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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Dev Cell. Author manuscript; available in PMC Aug 1, 2009.
Published in final edited form as:
PMCID: PMC2610489
NIHMSID: NIHMS66387
Hox and Senseless antagonism functions as a molecular switch to regulate EGF secretion in the Drosophila PNS
David Li-Kroeger,1 Lorraine M. Witt,1 H. Leighton Grimes,2 Tiffany A. Cook,1,3 and Brian Gebelein1,4
1Division of Developmental Biology, Cincinnati Children’s Hospital, 3333 Burnet Ave, MLC 7007, Cincinnati, OH 45229
2Division of Immunobiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA, 45229
3Department of Pediatric Ophthalmology, Cincinnati Children’s Hospital Medical Center, Cincinnati OH 45229
4Corresponding author: Brian Gebelein, 513-636-3366 phone, 513-636-4317 fax, brian.gebelein/at/cchmc.org
Hox factors are key regulators of distinct cells, tissues, and organs along the body plan. However, little is known about how Hox factors regulate cell-specific gene expression to pattern diverse tissues. Here, we show an unexpected Hox transcriptional mechanism: the permissive regulation of EGF secretion, and thereby cell specification, by antagonizing the Senseless transcription factor in the peripheral nervous system. rhomboid expression in a subset of sensory cells stimulates EGF secretion to induce hepatocyte-like cell development. We identified a rhomboid enhancer that is active in these cells and show that an abdominal Hox complex directly competes with Senseless for enhancer binding with the transcriptional outcome dependent upon their relative binding activities. Thus, Hox-Senseless antagonism forms a molecular switch that integrates neural and anteriorposterior positional information. As the vertebrate senseless homologue is essential for neural development as well as hematopoiesis, we propose Hox-Senseless antagonism will broadly control cell fate decisions.
Developmental genetics has identified several gene families that assign cell fates along the body axes (Mann and Morata, 2000). Perhaps the best studied of these determinants are the highly conserved Hox transcription factors. Organisms throughout the animal kingdom contain at least one cluster of Hox-encoding genes that are differentially expressed along their anterior-posterior (A-P) axis to specify distinct cell fates (Carroll, 1995; Carroll et al., 2001; McGinnis and Krumlauf, 1992; Pearson et al., 2005). Indeed, alterations in Hox expression and/or function cause homeotic transformations, and have been associated with the diversity of appendages and body types found across animal phyla (Galant and Carroll, 2002; Gellon and McGinnis, 1998; Ronshaugen et al., 2002; Weatherbee and Carroll, 1999). Thus, Hox factors are regional selector genes that sculpt the body plan by instructing the development of complex morphological structures. Determining how Hox factors function is therefore of fundamental importance to understand both animal development and evolution.
Hox genes encode homeodomain proteins that bind DNA with relatively low specificity and selectivity in vitro However, individual Hox mutations have specific phenotypes, indicating that each Hox factor regulates a unique combination of target genes (Carroll et al., 2001; Graba et al., 1997; Pearson et al., 2005). Hox factors enhance target selection by forming complexes with other transcription factors, such as Extradenticle (Exd) and Homothorax (Hth) in Drosophila and their vertebrate homologues, Pbx and Meis, respectively (Burglin, 1997; Mann and Affolter, 1998; Mann and Chan, 1996; Moens and Selleri, 2006; Peifer and Wieschaus, 1990). exd and hth encode homeodomain proteins that are required for each other’s functions: Hth imports Exd into the nucleus and Exd stabilizes Hth (Abu-Shaar et al., 1999; Rieckhof et al., 1997). Exd and Hth heterodimers also cooperatively bind DNA with Hox proteins to form large protein complexes that enhance target selectivity (Ebner et al., 2005; Gebelein et al., 2004; Jacobs et al., 1999; Merabet et al., 2003; Merabet et al., 2007; Ryoo and Mann, 1999; Ryoo et al., 1999). However, a major question remains: how do these broadly expressed factors regulate transcription in a tissue- or cell-specific manner? For instance, the Drosophila Abdominal-A (Abd-A) Hox factor suppresses leg development by repressing Distal-less (Dll) in the ectoderm, modifies gut formation by regulating decapentaplegic (dpp) and wingless (wg) in the mesoderm, and affects sensory organ number by activating rhomboid (rho) in the peripheral nervous system (PNS) (Brodu et al., 2002; Capovilla et al., 1994; Grienenberger et al., 2003; Merabet et al., 2003; Vachon et al., 1992).
This study focuses on understanding how Abd-A activates rho in a subset of stretch receptors (chordotonal (ch) organs) in the PNS. Like all sensory organs in Drosophila, ch organs form from sensory organ precursor (SOP) cells specified by proneural genes encoding basic Helix-Loop-Helix (bHLH) transcription factors (Bertrand et al., 2002; Jan and Jan, 1994). Ch organ SOP cells are specified by atonal (ato), which with its two vertebrate homologues (Ath1/Ath5) defines a subfamily of proneural genes (Hassan and Bellen, 2000; Jarman et al., 1993). ato and Ath specify sensory organs for proprioception, hearing, and balance in their respective organisms, and their functional conservation has been demonstrated through cross-species studies (Ben-Arie et al., 1997; Ben-Arie et al., 2000; Ben-Arie et al., 1996; Bermingham et al., 1999). ato and Ath also activate a common target gene, the senseless (sens, Drosophila) and Growth factor independence-1 (Gfi1, vertebrates) zinc finger orthologues that are required for PNS development (Jafar-Nejad et al., 2003; Jafar-Nejad and Bellen, 2004; Kazanjian et al., 2006; Nolo et al., 2000). In fact, like Math1 mutant mice, Gfi1 mutants are deaf, ataxic, and show a loss in inner hair cells (Wallis et al., 2003). Thus, ato/Ath and sens/Gfi1 comprise a conserved pathway for sensory organ development.
While ato and sens are necessary for ch organ development, ch organ number and location varies along the A-P axis in a Hox-dependent manner (Heuer and Kaufman, 1992; Wong and Merritt, 2002). For example, the serially homologous thoracic dch3 and abdominal lch5 ch organs initially arise from three 1° SOP cells (C1–C3) within each segment (Fig 1A). However, only the abdominal SOP cells express abd-A, which stimulates the expression of the rho protease to promote Spitz (an EGF ligand) secretion. Neighboring ectodermal cells that receive the EGF signal are induced to form two 2° SOP cells and a cluster of abdominal hepatocyte-like cells (oenocytes, Fig 1A) (Elstob et al., 2001; Lage et al., 1997; Okabe and Okano, 1997; Rusten et al., 2001; Shilo, 2005;). Thus, differences in ch organ number and oenocyte formation between segments are dependent upon abd-A stimulating rho in the PNS (Fig 1B).
Figure 1
Figure 1
Model for the induction of oenocytes and SOP cells by abd-A
Here, we identified a rho cis-regulatory element that rescues rho function in a subset of abdominal SOP cells. Moreover, we show that an Exd/Hth/Abd-A complex regulates this enhancer through a unexpected mechanism: by directly competing for DNA binding with the Sens repressor protein. Since sens is essential for PNS development (Nolo et al., 2000), our data reveal a simple molecular switch through which neural and A-P positional information are integrated by a cis-regulatory element. Because Hox and Sens are highly conserved in vertebrates to regulate neural development and hematopoiesis, Hox-Sens antagonism has implications for tissue-specific gene regulation in multiple cell types and organisms.
Identification of a rho enhancer that rescues oenocyte formation
abd-A induces hepatocyte-like cells (oenocytes) as well as 2° SOP cells by stimulating EGF secretion via the up-regulation of rho in abdominal SOP cells (Fig 1A) (Brodu et al., 2002; Lage et al., 1997; Okabe and Okano, 1997). To determine the mechanism by which abd-A activates rho, we used bioinformatics and transgenic reporter assays to identify Hox-regulatory elements within the rho locus. As Abd-A binds DNA with Exd and/or Hth, we created position-based weight matrices for Exd/Hox and Hth/Hox sites using Target Explorer (http://trantor.bioc.columbia.edu/Target_Explorer/) (Sosinsky et al., 2003). Through this approach, we identified Rho654-lacZ, which contains a highly conserved Exd/Hth/Hox site and, like rho, is expressed in abdominal SOP cells (Fig 2A–C). Co-staining with Atonal (Ato) reveals Rho654-lacZ expression in the dorsal-most (C1) but not the ventral ch organ SOP cells (Fig 2C). In addition, β-gal labels the C1 lineage of mature lch5 organs with inconsistent staining in other abdominal ch organ lineages (Fig 2E). This pattern is interesting as the C1 differs from the C2 and C3 SOPs by its ability to induce oenocytes rather than 2° SOP cells (Fig 1). In fact, the β-gal positive C1 SOPs of Rho654-lacZ embryos are surrounded by developing oenocytes (marked by high Spalt-major (Salm) levels, Fig 2D) (Elstob et al., 2001; Rusten et al., 2001). Thus, Rho654 is expressed in the C1 but not other ch organ SOP cells that express rho (Fig 1A).
Figure 2
Figure 2
Rho654 rescues oenocyte formation in the abdomen
The abdominal C1-SOP expression of Rho654 suggests this regulatory element activates rho to induce oenocytes (Fig 1A). To determine if Rho654 can rescue oenocyte formation, we used Rho654-Gal4 to re-supply rho expression (UAS-rho) in rho7M43 null embryos that fail to develop either oenocytes (loss of Salm) or 2° SOP cells (loss of two mAb22C10 positive neurons, Fig 2F) (Bier et al., 1990; Elstob et al., 2001; Lage et al., 1997; Okabe and Okano, 1997; Rusten et al., 2001). As shown in Fig 2G, the oenocyte defect but not 2° SOPs were rescued in UAS-rho;Rho654-Gal4;rho7M43 embryos. Thus, Rho654 recapitulates abdominal C1-specific rho expression and function to induce oenocytes in the abdomen.
abd-A and hth regulate Rho654 activity in vivo
Brodu et al (2002) reported that abd-A is required for rho expression in abdominal SOP cells. We found that Rho654 similarly requires abd-A, as β-gal is greatly reduced in abd-A null embryos (Fig 3B). To determine if abd-A can activate Rho654-lacZ in the thorax, we mis-expressed Abd-A (Myc-Abd-A) using Paired-Gal4 (PrdG4). PrdG4 is ideal for this purpose as it is expressed in every other segment during the time SOP cells form, allowing for direct comparisons with wild type segments in the same embryo. As shown in Fig 3, Myc-Abd-A activated Rho654-lacZ(T2 in Fig 3D) as well as induced oenocytes and lch5 formation in the second thoracic segment (Fig 3F). In contrast, similar levels of a thoracic Hox factor, Antennapedia (Myc-Antp), failed to significantly stimulate Rho654-lacZ, oenocyte formation, or 2° SOP formation (Fig 3E, data not shown). Thus, like rho, Rho654-lacZ is regulated by abd-A in SOP cells.
Figure 3
Figure 3
abd-A and hth regulate Rho654-lacZ in abdominal SOP cells
We next wanted to address the role of Hox co-factors in Rho654 regulation. exd and hth function are dependent upon each other, as the genetic removal of one affects the localization or stability of the other protein (Abu-Shaar et al., 1999; Rieckhof et al., 1997). Unfortunately, analysis of Rho654 activity in maternal and zygotic exd mutants is complicated by segmentation defects that disrupt ch organ specification during early embryogenesis ((Peifer and Wieschaus, 1990), data not shown). However, embryos carrying hypomorphic alleles of hth (hthp2) undergo segmentation and specify 1° ch organ SOP cells (Kurant et al., 1998). hthp2 embryos also behave like abd-A mutants as most abdominal lch5 organs transform into a dch3 fate, consistent with a loss of rho expression ((Kurant et al., 1998), Fig 3G). Analysis of Rho654-lacZ activity and oenocyte formation in hthp2 embryos reveals that both are severely disrupted (Fig 3C and 3G). Altogether, these data indicate that abd-A requires Hox co-factors to stimulate Rho654 activity and oenocyte formation.
An Exd/Hth/Abd-A complex regulates Rho654 through a conserved binding site
Rho654 contains an Exd/Hth/Hox binding site within the conserved RhoA sequence (Fig 2A). To determine if Abd-A directly binds RhoA with Exd/Hth, we performed electromobility shift assays (EMSAs) using purified proteins and found that Exd/Hth heterodimers bind RhoA, and Abd-A cooperatively binds RhoA with Exd/Hth (Fig 4B). Consistent with Antp’s failure to stimulate Rho654 in vivo, equi-molar amounts of Antp bind five-fold less RhoA with Exd/Hth than Abd-A (data not shown). We next assayed the contribution of each binding site for Hox complex formation using mutations in the Exd, Hth, and Hox sites (Fig 4A). As expected, the Hth mutation (HthM) results in a loss of Exd/Hth and Exd/Hth/Abd-A binding, whereas HoxM causes a loss of Exd/Hth/Abd-A complexes but not Exd/Hth binding (Fig 4B). Surprisingly, ExdM decreased Exd/Hth binding but not Exd/Hth/Abd-A, suggesting that Hth and AbdA mediate most of the cooperative binding to RhoA (Fig 4B). This result is consistent with the Hth and Hox sites being directly juxtaposed in RhoA, unlike many other Hox target genes that have neighboring Hox/Exd sites (Pearson et al., 2005). To better test the relative contribution of Hth and Exd binding to RhoA, we created specific homeodomain mutations within each that disrupt DNA interactions (Asn51 to Ala) (Gehring et al., 1994) and found that Hth51A, but not Exd51A, abolished the majority of Exd/Hth/Abd-A binding to RhoA (Fig 4D). Importantly, however, supershift assays show that all three proteins, including Exd, are part of the Hox complex on RhoA (Fig 4C). In addition, Exd does contribute to Hox complex formation on RhoA, as Hth and Abd-A bind poorly to RhoA in the complete absence of an Exd protein compared to in the presence of Exd51A (data not shown). Thus, these findings suggest Exd stabilizes Hox complex formation on RhoA through protein-protein interactions with Hth and/or Abd-A.
Figure 4
Figure 4
RhoA has conserved Sens and Exd/Hth/Abd-A binding sites essential for proper SOP gene expression
The DNA binding data suggests that Abd-A complexes stimulate rho in abdominal SOP cells through RhoA. We next tested specific RhoA mutations within a minimal Rho enhancer (RhoBAD-lacZ, see Fig 2A for schematic) that retains C1-specific activity (Fig 4F). As expected, the HoxM mutation significantly reduces reporter activity in abdominal SOP cells (Fig 4I). Surprisingly, ExdM and HthM do not affect abdominal expression but rather increased reporter activity in thoracic SOPs (arrows, Fig 4G–H). Because opposite results were seen with HoxM versus ExdM and HthM, we created a double mutation (HthM/HoxM) and again observed de-repression in the thorax (data not shown). These findings reveal two unanticipated aspects of Rho enhancer activity: First, Exd/Hth/Abd-A binding to RhoA is not essential for gene activation. Second, derepression of specific mutants in the thorax suggests thoracic SOP factors bind RhoA to repress gene expression.
The Sens transcription factor binds RhoA to repress gene expression
To identify factors that repress reporter expression in the thorax, we analyzed RhoA for additional transcription factor binding sites and found a potential Senseless (Sens) site overlapping the Exd/Hth sites (Fig 4A). sens is a good candidate to regulate rho as it encodes a zinc finger protein that represses gene expression when bound to DNA, and is essential for PNS development (Acar et al., 2006; Jafar-Nejad et al., 2003; Jafar-Nejad and Bellen, 2004; Nolo et al., 2000). Using EMSA analysis, we found that: 1) Sens binds RhoA, 2) the ExdM and HthM mutations, which are de-repressed in vivo (Fig 4G–H), decrease Sens binding in vitro, and 3) the HoxM mutation has no affect on Sens binding (Fig 4E). To clearly distinguish between Sens and Hox binding RhoA and thoracic derepression in vivo, we created a mutation (SensM, Fig 4A) that abolishes Sens but not Exd/Hth or Exd/Hth/Abd-A DNA binding (Fig 4B and 4E). When tested in vivo, RhoBADsensm-lacZ was de-repressed in thoracic SOP cells (Fig 4J), indicating Sens binding represses RhoBAD-lacZ in the thorax.
sens is expressed in both thoracic and abdominal SOP cells of the Drosophila embryo (Fig 5A) (Nolo et al., 2000). If Sens represses rho in the thorax, then the genetic removal of sens should allow RhoBAD-lacZ expression in both thoracic and abdominal SOP cells. In fact, we observe weak RhoBAD-lacZ activity in thoracic and abdominal segments of sensE2 embryos (Fig 5B). However, the majority of abdominal RhoBAD-lacZ activity is lost in sensE2 embryos. Because sens stimulates the expression of proneural genes required for SOP development (Acar et al., 2006; Jafar-Nejad et al., 2003), we resupplied the ato gene in sensE2 embryos (EnG4,UAS-GFP/UAS-Ato;RhoBAD-lacZ,sensE2) and analyzed RhoBAD-lacZ activity. As shown in Fig 5C, RhoBAD-lacZ activity is significantly rescued in both the thorax and abdomen. These results are consistent with Sens indirectly activating RhoBAD-lacZ through ato regulation and directly repressing RhoBAD-lacZ by binding RhoA.
Figure 5
Figure 5
sens activity is required for proper RhoBAD-lacZ expression
Sens and Exd/Hth/Abd-A compete for RhoA to dictate the transcriptional outcome
As the Sens and Exd/Hth/Abd-A sites in RhoA overlap, it is possible that these factors either: 1) interact to form higher order transcription factor complexes, or 2) compete for the same DNA binding site. To discriminate between these possibilities, we performed EMSA analysis with all four proteins. Using several Sens proteins, including full-length Sens, we did not observe higher order transcription factor complexes, and supershift assays did not reveal Sens as part of the Hox complex (Fig 6D, Suppl Fig1). In contrast, we do observe Sens and Exd/Hth/Abd-A competition for RhoA. Using a constant amount of Exd/Hth/Abd-A in the absence or presence of Sens, we found that less Sens binds RhoA in the presence of Exd/Hth/Abd-A. In conjunction with the mutation analysis of RhoBAD reporters in vivo, this data suggests the primary role of Exd/Hth/Abd-A binding to RhoA is to exclude Sens. To test this idea in vivo, we generated a reporter containing mutations that abolish both Exd/Hth/Abd-A and Sens binding (RhoBADsensm/hoxm-lacZ). As shown in Fig 4K, RhoBADsensm/hoxm-lacZ behaves like RhoBADsensm-lacZ with β-gal expressed in both thoracic and abdominal segments. In contrast, RhoBADhoxm-lacZ, which does not affect Sens binding, is repressed throughout the embryo. These data are consistent with Exd/Hth/Abd-A activating transcription through a permissive mechanism: by interfering with the binding of the Sens repressor protein.
Figure 6
Figure 6
Sens competes with Exd/Hth/Abd-A for RhoA
We further reasoned that altering the Sens site (SensS, Fig 6A) to better match a consensus binding site for its vertebrate homolog, Gfi1 (Zweidler-Mckay et al., 1996), should favor Sens binding at the expense of Hox binding. EMSAs using wild type RhoA, SensS, or SensM as cold competitors revealed that Sens binds SensS with higher activity than wild type RhoA (Fig 6B, Suppl Fig 1). Importantly, SensS does not alter Exd/Hth/Abd-A binding to RhoA (Fig 6C), and Sens significantly out-competes the Hox complex for SensS compared to wild type RhoA (Fig 6D). To determine if enhanced Sens binding favors a repressive complex in vivo, we generated flies containing RhoBADsenss-lacZ. As shown in Fig 6E, RhoBADsenss-lacZ activity is strongly decreased in abdominal SOP cells, suggesting Sens out-competes Exd/Hth/Abd-A for this site to repress gene expression.
We next used a cell culture system to test Hox-Sens antagonism. Three copies of RhoA were cloned upstream of a minimal promoter and luciferase reporter (RhoAAA-luciferase). Consistent with a permissive role of Hox binding RhoA, co-transfection of Drosophila S2 cells with Abd-A and Hth (S2 cells express Exd, (Abu-Shaar et al., 1999)) has no affect on RhoAAA-luciferase activity (data not shown). To circumvent the lack of Hox activation, we used a Vp16Abd-A fusion protein with Hth to stimulate RhoAAA-luciferase expression (Fig 6F). If Sens competes with the Hox complex for this site, than co-transfection should decrease reporter activity in a Sens DNA binding-dependent manner. As shown in Fig 6F, Sens decreases wild type RhoAAA-luciferase, but has little affect on a RhoAAA-luciferase reporter lacking a functional Sens binding site (SensM). In addition, Sens strongly represses a reporter containing the high-affinity Sens binding site (SensS). As luciferase activation in this assay is directly linked to Vp16Abd-A DNA binding, these data further support a Hox-Sens competition model of gene regulation.
Hox genes have long been known to specify distinct cell types along the body axes of both vertebrates and invertebrates (Carroll et al., 2001; Mann and Morata, 2000; McGinnis and Krumlauf, 1992; Pearson et al., 2005). However, it has remained elusive how Hox factors regulate transcription in a tissue- or cell-specific manner. In this study, we identified a Hox-regulated enhancer (Rho654) active within a subset of PNS cells. We demonstrate Rho654 drives gene expression in abdominal C1-SOP cells to induce oenocytes, and we show that an Exd/Hth/Abd-A complex stimulates gene expression by directly competing with Sens for this enhancer. These findings have three main implications: 1) They demonstrate how a Hox selector gene integrates A-P positional information with a PNS factor to differentially regulate gene expression along the body plan. 2) They uncover a permissive rather than instructive role for Hox factors in regulating transcription. 3) As Hox and Sens binding sites share a common core sequence, they suggest additional target genes will be regulated through this mechanism. Moreover, genetic studies in mice have linked Gfi1 and Hox factors to both neural and blood cell development (Borrow et al., 1996; Duan and Horwitz, 2005; Kazanjian et al., 2006; Lawrence et al., 2005; Thorsteinsdottir et al., 2002), and we found that vertebrate Hox and Gfi1 factors compete for binding sites in blood cells (Horman et al, unpublished results).
Direct integration of an A-P selector gene with a PNS transcription factor
Sensory organs within the fly head, thorax, and abdomen require sens for their development (Fig 7A) (Nolo et al., 2000). However, the type, location, and number of sensory organs that form in different body regions are regulated, at least in part, by Hox factors (Heuer and Kaufman, 1992; Wong and Merritt, 2002). Our results provide new insight into how Hox factors provide positional information to modify gene expression in sensory cells. We used a series of point mutations to demonstrate that Hox-Sens competition forms a molecular switch whose outcome correlates with the binding activity of each factor (Fig 7B). Intrinsic to this model is the following prediction: if Hox factors differ in their ability to interact with composite sites, than A-P differences in Hox-Sens target expression will be observed. Previous biochemical studies revealed that posterior Hox factors have higher affinity for DNA when bound with Pbx (Exd) than anterior Hox proteins (LaRonde-LeBlanc and Wolberger, 2003). Consistent with these results, we found that a posterior Hox complex (Abd-A/Hth/Exd) that stimulates Rho654 binds five-fold more RhoA than an anterior Hox complex (Antp/Hth/Exd) that fails to stimulate Rho654. Thus, differences in binding activities between Hox factors for Hox-Sens composite sites result in the differential regulation of gene expression along the A-P axis of the sensory system.
Figure 7
Figure 7
Model for integration of Sens and Hox inputs on regulatory elements
A permissive role for Hox complexes in regulating gene expression
Hox proteins instructively regulate gene expression by either activating and/or repressing transcription (Graba et al., 1997; Pearson et al., 2005). In fact, the same Hox factor can perform both functions. Abd-A directly binds regulatory elements to activate wingless (wg) and repress decapentaplegic (dpp) in the same cells of the visceral mesoderm (Capovilla and Botas, 1998; Capovilla et al., 1994; Grienenberger et al., 2003). So what determines if a Hox factor activates or represses transcription? Two recent studies revealed that the transcriptional outcome depends upon the binding of additional transcription factors (Gebelein et al., 2004; Walsh and Carroll, 2007). The repression of Distal-less (Dll) by the Abd-A and Ultrabithorax (Ubx) Hox factors requires the binding of two transcription factors in addition to Exd and Hth. In posterior compartment cells, the Engrailed (En) protein collaborates with Abd-A/Exd/Hth to bind DNA and repress Dll. In anterior compartment cells, the Sloppy-paired (Slp) protein binds DNA near the Hox complex to repress Dll (Gebelein et al., 2004). As both En and Slp interact with the Groucho (Gro) co-repressor, their recruitment by Hox factors suggests a mechanism to repress transcription (Andrioli et al., 2004; Jimenez et al., 1997; Tolkunova et al., 1998). Similarly, Walsh and Carroll found that Ubx and Smad binding are required to repress spalt-major (salm) in the wing. In this case, the Smad proteins recruit the Schnurri corepressor to inhibit transcription (Walsh and Carroll, 2007). Thus, Hox factors collaborate with additional factors to determine the transcriptional outcome.
Our studies on Abd-A stimulation of a rho enhancer reveal an unexpected mechanism by which Hox factors control gene expression: through competition with the Sens repressor for DNA binding sites. We found that Sens binds RhoA to repress thoracic gene expression, whereas in the abdomen Exd/Hth/Abd-A is permissive for activation by out-competing Sens. Importantly, mutations that disrupt both Sens and Hox binding to RhoA (SensM/HoxM) are expressed in the thorax and abdomen, revealing that Exd/Hth/Abd-A binding is not required to activate gene expression. In addition, co-expression of Exd, Hth, and Abd-A in cultured cells failed to stimulate Rho654- or RhoAAA-luciferase unless Abd-A is fused to a potent activation domain. Thus, unlike other Hox target genes, Hox complexes on RhoA are permissive rather than instructive and stimulate Rho654 by interfering with the binding of a transcriptional repressor (Fig 7A).
Shared core binding sites: Implications for Hox-Sens/Gfi1 target genes in neuronal and blood cell lineages
A comparison of consensus Sens, Hox/Exd, and Exd/Hth sites reveal a shared core sequence (Fig 7b), suggesting that additional target genes will be regulated through Hox-Sens antagonism. In fact, bioinformatics reveals many Hox-Sens composite sites throughout the Drosophila and mammalian genomes (data not shown). However, both the Sens and Hox sites extend beyond this core sequence, indicating only a subset of target genes will comprise composite sites. Thus, we propose three types of target genes for those factors: 1) those regulated by only Hox factors, 2) those regulated by only Sens/Gfi1, and 3) those regulated by both Hox-Sens/Gfi1. For example, many of the previously characterized Hox target genes in the Drosophila embryo are controlled in tissues that do not express Sens, suggesting they are only regulated by Hox genes. However, the Hox and Sens/Gfi1 factors are co-expressed in many neural cells of the developing PNS in both flies and vertebrates indicating that similarly to rho regulation in abdominal SOP cells, additional targets will be co-regulated by Hox and Sens.
Like Hox genes, the Sens gene family is conserved in C elegans (Pag-3), Drosophila, and vertebrates (Gfi1 and Gfi1b) (Jafar-Nejad and Bellen, 2004; Kazanjian et al., 2006). These zinc finger transcription factors are essential for nervous system development in all three organisms. In addition, Gfi1 plays a critical role in hematopoiesis, where it participates in regulating stem cell renewal as well as specific blood cell lineages (Duan and Horwitz, 2005). Interestingly, Hox factors also regulate blood cell differentiation, proliferation and stem cell renewal. HoxA9, for example, is required for normal hematopoiesis in mice and alterations in HoxA9 expression have been implicated in acute myeloid leukemia (AML) (Borrow et al., 1996; Lawrence et al., 2005; Thorsteinsdottir et al., 2002). In fact, a study analyzing the expression profile of 6817 genes in AML patients who either responded or did not respond to treatment found the highest correlated gene associated with poor prognosis is HoxA9 (Golub et al., 1999). To determine if the Hox-Sens mechanism we uncovered in Drosophila is conserved in mammals, we used in vitro DNA binding assays to show that HoxA9 forms a complex with Pbx and Meis that competes with Gfi1 for common binding sites (Horman et al, unpublished results). Moreover, mouse genetic studies support the hypothesis that Hox-Gfi1 factors antagonize each other to regulate gene expression and blood cell development. Thus, Hox-Sens/Gfi1 competition for composite binding sites is likely a conserved mechanism for the regulation of gene expression in organisms from flies to humans.
Plasmids and transgenic fly generation
Rho654 was PCR amplified (details upon request) from Drosophila melanogaster genomic DNA and cloned into the hs43-nuc-lacZ or hs43-Gal4 P-element vectors. Deletion constructs and specific mutations were generated by PCR and confirmed by DNA sequencing. Transgenic fly lines were established using standard P-element transformation (Rainbow Transgenic Flies).
Protein purification and EMSAs
Abd-A, Exd, Hth, and a Sens protein containing the zinc finger motifs (348–541aa) were purified from BL21 bacteria as His-tagged fusions using Ni-chromatography (Gebelein et al., 2002; Gebelein et al., 2004; Xie et al., 2007). Exd51A and Hth51A were made using PCR based site-directed mutagenesis, cloned into pET14b (Novagen), and purified like wild type Exd/Hth. Protein concentrations were measured by the Bradford assay and confirmed by SDS PAGE and Coomassie blue analysis. EMSAs were performed as described (Gebelein et al., 2002). Protein amounts and sequences of each probe are noted in the figures.
Fly stocks, antibody production, and embryo staining
Fly lines include: abd-AM1, hthp2, UAS-MycAbdA and UAS-MycAntp (Dr. Richard Mann), sensE2 (Dr. Hugo Bellen), PrdG4, enGal4, and UAS-GFP (Bloomington Stock Center), UAS-Rho and rho7M43 (Dr. Gary Struhl), and UAS-Ato (Dr. Andrew Jarman). Gal4-UAS experiments were performed at 25°C and mutant embryos were identified by immunostaining. An Abd-A protein (79–330aa) was used to immunize guinea pigs (Cocalico Biologicals) and specificity was determined using abd-AM1 embryos. Expression of lacZ (anti-β-gal, Abcam, 1:1000), Abd-A (GP4, 1:500), mAb22C10 (DSHB, 1:50), Ato (1:5000) (Jarman et al., 1993), Sens (1:200) (Xie et al., 2007), and Salm (1:600) (Xie et al., 2007) were detected by fluorescent staining and a Zeiss microscope as described (Xie et al., 2007).
Cell culture and luciferase assays
Reporter vectors were assembled by cloning the hsp70 minimal promoter (hs43) upstream of luciferase in the pGL3 basic vector (Promega). Three copies of WT, SensM, or SensS RhoA were cloned upstream of the minimal promoter. Drosophila S2 cells were cultured in HyClone serum free media (Fisher Scientific). For transfections; 0.6 × 106 cells were cultured in 12 well plates 24 hr prior to transfection. Each well was transfected with a total of 0.75 ug of DNA (12.5 ng of luciferase reporter, 125 ng of pAc5.1-lacZ, and 150 ng of pAc5.1-Vp16AbdA, 150ng of pAc5.1-Hth, and/or 300ng of pAc5.1-Sens as indicated) using 2 uls of Fugene (Roche). When required, the pAc5.1 vector was added to bring the total DNA to 0.75 ug for each well. 48 hrs post-transfection, cells were harvested, lysates isolated, and luciferase activity determined as described (Xie et al., 2007). Transfection efficiencies were normalized to β-gal using standard ONPG methods. Each experiment was performed at least three times in triplicate with similar results. Results were compared using ANOVA (Systat V.12).
Supplementary Material
01
Acknowledgements
We thank Andrew Jarman, Hugo Bellen, Yuh Nung Jan, Richard Mann, Gary Struhl, and the Developmental Studies Hybridoma Bank (Univ of Iowa) for reagents. We thank Richard Mann, Barbara Noro, Dan McKay, Jill Wildonger, Michael Crickmore, Dragana Rogulja, Steve Potter, Nadean Brown, and Kenny Campbell for comments on the manuscript. This work was supported by a MOD Basil O’Connor Award, an ACS Ohio Pilot Grant, a Barrett Cancer Center Grant, and an NIH grant (RGM079428A) to B.G.
Footnotes
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anti-V5 or IgG antibodies as indicated. Note that the V5 antibody supershifts the majority of SensV5 protein, but did not alter Exd/Hth/Abd-A binding. These data indicate that Sens does not form part of a larger complex with the Exd/Hth/Hox factors on RhoA.
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