Chromatin accessibility has been shown to play an important role in dictating transcription factor binding. In this regard, integration of HIF1 alpha Cinoxacin binding locations in U87 and HepG2 cells with gene expression data in the same cell types revealed a preference for HIF1 binding to map to transcriptionally active genes in normoxia, therefore 
suggesting that chromatin accessibility, as indirectly evidenced by basal Lomitapide Mesylate transcriptional activity, determines HIF1 binding. As an independent approach to test this hypothesis, we looked at the correlation of normoxic gene expression and induction of known HIF targets in publicly available microarray datasets of hypoxic cell cultures. In agreement, we found a statistically significant association between basal expression and hypoxia inducibility of known targets. Furthermore, comparison of HIF1a and HIF2a binding locations in MCF-7 cells with DNAse hypersentitivity data in the same cell type also revealed a significant association of HIF binding with normoxic DNAse hypersensitive sites, again pointing at an important role of open chromatin regions in dicating HIF binding. However, when conserved RCGTG HIF binding consensus motifs are identified in non-coding regions of genes showing basal expression, a majority of these are not induced by hypoxia. Therefore, although chromatin accessibility clearly favors HIF1 binding, additional mechanisms are likely needed to fully specify HIF target selectivity. DNA methylation of a HIF binding site was originally shown to block HIF1a binding to the 39 erythropoietin enhancer, and indeed erythropoietin expression appears to be restricted to cell types in which the hypoxia response element is unmethylated. Altered HIF binding due to methylation changes in HREs has been further validated in additional target genes, such as BNIP3 or HIF1A, and is often associated with cancer progression. However, a global view on the effects of DNA methylation in HIF binding selectivity is lacking, and may be challenging to analyze in view of recent evidence arguing for dynamic DNA methylation in hypoxia. Additional transcription factors binding in the proximity of a HIF1 binding site could impact either HIF1 binding or transcriptional modulation of the target gene. In agreement with this possibility, a recent study addressing the functional validation of common genetic variants at a renal cancer susceptibility locus found HIF2 binding to be dependent on a polymorphism falling outside the RCGTG HIF binding consensus, strongly suggesting that sequences outside the HIF binding site can be functionally important in determining HIF binding. We tested this hypothesis by computational prediction of transcription factor binding sites enriched in a core set of bona fide HIF binding regions. These were obtained through integration of HIF1a ChIP-chip data with a gene expression meta-analysis of hypoxic cell cultures, thereby combining multiple HIF DNA binding and hypoxic gene expression datasets. Our approach has the advantage of using an integrated set of sequences that could overcome the limitations of analyses based on a single dataset, where a proportion of binding sites could potentially correspond to false positives or non-functional sites. In addition to HIF matrices, we observed additional sequence motifs that were enriched in core HIF binding regions and that could potentially impact HIF binding and transactivation selectivity. Of note, the transcriptional activity of several of these proteins, such as AP-1, CREB, EGR-2 or CEBPB is known to be induced by hypoxia. Nevertheless, and in agreement with previous predictions of enriched TFBSs in the vicinity of experimentally or computationally identified HIF binding sites, the statistical significance of these predictions is relatively low and, even on an integrated dataset, no single collaborating TF stands out.