Phd in GENETICS AND MOLECULAR BIOLOGY

Thesis title: Understanding the role of CENP-A in modulating responses to DNA damage and R-loops formation in human cancers

The human centromere is the primary constriction within mitotic chromosomes and is one of the most diverse and rapidly evolving regions of the genome. In the human genome, the centromeres are made up of repetitive ~171 bp alpha-satellite DNA hierarchically organized in megabase-long arrays of near-identical higher order repeats (HORs). Centromeres are epigenetically specified by the presence of the centromere-specific histone H3 variant, CENP-A, which enables the assembly of the kinetochore for microtubule attachment. The correct assembly of the kinetochore machinery is fundamental to ensure the proper distribution of the genetic material to the daughter cells during cell division, making centromeres critical for genome stability, fertility, and healthy development. The correct chromosome segregation is also ensured by the maintenance of centromere integrity, allowing to preserve the stability of intra and inter-generational inheritance of the genetic information. When errors occur at centromere level, the cells undergo aneuploidy, a major contributor to cancer development. Despite being recognized as a hallmark of human cancer, the exact role of aneuploidy as a “driver” is still largely unknown. In this work we present a model for centromere chromatin organization which is functional for the binding of the kinetochore and for chromosome segregation. Using a combination of the Centromere Chromosome Orientation Fluorescence In Situ Hybridization (Cen-CO-FISH) technique together with Structured Illumination Microscopy (SIM) we observed the alpha-satellite DNA to be arranged in a ring-like organization within prometaphase chromosomes, in the presence or absence of spindle’s microtubules. Cen-CO-FISH allows the differential labeling of the sister chromatids without the heat denaturation step used in conventional FISH that may affect DNA structure. We also used Expansion Microscopy (ExM) to observe CENP-A organization within mitotic chromosomes. The H3 variant gets deposited within centromeric chromatin following a rounded pattern similar to that of alpha-satellite DNA, often visible as a ring thicker at the outer surface oriented toward the kinetochore-microtubule interface. Because the stability of centromere sequence and of chromatin organization is fundamental for its activity, interfering with the proper interaction of the components of the kinetochore can have deleterious consequences on chromosome segregation. To investigate such consequences I used two different model systems. I employed two different model systems. The first model system was used to interfere with the centromere-kinetochore stability. Specifically, I used U2OS tumor cells expressing with a Doxycycline-inducible catalytically dead Cas9 (dCas9) and a sgRNA designed to target the centromeres. After dCas9 induction, I observed the formation of DNA damage at centromere level. The second model system was used to study the consequences of centromere instability in chromosome segregation after inducing replication stress and R-loop formation at the centromere, with the aim to observe the cell response during and after mitotic segregation. Specifically, I used hTERT RPE-1 normal cells expressing a Doxycycline inducible RNaseH1 to remove R-loops (DNA-RNA hybrids that accumulates after replication stress), and an Auxin inducible degron to control the degradation of CENP-A. After inducing replication stress with Hydroxyurea, I observed a non-random segregation of the DNA damage marker γH2AX. This phenotype was increased after the depletion of CENP-A and reduced after the removal of R-loops. I also observed the non-random segregation of the DNA damage repair protein Rad51. Both phenotypes were present also in the BJ fibroblasts. After using a chemical inhibitor of the catalytic activity of Rad51, I observed a drop of cells segregating the DNA damage non-randomly, without a change in the total amount of damaged cells, implying an involvement of the Homologous Recombination (HR) pathway in accumulating γH2AX in only one chromatid. Taken together, here, we propose a new model of centromeric chromatin folding that reconciles the mitotic ring-like centromere structure that we observed with Cen-CO-FISH and 3D SIM with information from previous models. We took into consideration the recent advances in understanding mitotic chromosomes folding through chromatin loops, the interposition of CENP-A-containing nucleosomes with H3-containing nucleosomes, the latest genomic data, and the concept that the mammalian kinetochore is based on the repetition of the budding yeast single structure. We then observed that the replication stress triggers the HR pathway to accumulate the damaged newly synthetized strands in only one of the two sister chromatids. We also hypothesized that the accumulation of R-loops affects how the kinetochore proteins bind to the centromere inducing mechanical and/or epigenetic signals that influence the orientation of the sister chromatids on the metaphase plate. We also observed that this interaction can be destabilized by tethering it with the dCas9 inducing centromeric DNA damage.