Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T

Abstract

Centromeres are highly specialized chromatin domains that enable chromosome segregation and orchestrate faithful cell division. Human centromeres are composed of tandem arrays of α-satellite DNA, which spans up to several megabases. Little is known about the mechanisms that maintain integrity of the long arrays of α-satellite DNA repeats. Here, we monitored centromeric repeat stability in human cells using chromosome-orientation fluorescent in situ hybridization (CO-FISH). This assay detected aberrant centromeric CO-FISH patterns consistent with sister chromatid exchange at the frequency of 5% in primary tissue culture cells, whereas higher levels were seen in several cancer cell lines and during replicative senescence. To understand the mechanism(s) that maintains centromere integrity, we examined the contribution of the centromere-specific histone variant CENP-A and members of the constitutive centromere-associated network (CCAN), CENP-C, CENP-T, and CENP-W. Depletion of CENP-A and CCAN proteins led to an increase in centromere aberrations, whereas enhancing chromosome missegregation by alternative methods did not, suggesting that CENP-A and CCAN proteins help maintain centromere integrity independently of their role in chromosome segregation. Furthermore, superresolution imaging of centromeric CO-FISH using structured illumination microscopy implied that CENP-A protects α-satellite repeats from extensive rearrangements. Our study points toward the presence of a centromere-specific mechanism that actively maintains α-satellite repeat integrity during human cell proliferation.centromere | genome integrity | α-satellite repeats | cancer | CO-FISH

Precarious maintenance of simple DNA repeats in eukaryotes

“…Studies in fission yeast (which have different centromere sequences but similar centromeric chromatin to humans) have identified several of the replication fork restart proteins, including Smc5/6 and Brc1, as crucial to duplicating and suppressing crossover recombination within centromeric regions (for a recent review see [118]). Mitotic recombination at centromeres in mammalian cells may be quite common and is also suppressed by centromere associated histone proteins and epigenetic silencing [119,120]. …”

DNA replication stress: oncogenes in the spotlight

“…Sophisticated protein complexes regulate telomere and centromere stability and function. Disruption of several telomere-and centromere-binding proteins has been shown to impair resolution of DNA secondary structures, induce replication fork stalling, and cause fragility at these loci (Martínez et al, 2009;Sfeir et al, 2009;Aze et al, 2016;Giunta and Funabiki, 2017). In addition, oncogene activation has been demonstrated to induce chromosome breaks at centromeres and generate aberrant structures at telomeres in response to replication stress (Suram et al, 2012;Miron et al, 2015).…”

The String Decomposition Problem and its Applications to Centromere Assembly

“…Recent advancements in long read sequencing technologies, such as Pacific Biosciences (PB) and Oxford Nanopore Technologies (ONT), led to a substantial increase in the contiguity of genome assemblies (Koren et al, 2017;Kolmogorov et al, 2019;Ruan and Li, 2019;Shafin et al, 2019) and opened a possibility to resolve extra-long tandem repeats ( ETRs ), the problem that was viewed as intractable until recently. Assembling ETRs is important since variations in ETR have been linked to cancer and infertility (Barra and Fachinetti, 2018;Black and Giunta, 2018;Ferreira et al, 2015;Giunta and Funabiki, 2017;Miga et al, 2019;Smurova and De Wulf, 2018;Zhu et al, 2018) . ETR sequencing is also important for addressing open problems about centromere evolution (Alkan et al, 2007;Lower et al, 2018;Shepelev et al, 2009, Henikoff et al, 2015 .…”