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Although originally thought to be silent chromosomal regions, centromeres are instead 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 levels vary across cell lines and over the cell cycle, but do not remain associated with centromeres, displaying localization consistent with other long non-coding RNAs. Alpha-satellite expression occurs through RNA polymerase II-dependent transcription, but does not require established centromere or cell division components. Instead, our work implicates centromere–nucleolar interactions as repressing alpha-satellite expression. The fraction of nucleolar-localized centromeres inversely correlates with alpha-satellite transcripts levels across cell lines and transcript levels increase substantially when the nucleolus is disrupted. The control of alpha-satellite transcripts by centromere-nucleolar contacts provides a mechanism to modulate centromere transcription and chromatin dynamics across diverse cell states and conditions.

Understanding the basis for cellular growth, proliferation, and function requires determining the contributions of essential genes to diverse cellular processes. Here, we combined pooled CRISPR/Cas9-based functional screening of 5,072 fitness-conferring genes in human cells with microscopy-based visualization of DNA, DNA damage, actin, and microtubules. Analysis of >31 million individual cells revealed measurable phenotypes for >90% of genes. Using multi-dimensional clustering based on hundreds of quantitative phenotypic parameters, we identified co-functional genes across diverse cellular activities, revealing novel gene functions and associations. Pooled live-cell screening of ∼450,000 cell division events for 239 genes further identified functional contributions to chromosome segregation. Our work creates a resource for the phenotypic analysis of core cellular processes and defines the functional landscape of essential human genes.

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.

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