Author ORCID Identifier

https://orcid.org/0000-0001-6150-6361

Date of Graduation

12-2017

Document Type

Dissertation (PhD)

Program Affiliation

Cancer Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Min Gyu Lee

Committee Member

Xiaobing Shi

Committee Member

Jae-Il Park

Committee Member

Min Sup Song

Committee Member

Liuqing Yang

Abstract

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., Hoxa1 and Hoxa2) and neuron-related genes (e.g., Nestin and Pax6). SETD5 was upregulated during ESC differentiation. Additional results demonstrated that SETD5 bound to RAR-α upon RA treatment and was recruited to a retinoic acid response element (RARE) for Hoxa1 and Hoxa2 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.

In a related but independent study, we show that miR-221-3p and miR-221-5p, which are encoded by the miR-221 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 miR-221 deletion and anti-sense miR-221 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 miR-221 as novel anti-stemness regulators that play a pro-differentiation role in ESC differentiation.

Keywords

ES cells, SETD5, miR-221, Retinoic acid, differentiation, HCF1, stemness, pluripotent factors

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