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Precise gene expression is a fundamental aspect of organismal function and depends on the combinatorial interplay of transcription factors (TFs) with cis-regulatory DNA elements. While much is known about TF function in general, our understanding of their cell type-specific activities is still poor. To address how widely expressed transcriptional regulators modulate downstream gene activity with high cellular specificity, we have identified binding regions for the Hox TF Deformed (Dfd) in the Drosophila genome. Our analysis of architectural features within Hox cis-regulatory response elements (HREs) shows that HRE structure is essential for cell type-specific gene expression. We also find that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact with non-overlapping regions in vivo, despite their similar DNA binding preferences. While Dfd and Ubx HREs exhibit comparable design principles, their motif compositions and motif-pair associations are distinct, explaining the highly selective interaction of these Hox proteins with the regulatory environment. Thus, our results uncover the regulatory code imprinted in Hox enhancers and elucidate the mechanisms underlying functional specificity of TFs in vivo.

Interphase phosphorylation of S10 at histone H3 is linked to transcriptional activation of a specific subset of mammalian genes like HDAC1. Recently, 14-3-3 proteins have been described as detectors for this phosphorylated histone H3 form. Here, we report that 14-3-3 binding is modulated by combinatorial modifications of histone H3. S10 phosphorylation is necessary for an interaction, but additional H3K9 or H3K14 acetylation increases the affinity of 14-3-3 for histone H3. Histone H3 phosphoacetylation occurs concomitant with K9 methylation in vivo, suggesting that histone phosphorylation and acetylation can synergize to overcome repressive histone methylation. Chromatin immunoprecipitation experiments reveal recruitment of 14-3-3 proteins to the HDAC1 gene in an H3S10ph-dependent manner. Recruitment of 14-3-3 to the promoter is enhanced by additional histone H3 acetylation and correlates with dissociation of the repressive binding module HP1gamma. Finally, siRNA-mediated loss of 14-3-3 proteins abolishes the transcriptional activation of HDAC1. Together our data indicate that 14-3-3 proteins are crucial mediators of histone phosphoacetylation signals.