Alpha-satellite RNA transcripts are repressed by centromere-nucleolus associations

Abstract

Centromeres play a fundamental role in chromosome segregation. Although originally thought to be silent chromosomal regions, centromeres are actively transcribed.However, the behavior and contributions of centromere-derived RNAs have remained unclear. Here, we used single-molecule fluorescence in-situ hybridization (smFISH) to detect alpha-satellite RNA transcripts in intact human cells. We find that alpha-satellite RNA smFISH foci fluctuate in their levels over the cell cycle and do not remain associated with centromeres, displaying localization consistent with other long noncoding RNAs. Our results demonstrate that alpha-satellite expression occurs through RNA Polymerase II-dependent transcription, but does not require centromere proteins and other cell division components. Instead, our work implicates centromere-nucleolar associations as the major factor regulating alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels, explaining variations in alpha-satellite RNA between cell lines. In addition, alphasatellite transcript levels increase substantially when the nucleolus is disrupted.Together, our results are inconsistent with a direct, physical role for alpha-satellite transcripts in cell division processes, and instead support a role for ongoing transcription in promoting centromere chromatin dynamics. The control of alpha-satellite transcription by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions. Alexandrov, I., A. Kazakov, I. Tumeneva, V. Shepelev, and Y. Yurov. 2001. Alphasatellite DNA of primates: old and new families. Chromosoma. 110:253-266. . 2012. Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function. J Cell Sci. 125:411-421. . 2011. Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore. Embo J. 30:328-340. Biscotti, M.A., A. Canapa, M. Forconi, E. Olmo, and M. Barucca. 2015. Transcription of tandemly repetitive DNA: functional roles.

The chromatin-binding domain of Ki-67 together with p53 protects human chromosomes from mitotic damage

“…Rather, the rapid appearance of damage during progression through mitosis after acute depletion of Ki-67 (Figure 3) leads us to favor the idea that Ki-67 directly protects mitotic chromosomes. In this view, the appearance of γH2AX is one more aspect of defective chromatin maintenance upon Ki-67 depletion, consistent with previously described structural deformations of heterochromatin (11), loss of association and modification of heterochromatin at the nucleolar periphery (10–13), and reduced transcriptional silencing of pericentromeric alpha-satellite repeats (50).…”

CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy

“…R-loops, which form co-transcriptionally, are prevalent at replication-transcription conflicts (38,40). Centromeres are actively transcribed throughout the cell cycle (17,41), raising the hypothesis that CENP-A depletion causes R-loop formation due to convergence of replication and transcription machineries. Upon release from thymidine-mediated arrest, CENP-A depletion did not affect the levels of centromere transcripts during early S-phase, but when cells progressed to late S-phase, the RNA levels increased ( Figure 3F).…”

“…As CENP-A levels are reduced in senescent cells 59 (41) and favorize rearrangements at acrocentric chromosomes. During neocentromere formation, CENP-A can also be lost from the original alpha-satellite locus which has been reported to lead to centromere erosion at the repeats (64).…”

Centromeric Transcription: A Conserved Swiss-Army Knife

“…Recent work showed that knock-down of CENP-C resulted in increased centromeric transcription [ 117 ], whereas knock-down of centromeric transcripts resulted in increased levels of centromeric CENP-C [ 31 ]. These data suggest that there is a functional interaction between CENP-C and centromeric transcription.…”