Abstract

Understanding the information encoded in the human genome requires two genetic codes, the first code specifies how mRNA sequence is converted to protein sequence, and the second code determines where and when the mRNAs are expressed. Although the proteins that read the second, regulatory code – transcription factors (TFs) – have been largely identified, the code is poorly understood as it is not known which sequences TFs can bind in the genome. To understand the regulatory code, we have analyzed the sequence-specific binding of TFs to unmodified and epigenetically modified DNA using multiple different methods. Our findings indicate that DNA commonly mediates interactions between TFs, and that dimer formation results in changes in the binding preferences of TFs. We also found that CpG methylation has a major impact on TF binding. Binding of most major classes of TFs, including bHLH, bZIP, and ETS is inhibited by mCpG. In contrast, TFs that prefer to bind to methylated DNA mainly represent homeodomain, POU and NFAT proteins, and are enriched in TFs with central roles in embryonic and organismal development. Despite the extensive knowledge of TF binding preferences, reading the regulatory code remains a challenge. To address this, we have begun to identify the sources of this problem by performing several experiments that bridge the gap between in vivo analyses such as ChIP-seq and in vitro studies such as SELEX . These approaches include analysis of TF binding in the presence of the nucleosome, determining DNA -binding activities of all TFs from distinct cell types, and determining transcriptional activities of TF motifs in vivo. A binding model that is required to understand binding of TFs to the genome, which incorporates information about cellular TF activity, protein-protein interactions induced by DNA , and inheritance of epigenetic states across cell division will be discussed.