scholarly journals Nucleosome formation with the testis-specific histone H3 variant, H3t, by human nucleosome assembly proteins in vitro

2008 ◽  
Vol 36 (7) ◽  
pp. 2208-2218 ◽  
Author(s):  
H. Tachiwana ◽  
A. Osakabe ◽  
H. Kimura ◽  
H. Kurumizaka
Keyword(s):  
Histone H3 ◽  
Nucleosome Assembly ◽  
Nucleosome Formation ◽  
Histone H3 Variant ◽  
Assembly Proteins

scholarly journals Involvement of Nucleocytoplasmic Shuttling of Yeast Nap1 in Mitotic Progression

2003 ◽  
Vol 23 (18) ◽  
pp. 6672-6684 ◽  
Author(s):  
Mary Miyaji-Yamaguchi ◽  
Kohsuke Kato ◽  
Ryosuke Nakano ◽  
Tomohiro Akashi ◽  
Akihiko Kikuchi ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Temperature Sensitive ◽  
Nucleosome Assembly ◽  
Mitotic Progression ◽  
Nucleocytoplasmic Shuttling ◽  
Nucleosome Formation ◽  
Mitotic Cyclin ◽  
Carboxy Terminal

ABSTRACT Nucleosome assembly protein 1 (Nap1) is widely conserved from yeasts to humans and facilitates nucleosome formation in vitro as a histone chaperone. Nap1 is generally localized in the cytoplasm, except that subcellular localization of Drosophila melanogaster Nap1 is dynamically regulated between the cytoplasm and nucleus during early development. The cytoplasmic localization of Nap1 is seemingly incompatible with the proposed role of Nap1 in nucleosome formation, which should occur in the nucleus. Here, we have examined the roles of a putative nuclear export signal (NES) sequence in yeast Nap1 (yNap1). yNap1 mutants lacking the NES-like sequence were localized predominantly in the nucleus. Deletion of NAP1 in cells harboring a single mitotic cyclin gene is known to cause mitotic delay and temperature-sensitive growth. A wild-type NAP1 complemented these phenotypes while nap1 mutant genes lacking the NES-like sequence or carboxy-terminal region did not. These and other results suggest that yNap1 is a nucleocytoplasmic shuttling protein and that its shuttling is important for yNap1 function during mitotic progression. This study also provides a possible explanation for Nap1's involvement in nucleosome assembly and/or remodeling in the nucleus.

scholarly journals SUMO-targeted ubiquitin ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin

2016 ◽  
Vol 27 (9) ◽  
pp. 1500-1510 ◽  
Author(s):  
Kentaro Ohkuni ◽  
Yoshimitsu Takahashi ◽  
Alyona Fulp ◽  
Josh Lawrimore ◽  
Wei-Chun Au ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Genome Stability ◽  
Critical Role ◽  
Histone H3 ◽  
E3 Ligases ◽  
Physiological Conditions ◽  
Histone H3 Variant ◽  
Maintain Genome Stability

Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast and flies and tumorigenesis in human cells; thus defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast shows that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by the small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1, since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization than either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.

scholarly journals Chromatin assembly: Journey to the CENter of the chromosome

2016 ◽  
Vol 214 (1) ◽  
pp. 13-24 ◽  
Author(s):  
Chin-Chi Chen ◽  
Barbara G. Mellone
Keyword(s):  
Cell Survival ◽  
Histone H3 ◽  
Chromatin Assembly ◽  
Nucleosome Assembly ◽  
Histone Proteins ◽  
Chromatin Domains ◽  
Histone H3 Variant ◽  
Distinctive Type ◽  
Eukaryotic Genomes

All eukaryotic genomes are packaged into basic units of DNA wrapped around histone proteins called nucleosomes. The ability of histones to specify a variety of epigenetic states at defined chromatin domains is essential for cell survival. The most distinctive type of chromatin is found at centromeres, which are marked by the centromere-specific histone H3 variant CENP-A. Many of the factors that regulate CENP-A chromatin have been identified; however, our understanding of the mechanisms of centromeric nucleosome assembly, maintenance, and reorganization remains limited. This review discusses recent insights into these processes and draws parallels between centromeric and noncentromeric chromatin assembly mechanisms.

scholarly journals CENP-C recruits M18BP1 to centromeres to promote CENP-A chromatin assembly

2011 ◽  
Vol 194 (6) ◽  
pp. 855-871 ◽  
Author(s):  
Ben Moree ◽  
Corey B. Meyer ◽  
Colin J. Fuller ◽  
Aaron F. Straight
Keyword(s):  
Chromosome Segregation ◽  
Protein A ◽  
Histone H3 ◽  
Chromatin Assembly ◽  
Nucleosome Assembly ◽  
Chromosomal Locus ◽  
C Protein ◽  
Centromere Protein A ◽  
Histone H3 Variant ◽  
Complex Protein

Eukaryotic chromosomes segregate by attaching to microtubules of the mitotic spindle through a chromosomal microtubule binding site called the kinetochore. Kinetochores assemble on a specialized chromosomal locus termed the centromere, which is characterized by the replacement of histone H3 in centromeric nucleosomes with the essential histone H3 variant CENP-A (centromere protein A). Understanding how CENP-A chromatin is assembled and maintained is central to understanding chromosome segregation mechanisms. CENP-A nucleosome assembly requires the Mis18 complex and the CENP-A chaperone HJURP. These factors localize to centromeres in telophase/G1, when new CENP-A chromatin is assembled. The mechanisms that control their targeting are unknown. In this paper, we identify a mechanism for recruiting the Mis18 complex protein M18BP1 to centromeres. We show that depletion of CENP-C prevents M18BP1 targeting to metaphase centromeres and inhibits CENP-A chromatin assembly. We find that M18BP1 directly binds CENP-C through conserved domains in the CENP-C protein. Thus, CENP-C provides a link between existing CENP-A chromatin and the proteins required for new CENP-A nucleosome assembly.

scholarly journals Constitutive centromere-associated network contacts confer differential stability on CENP-A nucleosomes in vitro and in the cell

2018 ◽  
Vol 29 (6) ◽  
pp. 751-762 ◽  
Author(s):  
Shengya Cao ◽  
Keda Zhou ◽  
Zhening Zhang ◽  
Karolin Luger ◽  
Aaron F. Straight
Keyword(s):  
Protein A ◽  
Histone H3 ◽  
Intrinsic Property ◽  
Cell Cycles ◽  
Centromere Protein A ◽  
Histone H3 Variant ◽  
Differential Stability ◽  
The Stability

Eukaryotic centromeres are defined by the presence of nucleosomes containing the histone H3 variant, centromere protein A (CENP-A). Once incorporated at centromeres, CENP-A nucleosomes are remarkably stable, exhibiting no detectable loss or exchange over many cell cycles. It is currently unclear whether this stability is an intrinsic property of CENP-A containing chromatin or whether it arises from proteins that specifically associate with CENP-A chromatin. Two proteins, CENP-C and CENP-N, are known to bind CENP-A human nucleosomes directly. Here we test the hypothesis that CENP-C or CENP-N stabilize CENP-A nucleosomes in vitro and in living cells. We show that CENP-N stabilizes CENP-A nucleosomes alone and additively with CENP-C in vitro. However, removal of CENP-C and CENP-N from cells, or mutating CENP-A so that it no longer interacts with CENP-C or CENP-N, had no effect on centromeric CENP-A stability in vivo. Thus, the stability of CENP-A nucleosomes in chromatin does not arise solely from its interactions with CENP-C or CENP-N.

scholarly journals The ATAD2/ANCCA homolog Yta7 cooperates with Scm3HJURP to deposit Cse4CENP-A at the centromere in yeast

2020 ◽  
Vol 117 (10) ◽  
pp. 5386-5393 ◽  
Author(s):  
Sara Shahnejat-Bushehri ◽  
Ann E. Ehrenhofer-Murray
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Histone H3 ◽  
Nucleosome Assembly ◽  
Direct Role ◽  
Kinetochore Assembly ◽  
Histone H3 Variant ◽  
Assembly Factor

The AAA+ ATPase and bromodomain factor ATAD2/ANCCA is overexpressed in many types of cancer, but how it contributes to tumorigenesis is not understood. Here, we report that the Saccharomyces cerevisiae homolog Yta7ATAD2 is a deposition factor for the centromeric histone H3 variant Cse4CENP-A at the centromere in yeast. Yta7ATAD2 regulates the levels of centromeric Cse4CENP-A in that yta7∆ causes reduced Cse4CENP-A deposition, whereas YTA7 overexpression causes increased Cse4CENP-A deposition. Yta7ATAD2 coimmunoprecipitates with Cse4CENP-A and is associated with the centromere, arguing for a direct role of Yta7ATAD2 in Cse4CENP-A deposition. Furthermore, increasing centromeric Cse4CENP-A levels by YTA7 overexpression requires the activity of Scm3HJURP, the centromeric nucleosome assembly factor. Importantly, Yta7ATAD2 interacts in vivo with Scm3HJURP, indicating that Yta7ATAD2 is a cochaperone for Scm3HJURP. The absence of Yta7 causes defects in growth and chromosome segregation with mutations in components of the inner kinetochore (CTF19/CCAN, Mif2CENP-C, Cbf1). Since Yta7ATAD2 is an AAA+ ATPase and potential hexameric unfoldase, our results suggest that it may unfold the Cse4CENP-A histone and hand it over to Scm3HJURP for subsequent deposition in the centromeric nucleosome. Furthermore, our findings suggest that ATAD2 overexpression may enhance malignant transformation in humans by misregulating centromeric CENP-A levels, thus leading to defects in kinetochore assembly and chromosome segregation.

scholarly journals CENP-H–containing Complex Facilitates Centromere Deposition of CENP-A in Cooperation with FACT and CHD1

2009 ◽  
Vol 20 (18) ◽  
pp. 3986-3995 ◽  
Author(s):  
Masahiro Okada ◽  
Katsuya Okawa ◽  
Toshiaki Isobe ◽  
Tatsuo Fukagawa
Keyword(s):  
Chromatin Remodeling ◽  
Mutant Cell ◽  
Histone H3 ◽  
Dependent Manner ◽  
Centromeric Chromatin ◽  
Conditional Mutant ◽  
Histone H3 Variant ◽  
A Subunit

Centromere identity is thought to be determined by epigenetic mechanisms. The centromere-specific histone H3 variant CENP-A plays a central role in specifying the locus where the centromere is constructed. However, the precise mechanisms that target CENP-A to centromeric chromatin are poorly understood. Here, we show that facilitates chromatin transcription (FACT) localizes to centromeres in a CENP-H–containing complex-dependent manner. In conditional mutant cell lines for SSRP1, a subunit of FACT, centromere targeting of newly synthesized CENP-A is severely inhibited. The chromatin remodeling factor CHD1 binds to SSRP1 both in vivo and in vitro and associates with centromeres. The centromeric localization of CHD1 is lost in SSRP1-depleted cells. RNA interference knockdown of CHD1 leads to a decrease in the amount of centromere localized CENP-A. These findings indicate that the CENP-H–containing complex facilitates deposition of newly synthesized CENP-A into centromeric chromatin in cooperation with FACT and CHD1.

scholarly journals Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae

2013 ◽  
Vol 24 (12) ◽  
pp. 2034-2044 ◽  
Author(s):  
Lars Boeckmann ◽  
Yoshimitsu Takahashi ◽  
Wei-Chun Au ◽  
Prashant K. Mishra ◽  
John S. Choy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Histone H3 ◽  
In Vitro Assays ◽  
Histone H3 Variant

The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein–labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.

scholarly journals The N-terminal Tail of C. elegans CENP-A Interacts with KNL-2 and is Essential for Centromeric Chromatin Assembly

2020 ◽  
Author(s):  
Christian de Groot ◽  
Jack Houston ◽  
Bethany Davis ◽  
Adina Gerson-Gurwitz ◽  
Joost Monen ◽  
...  
Keyword(s):  
Histone H3 ◽  
Direct Interaction ◽  
Chromatin Assembly ◽  
Nucleosome Assembly ◽  
Centromeric Chromatin ◽  
C Elegans ◽  
Histone Fold ◽  
Kinetochore Assembly ◽  
Histone H3 Variant ◽  
Evolutionary Variation

ABSTRACTCentromeres are epigenetically defined by the presence of the centromere-specific histone H3 variant CENP-A. A specialized loading machinery, including the histone chaperone HJURP/Scm3, participates in CENP-A nucleosome assembly. However, Scm3/HJURP is missing from multiple lineages, including nematodes, which rely on a CENP-A-dependent centromere. Here, we show that the extended N-terminal tail of C. elegans CENP-A contains a predicted structured region that is essential for centromeric chromatin assembly. Removal of this region of the CENP-A N-Tail prevents loading, resulting in failure of kinetochore assembly and defective chromosome condensation. By contrast, the N-Tail mutant CENP-A localizes normally in the presence of endogenous CENP-A. The portion of the N-Tail containing the predicted structured region binds to KNL-2, a conserved SANTA and Myb domain-containing protein (referred to as M18BP1 in vertebrates), that is specifically involved in CENP-A chromatin assembly. This direct interaction is conserved in the related nematode C. briggsae, despite divergence of the N-Tail and KNL-2 primary sequences. Thus, the extended N-Tail of CENP-A is essential for CENP-A chromatin assembly in C. elegans and partially substitutes for the function of Scm3/HJURP, in that it mediates an interaction of the specialized histone fold of CENP-A with KNL-2. These results highlight an evolutionary variation on centromeric chromatin assembly in the absence of a dedicated CENP-A-specific chaperone/targeting factor of the Scm3/HJURP family.

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