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1.
Genes Dev ; 33(9-10): 565-577, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30808655

RESUMO

Heterochromatin protein 1 (HP1) proteins are key factors of eukaryotic heterochromatin that coordinate chromatin compaction and transcriptional gene silencing. Through their multivalency they act as adaptors between histone H3 Lys9 di/trimethyl marks in chromatin and effector complexes that bind to the HP1 chromoshadow domain. Most organisms encode for multiple HP1 isoforms and the molecular mechanisms that underpin their diverse functions in genome regulation remain poorly understood. In fission yeast, the two HP1 proteins Chp2 and Swi6 assume distinct roles and Chp2 is tightly associated with the nucleosome remodeling and deacetylation complex SHREC. Here we show that Chp2 directly engages the SHREC nucleosome remodeler subunit Mit1. The crystal structure of the interaction interface reveals an extraordinarily extensive and specific interaction between the chromoshadow domain of Chp2 and the N terminus of Mit1. The integrity of this interface is critical for high affinity binding and for heterochromatin formation. Comparison with Swi6 shows that the Chp2-Mit1 interface is highly selective and thereby provides the molecular basis for the functional specialization of an HP1 isoform.


Assuntos
Regulação Fúngica da Expressão Gênica , Inativação Gênica , Proteínas Repressoras/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Cristalização , Heterocromatina/metabolismo , Ligação Proteica , Isoformas de Proteínas , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética
2.
EMBO Rep ; 24(1): e55928, 2023 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-36408846

RESUMO

Methylation of histone H3 at lysine 9 (H3K9) is a hallmark of heterochromatin that plays crucial roles in gene silencing, genome stability, and chromosome segregation. In Schizosaccharomyces pombe, Clr4 mediates both di- and tri-methylation of H3K9. Although H3K9 methylation has been intensely studied in mitotic cells, its role during sexual differentiation remains unclear. Here, we map H3K9 methylation genome-wide during meiosis and show that constitutive heterochromatin temporarily loses H3K9me2 and becomes H3K9me3 when cells commit to meiosis. Cells lacking the ability to tri-methylate H3K9 exhibit meiotic chromosome segregation defects. Finally, the H3K9 methylation switch is accompanied by differential phosphorylation of Clr4 by the cyclin-dependent kinase Cdk1. Our results suggest that a conserved master regulator of the cell cycle controls the specificity of an H3K9 methyltransferase to prevent ectopic H3K9 methylation and to ensure faithful gametogenesis.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Metilação , Histonas/genética , Histonas/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fosforilação , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Gametogênese/genética
3.
Mol Cell ; 62(2): 207-221, 2016 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-27105116

RESUMO

Nucleosome remodeling and deacetylation (NuRD) complexes are co-transcriptional regulators implicated in differentiation, development, and diseases. Methyl-CpG binding domain (MBD) proteins play an essential role in recruitment of NuRD complexes to their target sites in chromatin. The related SHREC complex in fission yeast drives transcriptional gene silencing in heterochromatin through cooperation with HP1 proteins. How remodeler and histone deacetylase (HDAC) cooperate within NuRD complexes remains unresolved. We determined that in SHREC the two modules occupy distant sites on the scaffold protein Clr1 and that repressive activity of SHREC can be modulated by the expression level of the HDAC-associated Clr1 domain alone. Moreover, the crystal structure of Clr2 reveals an MBD-like domain mediating recruitment of the HDAC module to heterochromatin. Thus, SHREC bi-functionality is organized in two separate modules with separate recruitment mechanisms, which work together to elicit transcriptional silencing at heterochromatic loci.


Assuntos
Montagem e Desmontagem da Cromatina , Inativação Gênica , Heterocromatina/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Acetilação , Sítios de Ligação , Ilhas de CpG , DNA Fúngico/metabolismo , Regulação Fúngica da Expressão Gênica , Heterocromatina/química , Heterocromatina/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Modelos Moleculares , Nucleossomos/enzimologia , Nucleossomos/genética , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Fúngico/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica
4.
Hum Mol Genet ; 26(18): 3453-3465, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28911206

RESUMO

Congenital hyperinsulinism/hyperammonemia (HI/HA) syndrome gives rise to unregulated protein-induced insulin secretion from pancreatic beta-cells, fasting hypoglycemia and elevated plasma ammonia levels. Mutations associated with HI/HA were identified in the Glud1 gene, encoding for glutamate dehydrogenase (GDH). We aimed at identifying the molecular causes of dysregulation in insulin secretion and ammonia production conferred by the most frequent HI/HA mutation Ser445Leu. Following transduction with adenoviruses carrying the human GDH-wild type or GDH-S445L-mutant gene, immunoblotting showed efficient expression of the transgenes in all the investigated cell types. Enzymatic activity tested in INS-1E beta-cells revealed that the mutant was much more sensitive to the allosteric activator ADP, rendering it highly responsive to substrates. INS-1E cells expressing either the wild type or mutant GDH responded similarly to glucose stimulation regarding mitochondrial activation and insulin secretion. However, at basal glucose glutamine stimulation increased mitochondrial activity and insulin release only in the mutant cells. In mouse and human islets, expression of mutant GDH resulted in robust elevation of insulin secretion upon glutamine stimulation, not observed in control islets. Hepatocytes expressing either the wild type or mutant GDH produced similar levels of ammonia when exposed to glutamine, although alanine response was strongly elevated with the mutant form. In conclusion, the GDH-S445L mutation confers hyperactivity to this enzyme due to higher sensitivity to ADP allosteric activation. This renders beta-cells responsive to amino acid stimulation, explaining protein-induced hypoglycemia secondary to non-physiological insulin release. Hepatocytes carrying mutant GDH produced more ammonia upon alanine exposure, which underscores hyperammonemia developed by the patients.


Assuntos
Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Difosfato de Adenosina/metabolismo , Aminoácidos/genética , Animais , Glicemia/metabolismo , Hiperinsulinismo Congênito/genética , Glucose/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Hiperamonemia/genética , Hiperamonemia/metabolismo , Hiperinsulinismo/genética , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Polimorfismo de Nucleotídeo Único/genética
5.
Chromosoma ; 126(4): 443-455, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-27858158

RESUMO

The centromere is essential for the segregation of chromosomes, as it serves as attachment site for microtubules to mediate chromosome segregation during mitosis and meiosis. In most organisms, the centromere is restricted to one chromosomal region that appears as primary constriction on the condensed chromosome and is partitioned into two chromatin domains: The centromere core is characterized by the centromere-specific histone H3 variant CENP-A (also called cenH3) and is required for specifying the centromere and for building the kinetochore complex during mitosis. This core region is generally flanked by pericentric heterochromatin, characterized by nucleosomes containing H3 methylated on lysine 9 (H3K9me) that are bound by heterochromatin proteins. During mitosis, these two domains together form a three-dimensional structure that exposes CENP-A-containing chromatin to the surface for interaction with the kinetochore and microtubules. At the same time, this structure supports the tension generated during the segregation of sister chromatids to opposite poles. In this review, we discuss recent insight into the characteristics of the centromere, from the specialized chromatin structures at the centromere core and the pericentromere to the three-dimensional organization of these regions that make up the functional centromere.


Assuntos
Centrômero/química , Cromatina/química , Nucleossomos/química , Animais , Centrômero/fisiologia , Cromatina/fisiologia , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Heterocromatina/química , Heterocromatina/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Nucleossomos/fisiologia
6.
Mol Cell ; 34(1): 36-46, 2009 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-19362535

RESUMO

In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Sequência de Aminoácidos , Proteínas Argonautas , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , Lisina/metabolismo , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo
7.
Proc Natl Acad Sci U S A ; 111(5): 1795-800, 2014 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-24449894

RESUMO

Repressive histone H3 lysine 9 methylation (H3K9me) and its recognition by HP1 proteins are necessary for pericentromeric heterochromatin formation. In Schizosaccharomyces pombe, H3K9me deposition depends on the RNAi pathway. Cryptic loci regulator 4 (Clr4), the only known H3K9 methyltransferase in this organism, is a subunit of the Clr4 methyltransferase complex (CLRC), whose composition is reminiscent of a CRL4 type cullin-RING ubiquitin ligase (CRL) including its cullin Cul4, the RING-box protein Pip1, the DNA damage binding protein 1 homolog Rik1, and the DCAF-like protein delocalization of Swi6 1 (Dos1). Dos2 and Stc1 have been proposed to be part of the complex but do not bear similarity to canonical ubiquitin ligase components. CLRC is an active E3 ligase in vitro, and this activity is necessary for heterochromatin assembly in vivo. The similarity between CLRC and the CRLs suggests that the WD repeat protein Dos1 will act to mediate target recognition and substrate specificity for CLRC. Here, we present a pairwise interaction screen that confirms a CRL4-like subunit arrangement and further identifies Dos2 as a central component of the complex and recruiter of Stc1. We determined the crystal structure of the Dos1 WD repeat domain, revealing an eight-bladed ß-propeller fold. Functional mapping of the putative target-binding surface of Dos1 identifies key residues required for heterochromatic silencing, consistent with Dos1's role as the specificity factor for the E3 ubiquitin ligase.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Inativação Gênica , Heterocromatina/metabolismo , Metiltransferases/metabolismo , Complexos Multiproteicos/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Centrômero/metabolismo , Histona-Lisina N-Metiltransferase , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Coativadores de Receptor Nuclear/química , Fenótipo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Eletricidade Estática , Especificidade por Substrato
8.
BMC Plant Biol ; 16: 42, 2016 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-26864020

RESUMO

BACKGROUND: Plants perceive UV-B through the UV RESISTANCE LOCUS 8 (UVR8) photoreceptor and UVR8 activation leads to changes in gene expression such as those associated with UV-B acclimation and stress tolerance. Albeit functionally unrelated, UVR8 shows some homology with RCC1 (Regulator of Chromatin Condensation 1) proteins from non-plant organisms at the sequence level. These proteins act as guanine nucleotide exchange factors for Ran GTPases and bind chromatin via histones. Subsequent to the revelation of this sequence homology, evidence was presented showing that UVR8 activity involves interaction with chromatin at the loci of some target genes through histone binding. This suggested a UVR8 mode-of-action intimately and directly linked with gene transcription. However, several aspects of UVR8 chromatin association remained undefined, namely the impact of UV-B on the process and how UVR8 chromatin association related to the transcription factor ELONGATED HYPOCOTYL 5 (HY5), which is important for UV-B signalling and has overlapping chromatin targets. Therefore, we have investigated UVR8 chromatin association in further detail. RESULTS: Unlike the claims of previous studies, our chromatin immunoprecipitation (ChIP) experiments do not confirm UVR8 chromatin association. In contrast to human RCC1, recombinant UVR8 also does not bind nucleosomes in vitro. Moreover, fusion of a VP16 activation domain to UVR8 did not alter expression of proposed UVR8 target genes in transient gene expression assays. Finally, comparison of the Drosophila DmRCC1 and the Arabidopsis UVR8 crystal structures revealed that critical histone- and DNA-interaction residues apparent in DmRCC1 are not conserved in UVR8. CONCLUSION: This has led us to conclude that the cellular activity of UVR8 likely does not involve its specific binding to chromatin at target genes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fotorreceptores de Plantas/metabolismo , Genes de Plantas , Regiões Promotoras Genéticas , Ligação Proteica
9.
Nat Commun ; 13(1): 7759, 2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-36522330

RESUMO

Histone modifications are deposited by chromatin modifying enzymes and read out by proteins that recognize the modified state. BRD4-NUT is an oncogenic fusion protein of the acetyl lysine reader BRD4 that binds to the acetylase p300 and enables formation of long-range intra- and interchromosomal interactions. We here examine how acetylation reading and writing enable formation of such interactions. We show that NUT contains an acidic transcriptional activation domain that binds to the TAZ2 domain of p300. We use NMR to investigate the structure of the complex and found that the TAZ2 domain has an autoinhibitory role for p300. NUT-TAZ2 interaction or mutations found in cancer that interfere with autoinhibition by TAZ2 allosterically activate p300. p300 activation results in a self-organizing, acetylation-dependent feed-forward reaction that enables long-range interactions by bromodomain multivalent acetyl-lysine binding. We discuss the implications for chromatin organisation, gene regulation and dysregulation in disease.


Assuntos
Lisina , Proteínas Nucleares , Acetilação , Proteínas Nucleares/metabolismo , Lisina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cromatina
10.
Nature ; 436(7047): 138-41, 2005 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-16001076

RESUMO

DNA in eukaryotic chromosomes is organized in arrays of nucleosomes compacted into chromatin fibres. This higher-order structure of nucleosomes is the substrate for DNA replication, recombination, transcription and repair. Although the structure of the nucleosome core is known at near-atomic resolution, even the most fundamental information about the organization of nucleosomes in the fibre is controversial. Here we report the crystal structure of an oligonucleosome (a compact tetranucleosome) at 9 A resolution, solved by molecular replacement using the nucleosome core structure. The structure shows that linker DNA zigzags back and forth between two stacks of nucleosome cores, which form a truncated two-start helix, and does not follow a path compatible with a one-start solenoidal helix. The length of linker DNA is most probably buffered by stretching of the DNA contained in the nucleosome cores. We have built continuous fibre models by successively stacking tetranucleosomes one on another. The resulting models are nearly fully compacted and most closely resemble the previously described crossed-linker model. They suggest that the interfaces between nucleosomes along a single helix start are polymorphic.


Assuntos
Cromatina/química , Nucleossomos/química , Animais , Cromatina/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Modelos Moleculares , Conformação Molecular , Conformação de Ácido Nucleico , Nucleossomos/metabolismo , Xenopus laevis
11.
Curr Opin Struct Biol ; 71: 156-163, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34303934

RESUMO

Eukaryotic genomes are segregated into active euchromatic and repressed heterochromatic compartments. Gene regulatory networks, chromosomal structures, and genome integrity rely on the timely and locus-specific establishment of active and silent states to protect the genome and provide the basis for cell division and specification of cellular identity. Here, we focus on the mechanisms and molecular machinery that establish heterochromatin in Schizosaccharomyces pombe and compare it with Saccharomyces cerevisiae and the mammalian polycomb system. We present recent structural and mechanistic evidence, which suggests that histone acetylation protects active transcription by disrupting the positive feedback loops used by the heterochromatin machinery and that H2A and H3 monoubiquitination actively drives heterochromatin, whereas H2B monoubiquitination mobilizes the defenses to quench heterochromatin.


Assuntos
Heterocromatina , Schizosaccharomyces , Acetilação , Animais , Cromatina , Heterocromatina/genética , Histonas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Ubiquitinação
12.
Elife ; 102021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34524082

RESUMO

The SUV39 class of methyltransferase enzymes deposits histone H3 lysine 9 di- and trimethylation (H3K9me2/3), the hallmark of constitutive heterochromatin. How these enzymes are regulated to mark specific genomic regions as heterochromatic is poorly understood. Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast Schizosaccharomyces pombe, and recent evidence suggests that ubiquitination of lysine 14 on histone H3 (H3K14ub) plays a key role in H3K9 methylation. However, the molecular mechanism of this regulation and its role in heterochromatin formation remain to be determined. Our structure-function approach shows that the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby stimulates the enzyme by over 250-fold. Mutations that disrupt this mechanism lead to a loss of H3K9me2/3 and abolish heterochromatin silencing similar to clr4 deletion. Comparison with mammalian SET domain proteins suggests that the Clr4 SET domain harbors a conserved sensor for H3K14ub, which mediates licensing of heterochromatin formation.


Assuntos
Proteínas de Ciclo Celular , Heterocromatina , Código das Histonas/genética , Histona-Lisina N-Metiltransferase , Histonas , Proteínas de Schizosaccharomyces pombe , Domínio Catalítico/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Metilação de DNA/genética , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitinação/genética
13.
Cell Rep ; 35(7): 109137, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34010645

RESUMO

Oncogenic histone lysine-to-methionine mutations block the methylation of their corresponding lysine residues on wild-type histones. One attractive model is that these mutations sequester histone methyltransferases, but genome-wide studies show that mutant histones and histone methyltransferases often do not colocalize. Using chromatin immunoprecipitation sequencing (ChIP-seq), here, we show that, in fission yeast, even though H3K9M-containing nucleosomes are broadly distributed across the genome, the histone H3K9 methyltransferase Clr4 is mainly sequestered at pericentric repeats. This selective sequestration of Clr4 depends not only on H3K9M but also on H3K14 ubiquitylation (H3K14ub), a modification deposited by a Clr4-associated E3 ubiquitin ligase complex. In vitro, H3K14ub synergizes with H3K9M to interact with Clr4 and potentiates the inhibitory effects of H3K9M on Clr4 enzymatic activity. Moreover, binding kinetics show that H3K14ub overcomes the Clr4 aversion to H3K9M and reduces its dissociation. The selective sequestration model reconciles previous discrepancies and demonstrates the importance of protein-interaction kinetics in regulating biological processes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Heterocromatina/metabolismo , Histona Metiltransferases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitinação/imunologia , Mutação
14.
Essays Biochem ; 63(1): 123-132, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30967476

RESUMO

Nucleosomes cover eukaryotic genomes like beads on a string and play a central role in regulating genome function. Isolated strings of nucleosomes have the potential to compact and form higher order chromatin structures, such as the well-characterized 30-nm fiber. However, despite tremendous advances in observing chromatin fibers in situ it has not been possible to confirm that regularly ordered fibers represent a prevalent structural level in the folding of chromosomes. Instead, it appears that folding at a larger scale than the nucleosome involves a variety of random structures with fractal characteristics. Nevertheless, recent progress provides evidence for the existence of structural motifs in chromatin fibers, potentially localized to strategic sites in the genome. Here we review the current understanding of chromatin fiber folding and the emerging roles that oligonucleosomal motifs play in the regulation of genome function.


Assuntos
DNA/metabolismo , Genoma/fisiologia , Nucleossomos/metabolismo , Animais , DNA/química , Modelos Moleculares , Nucleossomos/química
15.
Sci Rep ; 8(1): 10930, 2018 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-30026545

RESUMO

The mechanism by which specific protein-DNA complexes induce programmed replication fork stalling in the eukaryotic genome remains poorly understood. In order to shed light on this process we carried out structural investigations on the essential fission yeast protein Sap1. Sap1 was identified as a protein involved in mating-type switching in Schizosaccharomyces pombe, and has been shown to be involved in programmed replication fork stalling. Interestingly, Sap1 assumes two different DNA binding modes. At the mating-type locus dimers of Sap1 bind the SAS1 sequence in a head-to-head arrangement, while they bind to replication fork blocking sites at rDNA and Tf2 transposons in a head-to-tail mode. In this study, we have solved the crystal structure of the Sap1 DNA binding domain and we observe that Sap1 molecules interact in the crystal using a head-to-tail arrangement that is compatible with DNA binding. We find that Sap1 mutations which alleviate replication-fork blockage at Tf2 transposons in CENP-B mutants map to the head-to-tail interface. Furthermore, several other mutations introduced in this interface are found to be lethal. Our data suggests that essential functions of Sap1 depend on its head-to-tail oligomerization.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Mutação , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Sítios de Ligação , Proteína B de Centrômero/genética , Cristalografia por Raios X , Replicação do DNA , DNA Fúngico/metabolismo , DNA Ribossômico/metabolismo , Proteínas de Ligação a DNA/genética , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Schizosaccharomyces/química , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética
16.
J Mol Biol ; 429(20): 3031-3042, 2017 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-28893533

RESUMO

Chromatin fiber organization is implicated in processes such as transcription, DNA repair and chromosome segregation, but how nucleosomes interact to form higher-order structure remains poorly understood. We solved two crystal structures of tetranucleosomes with approximately 11-bp DNA linker length at 5.8 and 6.7 Å resolution. Minimal intramolecular nucleosome-nucleosome interactions result in a fiber model resembling a flat ribbon that is compatible with a two-start helical architecture, and that exposes histone and DNA surfaces to the environment. The differences in the two structures combined with electron microscopy reveal heterogeneous structural states, and we used site-specific chemical crosslinking to assess the diversity of nucleosome-nucleosome interactions through identification of structure-sensitive crosslink sites that provide a means to characterize fibers in solution. The chromatin fiber architectures observed here provide a basis for understanding heterogeneous chromatin higher-order structures as they occur in a genomic context.


Assuntos
Cromatina/química , Cromatina/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Cristalografia por Raios X , Microscopia Eletrônica , Conformação de Ácido Nucleico , Conformação Proteica
17.
J Mol Biol ; 327(1): 85-96, 2003 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-12614610

RESUMO

We have developed a self-assembly system for nucleosome arrays in which recombinant, post-translationally unmodified histone proteins are combined with DNA of defined-sequence to form chromatin higher-order structure. The nucleosome arrays obtained are highly homogeneous and sediment at 53S when maximally folded in 1mM or 100mM MgCl(2). The folding properties are comparable to established systems. Analytical ultracentrifugation is used to determine the consequence of individual histone tail domain deletions on array folding. Fully compacted chromatin fibers are obtained with any one of the histone tails deleted with the exception of the H4 N terminus. The region of the H4 tail, which mediates compaction, resides in the stretch of amino acids 14-19.


Assuntos
Cromatina/química , Cromatina/metabolismo , Histonas/química , Histonas/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Cloreto de Magnésio/farmacologia , Peso Molecular , Nucleossomos/química , Nucleossomos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica/efeitos dos fármacos , Ultracentrifugação , Xenopus laevis
18.
Mol Cell Biol ; 35(4): 662-74, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25487573

RESUMO

Heterochromatin underpins gene repression, genome integrity, and chromosome segregation. In the fission yeast Schizosaccharomyces pombe, conserved protein complexes effect heterochromatin formation via RNA interference-mediated recruitment of a histone H3 lysine 9 methyltransferase to cognate chromatin regions. To identify small molecules that inhibit heterochromatin formation, we performed an in vivo screen for loss of silencing of a dominant selectable kanMX reporter gene embedded within fission yeast centromeric heterochromatin. Two structurally unrelated compounds, HMS-I1 and HMS-I2, alleviated kanMX silencing and decreased repressive H3K9 methylation levels at the transgene. The decrease in methylation caused by HMS-I1 and HMS-I2 was observed at all loci regulated by histone methylation, including centromeric repeats, telomeric regions, and the mating-type locus, consistent with inhibition of the histone deacetylases (HDACs) Clr3 and/or Sir2. Chemical-genetic epistasis and expression profiles revealed that both compounds affect the activity of the Clr3-containing Snf2/HDAC repressor complex (SHREC). In vitro HDAC assays revealed that HMS-I1 and HMS-I2 inhibit Clr3 HDAC activity. HMS-I1 also alleviated transgene reporter silencing by heterochromatin in Arabidopsis and a mouse cell line, suggesting a conserved mechanism of action. HMS-I1 and HMS-I2 bear no resemblance to known inhibitors of chromatin-based activities and thus represent novel chemical probes for heterochromatin formation and function.


Assuntos
Dioxanos/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Heterocromatina/efeitos dos fármacos , Compostos Heterocíclicos com 2 Anéis/farmacologia , Piperazinas/farmacologia , Piridinas/farmacologia , Schizosaccharomyces/efeitos dos fármacos , Tiofenos/farmacologia , Animais , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Metilação de DNA , Dioxanos/síntese química , Dioxanos/química , Heterocromatina/química , Compostos Heterocíclicos com 2 Anéis/síntese química , Compostos Heterocíclicos com 2 Anéis/química , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Camundongos , Piperazinas/síntese química , Piperazinas/química , Piridinas/síntese química , Piridinas/química , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/antagonistas & inibidores , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Tiofenos/síntese química , Tiofenos/química
19.
Cell Rep ; 3(1): 92-102, 2013 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-23318260

RESUMO

Chromodomain Helicase DNA binding protein 5 (CHD5) is a tumor suppressor mapping to 1p36, a genomic region that is frequently deleted in human cancer. Although CHD5 belongs to the CHD family of chromatin-remodeling proteins, whether its tumor-suppressive role involves an interaction with chromatin is unknown. Here we report that Chd5 binds the unmodified N terminus of H3 through its tandem plant homeodomains (PHDs). Genome-wide chromatin immunoprecipitation studies reveal preferential binding of Chd5 to loci lacking the active mark H3K4me3 and also identify Chd5 targets implicated in cancer. Chd5 mutations that abrogate H3 binding are unable to inhibit proliferation or transcriptionally modulate target genes, which leads to tumorigenesis in vivo. Unlike wild-type Chd5, Chd5-PHD mutants are unable to induce differentiation or efficiently suppress the growth of human neuroblastoma in vivo. Our work defines Chd5 as an N-terminally unmodified H3-binding protein and provides functional evidence that this interaction orchestrates chromatin-mediated transcriptional programs critical for tumor suppression.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Neoplasias/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Loci Gênicos/genética , Genoma/genética , Humanos , Lisina/metabolismo , Metilação , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Neoplasias/genética , Neoplasias/patologia , Neuroblastoma/genética , Neuroblastoma/patologia , Peptídeos/metabolismo , Ligação Proteica , Relação Estrutura-Atividade
20.
Z Med Phys ; 27(2): 75-77, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28457676
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