Chromatin landscape, transcription and DNA replication origin positioning

Murugarren Garrido, Nerea (2022). Chromatin landscape, transcription and DNA replication origin positioning. University of Birmingham. M.Sc.

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Abstract

DNA replication initiation is made possible by the assembly of pre- replication complexes (Pre- RCs) at genomic locations called DNA replication initiation sites (origins) during G1-phase. Although there are no conserved characteristics of metazoan origins on which Pre-RCs form, previous findings suggest that origins are strongly associated with open, active chromatin regions of the genome such as promoters, enhancers, and active histone marks. However, over a third of transcriptionally silent genes associate with DNA replication initiation activity in mESC cells, and most of these genes are bound and repressed by the Polycomb repressive complex 2 (PRC2) through the repressive H3K27me3 mark. This is the strongest observed association of an epigenetic regulator element with replication origin activity. Thus, I wished to ask whether Polycomb-mediated gene repression plays a direct role in the recruitment of DNA replication origins activity to silent genes. I comprehensively characterized the consequences of PRC2 catalytic activity (EZH2 subunit) depletion on the activity of DNA replication origins in mESC through a genome-wide evaluation. My findings suggest that absence of EZH2 results in increased DNA replication initiation activity at EZH2-bound sites. Interestingly, the increase in DNA replication initiation activity does not correlate with transcriptional de-repression or acquisition of the activating marks H3K4me3 and H3K27ac but loss of the repressive H3K27me3 mark leading to increased accessibility of chromatin.
DNA replication initiation and transcriptional initiation frequently co-localize at gene promoters. Numerous studies have examined the impact of transcription (and chromatin) and its ability to recruit DNA replication activity. In the final part (Part II) of this thesis, I began to investigate what impact the localization of DNA replication initiation may have on transcription. I used a mEF cell line in which a DNA replication origin is rendered inactive. My experiments in synchronized cells suggest that Pre-RC formation impedes transcription in a transient, time-specific manner during G1-phase of the cell cycle.

Type of Work:

Thesis (Masters by Research > M.Sc.)

Award Type:

Masters by Research > M.Sc.

Supervisor(s):