The genes encoded in DNA contain the instructions to make proteins and other molecules important for cell behavior. Only a fraction of genes are ‘expressed’ at any particular time; proteins called transcriptional repressors keep many in a silent state. One such repressor in the fruit fly is called Hairy, and its activity is essential for embryos to develop correctly. Similar Hairy-related proteins are crucial regulators of development in mammals.

A central mechanism of controlling gene expression involves the wrapping of DNA around histone proteins to form a structure called chromatin. Attaching chemical tags to histones changes how accessible the genes are within the chromatin—the more accessible the genes are, the more likely they are to be active. Some tags promote gene activation, while other tags block expression. Previous research showed that Hairy reduces gene expression by influencing which tags are added to, or removed from, the chromatin.

Kok et al. have now tracked the effects of the Hairy protein on the entire genome of Drosophila fruit fly embryos. This revealed the genes that Hairy directly targets and the corresponding effects this targeting has on chromatin structure. Hairy altered chromatin chemical tags over large blocks of DNA on silenced genes, with some of the changes being specific to particular genes. However, many areas of chromatin activity were not associated with changes in gene expression. Instead, many genes ignore Hairy-mediated changes in their vicinity, while in other cases chromatin changes occurred on genes that were already silent.

Previous studies have suggested that regulatory factors like Hairy frequently bind to many sites on the genome and have no function. Kok et al. now suggest that these sites—previously regarded as representing ‘inert’ sites—are biochemically very active. Genomic studies that label regulatory sites solely by changes to their chromatin modifications may be fooled by the apparent activity of such ‘errantly targeted’ sites, assuming that they are critical for gene regulation. At the same time, these sites may represent regions that are particularly likely to evolve regulatory properties. Kok et al. therefore propose that errant targeting by Hairy may help new regulatory elements to evolve that could eventually influence how genes are expressed.

Metazoan transcriptional circuitry features activation and repression signals that constitute robust regulatory networks important for the unfolding of developmental programs. In the Drosophila embryo, localized transcriptional repressors provide essential patterning information that establishes the primary anterior-posterior and dorsal–ventral axes of the organism. The action of transcriptional repressors is heterogeneous and can exhibit context effects; one of the most striking aspects involves the different classes of repressors that mediate distinct chromatin changes on target genes. Short-range acting proteins Snail and Knirps interfere with transcription only when their cognate binding sites are located within close range of the activator binding sites (Gray and Levine, 1996). These proteins interact with evolutionarily conserved corepressors that possess chromatin modifying activities (Nibu et al., 1998; Payankaulam and Arnosti, 2009). Paradoxically, these same cofactors are also recruited by another class of repressors, the long-range transcriptional repressors, exemplified by the Hairy factor (Paroush et al., 1994; Barolo and Levine, 1997; Poortinga et al., 1998). This protein is a founding member of the Hairy/Enhancer of Split (HES) transcription factors, which play essential roles in animal development, including segmental gene patterning in the early embryo and specification of neuronal differentiation in response to Notch signaling (Kageyama et al., 2007). Thus, elucidation of molecular mechanisms of Hairy activity will shed light on a number of important gene circuits that are prominently represented in key developmental pathways. The biochemical function of Hairy is associated with long-range chromatin modifications, which endow this factor with the ability to interfere with multiple cis-regulatory regions, including activators bound over 1 kb distal to the Hairy binding sites. The long-range effect has been proposed to be due to the recruitment of the corepressor Groucho (Gro), that can oligomerize to spread over large areas of the genome, and colocalization of HDAC to the target genes resulting in deacetylation of specific lysine residues in histones H3 and H4 (Courey and Jia, 2001; Martinez and Arnosti, 2008). In our previous studies, we showed that Hairy induced extensive tracts of deacetylation on ftz, a segmental patterning gene expressed early in embryogenesis (Li and Arnosti, 2011).

While potent in repression potential, Hairy and other long-range repressors are apparently restricted in their ability to exercise transcriptional effects by the local cis-regulatory context in which binding sites are located. Hairy was demonstrated to lack long-range effects on a distal RACE enhancer in the embryonic dorsal ectoderm, when Hairy binding motifs were situated in an element with activators that are restricted to mesoderm/neurectoderm regions. Furthermore, the Dorsal protein, when itself acting as a long-range repressor, is dependent on neighboring Cut and Dri transcription factor motifs to function, indicating that long-range repression complexes may require specific cis-regulatory grammar (Cai et al., 1996; Nibu et al., 2001).

The action of eukaryotic transcriptional repressors involves a number of biochemical activities, including direct antagonism of transcriptional activators and assembly of chromatin-associated factors that are correlated with gene silencing (Perissi et al., 2010). Specific types of covalent histone modifications, such as H3 and H4 deacetylation, H3K9 trimethylation and H3K27 trimethylation are correlated with repressed genes, but there is still no general understanding of how important in a quantitative sense such modifications are for inhibition of transcription at specific genes. Context effects for a particular transcriptional repressor can influence what sort and how much of a response will be generated. At a genome-wide level, specific chromatin features correlate with transcriptionally repressed genes (e.g., H3K9 and 27 methylation, reduced levels of H3 and H4 acetylation, binding of HP1), however these marks are also found within highly active loci (modENCODE Consortium et al., 2010). The epigenetic signature of transcriptional repression is thus context-dependent, consistent with a revised picture of the simple ‘histone code’ hypothesis. In the context of specific transcriptional repressors, we know little about how the context of distinct factors present at cis-regulatory elements shapes their action. Genome-wide information obtained from chromatin immunoprecipitation experiments should provide information about molecular targets and action of transcription factors, however, in addition to bona fide regulatory targets, metazoan transcription factors typically associate with a large number of in vivo binding sites of unknown significance. Recent studies have suggested that these interactions represent off-target genomic interactions, driven by low binding specificity of transcription factors and a general affinity for open chromatin of active enhancers (MacArthur et al., 2009). A survey of possible ‘off target’ binding elements suggested that these tend to be of lower affinity and are transcriptionally inert (Fisher et al., 2012). As noted above, previous studies of Hairy suggested that the protein is unable to mediate transcriptional repression in the absence of other factors co-occupying regulatory elements (Nibu et al., 2001).

Identification of functional properties of Hairy transcends the simple biochemical elucidation of repression; this protein is representative of the regulatory factors comprising conserved gene regulatory networks (GRN) that constitute the basis of animal development. Molecular studies have demonstrated that the acquisition or loss of binding sites or entire regulatory modules appears to drive significant changes in gene expression that initiate critical evolutionary transitions, such as elaboration of novel limb structures (Khila et al., 2009; Pavlopoulos et al., 2009; Tanaka et al., 2011). Significantly, although relatively subtle changes have been linked to such important evolutionary innovations, it appears that functional conservation of gene expression is also compatible with major changes in the structure of transcription control regions (Hare et al., 2008). The constraints for reorganization of existing cis-regulatory elements, or appearance of such elements de novo, are poorly understood; in some cases, the exact placement of multiple transcription factor motifs is essential for transcriptional function, while the composition of other genetic switches appears to be very loosely organized (Arnosti and Kulkarni, 2005). The existence of a large fraction of ‘off-target’ binding sites both complicates the analysis of important functional links, and the interpretation of potential evolutionary changes. Thus, elucidation of the functional targets and chromatin effects of Hairy can provide important insights on the basic substance of evolutionary variation. In this study, we use genetic tools to mediate induction of Hairy on a short time scale, permitting us to identify direct regulatory targets and chromatin effects on a genome-wide level. In addition to identifying common features of Hairy repression mechanisms across many targets, we also show that this protein exerts pervasive biochemical activity to change chromatin states at many loci unlinked to gene expression, revealing a possible pathway to evolution of novel gene regulatory connections.

By testing direct effects of the Hairy repressor in the embryo, we conclude that this protein coordinates a stereotypical set of chromatin modifications, modulated by local context, that underlie its function as a long-range repressor. Most remarkably, these changes on chromatin impact large segments of the genome that are not directly relevant to gene expression in this developmental context. We speculate that these off-target activities may provide an easy entry point for evolution of novel regulatory switches (Figure 9). Our mechanistic analysis of Hairy provides insights into likely mechanisms of related HES factors, as well as other transcriptional repressors that serve as scaffolds for chromatin modifying complexes. Hairy interacts with the widely utilized cofactors Gro, CtBP, and the Sir2 HDAC, and here we provide for the first time a genome-wide picture of the biochemical activities of this archetypal repressor.

Figure 9

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How is transcription actually controlled by Hairy? The associated chromatin modifications may be effects, rather than direct causes of gene silencing. Our previous studies indicated that Hairy modulated transcription independent of activator occupancy or SAGA co-activator occupancy (Martinez and Arnosti, 2008). These previous observations raised the possibility that Hairy acts through entirely independent pathways from that employed by activators to block transcription. Our work here indicates that Hairy does indeed directly reverse chromatin marks associated with activators, and may therefore work through a dynamic competition with these activators, undoing their positive influence on the chromatin environment that would be necessary for RNA polymerase initiation and/or elongation (Figure 9A). Indeed, Hairy repression is readily reversible, with genes showing reversion to an active state minutes after depletion of the overexpressed repressor (K Kok, unpublished results).

The genome-wide analysis of repression by Hairy revealed an unexpected facet of chromatin activity and highlights the need to consider the activity of ‘off target’ sites in generating novel elements, particularly because for Hairy at least (and likely other factors that employ the same cellular machinery) they are ‘shovel ready’ and not constrained by complex cis-regulatory grammar. Metazoan transcription factors typically interact with thousands of discrete sites in the genome, but only a small subset of these interactions correlate with observable effects on gene expression. In this study, we combined analysis of gene expression and chromatin dynamics in a way that allowed us to attribute effects directly to the induction of Hairy, inferences that would be difficult with a loss-of-function assay due to kinetics of depletion and secondary effects. In contrast, many other genome-wide data sets provide a static snapshot of the extant chromatin landscape or track complex changes through development, which represents the combined contributions of many activators and repressors. Previous studies have noted the presence of detectable but lowly-occupied sites, which have been suggested to reflect non-specific, non-functional interactions that are unavoidable by-products of proteins binding to large genomes (Fisher et al., 2012). Other studies have emphasized that transcription factors may have general nonspecific interaction with HOT sites that represent preferences for open chromatin (Gerstein et al., 2010; modENCODE Consortium et al., 2010). In general, the overall view is that whether or not these interactions are conserved, they may be of little functional consequence, and are not important for activity of GRNs (Cusanovich et al., 2014). Importantly, considering our finding that ‘off-target’ Hairy sites still appear to regulate chromatin structure, we should fundamentally reconsider how we interpret genome-wide data sets. Frequently, an increase in H3K27 acetylation is taken as an indication that the element is an active enhancer, without further functional tests (e.g., Villar et al., 2015). Of course, correlated gene expression measurements indicate that such elements are likely to be enhancers in many cases, but genomic consideration of chromatin marking must not automatically equate changes in certain active marks with enhancers.

Our study provides a new perspective on these previous observations, in that essentially trivial biological interactions may have consequences in evolutionary time. We show that Hairy is engaged apparently in errant targeting of chromatin on many loci during the period when it is expressed, and demonstrate that in many cases, little distinguishes the types of chromatin effects observed on functionally repressed targets compared to ‘non-functional’ interactions on other loci (Figure 9A,B). Thus, unlike an earlier model for Hairy action, in which the protein is active only when embedded in a previously active enhancer (Nibu et al., 2001), our work demonstrates that Hairy is able to mediate biochemical activities in most bound regions, indicating that there is little context necessary for the protein to function. Therefore, Hairy may be relatively nonselective about where it can attract chromatin-modifying agents across the genome. Much molecular biology research has emphasized the high degree of cooperativity necessary for metazoan transcription factors to work well. Enhanceosomes, patterning elements and other enhancers give aberrant readouts if correct stoichiometries and spacings are not respected. These findings suggest that random individual sites are less likely to generate a suitable transcriptional readout. At least for repressors such as Hairy, the demands for generating biochemical activity are lower than anticipated, indicating that enhancers may have a lower threshold for formation that we might have expected. Although some of the targeted genes that are not transcriptionally affected may represent ectopic binding events of the induced Hairy protein, most sites are found in ChIP analysis of endogenous Hairy. The unresponsive genes may in some cases represent later targets of Hairy, may be already repressed by endogenous Hairy, or may have responses too small to measure in this system, however it is likely that there are hundreds of changed chromatin regions that not formally part of the functional Hairy GRN. Thus, a large fraction of the genomic interactions are likely to be with regions that are not strongly selected on an evolutionary timescale. As long as the induced chromatin changes are inconsequential, these effects will not be selected against during genomic evolution. This biochemical activity, however, may provide a unique molecular exaptation to generate novel edges between nodes of a standing GRN (Figure 9B). Most enhancers involve the combined action of transcriptional activators and repressors, thus errant targeting may facilitate formation of new modules with gain of a few activator binding sites (Gould and Vrba, 1982) (Figure 9C).

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Author details

1. Kurtulus Kok

   Genetics Program, Michigan State University, East Lansing, United States

   Contribution

   KK, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Ahmet Ay

   Departments of Biology and Mathematics, Colgate University, Hamilton, United States

   Contribution

   AA, Performed machine learning analysis

   Competing interests

   The authors declare that no competing interests exist.

3. Li M Li

   Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States

   Contribution

   LML, Designed and acquired microarray data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. David N Arnosti

   1. Genetics Program, Michigan State University, East Lansing, United States
   2. Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, United States

   Contribution

   DNA, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   arnosti@msu.edu

   Competing interests

   The authors declare that no competing interests exist.

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