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

A Machine Learning Approach for Identifying Novel Cell Type–Specific Transcriptional Regulators of Myogenesis

Transcriptional enhancers integrate the contributions of multiple classes of transcription factors (TFs) to orchestrate the myriad spatio-temporal gene expression programs that occur during development. A molecular understanding of enhancers with similar activities requires the identification of both their unique and their shared sequence features. To address this problem, we combined phylogenetic profiling with a DNA–based enhancer sequence classifier that analyzes the TF binding sites (TFBSs) governing the transcription of a co-expressed gene set. We first assembled a small number of enhancers that are active in Drosophila melanogaster muscle founder cells (FCs) and other mesodermal cell types. Using phylogenetic profiling, we increased the number of enhancers by incorporating orthologous but divergent sequences from other Drosophila species. Functional assays revealed that the diverged enhancer orthologs were active in largely similar patterns as their D. melanogaster counterparts, although there was extensive evolutionary shuffling of known TFBSs. We then built and trained a classifier using this enhancer set and identified additional related enhancers based on the presence or absence of known and putative TFBSs. Predicted FC enhancers were over-represented in proximity to known FC genes; and many of the TFBSs learned by the classifier were found to be critical for enhancer activity, including POU homeodomain, Myb, Ets, Forkhead, and T-box motifs. Empirical testing also revealed that the T-box TF encoded by org-1 is a previously uncharacterized regulator of muscle cell identity. Finally, we found extensive diversity in the composition of TFBSs within known FC enhancers, suggesting that motif combinatorics plays an essential role in the cellular specificity exhibited by such enhancers. In summary, machine learning combined with evolutionary sequence analysis is useful for recognizing novel TFBSs and for facilitating the identification of cognate TFs that coordinate cell type–specific developmental gene expression patterns

Requirements for NK-2 binding sites for the full activities of multiple tested mesodermal enhancers.

<p>(A) Loss of ßgal reporter (green) in the Lb-expressing SBM (magneta) driven by a version of the <i>lbl</i> enhancer in which the Tin binding sites are inactivated (<i>lbl^noNK-2-lacZ</i>) in stage 14 embryos. Compare to the WT version of the <i>lbl</i> enhancer (<i>lbl^WT-lacZ</i>) in Figure 2A. (B) Normal GFP reporter (green) activity in the <i>ap</i> enhancer in which the Tin binding sites are mutated (<i>ap^noNK-2-GFP</i>) in stage 14 embryos. Compare to the WT version of the <i>ap</i> enhancer (<i>ap^WT-GFP</i>) in Figure 2C. (C) Attenuation of GFP (green) driven by a version of the <i>mib2</i> enhancer in which Tin binding sites are inactivated (<i>mib2^noNK-2-GFP</i>) as compared to ßgal (magneta) driven by a WT version of the <i>mib2</i> enhancer (<i>mib2^WT-lacZ</i>) in stage 12 embryos. Compare to WT versions of both GFP and lacZ reporters in Figure 2E. (D) Loss of GFP (green) driven by a version of the <i>Ndg</i> enhancer in which Tin binding sites are mutated (<i>Ndg^noNK-2-GFP</i>) as compared to ßgal (magneta) driven by a WT version of the <i>Ndg</i> enhancer (<i>Ndg^WT-lacZ</i>) in stage 12 embryos. The ventral <i>Ndg</i> reporter-expressing cells are not in this focal plane but do not express the GFP reporter (data not shown). Compare to WT versions of both GFP and lacZ reporters in Figure 2G.</p

I–HD binding sites are required for the full activities of all tested mesodermal enhancers.

<p>(A) Loss of ßgal reporter (green) in the Lb-expressing SBM (magneta) driven by a version of the <i>lbl</i> enhancer in which the I–HD binding sites are inactivated (<i>lbl^noI-HD-lacZ</i>) in stage 14 embryos. Compare to the WT version of the <i>lbl</i> enhancer (<i>lbl^WT-lacZ</i>) in Figure 2A. Asterix indicate ßgal-expressing myotube VT1. (B) Loss of ßgal reporter (green) in the <i>ap</i> enhancer in which the I–HD binding sites are mutated (<i>ap^noI-HD-lacZ</i>) in stage 14 embryos. Compare to the WT version of the <i>ap</i> enhancer (<i>ap^WT-lacZ</i>) in Figure 2C. (C) Attenuation of GFP (green) driven by a version of the <i>mib2</i> enhancer in which FCI-HD binding sites are inactivated (<i>mib2^noI-HD-GFP</i>) as compared to ßgal (magneta) driven by a WT version of the <i>mib2</i> enhancer (<i>mib2^WT-lacZ</i>) in stage 12 embryos. Compare to WT versions of both GFP and lacZ reporters in Figure 2E. (D) Loss of GFP (green) driven by a version of the <i>Ndg</i> enhancer in which I–HD binding sites are inactivated (<i>Ndg^noI-HD-GFP</i>) as compared to ßgal (magneta) driven by a WT version of the <i>Ndg</i> enhancer (<i>Ndg^WT-lacZ</i>) in stage 12 embryos. The ventral <i>Ndg</i> reporter-expressing cells are not present in the indicated focal plane but do not express the GFP reporter (data not shown). Compare to WT versions of both GFP and lacZ reporters in Figure 2G.</p

Requirements of Six binding sites for the activities of some but not all tested mesodermal enhancers.

<p>(A) Loss of GFP (green) reporter expression in the Lb-expressing SBM (magneta) driven by a version of the <i>lbl</i> enhancer in which the Six4 binding sites are inactivated (<i>lbl^noSix-GFP</i>) in stage 14 embryos. Compare to the WT version of the <i>lbl</i> enhancer (<i>lbl^WT-lacZ</i>) in Figure 2A. (B) The GFP (green) reporter driven by the WT <i>ap</i> enhancer (<i>ap^WT-GFP</i>) is active in a small subset of lateral Mef2-positive FCs (magenta) in stage 12 embryos. (C) De-repression of the GFP reporter (green) into additional Mef2-positive (magenta) mesodermal cells in a version of the <i>ap</i> enhancer in which the Six4 binding sites are mutated (<i>ap^noSix-GFP</i>) in stage 12 embryos. (D) The GFP (green) reporter driven by a version of the <i>mib2</i> enhancer in which Six4 binding sites are inactivated (<i>mib2^noSix-GFP</i>) co-expresses with ßgal (magneta) driven by a WT version of the <i>mib2</i> enhancer (<i>mib2^WT-lacZ</i>) in stage 12 embryos. Compare to WT versions of both GFP and lacZ reporters in Figure 2E. (E) The GFP (green) reporter driven by a version of the <i>Ndg</i> enhancer in which Six4 binding sites are mutated (<i>Ndg^noSix-GFP</i>) also co-expressed with ßgal (magneta) driven by a WT version of the <i>Ndg</i> enhancer (<i>Ndg^WT-lacZ</i>) in stage 12 embryos. The ventral <i>Ndg</i> reporter-expressing cells are not present in the indicated focal plane but do not express the GFP reporter (data not shown). Compare to WT versions of both GFP and lacZ reporters in Figure 2G.</p

Targeted mutagenesis of different classes of HD binding sites in the <i>ap</i> muscle enhancer.

<p>E-score (y-axis) binding profiles of the indicated HD TFs within a particular 22 base pair segment of the entire wild-type (WT) <i>Ndg</i> enhancer (A) and versions in which all I-HD plus Hox (“noHD”, B), all Hox (“noHox”, C), or all I-HD (“noI-HD”, D) binding sites are mutated. The mutant in which all NK-2 binding sites are mutated are wild-type for these other HD TFs (“noNK-2”, E). The horizontal black line represents a threshold E-score of 0.31 below which binding is not considered significant, and was chosen as described in the Materials and Methods [5]. The effects of E-score binding profiles for additional HD TFs, as well as additional mutants investigated in the current study, and the entirety of the <i>Ndg</i> enhancer are shown in Figures S1-S4. See Materials and Methods for details of mutagenesis design and Table S2 for the actual nucleotide sequences that were investigated.</p

Hox binding sites are required for the full activities of all tested mesodermal enhancers.

<p>(A) Loss of ßgal reporter (green) in the Lb-expressing SBM (magneta) driven by a version of the <i>lbl</i> enhancer in which the Hox binding sites are mutated (<i>lbl^noHox-lacZ</i>) in stage 14 embryos. Compare to the WT version of the <i>lbl</i> enhancer (<i>lbl^WT-lacZ</i>) in Figure 2A. (B) Loss of ßgal reporter (green) in the <i>ap</i> enhancer in which the Hox binding sites are mutated (<i>ap^noHox-lacZ</i>) in stage 14 embryos. Compare to the WT version of the <i>ap</i> enhancer (<i>ap^WT-lacZ</i>) in Figure 2C. (C) Attenuation of GFP (green) driven by a version of the <i>mib2</i> enhancer in which Hox binding sites are inactivated (<i>mib2^noHox-GFP</i>) as compared to ßgal (magneta) driven by a WT version of the <i>mib2</i> enhancer (<i>mib2^WT-lacZ</i>) in stage 12 embryos. Compare to WT versions of both GFP and lacZ reporters in Figure 2E. (D) Attenuation of GFP (green) driven by a version of the <i>Ndg</i> enhancer in which Hox binding sites are inactivated (<i>Ndg^noHox-GFP</i>) as compared to ßgal (magneta) driven by a WT version of the <i>Ndg</i> enhancer (<i>Ndg^WT-lacZ</i>) in stage 12 embryos. The ventral <i>Ndg</i> reporter-expressing cells are not in the indicated focal plane but do not express the GFP reporter (data not shown). Compare to WT versions of both GFP and lacZ reporters in Figure 2G.</p

Functional requirements for HD binding sites in all tested mesodermal enhancers.

<p>(A) ßgal (green) driven by the wild-type (WT) <i>lbl</i> enhancer (<i>lbl^WT-lacZ</i>) co-expresses with Lb protein (magenta) in the Lb-expressing SBM in stage 14 embryos. (B) Loss of ßgal reporter in the Lb-expressing SBM driven by a version of the <i>lbl</i> enhancer in which all I–HD plus Hox binding sites are selectively inactivated (<i>lbl^noHD-lacZ</i>) in stage 14 embryos. (C) The GFP (green) reporter driven by the WT <i>ap</i> enhancer (<i>ap^WT-GFP</i>) is active in stage 14 lateral transverse myotubes, two of which express Kr protein (magenta). (D) Loss of the GFP reporter in stage 14 lateral transverse myotubes by a version of the <i>ap</i> enhancer in which all I–HD plus Hox binding sites are inactivated (<i>ap^noHD-lacZ</i>). (E) GFP (green) and ßgal (magenta) are co-expressed in stage 12 embryos when driven by the WT <i>mib2</i> enhancer (<i>mib2</i>^<i>WT</i><i>-GFP</i> and <i>mib2</i>^<i>WT</i><i>-lacZ</i>, respectively). (F) GFP (green) expression driven by a version of the <i>mib2</i> enhancer in which all I–HD plus Hox binding sites are mutated (<i>mib2^noHD-GFP</i>) is significantly reduced compared to ßgal (magenta) driven by <i>mib2</i>^<i>WT</i><i>-lacZ</i> in stage 12 embryos. (G) GFP (green) and ßgal (magenta) are co-expressed when driven by the <i>Ndg</i> enhancer in stage 12 embryos (<i>Ndg</i>^<i>WT</i><i>-GFP</i> and <i>Ndg</i>^<i>WT</i><i>-lacZ</i>, respectively). The 1-2 non-co-expressing cells are due to ectopic reporter activity caused by the P-element insertion [22]. The ventral <i>Ndg</i> reporter-expressing cells are not present in the indicated focal plane. (H) Loss of GFP (green) driven by a version of the <i>Ndg</i> enhancer in which all I–HD plus Hox binding sites are mutated (<i>Ndg^noHD-GFP</i>) as compared to ßgal (magneta) driven by the WT <i>Ndg</i> enhancer (<i>Ndg^WT-lacZ</i>) in stage 12 embryos. The ventral <i>Ndg</i> reporter-expressing cells are not in this focal plane but do not express the GFP reporter (data not shown).</p