scholarly journals CAL1 is the Drosophila CENP-A assembly factor

2014 ◽  
Vol 204 (3) ◽  
pp. 313-329 ◽  
Author(s):  
Chin-Chi Chen ◽  
Mekonnen Lemma Dechassa ◽  
Emily Bettini ◽  
Mary B. Ledoux ◽  
Christian Belisario ◽  
...  
Keyword(s):  
Histone H3 ◽  
Specific Protein ◽  
Terminal Domain ◽  
Left Handed ◽  
C Terminus ◽  
Assembly Factors ◽  
Histone H3 Variant ◽  
Assembly Factor ◽  
Drosophila Cells ◽  
Assembly Activity

Centromeres are specified epigenetically by the incorporation of the histone H3 variant CENP-A. In humans, amphibians, and fungi, CENP-A is deposited at centromeres by the HJURP/Scm3 family of assembly factors, but homologues of these chaperones are absent from a number of major eukaryotic lineages such as insects, fish, nematodes, and plants. In Drosophila, centromeric deposition of CENP-A requires the fly-specific protein CAL1. Here, we show that targeting CAL1 to noncentromeric DNA in Drosophila cells is sufficient to heritably recruit CENP-A, kinetochore proteins, and microtubule attachments. CAL1 selectively interacts with CENP-A and is sufficient to assemble CENP-A nucleosomes that display properties consistent with left-handed octamers. The CENP-A assembly activity of CAL1 resides within an N-terminal domain, whereas the C terminus mediates centromere recognition through an interaction with CENP-C. Collectively, this work identifies the “missing” CENP-A chaperone in flies, revealing fundamental conservation between insect and vertebrate centromere-specification mechanisms.

scholarly journals Deposition of Centromeric Histone H3 Variant CENP-A/Cse4 into Chromatin Is Facilitated by Its C-Terminal Sumoylation

Genetics ◽  
2020 ◽  
Vol 214 (4) ◽  
pp. 839-854 ◽  
Author(s):  
Kentaro Ohkuni ◽  
Evelyn Suva ◽  
Wei-Chun Au ◽  
Robert L. Walker ◽  
Reuben Levy-Myers ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Histone Chaperones ◽  
Wild Type ◽  
Link Type ◽  
C Terminus ◽  
Genome Wide ◽  
Evolutionarily Conserved ◽  
Histone H3 Variant ◽  
Assembly Factor ◽  
Active Investigation

Centromeric localization of CENP-A (Cse4 in Saccharomyces cerevisiae, CID in flies, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of overexpressed CENP-A contributes to aneuploidy in yeast, flies, and humans, and is proposed to promote tumorigenesis in human cancers. Hence, defining molecular mechanisms that promote or prevent mislocalization of CENP-A is an area of active investigation. In budding yeast, evolutionarily conserved histone chaperones Scm3 and chromatin assembly factor-1 (CAF-1) promote localization of Cse4 to centromeric and noncentromeric regions, respectively. Ubiquitin ligases, such as Psh1 and Slx5, and histone chaperones (HIR complex) regulate proteolysis of overexpressed Cse4 and prevent its mislocalization to noncentromeric regions. In this study, we have identified sumoylation sites lysine (K) 215/216 in the C terminus of Cse4, and shown that sumoylation of Cse4 K215/216 facilitates its genome-wide deposition into chromatin when overexpressed. Our results showed reduced levels of sumoylation of mutant Cse4 K215/216R/A [K changed to arginine (R) or alanine (A)] and reduced interaction of mutant Cse4 K215/216R/A with Scm3 and CAF-1 when compared to wild-type Cse4. Consistent with these results, levels of Cse4 K215/216R/A in the chromatin fraction and localization to centromeric and noncentromeric regions were reduced. Furthermore, in contrast to GAL-CSE4, which exhibits Synthetic Dosage Lethality (SDL) in psh1∆, slx5∆, and hir2∆ strains, GAL-cse4 K215/216R does not exhibit SDL in these strains. Taken together, our results show that deposition of Cse4 into chromatin is facilitated by its C-terminal sumoylation.

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 The C-Terminal Domain of Sin1 Interacts with the SWI-SNF Complex in Yeast

1998 ◽  
Vol 18 (7) ◽  
pp. 4157-4164 ◽  
Author(s):  
José Pérez-Martín ◽  
Alexander D. Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Histone H3 ◽  
Full Length ◽  
Terminal Domain ◽  
C Terminus

ABSTRACT In the yeast Saccharomyces cerevisiae, the SWI-SNF complex has been proposed to antagonize the repressive effects of chromatin by disrupting nucleosomes. The SIN genes were identified as suppressors of defects in the SWI-SNF complex, and theSIN1 gene encodes an HMG1-like protein that has been proposed to be a component of chromatin. Specific mutations (sin mutations) in both histone H3 and H4 genes produce the same phenotypic effects as do mutations in the SIN1 gene. In this study, we demonstrate that Sin1 and the H3 and H4 histones interact genetically and that the C terminus of Sin1 physically associates with components of the SWI-SNF complex. In addition, we demonstrate that this interaction is blocked in the full-length Sin1 protein by the N-terminal half of the protein. Based on these and additional results, we propose that Sin1 acts as a regulatable bridge between the SWI-SNF complex and the nucleosome.

scholarly journals AtFKBP53: a chimeric histone chaperone with functional nucleoplasmin and PPIase domains

2019 ◽  
Vol 48 (3) ◽  
pp. 1531-1550
Author(s):  
Ajit Kumar Singh ◽  
Aritreyee Datta ◽  
Chacko Jobichen ◽  
Sheng Luan ◽  
Dileep Vasudevan
Keyword(s):  
Binding Sites ◽  
Histone H3 ◽  
Histone Chaperone ◽  
Nucleosome Assembly ◽  
Ppiase Activity ◽  
Terminal Domain ◽  
C Terminus ◽  
Ppiase Domain ◽  
Fk506 Binding Proteins ◽  
Discrete Complex

Abstract FKBP53 is one of the seven multi-domain FK506-binding proteins present in Arabidopsis thaliana, and it is known to get targeted to the nucleus. It has a conserved PPIase domain at the C-terminus and a highly charged N-terminal stretch, which has been reported to bind to histone H3 and perform the function of a histone chaperone. To better understand the molecular details of this PPIase with histone chaperoning activity, we have solved the crystal structures of its terminal domains and functionally characterized them. The C-terminal domain showed strong PPIase activity, no role in histone chaperoning and revealed a monomeric five-beta palm-like fold that wrapped over a helix, typical of an FK506-binding domain. The N-terminal domain had a pentameric nucleoplasmin-fold; making this the first report of a plant nucleoplasmin structure. Further characterization revealed the N-terminal nucleoplasmin domain to interact with H2A/H2B and H3/H4 histone oligomers, individually, as well as simultaneously, suggesting two different binding sites for H2A/H2B and H3/H4. The pentameric domain assists nucleosome assembly and forms a discrete complex with pre-formed nucleosomes; wherein two pentamers bind to a nucleosome.

scholarly journals Identification of Genes Encoding CENP-A and Heterochromatin Protein 1 of Lipomyces starkeyi and Functional Analysis Using Schizosaccharomyces pombe

Genes ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 769
Author(s):  
Yuko Takayama
Keyword(s):  
Histone H3 ◽  
Specific Protein ◽  
Lipomyces Starkeyi ◽  
Heterochromatin Protein 1 ◽  
Heterochromatin Protein ◽  
Heterochromatin Formation ◽  
Histone H3 Variant ◽  
Genes Encoding ◽  
Almost All ◽  
Heterochromatin Structure

Centromeres function as a platform for the assembly of multiple kinetochore proteins and are essential for chromosome segregation. An active centromere is characterized by the presence of a centromere-specific histone H3 variant, CENP-A. Faithful centromeric localization of CENP-A is supported by heterochromatin in almost all eukaryotes; however, heterochromatin proteins have been lost in most Saccharomycotina. Here, identification of CENP-A (CENP-AL.s.) and heterochromatin protein 1 (Lsw1) in a Saccharomycotina species, the oleaginous yeast Lipomyces starkeyi, is reported. To determine if these proteins are functional, the proteins in S. pombe, a species widely used to study centromeres, were ectopically expressed. CENP-AL.s. localizes to centromeres and can be replaced with S. pombe CENP-A, indicating that CENP-AL.s. is a functional centromere-specific protein. Lsw1 binds at heterochromatin regions, and chromatin binding is dependent on methylation of histone H3 at lysine 9. In other species, self-interaction of heterochromatin protein 1 is thought to cause folding of chromatin, triggering transcription repression and heterochromatin formation. Consistent with this, it was found that Lsw1 can self-interact. L. starkeyi chromatin contains the methylation of histone H3 at lysine 9. These results indicated that L. starkeyi has a primitive heterochromatin structure and is an attractive model for analysis of centromere heterochromatin evolution.

scholarly journals Chromatin Assembly Factor I Mutants Defective for PCNA Binding Require Asf1/Hir Proteins for Silencing

2002 ◽  
Vol 22 (2) ◽  
pp. 614-625 ◽  
Author(s):  
Denise C. Krawitz ◽  
Tamar Kama ◽  
Paul D. Kaufman
Keyword(s):  
Histone H3 ◽  
Chromatin Assembly ◽  
Nucleosome Assembly ◽  
Factor I ◽  
Chromatin Assembly Factor ◽  
Assembly Factor ◽  
Histone Deposition ◽  
Assembly Activity ◽  
Impaired Binding

ABSTRACT Chromatin assembly factor I (CAF-I) is a conserved histone H3/H4 deposition complex. Saccharomyces cerevisiae mutants lacking CAF-I subunit genes (CAC1 to CAC3) display reduced heterochromatic gene silencing. In a screen for silencing-impaired cac1 alleles, we isolated a mutation that reduced binding to the Cac3p subunit and another that impaired binding to the DNA replication protein PCNA. Surprisingly, mutations in Cac1p that abolished PCNA binding resulted in very minor telomeric silencing defects but caused silencing to be largely dependent on Hir proteins and Asf1p, which together comprise an alternative silencing pathway. Consistent with these phenotypes, mutant CAF-I complexes defective for PCNA binding displayed reduced nucleosome assembly activity in vitro but were stimulated by Asf1p-histone complexes. Furthermore, these mutant CAF-I complexes displayed a reduced preference for depositing histones onto newly replicated DNA. We also observed a weak interaction between Asf1p and Cac2p in vitro, and we hypothesize that this interaction underlies the functional synergy between these histone deposition proteins.

RNA secondary structure dependence in METTL3–METTL14 mRNA methylation is modulated by the N-terminal domain of METTL3

2020 ◽  
Vol 402 (1) ◽  
pp. 89-98
Author(s):  
Nathalie Meiser ◽  
Nicole Mench ◽  
Martin Hengesbach
Keyword(s):  
Secondary Structure ◽  
Rna Binding ◽  
Specific Protein ◽  
Structure Dependence ◽  
Terminal Domain ◽  
Methylation Assay ◽  
A Site ◽  
Methylation Activity

AbstractN6-methyladenosine (m6A) is the most abundant modification in mRNA. The core of the human N6-methyltransferase complex (MTC) is formed by a heterodimer consisting of METTL3 and METTL14, which specifically catalyzes m6A formation within an RRACH sequence context. Using recombinant proteins in a site-specific methylation assay that allows determination of quantitative methylation yields, our results show that this complex methylates its target RNAs not only sequence but also secondary structure dependent. Furthermore, we demonstrate the role of specific protein domains on both RNA binding and substrate turnover, focusing on postulated RNA binding elements. Our results show that one zinc finger motif within the complex is sufficient to bind RNA, however, both zinc fingers are required for methylation activity. We show that the N-terminal domain of METTL3 alters the secondary structure dependence of methylation yields. Our results demonstrate that a cooperative effect of all RNA-binding elements in the METTL3–METTL14 complex is required for efficient catalysis, and that binding of further proteins affecting the NTD of METTL3 may regulate substrate specificity.

scholarly journals The box C/D snoRNP assembly factor Bcd1 interacts with the histone chaperone Rtt106 and controls its transcription dependent activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Benoît Bragantini ◽  
Christophe Charron ◽  
Maxime Bourguet ◽  
Arnaud Paul ◽  
Decebal Tiotiu ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Histone H3 ◽  
Histone Chaperone ◽  
Genetic Interactions ◽  
X Ray ◽  
X Ray Crystallography ◽  
Dependent Activity ◽  
Interaction Interface ◽  
Assembly Factor ◽  
Encoding Gene

AbstractBiogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins initiates co-transcriptionally and requires the action of the assembly machinery including the Hsp90/R2TP complex, the Rsa1p:Hit1p heterodimer and the Bcd1 protein. We present genetic interactions between the Rsa1p-encoding gene and genes involved in chromatin organization including RTT106 that codes for the H3-H4 histone chaperone Rtt106p controlling H3K56ac deposition. We show that Bcd1p binds Rtt106p and controls its transcription-dependent recruitment by reducing its association with RNA polymerase II, modulating H3K56ac levels at gene body. We reveal the 3D structures of the free and Rtt106p-bound forms of Bcd1p using nuclear magnetic resonance and X-ray crystallography. The interaction is also studied by a combination of biophysical and proteomic techniques. Bcd1p interacts with a region that is distinct from the interaction interface between the histone chaperone and histone H3. Our results are evidence for a protein interaction interface for Rtt106p that controls its transcription-associated activity.

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