Understanding Transcriptional Control During Primordial Germ Cell DifferentiationDoctoral thesis
Date of Examination:2022-06-09
Date of issue:2022-07-08
Advisor:Dr. Ufuk Günesdogan
Referee:Dr. Ufuk Günesdogan
Referee:Prof. Dr. Gregor Bucher
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EnglishAP2γ, encoded by Tfap2c, is a transcription factor which both represses and activates target gene expression. It is required for the survival of the early mouse embryo; depending on the context of its depletion, the loss of the protein may prevent the correct formation of blastocysts, result in placental defects and embryonic death, or result in infertility of the adult mouse. AP2γ expression has been identified in the maintenance of adult tissues, and its expression has been localized to the skin, brain, and mammary gland. It has also been identified in numerous cancers, as a prognostic marker of both poor and good outcomes, depending upon the cancer and context. It is thus a fascinating transcription factor, as its expression or loss thereof can tip the scales of survival in both the embryo and adult. AP2γ was examined in this work in the context of its role as a transcription factor required for primordial germ cell (PGC) differentiation. PGCs are unipotent embryonic cells which give rise to either sperm or egg, and at the point of their induction are the only embryonic cells which will be able to transmit heritable genetic and epigenetic information. The knockout (KO) of AP2γ has been shown to impair PGC differentiation and result in sterility of otherwise healthy, adult mice. However, the transcriptional regulation exerted by AP2γ during PGC differentiation is poorly understood. One hypothesis for the strong transcriptional effect exerted by AP2γ is that it may be a pioneer factor, a protein which is capable of binding to and opening closed chromatin to allow other transcription factors and adapter proteins to bind and thus effect transcriptional regulation, however it is still unknown if AP2γ is truly a pioneer factor, and if so in which of its many biological contexts does it exert this effect. My work aimed to improve our understanding of the role of AP2γ in PGC differentiation in two ways: First, to characterize the role of AP2γ as a regulator of transcriptional activity and as a potential pioneer factor in embryonic stem cells (ESCs), epiblast-like cells (EpiLCs), and PGC-like cells (PGCLCs). These cells represent the in vitro recapitulation of the developmental process which allows for the differentiation of ESCs towards PGCLC fate, and thus allows for the examination of transcriptional events resulting in the correct differentiation of PGCLCs. To accomplish this, I generated AP2γ knockout (KO) ESCs and I applied RNA-sequencing (ribonucleic acid sequencing) transcriptomic analysis to identify changes in gene expression as I differentiated the cells towards PGCLCs. I was able to show that, in the absence of AP2γ, PGCLCs cannot be correctly specific and further, numerous marker genes of naïve pluripotency and somatic fate are misregulated, raising broader questions about the role of AP2γ not only in PGCLCs but also within the context of naïve pluripotent ESCs. I also examined the chromatin accessibility of the AP2γ-KO cells as they progressed from ESCs towards PGCLC fate by performing ATAC-sequencing (assay for transposase-accessible chromatin with high-throughput sequencing). I was able to identify differentially accessible chromatin in all examined cell types, however most dramatically in the AP2γ-KO ESCs, further underlining the potential role of AP2γ in naïve pluripotency. My second aim was to identify cis-regulatory elements (CREs) including enhancers, regulating the expression of AP2γ as the cells differentiated from ESCs towards PGCLC fate. To this end I employed a lentiviral CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) based screen approach, tiling the topologically-active domain of AP2γ with gRNAs to KO potential regulatory elements acting on the gene. Analysis of the screen results did not clearly indicate CREs regulating AP2γ expression, nevertheless interesting genomic sites were identified which may be investigated further as potential cell type-specific regulatory regions. This work broadens our understanding of AP2γ in the context of naïve pluripotent ESCs as well as during the process of PGCLC differentiation. It also raises questions about the role of AP2γ as a pioneer factor, and the relationship between AP2γ and the closely related protein AP2α. In addition, although the employed screen was not able to identify specific CREs driving AP2γ expression, I was able to identify inter- esting targets of further investigation including potential regulatory regions involved in cell survival during the process of differentiation towards PGCLC fate.
Keywords: Development; Tfap2c; AP2γ; Mouse; PGC; PGCLC; Primordial germ cell; Mus musculus; CRISPR/Cas9 screen