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.
Abstract
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.
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