Vertebrate GAGA factor associated insulator elements demarcate homeotic genes in the HOX clusters

Abstract

BackgroundHox genes impart segment identity to body structures along the anterior-posterior axis and are crucial for the proper development of all organisms. Multiple regulatory elements, best defined in Drosophila melanogaster, ensure that Hox expression patterns follow the spatial and temporal colinearity reflected in their tight genomic organization. However, the precise mechanisms that regulate colinear patterns of Hox gene expression remain unclear, especially in higher vertebrates where it is not fully determined how the distinct activation domains of the tightly clustered Hox genes are defined independently of each other. Here, we report the identification of a large number of novel cis-elements at mammalian Hox clusters that can help in regulating their precise expression pattern.ResultsWe have identified DNA elements at all four murine Hox clusters that show poor association with histone H3 in chromatin immunoprecipitation (ChIP)-chip tiling arrays. The majority of these elements lie in the intergenic regions segregating adjacent Hox genes; we demonstrate that they possess efficient enhancer-blocking activity in mammalian cells. Further, we find that these histone-free intergenic regions bear GA repeat motifs and associate with the vertebrate homolog of the GAGA binding boundary factor. This suggests that they can act as GAGA factor-dependent chromatin boundaries that create independent domains, insulating each Hox gene from the influence of neighboring regulatory elements.ConclusionsOur results reveal a large number of potential regulatory elements throughout the murine Hox clusters. We further demarcate the precise location of several novel cis-elements bearing chromatin boundary activity that appear to segregate successive Hox genes. This reflects a pattern reminiscent of the organization of homeotic genes in Drosophila, where such regulatory elements have been characterized. Our findings thus provide new insights into the regulatory processes and evolutionarily conserved epigenetic mechanisms that control homeotic gene expression.

Chromatin accessibility dynamics reveal novel functional enhancers in C. elegans

“…This is noteworthy given our findings that EOR-1 is found in less accessible and potentially even nucleosome-occupied regions of the genome and that the EOR-1 motif is predictive of increased accessibility in development. GAGA-factors are conserved regulators of gene expression (Ohtsuki and Levine 1998;Mahmoudi et al 2002;Petrascheck et al 2005;Srivastava et al 2013), and a GAGA motif has been found to be necessary for enhancer functionality in C. elegans (Harfe et al 1998). Indeed, two out of five of the novel validated enhancers identified in this study (nhr-25 and swip-10) contain an EOR-1/GAGA motif.…”

“…This GAGA/EOR-1 motif was also present in two of the five functional enhancers that we experimentally validated, including the nhr-25-associated enhancer described above. TFs binding GAGA motifs have been identified as modulators of chromatin structure dynamics from plants to humans (Lund et al 2013;Srivastava et al 2013;Hecker et al 2015), and the GAGA motif itself is required for full functionality in two previously defined C.…”

Epigenetic mechanisms and boundaries in the regulation of mammalian Hox clusters

“…Interspersed between the peaks of histone H3 enrichment, we observed a distinctive pattern of gaps, which we defined as histone free regions or HFRs (Srivastava et al, 2013). Using a bioinformatics approach to map the extent and location of the HFRs (Srivastava et al, 2014), we showed that the gaps in H3 binding mapped precisely to the intergenic regions separating adjacent Hox genes.…”

“…A boundary element characterized at the mouse Hoxd locus between the Evx2 and (Srivastava et al, 2013) showing unenriched HFR regions (black arrows) interspersed with histone H3 peaks. Y-axis shows the normalized log ratio (NLR) values for all probes tiled along the cluster (X-axis) with the position of the Hoxd genes marked as boxes below the graph (transcriptional orientation is indicated).…”

“…Such studies indicate that elements that define the domains of Hox gene expression are likely to be located in close proximity to them within the conserved clusters. In fact, we have recently defined a large number of putative intergenic cis regulatory elements within all four murine Hox clusters (Srivastava et al, 2013) which can allow for the tight organization of enhancer activity in a gene-specific manner. Besides facilitating coordinated regulation, interspersed coding and regulatory features may also promote enhancer sharing and promoter competition between vertebrate Hox genes.…”