acid sequence was 91% identical to the Ce-FTT-2, and was most closely related to FTT-2 from other nematodes. Ac-FTT-2 was expressed in HEK 293T cells, and was recognized by an antibody against human 14-3-3β isoform. Reciprocal co-immunoprecipitations using anti-epitope tag antibodies indicated that Ac-FTT-2 interacts with Ac-DAF-16 when co-expressed in serum-stimulated HEK 293T cells. This interaction requires intact Akt consensus phosphorylation sites at serine107 and threonine312, but not serine381. Ac-FTT-2 was undetectable by Western blot in excretory/secretory products from serum-stimulated (activated) L3 or adult A. caninum.
The results indicate that Ac-FTT-2 interacts with DAF-16 in a phosphorylation-site dependent manner, and suggests that Ac-FTT-2 mediates activation of L3 by binding Ac-DAF-16 during hookworm infection.