Engrailed cooperates directly with Extradenticle and Homothorax on a distinct class of homeodomain binding sites to repress sloppy paired.

Even skipped (Eve) and Engrailed (En) are homeodomain-containing transcriptional repressors with similar DNA binding specificities that are sequentially expressed in Drosophila embryos. The sloppy-paired (slp) locus is a target of repression by both Eve and En. At blastoderm, Eve is expressed in 7 stripes that restrict the posterior border of slp stripes, allowing engrailed (en) gene expression to be initiated in odd-numbered parasegments. En, in turn, prevents expansion of slp stripes after Eve is turned off. Prior studies showed that the two tandem slp transcription units are regulated by cis-regulatory modules (CRMs) with activities that overlap in space and time. An array of CRMs that generate 7 stripes at blastoderm, and later 14 stripes, surround slp1 (Fujioka and Jaynes, 2012). Surprisingly given their similarity in DNA binding specificity and function, responsiveness to ectopic Eve and En indicates that most of their direct target sites are either in distinct CRMs, or in different parts of coregulated CRMs. We localized cooperative binding sites for En, with the homeodomain-containing Hox cofactors Extradenticle (Exd) and Homothorax (Hth), within two CRMs that drive similar expression patterns. Functional analysis revealed two distinct, redundant sites within one CRM. The other CRM contains a single cooperative site that is both necessary and sufficient for repression in the en domain. Correlating in vivo and in vitro analysis suggests that cooperativity with Exd and Hth is a key ingredient in the mechanism of En-dependent repression, and that apparent affinity in vitro is an unreliable predictor of in vivo function

Prospero and Pax2 combinatorially control neural cell fate decisions by modulating Ras- and Notch-dependent signaling

<p>Abstract</p> <p>Background</p> <p>The concept of an equivalence group, a cluster of cells with equal potential to adopt the same specific fate, has served as a useful paradigm to understand neural cell type specification. In the <it>Drosophila </it>eye, a set of five cells, called the 'R7 equivalence group', generates a single photoreceptor neuron and four lens-secreting epithelial cells. This choice between neuronal versus non-neuronal cell fates rests on differential requirements for, and cross-talk between, Notch/Delta- and Ras/mitogen-activated protein kinase (MAPK)-dependent signaling pathways. However, many questions remain unanswered related to how downstream events of these two signaling pathways mediate distinct cell fate decisions.</p> <p>Results</p> <p>Here, we demonstrate that two direct downstream targets of Ras and Notch signaling, the transcription factors Prospero and dPax2, are essential regulators of neuronal versus non-neuronal cell fate decisions in the R7 equivalence group. Prospero controls high activated MAPK levels required for neuronal fate, whereas dPax2 represses Delta expression to prevent neuronal fate. Importantly, activity from both factors is required for proper cell fate decisions to occur.</p> <p>Conclusions</p> <p>These data demonstrate that Ras and Notch signaling are integrated during cell fate decisions within the R7 equivalence group through the combinatorial and opposing activities of Pros and dPax2. Our study provides one of the first examples of how the differential expression and synergistic roles of two independent transcription factors determine cell fate within an equivalence group. Since the integration of Ras and Notch signaling is associated with many developmental and cancer models, these findings should provide new insights into how cell specificity is achieved by ubiquitously used signaling pathways in diverse biological contexts.</p

Extradenticle and Homothorax Control Adult Muscle Fiber Identity in Drosophila

SummaryHere we identify a key role for the homeodomain proteins Extradenticle (Exd) and Homothorax (Hth) in the specification of muscle fiber fate in Drosophila. exd and hth are expressed in the fibrillar indirect flight muscles but not in tubular jump muscles, and manipulating exd or hth expression converts one muscle type into the other. In the flight muscles, exd and hth are genetically upstream of another muscle identity gene, salm, and are direct transcriptional regulators of the signature flight muscle structural gene, Actin88F. Exd and Hth also impact muscle identity in other somatic muscles of the body by cooperating with Hox factors. Because mammalian orthologs of exd and hth also contribute to muscle gene regulation, our studies suggest that an evolutionarily conserved genetic pathway determines muscle fiber differentiation

Gli3 utilizes Hand2 to synergistically regulate tissue-specific transcriptional networks.

Despite a common understanding that Gli TFs are utilized to convey a Hh morphogen gradient, genetic analyses suggest craniofacial development does not completely fit this paradigm. Using the mouse model (Mus musculus), we demonstrated that rather than being driven by a Hh threshold, robust Gli3 transcriptional activity during skeletal and glossal development required interaction with the basic helix-loop-helix TF Hand2. Not only did genetic and expression data support a co-factorial relationship, but genomic analysis revealed that Gli3 and Hand2 were enriched at regulatory elements for genes essential for mandibular patterning and development. Interestingly, motif analysis at sites co-occupied by Gli3 and Hand2 uncovered mandibular-specific, low-affinity, \u27divergent\u27 Gli-binding motifs (dGBMs). Functional validation revealed these dGBMs conveyed synergistic activation of Gli targets essential for mandibular patterning and development. In summary, this work elucidates a novel, sequence-dependent mechanism for Gli transcriptional activity within the craniofacial complex that is independent of a graded Hh signal

Contribution of Distinct Homeodomain DNA Binding Specificities to Drosophila Embryonic Mesodermal Cell-Specific Gene Expression Programs

Homeodomain (HD) proteins are a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, often acting within the same cell types, yet many members of this family paradoxically recognize similar DNA sequences. Thus, with multiple family members having the potential to recognize the same DNA sequences in cis-regulatory elements, it is difficult to ascertain the role of an individual HD or a subclass of HDs in mediating a particular developmental function. To investigate this problem, we focused our studies on the Drosophila embryonic mesoderm where HD TFs are required to establish not only segmental identities (such as the Hox TFs), but also tissue and cell fate specification and differentiation (such as the NK-2 HDs, Six HDs and identity HDs (I-HDs)). Here we utilized the complete spectrum of DNA binding specificities determined by protein binding microarrays (PBMs) for a diverse collection of HDs to modify the nucleotide sequences of numerous mesodermal enhancers to be recognized by either no or a single subclass of HDs, and subsequently assayed the consequences of these changes on enhancer function in transgenic reporter assays. These studies show that individual mesodermal enhancers receive separate transcriptional input from both I–HD and Hox subclasses of HDs. In addition, we demonstrate that enhancers regulating upstream components of the mesodermal regulatory network are targeted by the Six class of HDs. Finally, we establish the necessity of NK-2 HD binding sequences to activate gene expression in multiple mesodermal tissues, supporting a potential role for the NK-2 HD TF Tinman (Tin) as a pioneer factor that cooperates with other factors to regulate cell-specific gene expression programs. Collectively, these results underscore the critical role played by HDs of multiple subclasses in inducing the unique genetic programs of individual mesodermal cells, and in coordinating the gene regulatory networks directing mesoderm development.National Institutes of Health (U.S.) (Grant R01 HG005287

Mechanisms of Specificity for Hox Factor Activity

Metazoans encode clusters of paralogous Hox genes that are critical for proper development of the body plan. However, there are a number of unresolved issues regarding how paralogous Hox factors achieve specificity to control distinct cell fates. First, how do Hox paralogs, which have very similar DNA binding preferences in vitro, drive different transcriptional programs in vivo? Second, the number of potential Hox binding sites within the genome is vast compared to the number of sites bound. Hence, what determines where in the genome Hox factors bind? Third, what determines whether a Hox factor will activate or repress a specific target gene? Here, we review the current evidence that is beginning to shed light onto these questions. In particular, we highlight how cooperative interactions with other transcription factors (especially PBC and HMP proteins) and the sequences of cis-regulatory modules provide a basis for the mechanisms of Hox specificity. We conclude by integrating a number of the concepts described throughout the review in a case study of a highly interrogated Drosophila cis-regulatory module named “The Distal-less Conserved Regulatory Element” (DCRE)

A Hox Transcription Factor Collective Binds a Highly Conserved Distal-less cis-Regulatory Module to Generate Robust Transcriptional Outcomes.

cis-regulatory modules (CRMs) generate precise expression patterns by integrating numerous transcription factors (TFs). Surprisingly, CRMs that control essential gene patterns can differ greatly in conservation, suggesting distinct constraints on TF binding sites. Here, we show that a highly conserved Distal-less regulatory element (DCRE) that controls gene expression in leg precursor cells recruits multiple Hox, Extradenticle (Exd) and Homothorax (Hth) complexes to mediate dual outputs: thoracic activation and abdominal repression. Using reporter assays, we found that abdominal repression is particularly robust, as neither individual binding site mutations nor a DNA binding deficient Hth protein abolished cooperative DNA binding and in vivo repression. Moreover, a re-engineered DCRE containing a distinct configuration of Hox, Exd, and Hth sites also mediated abdominal Hox repression. However, the re-engineered DCRE failed to perform additional segment-specific functions such as thoracic activation. These findings are consistent with two emerging concepts in gene regulation: First, the abdominal Hox/Exd/Hth factors utilize protein-protein and protein-DNA interactions to form repression complexes on flexible combinations of sites, consistent with the TF collective model of CRM organization. Second, the conserved DCRE mediates multiple cell-type specific outputs, consistent with recent findings that pleiotropic CRMs are associated with conserved TF binding and added evolutionary constraints

Hox-mediated repression and activation through the DCRE requires multiple binding sites.

<p>(A) Schematic of sequences used in the <i>GD-lacZ</i> and <i>2xUD-lacZ</i> reporter assays. DCREwt shown in first line, dashes indicate unchanged bases and changed bases are written out. (B) Quantification of abdominal intensity (relative to T3) of <i>GD-lacZ</i> wild-type and mutant reporters reveals that the DCRE is capable of partial repression unless the DCRE contains mutations in two or more Hox binding sites (Hox01M, Hox02M, Hox12M or Hox012M). (C) Quantification of thoracic intensity (relative to A1) of <i>2xUD-lacZ</i> wild-type and mutant reporters demonstrates that the Hth and Hox2 binding site are not required for thoracic activation in this assay. (Statistics shown in tables below charts, Welch’s t-test. n.s. = not significant).</p

Antp and Hth are required for DCRE-mediated thoracic activation.

<p>(A) <i>ArmG4;2xUD-lacZ</i> embryo immunostained for Antp and β-gal demonstrates that DCRE-mediated thoracic activation is found in Antp+ cells. (B) <i>Antp</i>^<i>25</i> null embryo containing <i>ArmG4;2xUD-lacZ</i> demonstrates thoracic activation requires the Antp Hox factor. (C) <i>ArmG4;2xUD-lacZ</i> embryo immunostained for Hth and β-gal reveals significant co-expression. (D) Hth hypomorph (<i>Hth</i>^<i>P2</i>) embryo containing <i>ArmG4;2xUD-lacZ</i> demonstrates that thoracic activation requires the Hth transcription factor. (E) Wild-type <i>(Antp25/+)</i> embryo immunostained for Dll and Antp. (F) <i>Antp</i>^<i>25</i>/<i>Antp</i>^<i>25</i> null embryo immunostained for Dll reveals a reduction in Dll expression in the thorax. (G) <i>Antp</i>^<i>25</i><i>/+</i>;<i>DMX-lacZ</i> embryo immunostained for β-gal. (H) <i>Antp</i>^<i>25</i><i>/Antp</i>^<i>25</i> embryo containing <i>DMX-lacZ</i> reveals a substantial reduction in thoracic β-gal expression compared to the heterozygote. (I) <i>Antp</i>^<i>25</i><i>/+</i>;<i>DMEact-lacZ</i> embryo immunostained for β-gal. (J) <i>Antp</i>^<i>25</i><i>/Antp</i>^<i>25</i> embryo containing <i>DMEact-lacZ</i> reveals a slight reduction of thoracic β-gal expression compared to the heterozygote. (K) Quantification of Dll expression in <i>Antp</i>^<i>25</i><i>/ Antp</i>^<i>25</i> embryos relative to identically treated sibling <i>Antp</i>^<i>25</i><i>/+</i> embryos reveals that removal of Antp causes a significant reduction in Dll expression. (L) Quantification of β-gal expression from DMX or DMEact in <i>Antp</i>^<i>25</i><i>/ Antp</i>^<i>25</i> embryos compared to identically treated sibling <i>Antp</i>^<i>25</i><i>/+</i> embryos reveals that <i>DMX-lacZ</i> expresses substantially less when Antp is removed, while <i>DMEact-lacZ</i> is only slightly affected by Antp removal. All images are lateral views of Stage 11 embryos immunostained for β-gal (red or white), Antp (magenta), and Hth, or Dll (cyan or green) as indicated. (Statistics, * p<0.05, Welch’s t-test).</p