In order to elucidate the function of Myc in the maintenance of pluripotency and self-renewal in mouse embryonic stem cells (mESCs), we screened for novel ESC-specific interactors of Myc by mass spectrometry. Undifferentiated Embryonic Cell Transcription Factor 1 (Utf1) was identified in the screen as a putative Myc binding protein in mESCs. Utf1 is a chromatin-associated factor required for maintaining pluripotency and self-renewal in mESCs. It can also replace c-myc during induced pluripotent stem cell (iPSC) generation with relatively high efficiency, and shares target genes with Myc in mESCs highlighting a potentially redundant functional role between Myc and Utf1.
We validated that Myc and Utf1 directly interact in an in vitro context by GST-pull down. From in vitro truncation mutant studies of both proteins, a large region of Utf1 was found to be necessary for direct interaction with N-Myc, while the basic helix-loop-helix leucine zipper domain of N-Myc is required for direct interaction with Utf1. Utf1 was not found to directly interact with Myc's heterodimerizing partner, Max.
Utf1 and Myc complex formation could not be recapitulated in vivo, in both an ESC context and overexpressed in 293FT cells. Also, analysis of transcriptional function of Myc and Utf1 in 293FT cells using an E-box reporter construct did not result in changes to reporter expression levels, further supporting lack of complex formation in this cellular context.
Using pre-existing data sets two putative direct common target genes of Myc and Utf1, Slc27a2 and Ttyh1 were identified in mESCs. Due to the low level of data set overlap and technical variables that arise from overlapping data sets from independent studies a more optimal technical approach for identifying common genomic binding sites and target genes of both proteins was identified. Utf1, c-Myc, N-Myc, H3K27me3, and H3K4me3 ChIP-Seq, as well as RNA-Seq after diminished levels of Myc and/or Utf1 would lead to a better understanding of the combinatorial role of Myc and Utf1 in mESCs.
|Advisor:||Knoepfler, Paul S.|
|Commitee:||Chedin, Frederic L., Nolta, Jan A., Segal, David J.|
|School:||University of California, Davis|
|Department:||Biochemistry, Molecular, Cellular, and Developmental Biology|
|School Location:||United States -- California|
|Source:||DAI-B 75/01(E), Dissertation Abstracts International|
|Keywords:||Chromatin, Embryonic stem cells, Gene expression regulation, Myc, Pluripotency|
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