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Background   Hox  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.    Results  We 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.    Conclusions  Our 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.

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