Project description:The regulatory networks of differentiation programs have been partly characterized; however, the molecular mechanisms of lineage-specific gene regulation by highly similar transcription factors remain largely unknown. Here we compare the genome-wide binding and transcription profiles of NEUROD2-mediated neurogenesis with MYOD-mediated myogenesis. We demonstrate that NEUROD2 and MYOD bind a shared CAGCTG E-box motif and E-box motifs specific for each factor: CAGGTG for MYOD and CAGATG for NEUROD2. Binding at factor-specific motifs is associated with gene transcription, whereas binding at shared sites is associated with regional epigenetic modifications but not as strongly associated with gene transcription. Binding is largely constrained to E-boxes pre-set in an accessible chromatin context that determines the set of target genes activated in each cell type. These findings demonstrate that the differentiation program is genetically determined by E-box sequence whereas cell lineage epigenetically determines the availability of E-boxes for each differentiation program. Comparing NeuroD2 induced neurogenesis and MyoD induced myogenesis by examining NeuroD2/MyoD binding sites in fibroblasts and P19 cells and corresponding changes in AcH4 using Chip-Seq. Assess chromatin accessibility using PvuII assay followed by high throughput sequencing.
Project description:Pioneer transcription factors, by interacting with nucleosomes, scan silent, compact chromatin to target regulatory sequences, enabling cooperative binding events that modulate local chromatin structure and gene activity. However, not all cognate motifs are targeted by pioneer factors and dynamic scanning parameters required to scan compact chromatin are unknown. A combined genomics and single-molecule tracking approach shows that to target DNase-resistant, low-histone turnover sites, pioneer factors FOXA1 and SOX2 display opposite dynamics of chromatin scanning: slow, with low nucleoplasmic diffusion and stable interactions, versus fast, with high nucleoplasmic diffusion and transient interactions, respectively. Despite such differences, the ability of FOXA1 and SOX2 to scan low-mobility chromatin, mediated by protein domains outside of the respective DNA binding domains, leads to targeting silent chromatin. By contrast, non-pioneer HNF4A predominantly targets DNase-sensitive, nucleosome-depleted regions. We conclude that the targeting of compact chromatin sites by pioneer factors can be performed through diverse dynamic processes. Micrococcol nuclease digestion and sequencing (MNase-seq) of human BJ fibroblasts
