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Hutchinson‐Gilford Progeria syndrome (HGPS) is a lethal premature aging disorder caused by a de novo heterozygous mutation that leads to the accumulation of a splicing isoform of Lamin A termed progerin. Progerin expression deregulates the organization of the nuclear lamina and the epigenetic landscape. Progerin has also been observed to accumulate at low levels during normal aging in cardiovascular cells of adults that do not carry genetic mutations linked with HGPS. Therefore, the molecular mechanisms that lead to vascular dysfunction in HGPS may also play a role in vascular aging‐associated diseases, such as myocardial infarction and stroke. Here, we show that HGPS patient‐derived vascular smooth muscle cells (VSMCs) recapitulate HGPS molecular hallmarks. Transcriptional profiling revealed cardiovascular disease remodeling and reactive oxidative stress response activation in HGPS VSMCs. Proteomic analyses identified abnormal acetylation programs in HGPS VSMC replication fork complexes, resulting in reduced H4K16 acetylation. Analysis of acetylation kinetics revealed both upregulation of K16 deacetylation and downregulation of K16 acetylation. This correlates with abnormal accumulation of error‐prone nonhomologous end joining (NHEJ) repair proteins on newly replicated chromatin. The knockdown of the histone acetyltransferase MOF recapitulates preferential engagement of NHEJ repair activity in control VSMCs. Additionally, we find that primary donor‐derived coronary artery vascular smooth muscle cells from aged individuals show similar defects to HGPS VSMCs, including loss of H4K16 acetylation. Altogether, we provide insight into the molecular mechanisms underlying vascular complications associated with HGPS patients and normative aging. [ABSTRACT FROM AUTHOR]

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. Here, we defined the activated RNF8-Ubc13∼ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13 active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. These findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.

Introduction Over the past years, there has been remarkable progress in characterizing human and model systems' genomes and epigenomes. Basic health research in genomics and epigenomics is translated into improving [...]

Abstract Background Within chromatin, the histone variant H2A.Z plays a role in many diverse nuclear processes including transcription, preventing the spread of heterochromatin and epigenetic transcriptional memory. The molecular mechanisms of how H2A.Z mediates its effects are not entirely understood. However, it is now known that H2A.Z has two protein isoforms in vertebrates, H2A.Z-1 and H2A.Z-2, which are encoded by separate genes and differ by 3 amino acid residues. Results We report that H2A.Z-1 and H2A.Z-2 are expressed across a wide range of human tissues, they are both acetylated at lysine residues within the N-terminal region and they exhibit similar, but nonidentical, distributions within chromatin. Our results suggest that H2A.Z-2 preferentially associates with H3 trimethylated at lysine 4 compared to H2A.Z-1. The phylogenetic analysis of the promoter regions of H2A.Z-1 and H2A.Z-2 indicate that they have evolved separately during vertebrate evolution. Conclusions Our biochemical, gene expression, and phylogenetic data suggest that the H2A.Z-1 and H2A.Z-2 variants function similarly yet they may have acquired a degree of functional independence.

The Jumonji D2 proteins (JMJD2/KDM4) function to demethylate di- and trimethylated (me2/3) histone 3 lysine 9 (H3K9me2/3) and H3K36me3. Knockout mouse models for Kdm4b and Kdm4d have not resulted in gross abnormalities, while mouse models for Kdm4a and Kdm4c have not been reported. However, the KDM4 subfamily of demethylases are overexpressed in several tumor types. Overexpression of KDM4 proteins alters transcription and chromatin remodeling, driving cellular proliferation, anchorage-independent growth, invasion, and migration. Increased proliferation occurs through KDM4-mediated modification of cell cycle timing, as well as through increased numbers of replication forks. Recent evidence also suggests that KDM4C overexpression contributes to the maintenance of a pluripotent state. Together these data suggest that overexpression of KDM4 proteins induces numerous oncogenic effects. Key words: demethylase, H3K9me3, Jumonji, JMJD2/KDM4, cancer. Resume: Les proteines Jumonji D2 (JMJD2/KDM4) ont pour fonction de demethyler la lysine 9 de l'histone H3 di- et tri-methylee (H3K9me2/3) et la H3K36me3. Des modeles d'inactivation de kdm4b et kdm4d chez la souris ne resultent pas en anomalies majeures, alors que des modeles d'inactivation de kdm4a et kdm4c n'ont jamais ete rapportes. Cependant, la sous-famille de demethylases KDM4 est surexprimee dans plusieurs types de tumeurs. La surexpression des proteines KDM4 altere la transcription et le remodelage de la chromatine, induisant la proliferation cellulaire, la croissance independante de l'ancrage, l'invasion et la migration. Une proliferation accrue survient par l'intermediaire d'une modification du programme du cycle cellulaire dependante de KDM4 et de meme que par un nombre accru de fourches de replication. Des donnees recentes suggerent aussi que la surexpression de KDM4C contribue au maintien d'un etat pluripotent. L'ensemble de ces resultats suggere que la surexpression des proteines KDM4 induit de nombreux effets oncogeniques. [Traduit par la Redaction] Mots-cles: demethylase, H3K9me3, jumonji, JMJD2/KDM4, cancer., Introduction In the cell, DNA is packaged into chromatin. The basic building block of chromatin is a protein/DNA complex termed the nucleosome, a protein structure composed of 8 histones around [...]

Epigenetic programming is an important facet of biology, controlling gene expression patterns and the choice between developmental pathways. The Polycomb group proteins (PcGs) silence gene expression, allowing cells to both acquire and maintain identity. PcG silencing is important for stemness, X chromosome inactivation (XCI), genomic imprinting, and the abnormally silenced genes in cancers. Stem and cancer cells commonly share gene expression patterns, regulatory mechanisms, and signalling pathways. Many microRNA species have oncogenic or tumor suppressor activity, and disruptions in these networks are common in cancer; however, long non-coding (nc)RNA species are also important. Many of these directly guide PcG deposition and gene silencing at the HOX locus, during XCI, and in examples of genomic imprinting. Since inappropriate HOX expression and loss of genomic imprinting are hallmarks of cancer, disruption of long ncRNA-mediated PcG silencing likely has a role in oncogenesis. Aberrant silencing of coding and non-coding loci is critical for both the genesis and progression of cancers. In addition, PcGs are commonly abnormally overexpressed years prior to cancer pathology, making early PcG targeted therapy an option to reverse tumor formation, someday replacing the blunt instrument of eradication in the cancer therapy arsenal. Key words: Polycomb group proteins, non-coding RNA, EZH2, BMI1, stem cells, cancer. La programmation epigenetique constitue une facette importante de la biologie en controlant les patrons d'expression genique et le choix d'une voie de developpement specifique. Le groupe de proteines Polycom (PcG) inhibe l'expression genique, permettant aux cellules d'acquerir et de maintenir leur identite. Le silencage par les PcG est important au maintien de l'etat d'immaturite des cellules souches, a l'inactivation du chromosome X, a l'empreinte genomique, et au silencage anormal de genes dans le cancer. Les cellules souches et les cellules cancereuses partagent des patrons d'expression genique, des mecanismes de regulation et des voies de signalisation. Plusieurs especes de microARN possedent des activites oncogenes ou de suppresseurs de tumeur, et la perturbation de leurs activites au sein de ces voies est frequente dans le cancer; cependant, les especes d'ARN longs non codants sont aussi importantes. Plusieurs d'entre elles guident directement le recrutement des PcG et le silencage genique au locus HOX dont la regulation definit l'identite segmentaire, durant l'inactivation du chromosome X et dans differents exemples d'empreinte genomique. Puisque l'expression inadequate des HOX et la perte d'empreinte genomique sont des caracteristiques du cancer, la perturbation du silencage par les PcG causee par des ARN longs non codants jouerait probablement un role dans l'oncogenese. Le silencage inadequat de locus codants et non codants est critique a la genese et a la progression du cancer. De plus, les PcG sont frequemment exprimes de facon anormalement elevee, des annees avant l'apparition du cancer, ce qui rendrait une therapie precoce ciblee vers les PcG interessante pour renverser la formation tumorale, remplacant un jour certains outils de l'arsenal therapeutique anticancereux. Mots-cles : proteines du groupe Polycomb, ARN non codant, EZH2, BMI1, cellules souches, cancer. [Traduit par la Redaction] Biochem. Cell Biol. 87: 711-746 (2009) doi: 10.1139/O09-057 Published by NRC Research Press, Introduction Epigenetics is the study of heritable changes in gene expression not coded by the DNA sequence itself. Epigenetic regulation consists of alterations in chromatin structure mediated, primarily, by post-translational [...]

Actin exists as a dynamic equilibrium of monomers and polymers within the nucleus of living cells. It is utilized by the cell for many aspects of gene regulation, including mRNA processing, chromatin remodelling, and global gene expression. Polymeric actin is now specifically linked to transcription by RNA polymerase I, II, and III. An active process, requiring both actin polymers and myosin, appears to drive RNA polymerase I transcription, and is also implicated in long-range chromatin movement. This type of mechanism brings activated genes from separate chromosomal territories together, and then participates in their compartmentalization near nuclear speckles. Nuclear speckle formation requires polymeric actin, and factors promoting polymerization, such as profilin and PIP2, are concentrated there. A review of the literature shows that a functional population of G-actin cycles between the cytoplasm and the nucleoplasm. Its nuclear concentration is dependent on the cytoplasmic G-actin pool, as well as on the activity of import and export mechanisms and the availability of interactions that sequester it within the nucleus. The N-WASP-Arp2/3 actin polymernucleating mechanism functions in the nucleus, and its mediators, including NCK, PIP2, and Racl, can be found in the nucleoplasm, where they likely influence the kinetics of polymer formation. The actin polymer species produced are tightly regulated, and may take on conformations not easily recognized by phalloidin. Many of the factors that cleave F-actin in the cytoplasm are present at high levels in the nucleoplasm, and are also likely to affect actin dynamics there. The absolute and relative G-actin content in the nucleoplasm and the cytoplasm of a cell contains information about the homeostatic state of that cell. We propose that the cycling of G-actin between the nucleus and cytoplasm represents a signal transduction mechanism that can function through both extremes of global cellular G-actin content. MAL signalling within the serum response factor pathway, when G-actin levels are low, represents a well-studied example of actin functioning in signal transduction. The translocation of NCK into the nucleus, along with G-actin, during dissolution of the cytoskeleton in response to DNA damage represents another instance of a unique signalling mechanism operating when G-actin levels are high. Key words: actin dynamics, polymeric actin, gene regulation, active transport, signal transduction. Les formes monomeres et polymeres de l'actine existent en equilibre dynamique dans le noyau des cellules vivantes. L'actine est utilisee par la cellule dans plusieurs etapes de la regulation genique dont la maturation de l'ARNm, le remodelage de la chromatine et l'expression genique globale. L'actine polymerique est maintenant specifiquement associee a la transcription par YARN polymerase I, II et III. Un processus actif necessitant les polymeres d'actine et la myosine semble actionner la transcription par YARN polymerase I et il celui-ci est aussi implique dans les mouvements de la chromatine sur de longues distances. Ce type de mecanisme amene a proximite l'un de l'autre des genes actives, localises sur differents territoires chromosomiques, et il participe ainsi a leur compartimentation pres des corps nucleaires. La formation des corps nucleaires requiert l'actine polymerique et les facteurs qui favorisent la polymerisation, comme la profiline et PIP2, sont concentres a cet endroit. Une revue de la litterature montre qu'une population fonctionnelle de G-actine voyage entre le cytoplasme et le nucleoplasme. Sa concentration nucleaire est dependante du pool de G-actine cytoplasmique ainsi que de l'activite des processus d'import-export et de la disponibilite des interactions qui la sequestrent dans le noyau. Le mecanisme de nucleation du polymere d'actine impliquant N-WASP-Arp2/3 fonctionne dans le noyau et ses mediateurs dont NCK, PIP2 et Racl peuvent se retrouver dans le nucleoplasme ou ils influencent probablement la cinetique de formation du polymere. Les especes de polymere d' actine produites sont etroitement reglees et elles peuvent adopter une conformation qui n'est pas facilement reconnue par la phalloidine. Plusieurs des facteurs qui clivent la F-actine dans le cytoplasme sont presents en grande quantite dans le nucleoplasme et ceux-ci peuvent aussi probablement affecter la dynamique d'actine a cet endroit. Les contenus absolu et relatif de G-actine du nucleoplasme et du cytoplasme d'une cellule vehiculent l'information de l'etat homeostatique de cette cellule. Nous proposons que le va-et-vient de la G-actine entre le noyau et le cytoplasme represente un mecanisme de transduction de signaux qui peut fonctionner aux extremites du spectre relativement au contenu global cellulaire en G-actine. La signalisation par MAL dans la voie du SRF (<< serum response factor >>), lorsque le contenu en G-actine est faible, represente un exemple bien connu de l'implication de l'actine dans la transduction de signaux. La translocation de la NCK dans le noyau avec dactine lors de la dissolution du cytosquelette en reponse a un dommage a l'ADN represente un autre exemple des mecanismes de signalisation uniques qui oeuvrent lorsque les niveaux de G-actine sont eleves. Mots-cles : dynamique d' actine, actine polymerique, regulation genique, transport actif, transduction de signaux. [Traduit par la Redaction], Introduction The controversial 40 year debate surrounding the existence of a functional actin population in the cell nucleus has been supplanted in recent years by a growing body of work [...]

The histone H1 family of nucleoproteins represents an important class of structural and architectural proteins that are responsible for maintaining and stabilizing higher-order chromatin structure. Essential for mammalian cell viability, they are responsible for gene-specific regulation of transcription and other DNA-dependent processes. In this review, we focus on the wealth of information gathered on the molecular kinetics of histone H1 molecules using novel imaging techniques, such as fluorescence recovery after photobleaching. These experiments have shed light on the effects of H1 phosphorylation and core histone acetylation in influencing chromatin structure and dynamics. We also delineate important concepts surrounding the C-terminal domain of H1, such as the intrinsic disorder hypothesis, and how it affects H1 function. Finally, we address the biochemical mechanisms behind low-affinity H1 binding. Key words: Histone H1, FRAP, chromatin, phosphorylation, acetylation. Les nucleoproteines de la famille de l'histone H1 representent une classe importante de proteines structurales et architecturales responsables du maintien et de la stabilite de la chromatine d'ordre superieur. Essentielles a la viabilite des cellules mammiferes, elles sont responsables de la regulation de la transcription specifique des genes et d'autres processus dependants de l'ADN. Dans cet article de synthese, nous nous concentrons sur le gisement d'informations recueillies sur la cinetique des molecules d'histones H1 a l'aide de nouvelles techniques d'imagerie telles le FRAP (<< fluorescence recovery after photobleaching >>). Ces experiences ont leve le voile sur les effets de la phosphorylation de H1 et de l'acetylation des histones centrales sur la structure et la dynamique de la chromatine. Nous decrivons aussi d'importants concepts touchant le domaine C-terminal de H1, comme l'hypothese de desordre intrinseque et la facon par laquelle il affecte la fonction de H1. Finalement, nous abordons certains mecanismes biochimiques qui sous-tendent la liaison de faible affinite de H1. Mots-cles : H1, FRAP, chromatine, phosphorylation, acetylation. [Traduit par la Redaction], Introduction The incredible amount of genetic information in eukaryotes necessitated the highly complex, organized, yet dynamic system of nucleoproteins that orchestrates compaction of this vast amount of DNA into the [...]

Abstract: During interphase, histone amino-terminal tails play important roles in regulating the extent of DNA compaction. Post-translational modifications of the histone tails are intimately associated with regulating chromatin structure: phosphorylation [...]

Abstract: In the midst of an increasingly detailed understanding of the molecular basis of genome regulation, we still only vaguely understand the relationship between molecular biochemistry and the structure of [...]

[beta]-Actin, once thought to be an exclusively cytoplasmic protein, is now known to have important functions within the nucleus. Nuclear [beta]-actin associates with and functions in chromatin remodeling complexes, ribonucleic acid polymerase complexes, and at least some ribonucleoproteins. Proteins involved in regulating actin polymerization are also found in the interphase nucleus. We define the dynamic properties of nuclear actin molecules using fluorescence recovery after photobleaching. Our results indicate that actin and actin-containing complexes are reduced in their mobility through the nucleoplasm diffusing at ~0.5 [micro][m.sup.2] [s.sup.-1]. We also observed that ~20% of the total nuclear actin pool has properties of polymeric actin that turns over rapidly. This pool could be detected in endogenous nuclear actin by using fluorescent polymeric actin binding proteins and was sensitive to drugs that alter actin polymerization. Our results validate previous reports of polymeric forms of nuclear actin observed in fixed specimens and reveal that these polymeric forms are very dynamic.