scholarly journals N-terminal sumoylation of centromeric histone H3 variant Cse4 regulates its proteolysis to prevent mislocalization to non-centromeric chromatin

2017 ◽  
Author(s):  
Kentaro Ohkuni ◽  
Reuben Levy-Myers ◽  
Jack Warren ◽  
Wei-Chun Au ◽  
Yoshimitsu Takahashi ◽  
...  
Keyword(s):  
Genome Stability ◽  
Histone H3 ◽  
Physiological Role ◽  
Centromeric Chromatin ◽  
E3 Ligases ◽  
Physiological Conditions ◽  
Evolutionarily Conserved ◽  
Histone H3 Variant

AbstractStringent regulation of cellular levels of evolutionarily conserved centromeric histone H3 variant (CENP-A in humans, CID in flies, Cse4 in yeast) prevents its mislocalization to non-centromeric chromatin. Overexpression and mislocalization of CENP-A has been observed in cancers and leads to aneuploidy in yeast, flies, and human cells. Ubiquitin-mediated proteolysis of Cse4 by E3 ligases such as Psh1 and Sumo-Targeted Ubiquitin Ligase (STUbL) Slx5 prevent mislocalization of Cse4. Previously, we identified Siz1 and Siz2 as the major E3 ligases for sumoylation of Cse4. In this study, we identify lysine 65 (K65) in Cse4 as a SUMO site and show that sumoylation of Cse4 K65 regulates its ubiquitin-mediated proteolysis by Slx5. Strains expressing cse4 K65R exhibit reduced levels of sumoylated and ubiquitinated Cse4 in vivo. Furthermore, co-immunoprecipitation experiments reveal reduced interaction of cse4 K65R with Slx5. Defects in sumoylation of cse4 K65R contribute to increased stability and mislocalization of cse4 K65R under normal physiological conditions. Based on the increased stability of cse4 K65R in psh1∆ strains but not in slx5∆ strains, we conclude that Slx5 targets sumoylated Cse4 K65 for ubiquitination-mediated proteolysis independent of Psh1. In summary, we have identified and characterized the physiological role of Cse4 sumoylation and determined that sumoylation of Cse4 K65 regulates ubiquitin-mediated proteolysis and prevents mislocalization of Cse4 which is required for genome stability.

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 Dbf4-Dependent Kinase (DDK)-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A in Saccharomyces cerevisiae

2020 ◽  
Vol 10 (6) ◽  
pp. 2057-2068 ◽  
Author(s):  
Jessica R. Eisenstatt ◽  
Lars Boeckmann ◽  
Wei-Chun Au ◽  
Valerie Garcia ◽  
Levi Bursch ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Initiation ◽  
Centromeric Chromatin ◽  
Dna Replication Initiation ◽  
Link Type ◽  
Genome Wide ◽  
A Genome ◽  
Evolutionarily Conserved ◽  
Histone H3 Variant

The evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of CENP-A to non-centromeric chromatin contributes to chromosomal instability (CIN) in yeast, fly, and human cells and CENP-A is highly expressed and mislocalized in cancers. Defining mechanisms that prevent mislocalization of CENP-A is an area of active investigation. Ubiquitin-mediated proteolysis of overexpressed Cse4 (GALCSE4) by E3 ubiquitin ligases such as Psh1 prevents mislocalization of Cse4, and psh1Δ strains display synthetic dosage lethality (SDL) with GALCSE4. We previously performed a genome-wide screen and identified five alleles of CDC7 and DBF4 that encode the Dbf4-dependent kinase (DDK) complex, which regulates DNA replication initiation, among the top twelve hits that displayed SDL with GALCSE4. We determined that cdc7-7 strains exhibit defects in ubiquitin-mediated proteolysis of Cse4 and show mislocalization of Cse4. Mutation of MCM5 (mcm5-bob1) bypasses the requirement of Cdc7 for replication initiation and rescues replication defects in a cdc7-7 strain. We determined that mcm5-bob1 does not rescue the SDL and defects in proteolysis of GALCSE4 in a cdc7-7 strain, suggesting a DNA replication-independent role for Cdc7 in Cse4 proteolysis. The SDL phenotype, defects in ubiquitin-mediated proteolysis, and the mislocalization pattern of Cse4 in a cdc7-7 psh1Δ strain were similar to that of cdc7-7 and psh1Δ strains, suggesting that Cdc7 regulates Cse4 in a pathway that overlaps with Psh1. Our results define a DNA replication initiation-independent role of DDK as a regulator of Psh1-mediated proteolysis of Cse4 to prevent mislocalization of Cse4.

scholarly journals CENP-A nucleosome—a chromatin-embedded pedestal for the centromere: lessons learned from structural biology

2020 ◽  
Vol 64 (2) ◽  
pp. 205-221
Author(s):  
Ahmad Ali-Ahmad ◽  
Nikolina Sekulić
Keyword(s):  
Cell Division ◽  
Structural Biology ◽  
Histone H3 ◽  
Lessons Learned ◽  
Centromeric Chromatin ◽  
Centromere Proteins ◽  
Molecular Determinants ◽  
Histone H3 Variant ◽  
Segregation Of Chromosomes

Abstract The centromere is a chromosome locus that directs equal segregation of chromosomes during cell division. A nucleosome containing the histone H3 variant CENP-A epigenetically defines the centromere. Here, we summarize findings from recent structural biology studies, including several CryoEM structures, that contributed to elucidate specific features of the CENP-A nucleosome and molecular determinants of its interactions with CENP-C and CENP-N, the only two centromere proteins that directly bind to it. Based on those findings, we propose a role of the CENP-A nucleosome in the organization of centromeric chromatin beyond binding centromeric proteins.

scholarly journals Evolutionarily conserved roles of Caenorhabditis elegans perilipin in lipolysis

2019 ◽  
Author(s):  
Filip Kaššák ◽  
Ahmed A Chughtai ◽  
Marta Kostrouchová
Keyword(s):  
Neutral Lipids ◽  
Physiological Role ◽  
Hormone Sensitive Lipase ◽  
Functional Connection ◽  
Regulatory Pathways ◽  
C Elegans ◽  
Evolutionarily Conserved ◽  
Eukaryotic Organisms

Neutral lipids and namely triacyl-glycerols (TAGs) are the prevalent excess energy storage molecules in all eukaryotic organisms. They are universally organized in active cytoplasmic organelles called lipid droplets (LDs) and their breakdown is performed and regulated in an evolutionarily conserved manner. In mammals, two distinct but inter-connected pathways are believed to mediate this catabolism: conventional cytoplasmic lipolysis with effector neutral lipases; and lipophagy, a specific kind of autophagy exploiting lysosomal acidic lipases. Central molecules in this regulation are LD-resident proteins, perilipins (PLINs). Our recent discovery of a sole PLIN orthologue in C. elegans offers a unique opportunity to study these regulatory pathways, provided that the interactive mechanisms are orthologous. To determine this, we employed classical genetics with genome editing tools and in vivo microscopy to provide three lines of evidence demonstrating the conserved role of the C. elegans perilipin. Firstly, we proved the common presence of a standard lipolytic apparatus on LDs. Next, we ascertained a functional connection between nematode PLIN-1 and the effector enzyme, hormone-sensitive lipase (HOSL-1). Finally, we identified lipophagy as a secondary lipolytic pathway, which is consistent with the mammalian model. Our data provide not only a proof of concept but also suggests interesting implications by questioning the physiological role of lipophagy in lipolysis.

scholarly journals Epigenetic inheritance of centromere identity in a heterologous system

2019 ◽  
Author(s):  
Virginie Roure ◽  
Bethan Medina-Pritchard ◽  
Eduard Anselm ◽  
A. Arockia Jeyaprakash ◽  
Patrick Heun
Keyword(s):  
Chromosome Segregation ◽  
Chromosomal Region ◽  
Histone H3 ◽  
Epigenetic Inheritance ◽  
Centromeric Chromatin ◽  
Centromere Proteins ◽  
Heterologous System ◽  
Histone H3 Variant ◽  
Self Association

SUMMARYThe centromere is an essential chromosomal region required for accurate chromosome segregation. Most eukaryotic centromeres are defined epigenetically by the histone H3 variant, CENP-A, yet how its self-propagation is achieved remains poorly understood. Here we developed a heterologous system to reconstitute epigenetic inheritance of centromeric chromatin by ectopically targeting the Drosophila centromere proteins dCENP-A, dCENP-C and CAL1 to LacO arrays in human cells. Dissecting the function of these three components uncovers the key role of self-association of dCENP-C and CAL1 for their mutual interaction and dCENP-A deposition. Importantly, we identify the components required for dCENP-C loading onto chromatin, involving a cooperation between CAL1 and dCENP-A nucleosomes, thus closing the epigenetic loop to ensure dCENP-C and dCENP-A replenishment during the cell division cycle. Finally, we show that all three Drosophila factors are sufficient for dCENP-A propagation and propose a model for the epigenetic inheritance of centromere identity.

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 The ratio between centromeric proteins CENP-A and CENP-C maintains homeostasis of human centromeres

2019 ◽  
Author(s):  
Daniël P. Melters ◽  
Tatini Rakshit ◽  
Minh Bui ◽  
Sergei A. Grigoryev ◽  
David Sturgill ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
De Novo ◽  
Histone H3 ◽  
Chromosomal Locus ◽  
Centromeric Chromatin ◽  
Dna Accessibility ◽  
Nucleosome Dynamics ◽  
Histone H3 Variant ◽  
Correct Ratio

AbstractThe centromere is the chromosomal locus that seeds the kinetochore, allowing for a physical connection between the chromosome and the mitotic spindle. At the heart of the centromere is the centromere-specific histone H3 variant CENP-A/CENH3. Throughout the cell cycle the constitutive centromere associated network is bound to CENP-A chromatin, but how this protein network modifies CENP-A nucleosome dynamics in vivo is unknown. Here, we purify kinetochore associated native centromeric chromatin and analyze its biochemical features using a combinatorial approach. We report that kinetochore bound chromatin has strongly reduced DNA accessibility and a distinct stabilized nucleosomal configuration. Disrupting the balance between CENP-A and CENP-C result in reduced centromeric occupancy of RNA polymerase 2 and impaired de novo CENP-A loading on the centromeric chromatin fiber, correlating with significant mitotic defects. CENP-A mutants that restore the ratio rescue the mitotic defects. These data support a model in which CENP-C bound centromeric nucleosomes behave as a barrier to the transcriptional machinery and suggest that maintaining the correct ratio between CENP-A and CENP-C levels is critical for centromere homeostasis.

scholarly journals Evolutionarily conserved roles of Caenorhabditis elegans perilipin in lipolysis

2019 ◽  
Author(s):  
Filip Kaššák ◽  
Ahmed A Chughtai ◽  
Marta Kostrouchová
Keyword(s):  
Neutral Lipids ◽  
Physiological Role ◽  
Hormone Sensitive Lipase ◽  
Functional Connection ◽  
Regulatory Pathways ◽  
C Elegans ◽  
Evolutionarily Conserved ◽  
Eukaryotic Organisms

Neutral lipids and namely triacyl-glycerols (TAGs) are the prevalent excess energy storage molecules in all eukaryotic organisms. They are universally organized in active cytoplasmic organelles called lipid droplets (LDs) and their breakdown is performed and regulated in an evolutionarily conserved manner. In mammals, two distinct but inter-connected pathways are believed to mediate this catabolism: conventional cytoplasmic lipolysis with effector neutral lipases; and lipophagy, a specific kind of autophagy exploiting lysosomal acidic lipases. Central molecules in this regulation are LD-resident proteins, perilipins (PLINs). Our recent discovery of a sole PLIN orthologue in C. elegans offers a unique opportunity to study these regulatory pathways, provided that the interactive mechanisms are orthologous. To determine this, we employed classical genetics with genome editing tools and in vivo microscopy to provide three lines of evidence demonstrating the conserved role of the C. elegans perilipin. Firstly, we proved the common presence of a standard lipolytic apparatus on LDs. Next, we ascertained a functional connection between nematode PLIN-1 and the effector enzyme, hormone-sensitive lipase (HOSL-1). Finally, we identified lipophagy as a secondary lipolytic pathway, which is consistent with the mammalian model. Our data provide not only a proof of concept but also suggests interesting implications by questioning the physiological role of lipophagy in lipolysis.

scholarly journals BRCA1 prevents R-loop-associated centromeric instability

2021 ◽  
Vol 12 (10) ◽  
Author(s):  
Carine Racca ◽  
Sébastien Britton ◽  
Sabrine Hédouin ◽  
Claire Francastel ◽  
Patrick Calsou ◽  
...  
Keyword(s):  
Genome Stability ◽  
Essential Role ◽  
Histone H3 ◽  
Centromeric Chromatin ◽  
Induce Cell Death ◽  
Satellite Sequences ◽  
Centromeric Function ◽  
Satellite Repeats ◽  
Histone H3 Variant ◽  
Centromeric Instability

AbstractCentromeres are defined by chromatin containing the histone H3 variant CENP-A assembled onto repetitive α-satellite sequences, which are actively transcribed throughout the cell cycle. Centromeres play an essential role in chromosome inheritance and genome stability through coordinating kinetochores assembly during mitosis. Structural and functional alterations of the centromeres cause aneuploidy and chromosome aberrations which can induce cell death. In human cells, the tumor suppressor BRCA1 associates with centromeric chromatin in the absence of exogenous damage. While we previously reported that BRCA1 contributes to proper centromere homeostasis, the mechanism underlying its centromeric function and recruitment was not fully understood. Here, we show that BRCA1 association with centromeric chromatin depends on the presence of R-loops, which are non-canonical three-stranded structures harboring a DNA:RNA hybrid and are frequently formed during transcription. Subsequently, BRCA1 counteracts the accumulation of R-loops at centromeric α-satellite repeats. Strikingly, BRCA1-deficient cells show impaired localization of CENP-A, higher transcription of centromeric RNA, increased breakage at centromeres and formation of acentric micronuclei, all these features being R-loop-dependent. Finally, BRCA1 depletion reveals a Rad52-dependent hyper-recombination process between centromeric satellite repeats, associated with centromere instability and missegregation. Altogether, our findings provide molecular insights into the key function of BRCA1 in maintaining centromere stability and identity.

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