The Functions of Setd5 and Mir-221 In Embryonic Stem Cell Differentiation

<p>Embryonic stem cells (ESCs) are a widely used model system to study cellular differentiation because of their pluripotent characteristics, and ESC differentiation is an epigenetic process. In an effort to identify a new epigenetic factor that is required for ESC differentiation, the function of SETD5 in ESCs was studied for this thesis. Results show that SETD5 is essential for retinoic acid (RA)-induced differentiation of mouse ESCs and for RA-induced expression of critical developmental genes (e.g., <em>Hoxa1</em> and <em>Hoxa2</em>) and neuron-related genes (e.g., <em>Nestin </em>and <em>Pax6</em>). SETD5 was upregulated during ESC differentiation. Additional results demonstrated that SETD5 bound to RAR-α upon RA treatment and was recruited to a<em> </em>retinoic acid response element (RARE) for <em>Hoxa1</em> and <em>Hoxa2 </em>activation. Methyltransferase assay using recombinant SETD5 and SETD5 complex showed that SETD5 was catalytically inactive, although it has a putative catalytic domain called SET. The transcription coactivator HCF1 was identified as a major SETD5-interacting protein. Depletion of HCF1 inhibited RA-induced ESC differentiation and RA-induced expression of SETD5-regulated genes. Chromatin immunoprecipitation assay provided evidence that HCF1 was localized to the SETD5-bound RARE region after RA treatment. These findings reveal a previously unknown ESC differentiation mechanism in which SETD5 facilitates mouse ESC differentiation by activating differentiation-specific genes via cooperation with HCF1.</p> <p>In a related but independent study, we show that miR-221-3p and miR-221-5p, which are encoded by the <em>miR-221</em> gene, are new anti-stemness miRNAs whose expression levels in mouse ESCs are directly repressed by the epigenetic modifier and pluripotent factor PRMT7. Notably, both miR-221-3p and miR-221-5p can target the 3’ untranslated regions of the major pluripotent factors Oct4, Nanog and Sox2 to antagonize mouse ESC stemness while miR-221-5p additionally silences expression of its transcriptional repressor PRMT7. Transfection of miR-221-3p and miR-221-5p mimics induced spontaneous differentiation of mouse ESCs. CRISPR-mediated <em>miR-221 </em>deletion and anti-sense <em>miR-221</em> inhibitors inhibited spontaneous differentiation of PRMT7-depleted mouse ESCs. These results reveal that PRMT7-mediated repression of miR-221-3p and miR-221-5p is critical for maintaining mouse ESC stemness. Taken together, these two studies establish SETD5 and <em>miR-221</em> as novel anti-stemness regulators that play a pro-differentiation role in ESC differentiation.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation (PhD)

Year dc:date.available

2017

Authors dc:creator

• Chen, Tsai-Yu
• <p>https://orcid.org/0000-0001-6150-6361</p>

Contributors dc:contributor

• Min Gyu Lee
• Xiaobing Shi
• Jae-Il Park

• ES cells
• SETD5
• miR-221
• Retinoic acid
• differentiation
• HCF1
• stemness
• pluripotent factors
• Medicine and Health Sciences
• Molecular Biology
• Other Cell and Developmental Biology

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/817

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-1861