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 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 Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions

2016 ◽  
Vol 91 (3) ◽  
Author(s):  
Jolene Ramsey ◽  
Emily C. Renzi ◽  
Randy J. Arnold ◽  
Jonathan C. Trinidad ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Plasma Membrane ◽  
Sindbis Virus ◽  
Molecular Mechanisms ◽  
Wild Type Virus ◽  
Membrane Localization ◽  
Clear Understanding ◽  
Wild Type ◽  
Plasma Membrane Localization ◽  
C Terminus ◽  
N Terminus

ABSTRACT Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a −1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. IMPORTANCE Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo. Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

scholarly journals How Not To Be in the Wrong Place at the Wrong Time: An Education Primer for Use with “Deposition of Centromeric Histone H3 Variant CENP-A/Cse4 into Chromatin Is Facilitated by Its C-Terminal Sumoylation”

Genetics ◽  
2020 ◽  
Vol 216 (2) ◽  
pp. 333-342
Author(s):  
Yee Mon Thu
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Diseases ◽  
Histone H3 ◽  
Model Organism ◽  
Post Translational Modification ◽  
Genetic Changes ◽  
Equal Distribution ◽  
Link Type ◽  
Histone H3 Variant ◽  
Big Picture

Recent work by Kentaro Ohkuni and colleagues exemplifies how a series of molecular mechanisms contribute to a cellular outcome—equal distribution of chromosomes. Failure to maintain structural and numerical integrity of chromosomes is one contributing factor in genetic diseases such as cancer. Specifically, the authors investigated molecular events surrounding centromeric histone H3 variant Cse4 deposition—a process important for chromosome segregation, using Saccharomyces cerevisiae as a model organism. This study illustrates an example of a post-translational modification—sumoylation—regulating a cellular process and the concept of genetic interactions (e.g., synthetic dosage lethality). Furthermore, the study highlights the importance of using diverse experimental approaches in answering a few key research questions. The authors used molecular biology techniques (e.g., qPCR), biochemical experiments (e.g., Ni-NTA/8His protein purification), as well as genetic approaches to understand the regulation of Cse4. At a big-picture level, the study reveals how genetic changes can lead to subsequent molecular and cellular changes.

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 Mutation of a palmitoyltransferase ZDHHC18-like gene is responsible for the minute wing mutation in the silkworm (Bombyx mori)

2017 ◽  
Author(s):  
Ye Yu ◽  
Xiaojing Liu ◽  
Xiao Ma ◽  
Zhongjie Zhang ◽  
Na Liu ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Positional Cloning ◽  
Molecular Mechanisms ◽  
Adult Stage ◽  
Significant Proportion ◽  
Gene Promoter ◽  
Wild Type ◽  
Evolutionarily Conserved ◽  
In The Wild ◽  
Hippo Signalling

AbstractPulpal- and adult-stage minute wing (mw) mutants of the silkworm Bombyx mori have much smaller wings than those of the wild type. Herein, we report the genetic and molecular mechanisms underling the formation of the minute wing. Fine mapping and positional cloning revealed that a palmitoyltransferase ZDHHC18-like gene (BmAPP）was responsible for the mw mutation. CRISPR/Cas9 screening of the BmAPP gene in wild-type embryos revealed that a significant proportion of adults had the mw mutation. In an mw mutant strain, u11, a 10-bp insertion in the promoter of a novel gene resulted in low-level translation of a protein belonging to the DHHC family. A PiggyBac-based transgenic analysis showed that the promoter induced the expression of aBmAPP gene promoter in the wild type but not the mw mutant. These findings indicate that functional deletion of this gene promoter accounts for the mw mutation in silkworm. The possibility that BmAPP gene is involved in Hippo signalling pathway, an evolutionarily conserved signaling pathway that controls organ size, is discussed.

scholarly journals Molecular Basis of p53 Functional Inactivation by the Leukemic Protein MLL-ELL

2003 ◽  
Vol 23 (12) ◽  
pp. 4230-4246 ◽  
Author(s):  
Dmitri Wiederschain ◽  
Hidehiko Kawai ◽  
JiJie Gu ◽  
Ali Shilatifard ◽  
Zhi-Min Yuan
Keyword(s):  
Molecular Basis ◽  
Molecular Mechanisms ◽  
Cellular Transformation ◽  
Wild Type ◽  
Efficient Inhibitor ◽  
C Terminus ◽  
Functional Inactivation ◽  
Necessary And Sufficient ◽  
First Time

ABSTRACT The Eleven Lysine-rich Leukemia (ELL) gene undergoes translocation and fuses in frame to the Multiple Lineage Leukemia (MLL) gene in a substantial proportion of patients suffering from acute forms of leukemia. Molecular mechanisms of cellular transformation by the MLL-ELL fusion are not well understood. Although both MLL-ELL and wild-type ELL can reduce functional activity of p53 tumor suppressor, our data reveal that MLL-ELL is a much more efficient inhibitor of p53 than is wild-type ELL. We also demonstrate for the first time that ELL extreme C terminus [ELL(eCT)] is required for the recruitment of p53 into MLL-ELL nuclear foci and is both necessary and sufficient for the MLL-ELL inhibition of p53-mediated induction of p21 and apoptosis. Finally, our results demonstrate that MLL-ELL requires the presence of intact ELL(eCT) in order to disrupt p53 interactions with p300/CBP coactivator and thus significantly reduce p53 acetylation in vivo. Since ELL(eCT) has recently been shown to be both necessary and sufficient for MLL-ELL-mediated transformation of normal blood progenitors, our data correlate ELL(eCT) contribution to MLL-ELL transformative effects with its ability to functionally inhibit p53.

scholarly journals Coxsackievirus A9 VP1 Mutants with Enhanced or Hindered A Particle Formation and Decreased Infectivity

2001 ◽  
Vol 75 (2) ◽  
pp. 952-960 ◽  
Author(s):  
Antero Airaksinen ◽  
Merja Roivainen ◽  
Tapani Hovi
Keyword(s):  
Single Amino Acid ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Wild Type ◽  
Functional Flexibility ◽  
C Terminus ◽  
Evolutionarily Conserved ◽  
Coxsackievirus A9 ◽  
Stable Mutant ◽  
Infectious Progeny Virus

ABSTRACT We have studied coxsackievirus A9 (CAV9) mutants that each have a single amino acid substitution in the conserved 29-PALTAVETGHT-39 motif of VP1 and a reduced capacity to produce infectious progeny virus. After uncoating, all steps in the infection cycle occurred according to the same kinetics as and similar efficiency to the wild-type virus. However, the particle/infectious unit ratio in the progeny was significantly increased. The differences were apparently due to altered stability of the capsid: there were mutant viruses with enhanced or hindered uncoating, and both of these characteristics were found to reduce fitness under standard passaging conditions. At 32°C the instable mutants had an advantage, while the wild-type and the most stable mutant grew poorly. When comparing the newly published CAV9 structure and the other enterovirus structures, we found that the PALTAVETGHT motif is always in exactly the same position, in a cavity formed by the 3 other capsid proteins, with the C terminus of VP4 between this motif and the RNA. In the 7 enterovirus structures determined to date, the most conserved residues of the studied motif have identical contacts to neighboring residues of VP2, VP3, and VP4. We conclude that (i) the mutations affect the uncoating step necessary for infection, resulting in an untimely or hindered externalization of the VP1 N terminus together with the VP4, and (ii) the reason for the studied motif being evolutionarily conserved is its role in maintaining an optimal balance between the protective stability and the functional flexibility of the capsid.

scholarly journals SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage, while its absence leads to synthetic lethality with ATR/CHK1 inhibitors

2021 ◽  
Vol 118 (6) ◽  
pp. e2015654118
Author(s):  
Ukhyun Jo ◽  
Yasuhisa Murai ◽  
Sirisha Chakka ◽  
Lu Chen ◽  
Ken Cheng ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Molecular Mechanisms ◽  
Synthetic Lethality ◽  
E3 Ubiquitin Ligase ◽  
Replication Forks ◽  
Atpase Domain ◽  
C Terminus ◽  
Genome Wide ◽  
Schlafen 11

Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1–CUL4CDT2 ubiquitin ligase.

scholarly journals FinaleDB: a browser and database of cell-free DNA fragmentation patterns

2020 ◽  
Author(s):  
Haizi Zheng ◽  
Michelle S Zhu ◽  
Yaping Liu
Keyword(s):  
Molecular Mechanisms ◽  
Cell Types ◽  
Cell Free Dna ◽  
Link Type ◽  
Diagnosis And Prognosis ◽  
Dna Database ◽  
Free Dna ◽  
Genome Wide ◽  
Fragmentation Patterns ◽  
Different Cell Types

AbstractSummaryCirculating cell-free DNA (cfDNA) is a promising biomarker for the diagnosis and prognosis of many diseases, including cancer. The genome-wide non-random fragmentation patterns of cfDNA are associated with the nucleosomal protection, epigenetic environment, and gene expression in the cell types that contributed to cfDNA. However, current progress on the development of computational methods and understanding of molecular mechanisms behind cfDNA fragmentation patterns is significantly limited by the controlled-access of cfDNA whole-genome sequencing (WGS) dataset. Here, we present FinaleDB (FragmentatIoN AnaLysis of cEll-free DNA DataBase), a comprehensive database to host thousands of uniformly processed and curated de-identified cfDNA WGS datasets across different pathological conditions. Furthermore, FinaleDB comes with a fragmentation genome browser, from which users can seamlessly integrate thousands of other omics data in different cell types to experience a comprehensive view of both gene-regulatory landscape and cfDNA fragmentation patterns.Availability and implementationFinaleDB service: http://finaledb.research.cchmc.org/ FinaleDB source code: https://github.com/epifluidlab/finaledb_portal and https://github.com/epifluidlab/[email protected]

scholarly journals The chromatin remodeler LSH controls genome-wide cytosine hydroxymethylation

2020 ◽  
Author(s):  
Maud de Dieuleveult ◽  
Martin Bizet ◽  
Laurence Colin ◽  
Emilie Calonne ◽  
Martin Bachman ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Direct Consequence ◽  
Wild Type ◽  
Embryonic Fibroblasts ◽  
Knock Out ◽  
Tet Proteins ◽  
Genome Wide ◽  
Specific Factors ◽  
Epigenetic Repression

ABSTRACTTET proteins convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), leading to a dynamic epigenetic state of DNA that can influence transcription. While TET proteins have been associated with either epigenetic repression or activation complexes, the overall understanding of the molecular mechanisms involved in TET-mediated regulation of gene transcription still remains limited. Here, we show that TET proteins interact with lymphoid-specific helicase (LSH), a chromatin remodeling factor belonging to the SNF2 super family. Lsh knock-out leads to a significant reduction of 5-hydroxymethylation global level in mouse embryonic fibroblasts (MEFs) and in embryonic stem cells (ESC). Whole genome sequencing of 5hmC in wild-type versus Lsh knock-out MEFs and ESCs showed that in absence of Lsh, some regions of the genome gain 5hmC while others lose it, with not much effect on gene expression. We further show that 5hmC modifications upon Lsh loss is not a direct consequence of 5mC decrease, as differentially hydroxymethylated regions (DhMR) did not overlap with DMR (differentially methylated regions), underlying that these modifications occurred at different genomic loci. Altogether, our results suggest that LSH is a key regulator of 5hmC in both MEFs and ESC and that TET proteins rely on specific factors to establish genome-wide 5hmC patterns.

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