Molecular and regulatory profiling of murine transcription factor CTCFL in non-germ cells and male germline stem cells

Ab Samad, Maisarah (2022) Molecular and regulatory profiling of murine transcription factor CTCFL in non-germ cells and male germline stem cells. Masters thesis, Universiti Sains Malaysia.

PDF - Submitted Version
Download (994kB) | Preview


Testis-specific CCCTC-binding factor-like (CTCFL) is a transcription factor expressed in male germ cells and essential for spermatogenesis. CTCFL is the paralog of CCCTC-binding factor (CTCF), a transcription factor and chromatin architectural protein. They share a similar zinc-finger DNA binding domain but different end termini. CTCFL is repressed in somatic tissues; however, the aberrant expression in the non-germ cells is associated with cancer. The regulatory functions of CTCFL in spermatogenesis and tumourigenesis are still unclear. This study aimed to elucidate the regulatory profile of murine CTCFL in non-germ cells and male germ cells. To investigate the transcriptional effects of CTCFL aberrant expression in non-germ cells, ectopic FLAG-tagged CTCFL (3×FLAG-CTCFL) was expressed in the pluripotent mouse embryonic stem cells (ESCs) and JK1 testicular stromal cell line (JK1) for 24 hours. To discover the roles of CTCFL in the germ cells, the expression of endogenous Ctcfl in male germline stem cells (GSCs) was reduced by RNA interference for 48 hours. Global changes in the transcriptome were measured by microarray and RNA sequencing, respectively. The functional annotation analysis observed altered developmental genes and processes in CTCFL expressing ESCs, ESC-CTCFL(DOX), and JK1 cells, JK1-CTCFL(DOX). Ctcfl knockdown in GSCs repressed the genes involved in cell death regulation and cellular processes in spermatogonia. The regulatory mode of CTCFL was inferred based on the genomewide protein-DNA binding profile in ESC-CTCFL(DOX). Preliminary in silico analysis highlighted the association of the upregulated genes in ESC-CTCFL(DOX) with the enrichment of CTCFL, CTCF, and Polycomb Repressor Complex 2 (PRC2) components, including Suppressor of zeste 12 protein (SUZ12). The enrichment of CTCFL, CTCF, and SUZ12 to the genome in ESC-CTCFL(DOX) were analysed by chromatin immunoprecipitation and sequencing (ChIP-seq). The results validated the intersection of CTCFL binding with CTCF and SUZ12 enrichment sites that dominated the promoter of developmental regulators. CTCF and CTCFL co-binding at CTCF&CTCFL sites could drive the activation of germline genes in the non-germ cells. SUZ12 enrichment was reduced at the CTCF&CTCFL sites near the promoter of upregulated developmental genes, inferring alteration of PRC2 repression by CTCFL. In summary, CTCFL may play a role in developmental regulation by modifying the processes in non-germ cells and regulating spermatogonia's differentiation. CTCF and PRC2 activity dysregulation could be the CTCFL regulatory mode in tumourigenesis and warrant further investigations.

Item Type: Thesis (Masters)
Uncontrolled Keywords: Transcription factors
Subjects: R Medicine
Divisions: Kampus Kesihatan (Health Campus) > Pusat Pengajian Sains Perubatan (School of Medical Sciences) > Thesis
Depositing User: Mr Abdul Hadi Mohammad
Date Deposited: 18 Sep 2022 07:11
Last Modified: 18 Sep 2022 07:11

Actions (login required)

View Item View Item