Cytosine modifications modulate the chromatin architecture of transcriptional enhancers

The dynamics of cytosine modifications such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) have been correlated with activation of transcriptional enhancers. However, their functional relationship with the regulation of enhancer activity by pioneer transcription factors remains elusive. To gain insights into their function, we interrogated chromatin characteristics of enhancers bound by the pioneer factors FOXA1, MEIS1 and PBX1at different ime points during differentiation of mouse embryonal carcinoma cells into neural progenitors. Overall design: Genomic DNA or chromatin from P19 mouse embryonal carcinoma (EC) cells treated with retinoic acid (RA) and collected at 0 (vehicle, veh), 6, 12, 18, 24 or 48 hrs, were extracted and submitted to immunoprecipitation with 5mC (MeDIP) or 5hmC (hMeDIP) antibodies for genomic DNA or with H3K4me1, H3K27ac, FoxA1, Meis1, Pbx1 and Tet2 antibodies for chromatin. In addition, Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE) was run with chromatin in order to map open chromatin regions. Reads were mapped to the mouse mm8 genome.