In a widely held classical model, the E2F family has been regarded to play a critical role in regulating cell cycle progression upon binding to the promoters of its target genes via the consensus motif TTTSSCGC (where S=C or G). However, using chromatin immunoprecipitation coupled with DNA microarrays (ChIP-chip), our lab had demonstrated that the great majority of E2F targets lack this E2F motif in both normal and tumor cells in vivo. To follow up on these surprising findings, this thesis work has expanded previous studies to encompass all human promoters to better characterize 1) Evolutionary conservation of E2F binding, 2) E2F consensus sites that are not bound by E2F1, and 3) E2F targets that lack the consensus motif but are still bound by members of the E2F family. To address the first question, E2F4 ChIP-chip experiments conducted in rodent NIH3T3 cells demonstrated that the presence of the E2F consensus motif is a poor indicator of E2F targets in both human and mouse species. To address the second question, ChIP-chip experiments using antibodies to H3me3K9, H3me3K27, methylated DNA, or other E2F family members showed that neither marks of silenced chromatin nor other E2Fs are strongly associated with a lack of E2F recruitment to consensus motifs.
To address the third question, three possible mechanisms that could be responsible for the genomic recruitment of E2F1 to target sites that lack a consensus motif were investigated: 1) Indirect DNA binding by "piggy backing" on a transcription factor partner, 2) DNA looping, and 3) Direct "assisted" binding to DNA with a transcription factor partner. Because the results suggested that "assisted" binding was occurring, ChIP-seq experiments were performed to assess which domains of the E2F1 protein play an important role in E2F recruitment.
Together, these studies have provided the most comprehensive, in-depth comparison of E2F binding to consensus versus non-consensus sites to date, and have helped to further our understanding of the possible mechanisms that enable E2F1 to bind to human promoters.
|Advisor:||Farnham, Peggy J.|
|Commitee:||Furlow, John D., Segal, David J.|
|School:||University of California, Davis|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- California|
|Source:||DAI-B 71/12, Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology|
|Keywords:||ChIP-chip, ChIP-seq, Consensus motifs, Genomic recruitment, Transcription factor binding|
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