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

Leah Bury,Brittania Moodie,Liliana S Mckay,Karen Hh Miga,Jimmy Ly,Iain M Cheeseman

doi:10.7554/elife.59770

Abstract

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.

Highlights

Chromosome segregation requires the function of a macromolecular kinetochore structure to connect chromosomal DNA and spindle microtubule polymers
Despite the lack of strict sequence requirements, centromere regions are typically characterized by repetitive DNA sequences, such as the alpha-satellite repeats found at human centromeres
We first designed targeted probe sets to detect RNAs derived from centromere regions across multiple chromosomes: (1) Sequences complementary to a pan-chromosomal consensus alpha-satellite sequence, (2) sequences that target supra-chromosomal family 1 (SF1) higher-order arrays, present on chromosomes 1, 3, 5, 6, 7, 10, 12, 16, and 19 (Alexandrov et al, 2001; Uralsky et al, 2019), and (3) sequences that are enriched for transcripts from the Supra-Chromosomal family three higher-order arrays present on chromosomes 1, 11, 17 and X, with an increased number of targets on chromosome 17 (D17Z1) (Willard and Waye, 1987a)

Summary

Introduction

Chromosome segregation requires the function of a macromolecular kinetochore structure to connect chromosomal DNA and spindle microtubule polymers. Prior work has detected a-satellite transcription at centromere and pericentromere regions based on the localization of RNA polymerase II (Bergmann et al, 2012; Chan et al, 2012) and the production of centromere RNA transcripts (Chan et al, 2012; Saffery et al, 2003; Wong et al, 2007). Centromere transcription and the resulting RNA transcripts have been proposed to play diverse roles in kinetochore assembly and function (Biscotti et al, 2015; Blower, 2016; Fachinetti et al, 2013; Ferri et al, 2009; Grenfell et al, 2016; Ideue et al, 2014; McNulty et al, 2017; Quenet and Dalal, 2014; Rosicand Erhardt, 2016; Wong et al, 2007). We find that the predominant factor controlling alpha-satellite transcription is the presence of centromere–nucleolar contacts, providing a mechanism to modulate centromere transcription and the underlying chromatin dynamics across diverse cell states and conditions

Results and discussion

C ASAT antisense

G H ASAT 6

G Ki67 KO

Materials and methods

Funding Funder