Configuration-specific cofactors of Oct4

点击翻译按钮获取中文摘要

PROJECT SUMMARY Within the same cells, different target genes are frequently differentially regulated by the same transcription factor. While distinct chromatin neighborhoods and co-bound factors are known to alter cofactor association and hence transcription output, the role of DNA binding modality or transcription factor conformation in these processes is far less clear. The transcription factor Oct4 is a master regulator of pluripotency with key roles in early embryonic development, reprogramming and germ cell specification. Gaps in our understanding of Oct4’s functions limit our ability to reprogramming efficiency, our ability to more sculpt differentiation in vitro, and our ability to target Oct4 in different diseases. The fundamental hypothesis for this proposal is that the transcription factor Oct4, bound to DNA in different configurations, recruits different cofactors resulting in distinct transcription outputs. Oct4 can bind to DNA in different configurations depending on the specific DNA sequence recognized. Using an affinity purification/mass spectrometry approach, we identified transcription cofactors whose affinity for Oct4 is altered by the DNA sequence. One of these is the HBO1-Jade1 histone acetyltransferase complex, which binds preferentially to Oct4 homodimers associated with binding elements known as a MOREs (More palindromic Octamer-Related Elements). We recapitulated this result using purified proteins, affording us the opportunity to conduct structure/function studies. MORE sites are present in a unique class of broadly expressed genes in which Oct4 prevents inhibition that would otherwise be caused by oxidative stress, e.g. the oxidative stress associated with blastocyst implantation. The HBO1 complex acetylates multiple histone H3 and H4 lysines with little specificity, however in the context of Oct4 bound to a specific DNA site near a nucleosome, we found that Oct4 recruitment of HBO1-Jade1 resulted in specific enhancement of H3K9 acetylation, a key transcription activation mark. Our goals with this project are threefold: to identify the key features of the configuration-dependent interaction between Oct4 and the HBO1 complex (Aim 1), to determine if HBO1-Jade1 is associated with MORE-containing Oct4 target genes in pluripotent cells (Aim 2) and to determine the structure of Oct4 dimers bound to MORE sites in complex with HBO1-Jade1 (Aim 3).