CENP-B initiates the CENP-A self-assembly epigenetic loop by recruiting CENP-C at native human centromeres

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Post Translational Modifications of CENP-A nucleosomes at the centromere

Since CENP-A molecules are found in a greater number outside of the centromere region it is tempting to speculate that CENP-A PTMs found exclusively at the centromere could restrict CENP-A’s function to centromeric regions. In the case of the point centromeres of budding yeast for example the methylation of K37 is indeed described to be a key PTM for CCAN formation (Samel et al., 2012).
In species with regional centromeres a CENP-A methylation site with similar importance has not been described to my knowledge. However, in human, several CENP-A modifications (phosphorylation, acetylation, methylation, ubiquitination) have been reported and functional relevancies of these PTMs have been proposed. Phosphorylation of S68, acetylation/ubiquitination of K124 were suggested to be important for CENP-A deposition at the centromere (in the case of ubiquitination this mark was even proposed to be propagated by CENP-A dimerization at the centromere) and S7 phosphorylation for CENP-C recruitment or Aurora B recruitment to the centromere in human (Eot-Houllier et al., 2018; Goutte-Gattat et al., 2013; Kunitoku et al., 2003; Niikura et al., 2019, 2016, 2015; Yu et al., 2015; Zhao et al., 2016). Neither K124 nor S68 are part of the CENP-A CATD domain nor is S7 even close to the binding site for CENP-C and importantly, all functional relevancies of these modifications were substantially contradicted by our lab and the Black lab (Barra et al., 2019; Fachinetti et al., 2017). There is an ongoing controversy for the importance of K124 ubiquitination, which can be compensated by a protein tag in a recent publication from the Kitagawa lab (Niikura et al. 2019). Phosphorylation of the residues S16 and S18 are crucial and present in pre-nucleosomal CENP-A. Their absence or hyper- phosphorylation leads to chromosome missegregation (Bailey et al., 2013; Barra et al., 2019).
Additionally, in the same report that described S16 and S18 phosphorylation of CENP-A, amino-terminal tri-methylation of glycine was discovered (glycine is the N-terminal amino acid since Methionine, translated from the start codon, is post-translationally removed from CENP-A) (Bailey et al., 2013). Loss of N-terminal tri-methylation of CENP-A leads to a reduction of CENP-T and CENP-I at the centromere and causes chromosome segregation errors. (Sathyan et al., 2017). Overall, little is known about the regulation of CENP-A PTMs and only S16/18 phosphorylation have clear functional relevance before CENP-A is deposited at the centromere. Whether there are any functional implications of PTMs on centromeric CENP-A remains unclear.

Centromere nucleosome sequence and centromere architecture

An important question to better understand the structural organization of the centromere is the identification of the nucleosome sequence and the 3-D organization of centrochromatin. ChIP-sequencing and chromatin fiber staining techniques have been applied to investigate the distribution of H3 and CENP-A nucleosomes at the centromere. In the chromatin fiber analysis, centromeric chromatin is physically stretched to 50-100 times of its normal interphase length after cell lysis providing a resolution of approximately 20 kb. Using this chromatin unfolding technique, CENP-A molecules and H3 nucleosomes were found to be interspersed at the centromere in flies and human (Blower et al., 2002; Sullivan and Karpen, 2004). However, local CENP-A clusters with higher occupancy have been observed as well (Blower et al., 2002). CENP-A clustering may be even more pronounced in the 3-D architecture of the centromere as indicated by high resolution microscopy (Andronov et al., 2019).
ChIP-Seq is a powerful tool to study epigenetic modification genome-wide but currently does not provide single cell resolution like the chromatin fiber technique. Moreover, studying CENP-A distribution at native centromeres using ChIP-Seq is limited by the repetitive nature of the centromere. Sequencing reads cannot unambiguously be mapped to centromeric DNA in the way it would be required to determine a CENP-A nucleosome sequence. However, ChIP-Seq on chromosomes that are not characterized by highly repetitive DNA sequences (such as the Z chromosome of chicken or human neocentromeres (see section 2.4 and 2.6)), where sequence mapping is feasible have been used to study CENP-A nucleosome occupancy at the centromere (Bodor et al., 2014; Shang et al., 2013, 2010). These studies revealed that, in agreement with the chromatin fiber analysis, CENP-A nucleosomes are indeed not found in one major cluster but smaller sets of local clusters with very high CENP-A occupancy seeming to exist within the centromere. Recent advances in the chromatin fiber technique and ChIP approaches describe the presence of two distinct CENP-A populations at the centromere. Only one population appears to be strongly associated with CENP-C and is likely directly involved in kinetochore formation (Kyriacou and Heun, 2018; Melters et al., 2019) while the second population might have a different function in CENP-A homeostasis (Melters et al., 2019). This is in agreement with the estimated average molecule number of CENP-C (215 molecules) and CENP-A (400 molecules) (Bodor et al., 2014; Suzuki et al., 2015).

The Never-ending Story of Centromere Identity – CENP-A’s Epigenetic Self-Assembly Loop

In human and most species studied so far, centromeres can propagate through an epigenetic mechanism based on the epigenetic mark CENP-A (Zasadzińska and Foltz, 2017). New CENP-A chromatin assembly is ultimately directed by the presence of preexisting CENP-A nucleosomes which generates an epigenetic self-assembly loop (Figure 3).
Centromeric CENP-A nucleosomes are further described to be remarkably stable (see section 2.3d) and CENP-A molecules are very efficiently recycled at the centromere when cells undergo S-phase (unlike other H3 histones) (Bodor et al., 2013; Shelby et al., 2000; Zasadzińska et al., 2018).
DNA replication however, causes dilution of CENP-A nucleosomes to both daughter strands, which commonly occurs in a random fashion (Bodor et al., 2013; Shelby et al., 2000) with most likely some interesting exceptions discovered in the context of stem cell differentiation of the Drosophila mid-gut (Arco et al., 2018). CENP-A nucleosome replication does not occur immediately in S-phase but CENP-A is replenished in many species studied so far in a different cell cycle phase (Bernad et al., 2011; Black et al., 2007; Erhardt et al., 2008; Takayama et al., 2008). This is however different in budding yeast where CENP-ACse4 is replenished during S-phase (Pearson et al., 2004; Wisniewski et al., 2014) and in fission yeast that can replenish CENP-ACnp1 in S-phase and G2-phase (Takayama et al., 2008).
In human, the timing of new CENP-A reloading was uncovered by taking advantage of the SNAP-tag technology (Jansen et al., 2007a). At a time point zero preexisting SNAP-tagged proteins in the cell can be chemically quenched using a non-fluorescent benzylguanine derivative which rapidly and irreversibly binds to the SNAP-tag. Following a chase period, newly synthesized SNAP-tagged proteins can be labeled using a different, this time fluorescent benzylguanine derivative allowing exclusive detection of newly synthesized proteins by fluorescence microscopy. Using this technique Jansen et al. revealed that CENP-ASNAP is newly deposited at the centromere in late telo-/early G1 phase (Jansen et al., 2007a) (Figure 3A).

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The CCAN – the reader of the epigenetic mark

Although in frogs and chicken the CCAN may not be required for HJURP recruitment and CENP-A assembly, in human, CENP-C (and maybe CENP-I) seems to be very important to function as a “reader” of CENP-A nucleosomes (Dambacher et al., 2012; Hori et al., 2012; Moree et al., 2011; Shono et al., 2015; Stellfox et al., 2016).
As described above CENP-C interacts via its C-terminal domain with M18BP1 and Mis18β (Dambacher et al., 2012; Hori et al., 2012; Moree et al., 2011; Shono et al., 2015; Stellfox et al., 2016).
When bound to an ectopic site CENP-C and CENP-I are indeed individually sufficient to recruit the Mis18 complex and trigger the incorporation of endogenous CENP-A at this locus even though with a rather low efficiency (~30%) (Hori et al., 2012; Shono et al., 2015).

Table of contents :

1. ACKNOWLEDGEMENTS
2. RÉSUMÉ
3. ABSTRACT
4. INTRODUCTION
I. The Nucleus
I.1. Histones, Nucleosomes and Chromatin
I.3 Chromatin and Epigenetics
I.4 Chromosome Segregation
I.5 Chromosome Segregation Error
II The Centromere
II.1 The “Meshwork” and its “Blueprint” – Organization and Function of the Centromere Protein Network
a) CCAN organization and assembly
b) Inner kinetochore (CCAN) to outer kinetochore connections
II.2 A region like no other? – Centromeric Chromatin/Epigenetic Environment
a) The Histone H3 variant CENP-A
b) The CENP-A Nucleosome
c) Post Translational Modifications of CENP-A nucleosomes at the centromere
d) Centrochromatin beyond CENP-A
e) Pericentromeric chromatin
f) Centromere nucleosome sequence and centromere architecture
II.3 The Never-ending Story of Centromere Identity – CENP-A’s Epigenetic Self-Assembly Loop26
a) HJURP – CENP-A’s chaperone
b) Mis18 complex – the licensing factor
c) The CCAN – the reader of the epigenetic mark
d) CENP-A – the epigenetic mark
II.4 The Curious Case of Centromeric DNA (CenDNA)
a) Centromere Evolution
b) Centromere DNA sequence organization
c) Contribution of DNA sequence to the centromere architecture
II.5 CENP-B
a) CENP-B’s functions
b) Regulation of CENP-B binding at the centromere
II.6 De Novo Centromere Formation
a) Human Neocentromeres and inactivated centromeres
b) Artificial Generation of Neocentromeres
III.1 Summary and aims of this thesis
III.2 Résumé et objectifs de cette thèse
Previously deposited CENP-A is not essential for new CENP-A deposition at endogenous centromeres
De novo CENP-A localization remains unaltered in absence of old centromeric CENP-A
De novo CENP-A deposition is impaired in CENP-B deficient cells
Neocentromere formation occurs on the Y chromosome under selective pressure.
CENP-B bound to an ectopic location promotes CENP-C recruitment independently of CENP-A
CENP-B marks native centromere position to promote de novo CENP-A/C reloading
CENP-B initiates the CENP-A self-assembly epigenetic loop by recruiting CENP-C at native human centromeres
CENP-A-negative resting CD4+ T cells re-assemble CENP-A de novo upon cell cycle entry
6. RESULTATS (Résumé en français)
7. DISCUSSION
8. DISCUSSION (Résumé en français)
9. METHODS
Cell culture
Gene Targeting
Generation of Stable Cell Lines
siRNA, transient transfections, EdU staining and Colony Formation Assay
Immunoblotting
Immunofluorescence, Chromosome Spreads, IF-FISH and Live-Cell Microscopy
Single molecule microscopy
CUT&RUN-sequencing and -qPCR
Bioinformatic analysis
IF-FISH chromatin fiber
Intensity quantifications in microscopy experiments
Cloning, Expression, and protein Purification
GST pull down assay
CD4+ T cell staining and sorting
Data availability
10. CONTRIBUTIONS
11. REFERENCES
12. ABBREVIATION REGISTER
13. CURRICULUM VITAE
14. PUBLICATIONS

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