Centromere transcription allows CENP-A to transit from chromatin association to stable incorporation

Centromeres are essential for chromosome segregation and are specified epigenetically by the presence of the histone H3 variant CENP-A. In flies and humans, replenishment of the centromeric mark is uncoupled from DNA replication and requires the removal of H3 “placeholder” nucleosomes. Although transcription at centromeres has been previously linked to the loading of new CENP-A, the underlying molecular mechanism remains poorly understood. Here, we used Drosophila melanogaster tissue culture cells to show that centromeric presence of actively transcribing RNA polymerase II temporally coincides with de novo deposition of dCENP-A. Using a newly developed dCENP-A loading system that is independent of acute transcription, we found that short inhibition of transcription impaired dCENP-A incorporation into chromatin. Interestingly, initial targeting of dCENP-A to centromeres was unaffected, revealing two stability states of newly loaded dCENP-A: a salt-sensitive association with the centromere and a salt-resistant chromatin-incorporated form. This suggests that transcription-mediated chromatin remodeling is required for the transition of dCENP-A to fully incorporated nucleosomes at the centromere.

[1]  Shannon M. McNulty,et al.  Human Centromeres Produce Chromosome-Specific and Array-Specific Alpha Satellite Transcripts that Are Complexed with CENP-A and CENP-C. , 2017, Developmental cell.

[2]  Oscar Molina,et al.  Epigenetic engineering reveals a balance between histone modifications and transcription in kinetochore maintenance , 2016, Nature Communications.

[3]  R. Heald,et al.  Mitotic noncoding RNA processing promotes kinetochore and spindle assembly in Xenopus , 2016, The Journal of cell biology.

[4]  T. Nagase,et al.  KAT7/HBO1/MYST2 Regulates CENP-A Chromatin Assembly by Antagonizing Suv39h1-Mediated Centromere Inactivation , 2016, Developmental cell.

[5]  K. Sullivan,et al.  The CENP-T/-W complex is a binding partner of the histone chaperone FACT , 2016, Genes & development.

[6]  H. Kimura,et al.  Epigenetic engineering shows that a human centromere resists silencing mediated by H3K27me3/K9me3 , 2016, Molecular biology of the cell.

[7]  T. Nagase,et al.  CENP-C and CENP-I are key connecting factors for kinetochore and CENP-A assembly , 2015, Journal of Cell Science.

[8]  R. O’Neill,et al.  Establishment of Centromeric Chromatin by the CENP-A Assembly Factor CAL1 Requires FACT-Mediated Transcription. , 2015, Developmental cell.

[9]  R. Allshire,et al.  Sequence Features and Transcriptional Stalling within Centromere DNA Promote Establishment of CENP-A Chromatin , 2015, PLoS genetics.

[10]  J. Langowski,et al.  Opposing roles of H3- and H4-acetylation in the regulation of nucleosome structure—a FRET study , 2015, Nucleic acids research.

[11]  S. Erhardt,et al.  Repetitive centromeric satellite RNA is essential for kinetochore formation and cell division , 2014, The Journal of Cell Biology.

[12]  S. Biggins,et al.  The FACT complex interacts with the E3 ubiquitin ligase Psh1 to prevent ectopic localization of CENP-A , 2014, Genes & development.

[13]  Y. Dalal,et al.  A long non-coding RNA is required for targeting centromeric protein A to the human centromere , 2014, eLife.

[14]  K. Luger,et al.  CAL1 is the Drosophila CENP-A assembly factor , 2014, The Journal of cell biology.

[15]  C. Lehner,et al.  Distinct modes of centromere protein dynamics during cell cycle progression in Drosophila S2R+ cells , 2013, Journal of Cell Science.

[16]  G. Karpen,et al.  The Cell Cycle Timing of Centromeric Chromatin Assembly in Drosophila Meiosis Is Distinct from Mitosis Yet Requires CAL1 and CENP-C , 2012, PLoS biology.

[17]  Eun Shik Choi,et al.  Factors That Promote H3 Chromatin Integrity during Transcription Prevent Promiscuous Deposition of CENP-ACnp1 in Fission Yeast , 2012, PLoS genetics.

[18]  V. Noskov,et al.  Breaking the HAC Barrier: Histone H3K9 acetyl/methyl balance regulates CENP-A assembly , 2012, The EMBO journal.

[19]  Owen J. Marshall,et al.  Active transcription and essential role of RNA polymerase II at the centromere during mitosis , 2012, Proceedings of the National Academy of Sciences.

[20]  H. Kimura,et al.  Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function , 2012, Journal of Cell Science.

[21]  K. Kitagawa,et al.  Endogenous Transcription at the Centromere Facilitates Centromere Activity in Budding Yeast , 2011, Current Biology.

[22]  S. Saccani,et al.  Heterochromatin boundaries are hotspots for de novo kinetochore formation , 2011, Nature Cell Biology.

[23]  O. Bensaude,et al.  Inhibiting eukaryotic transcription. Which compound to choose? How to evaluate its activity? , 2011, Transcription.

[24]  G. Karpen,et al.  Assembly of Drosophila Centromeric Chromatin Proteins during Mitosis , 2011, PLoS genetics.

[25]  Eun Shik Choi,et al.  Identification of Noncoding Transcripts from within CENP-A Chromatin at Fission Yeast Centromeres* , 2011, The Journal of Biological Chemistry.

[26]  G. Karpen,et al.  H3.3 is deposited at centromeres in S phase as a placeholder for newly assembled CENP-A in G1 phase , 2011, Nucleus.

[27]  O. Rando,et al.  Overlapping Regulation of CenH3 Localization and Histone H3 Turnover by CAF-1 and HIR Proteins in Saccharomyces cerevisiae , 2011, Genetics.

[28]  Hiroshi Kimura,et al.  Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore , 2010, The EMBO journal.

[29]  C. Lehner,et al.  Detrimental incorporation of excess Cenp-A/Cid and Cenp-C into Drosophila centromeres is prevented by limiting amounts of the bridging factor Cal1 , 2010, Journal of Cell Science.

[30]  V. Studitsky,et al.  RNA polymerase complexes cooperate to relieve the nucleosomal barrier and evict histones , 2010, Proceedings of the National Academy of Sciences.

[31]  S. Chávez,et al.  FACT Prevents the Accumulation of Free Histones Evicted from Transcribed Chromatin and a Subsequent Cell Cycle Delay in G1 , 2010, PLoS genetics.

[32]  H. Ovaa,et al.  Recombination-induced tag exchange to track old and new proteins , 2009, Proceedings of the National Academy of Sciences.

[33]  M. Nakano,et al.  Hierarchical inactivation of a synthetic human kinetochore by a chromatin modifier. , 2009, Molecular biology of the cell.

[34]  T. Fukagawa,et al.  CENP-H-containing complex facilitates centromere deposition of CENP-A in cooperation with FACT and CHD1. , 2009, Molecular biology of the cell.

[35]  Adil Jamai,et al.  Histone chaperone spt16 promotes redeposition of the original h3-h4 histones evicted by elongating RNA polymerase. , 2009, Molecular cell.

[36]  C. Obuse,et al.  Active establishment of centromeric CENP-A chromatin by RSF complex , 2009, The Journal of cell biology.

[37]  Gary H. Karpen,et al.  Genome-wide analysis reveals a cell cycle–dependent mechanism controlling centromere propagation , 2008, The Journal of cell biology.

[38]  O. Rando,et al.  Altered Dosage and Mislocalization of Histone H3 and Cse4p Lead to Chromosome Loss in Saccharomyces cerevisiae , 2008, Genetics.

[39]  V. Noskov,et al.  Inactivation of a Human Kinetochore by Specific Targeting of Chromatin Modifiers , 2008, Developmental cell.

[40]  S. Diekmann,et al.  Dynamics of inner kinetochore assembly and maintenance in living cells , 2008, The Journal of cell biology.

[41]  J. Tyler,et al.  Transcriptional regulation by chromatin disassembly and reassembly. , 2007, Current opinion in genetics & development.

[42]  B. E. Black,et al.  Propagation of centromeric chromatin requires exit from mitosis , 2007, The Journal of cell biology.

[43]  N. Nomura,et al.  Comprehensive analysis of the ICEN (Interphase Centromere Complex) components enriched in the CENP‐A chromatin of human cells , 2006, Genes to cells : devoted to molecular & cellular mechanisms.

[44]  John R. Yates,et al.  The human CENP-A centromeric nucleosome-associated complex , 2006, Nature Cell Biology.

[45]  D. Trouche,et al.  Histone H3.3 deposition at E2F‐regulated genes is linked to transcription , 2006, EMBO reports.

[46]  P. Chambon,et al.  Temporally controlled targeted somatic mutagenesis in embryonic surface ectoderm and fetal epidermal keratinocytes unveils two distinct developmental functions of BRG1 in limb morphogenesis and skin barrier formation , 2005, Development.

[47]  D. Schübeler,et al.  Variant histone H3.3 is deposited at sites of nucleosomal displacement throughout transcribed genes while active histone modifications show a promoter-proximal bias. , 2005, Genes & development.

[48]  Kevin Struhl,et al.  Evidence for Eviction and Rapid Deposition of Histones upon Transcriptional Elongation by RNA Polymerase II , 2004, Molecular and Cellular Biology.

[49]  A. Kristjuhan,et al.  Evidence for distinct mechanisms facilitating transcript elongation through chromatin in vivo , 2004, The EMBO journal.

[50]  G. Karpen,et al.  Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin , 2004, Nature Structural &Molecular Biology.

[51]  J. Lieb,et al.  Evidence for nucleosome depletion at active regulatory regions genome-wide , 2004, Nature Genetics.

[52]  James Allan,et al.  Formation of facultative heterochromatin in the absence of HP1 , 2003, The EMBO journal.

[53]  Craig D. Kaplan,et al.  Transcription Elongation Factors Repress Transcription Initiation from Cryptic Sites , 2003, Science.

[54]  G. Orphanides,et al.  FACT Facilitates Transcription-Dependent Nucleosome Alteration , 2003, Science.

[55]  Gary H Karpen,et al.  Sequence analysis of a functional Drosophila centromere. , 2003, Genome research.

[56]  S. Henikoff,et al.  Histone H3 variants specify modes of chromatin assembly , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[57]  S. Henikoff,et al.  The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. , 2002, Molecular cell.

[58]  K. Yoda,et al.  CENP-A, -B, and -C Chromatin Complex That Contains the I-Type α-Satellite Array Constitutes the Prekinetochore in HeLa Cells , 2002, Molecular and Cellular Biology.

[59]  Gary H Karpen,et al.  Conserved organization of centromeric chromatin in flies and humans. , 2002, Developmental cell.

[60]  S. Henikoff,et al.  Heterochromatic deposition of centromeric histone H3-like proteins. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[61]  G. Orphanides,et al.  Requirement of RSF and FACT for transcription of chromatin templates in vitro. , 1998, Science.

[62]  G. Orphanides,et al.  FACT, a Factor that Facilitates Transcript Elongation through Nucleosomes , 1998, Cell.

[63]  G. Karpen,et al.  Molecular Structure of a Functional Drosophila Centromere , 1997, Cell.

[64]  G. Karpen,et al.  The case for epigenetic effects on centromere identity and function. , 1997, Trends in genetics : TIG.

[65]  D. Forbes,et al.  Mitotic repression of the transcriptional machinery. , 1997, Trends in biochemical sciences.

[66]  P Chambon,et al.  Ligand-activated site-specific recombination in mice. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[67]  A. Hilliker,et al.  Mapping simple repeated DNA sequences in heterochromatin of Drosophila melanogaster. , 1993, Genetics.

[68]  D. Luse,et al.  Transcription on nucleosomal templates by RNA polymerase II in vitro: inhibition of elongation with enhancement of sequence-specific pausing. , 1991, Genes & development.

[69]  R. Bravo,et al.  Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites , 1987, The Journal of cell biology.

[70]  K. Bloom,et al.  Genetic manipulation of centromere function , 1987, Molecular and cellular biology.

[71]  R. Kornberg,et al.  Nucleosomes inhibit the initiation of transcription but allow chain elongation with the displacement of histones , 1987, Cell.

[72]  J. Knezetic,et al.  The presence of nucleosomes on a DNA template prevents initiation by RNA polymerase II in vitro , 1986, Cell.

[73]  Michael D. Weiss,et al.  Peer Reviewed Title: Mislocalization of the Drosophila centromere-specific histone CID promotes formation of functional ectopic kinetochores , 2006 .

[74]  B. Migeon,et al.  Three related centromere proteins are absent from the inactive centromere of a stable isodicentric chromosome , 2004, Chromosoma.