GSE28286
GSE28286: Genome-wide analysis of transcription factor E2F1 mutant proteins reveals that N- and C-terminal protein interaction domains do not participate in targeting E2F1 to the human genome.
Summary: Previous studies of E2F family members have suggested that protein-protein interactions may be the mechanism by which E2Fs are recruited to specific genomic regions. We have addressed this hypothesis on a genome-wide scale using ChIP-seq analysis of MCF7 cell lines that express tagged wildtype and mutant E2F1 proteins.  First, we performed ChIP-seq for tagged wt E2F1.  Then, we analyzed E2F1 proteins that lacked the N terminal SP1 and cyclin A binding domains, the C terminal transactivation and pocket protein binding domains, and the internal marked box domain. Surprisingly, we found that the ChIP-seq patterns of the mutant proteins were identical to that of wt E2F1.  However, mutation of the DNA binding domain abrogated all E2F1 binding to the genome.  These results suggested that the interaction between the E2F1 DNA binding domain and a consensus motif may be the primary determinant of E2F1 recruitment.  To address this possibility, we analyzed the in vivo binding sites for the in vitro-derived consensus E2F1 motif (TTTSSCGC) and also performed de novo motif analysis. We found that only 12% of the ChIP-seq peaks contained the TTTSSCGC motif. De novo motif analysis indicated that most of the in vivo sites lacked the 5â?? half of the in vitro derived consensus, having instead the in vivo consensus of CGCGC. In summary, our findings do not provide support for the model that protein-protein interactions are involved in recruiting E2F1 to the genome, but rather suggest that recognition of a motif found at most human promoters is the critical determinant.  Overall Design: 9 total ChIP-seq datasets; four different HA-ER-E2F1 mutants, and one HA ER E2F1 wild type dataset done in duplicate, from 5 different stable cell lines derived from MCF7 cells;  Three Input replicates from 2 different stable cell lines derived from MCF7 cells; HA ER E2F1 wild type duplicate dataset from MCF7 stable cells; 1 HA ER E2F1 DBDmut replicate from MCF7 stable cells cells, 1 HA ER E2F1Î?C replicate from MCF7 stable cells cells, 1 HA ER E2F1Î?N/C replicate from MCF7 stable cells cells, 1 HA ER E2F1Î?MB replicate from MCF7 stable cells cells, 1 HA ER E2F1Î?MB replicate from MCF7 stable cells cells, 3 Input replicates from MCF7 stable cells cells.
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