RESUMO
The chromatin-remodeling enzyme helicase lymphoid-specific (HELLS) interacts with cell division cycle-associated 7 (CDCA7) on nucleosomes and is involved in the regulation of DNA methylation in higher organisms. Mutations in these genes cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, which also results in DNA hypomethylation of satellite repeat regions. We investigated the functional domains of human CDCA7 in HELLS using several mutant CDCA7 proteins. The central region is critical for binding to HELLS, activation of ATPase, and nucleosome sliding activities of HELLS-CDCA7. The N-terminal region tends to inhibit ATPase activity. The C-terminal 4CXXC-type zinc finger domain contributes to CpG and hemimethylated CpG DNA preference for DNA-dependent HELLS-CDCA7 ATPase activity. Furthermore, CDCA7 showed a binding preference to DNA containing hemimethylated CpG, and replication-dependent pericentromeric heterochromatin foci formation of CDCA7 with HELLS was observed in mouse embryonic stem cells; however, all these phenotypes were lost in the case of an ICF syndrome mutant of CDCA7 mutated in the zinc finger domain. Thus, CDCA7 most likely plays a role in the recruitment of HELLS, activates its chromatin remodeling function, and efficiently induces DNA methylation, especially at hemimethylated replication sites.
RESUMO
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common blood disorder, presenting multiple symptoms, including hemolytic anemia. It affects 400 million people worldwide, with more than 160 single mutations reported in G6PD. The most severe mutations (about 70) are classified as class I, leading to more than 90% loss of activity of the wild-type G6PD. The crystal structure of G6PD reveals these mutations are located away from the active site, concentrating around the noncatalytic NADP+-binding site and the dimer interface. However, the molecular mechanisms of class I mutant dysfunction have remained elusive, hindering the development of efficient therapies. To resolve this, we performed integral structural characterization of five G6PD mutants, including four class I mutants, associated with the noncatalytic NADP+ and dimerization, using crystallography, small-angle X-ray scattering (SAXS), cryogenic electron microscopy (cryo-EM), and biophysical analyses. Comparisons with the structure and properties of the wild-type enzyme, together with molecular dynamics simulations, bring forward a universal mechanism for this severe G6PD deficiency due to the class I mutations. We highlight the role of the noncatalytic NADP+-binding site that is crucial for stabilization and ordering two ß-strands in the dimer interface, which together communicate these distant structural aberrations to the active site through a network of additional interactions. This understanding elucidates potential paths for drug development targeting G6PD deficiency.
Assuntos
Coenzimas/química , Glucosefosfato Desidrogenase/química , Leucina/química , Mutação , NADP/química , Prolina/química , Sítios de Ligação , Clonagem Molecular , Coenzimas/metabolismo , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Deficiência de Glucosefosfato Desidrogenase/enzimologia , Deficiência de Glucosefosfato Desidrogenase/genética , Deficiência de Glucosefosfato Desidrogenase/patologia , Humanos , Cinética , Leucina/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , NADP/metabolismo , Prolina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Nucleosome dynamics, such as nucleosome sliding and DNA unwrapping, are important for gene regulation in eukaryotic chromatin. H2A.Z, a variant of histone H2A that is highly evolutionarily conserved, participates in gene regulation by forming unstable multipositioned nucleosomes in vivo and in vitro. However, the subsecond dynamics of this unstable nucleosome have not been directly visualized under physiological conditions. Here, we used high-speed atomic force microscopy (HS-AFM) to directly visualize the subsecond dynamics of human H2A.Z.1-nucleosomes. HS-AFM videos show nucleosome sliding along 4 nm of DNA within 0.3 s in any direction. This sliding was also visualized in an H2A.Z.1 mutant, in which the C-terminal half was replaced by the corresponding canonical H2A amino acids, indicating that the interaction between the N-terminal region of H2A.Z.1 and the DNA is responsible for nucleosome sliding. These results may reveal the relationship between nucleosome dynamics and gene regulation by histone H2A.Z.
Assuntos
Histonas , Nucleossomos , Humanos , Histonas/química , Microscopia de Força Atômica , Cromatina , DNA/químicaRESUMO
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic trait that can cause hemolytic anemia. To date, over 150 nonsynonymous mutations have been identified in G6PD, with pathogenic mutations clustering near the dimer and/or tetramer interface and the allosteric NADP+-binding site. Recently, our lab identified a small molecule that activates G6PD variants by stabilizing the allosteric NADP+ and dimer complex, suggesting therapeutics that target these regions may improve structural defects. Here, we elucidated the connection between allosteric NADP+ binding, oligomerization, and pathogenicity to determine whether oligomer stabilization can be used as a therapeutic strategy for G6PD deficiency (G6PDdef). We first solved the crystal structure for G6PDK403Q, a mutant that mimics the physiological acetylation of wild-type G6PD in erythrocytes and demonstrated that loss of allosteric NADP+ binding induces conformational changes in the dimer. These structural changes prevent tetramerization, are unique to Class I variants (the most severe form of G6PDdef), and cause the deactivation and destabilization of G6PD. We also introduced nonnative cysteines at the oligomer interfaces and found that the tetramer complex is more catalytically active and stable than the dimer. Furthermore, stabilizing the dimer and tetramer improved protein stability in clinical variants, regardless of clinical classification, with tetramerization also improving the activity of G6PDK403Q and Class I variants. These findings were validated using enzyme activity and thermostability assays, analytical size-exclusion chromatography (SEC), and SEC coupled with small-angle X-ray scattering (SEC-SAXS). Taken together, our findings suggest a potential therapeutic strategy for G6PDdef and provide a foundation for future drug discovery efforts.
Assuntos
Deficiência de Glucosefosfato Desidrogenase , Glucosefosfato Desidrogenase , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Deficiência de Glucosefosfato Desidrogenase/genética , Humanos , Mutação , NADP/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Chromatin is the fundamental structure of genomic DNA in eukaryotic cells. The nucleosome, the primary unit of chromatin, consists of DNA and histone proteins, and is important for the maintenance of genomic DNA. Histone mutations are present in many types of cancers, suggesting that chromatin and/or nucleosome structures could be closely related to cancer development. Histone modifications and histone variants are also involved in regulating chromatin and nucleosome structures. Chromatin structures are dynamically changed by nucleosome binding proteins. In this review article, we discuss the current progress toward understanding the relationship between chromatin structure and cancer development.
Assuntos
Histonas , Nucleossomos , Humanos , Histonas/genética , Histonas/metabolismo , Cromatina/genética , DNA/química , Carcinogênese/genéticaRESUMO
Immunopathologies caused by Leishmania cause severe human morbidity and mortality. This protozoan parasite invades and persists inside host cells, resulting in disease development. Leishmania modifies the epigenomic status of the host cells, thus probably averting the host cell defense mechanism. To accomplish this, Leishmania may change the host cell chromatin structure. However, the mechanism by which the parasite changes the host cell chromatin has not been characterized. In the present study, we found that ectopically produced Leishmania histone H3, LmaH3, which mimics the secreted LmaH3 in infected cells, is incorporated into chromatin in human cells. A crystallographic analysis revealed that LmaH3 forms nucleosomes with human histones H2A, H2B and H4. We found that LmaH3 was less stably incorporated into the nucleosome, as compared to human H3.1. Consistently, we observed that LmaH3-H4 association was remarkably weakened. Mutational analyses revealed that the specific LmaH3 Trp35, Gln57 and Met98 residues, which correspond to the H3.1 Tyr41, Arg63 and Phe104 residues, might be responsible for the instability of the LmaH3 nucleosome. Nucleosomes containing LmaH3 resisted the Mg2+-mediated compaction of the chromatin fiber. These distinct physical characteristics of LmaH3 support the possibility that histones secreted by parasites during infection may modulate the host chromatin structure.
Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Leishmania major/imunologia , Nucleossomos/metabolismo , Linhagem Celular Tumoral , Células HeLa , Histonas/genética , Humanos , Leishmania major/genética , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
Mutations of the Glu76 residue of canonical histone H2B are frequently found in cancer cells. However, it is quite mysterious how a single amino acid substitution in one of the multiple H2B genes affects cell fate. Here we found that the H2B E76K mutation, in which Glu76 is replaced by Lys (E76K), distorted the interface between H2B and H4 in the nucleosome, as revealed by the crystal structure and induced nucleosome instability in vivo and in vitro. Exogenous production of the H2B E76K mutant robustly enhanced the colony formation ability of the expressing cells, indicating that the H2B E76K mutant has the potential to promote oncogenic transformation in the presence of wild-type H2B. We found that other cancer-associated mutations of histones, H3.1 E97K and H2A.Z.1 R80C, also induced nucleosome instability. Interestingly, like the H2B E76K mutant, the H3.1 E97K mutant was minimally incorporated into chromatin in cells, but it enhanced the colony formation ability. In contrast, the H2A.Z.1 R80C mutant was incorporated into chromatin in cells, and had minor effects on the colony formation ability of the cells. These characteristics of histones with cancer-associated mutations may provide important information toward understanding how the mutations promote cancer progression.
Assuntos
Histonas/química , Neoplasias/genética , Nucleossomos/genética , Cromatina/genética , Histonas/genética , Humanos , Mutação , Nucleossomos/química , Dobramento de ProteínaRESUMO
The nucleosome containing the histone H2A.Z.1 variant is unstable, as compared to the canonical nucleosome in vitro, and the incorporation of H2A.Z.1 into chromatin is less stable than that of the canonical H2A in vivo. In the present study, we designed a human H2A.Z.1(S42R) mutant, in which the Ser42 residue is replaced by Arg. In the crystal structure of the nucleosome containing H2A.Z.1(S42R), the Arg residue inserted at the H2A.Z.1-Ser42 position forms additional hydrogen bonds and electrostatic interactions with the DNA backbone phosphates. The Arg42 residue is located in the L1-loop region of H2A.Z.1, but the backbone geometry of the L1-loop is not affected by the H2A.Z.1(S42R) substitution. The nucleosome containing H2A.Z.1(S42R) exhibited enhanced thermal stability, as compared to that containing wild-type H2A.Z.1 in vitro. Fluorescence recovery after photobleaching experiments revealed that H2A.Z.1(S42R) was more stably incorporated in chromatin than wild-type H2A.Z.1 in living cells. Therefore, the H2A.Z.1(S42R) mutant stabilizes the nucleosome in vitro and in vivo, and may be useful as a tool to study the functional significance of the unstable nature of the H2A.Z.1 nucleosome.
Assuntos
Histonas/química , Mutação , Nucleossomos/química , Engenharia de Proteínas/métodos , Cromatina/química , DNA/química , Dano ao DNA , Células HeLa , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Isoformas de Proteínas , TemperaturaRESUMO
Disruption of cyclic adenosine monophosphate response element binding protein (CREB) provides a potential new strategy to address acute leukemia, a disease associated with poor prognosis, and for which conventional treatment options often carry a significant risk of morbidity and mortality. We describe the structure-activity relationships (SAR) for a series of XX-650-23 derived from naphthol AS-E phosphate that disrupts binding and activation of CREB by the CREB-binding protein (CBP). Through the development of this series, we identified several salicylamides that are potent inhibitors of acute leukemia cell viability through inhibition of CREB-CBP interaction. Among them, a biphenyl salicylamide, compound 71, was identified as a potent inhibitor of CREB-CBP interaction with improved physicochemical properties relative to previously described derivatives of naphthol AS-E phosphate.
Assuntos
Antineoplásicos/farmacologia , Proteína de Ligação a CREB/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Salicilamidas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proteína de Ligação a CREB/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HL-60 , Humanos , Leucemia Mieloide Aguda/metabolismo , Estrutura Molecular , Salicilamidas/síntese química , Salicilamidas/química , Relação Estrutura-AtividadeRESUMO
Histone H3.Y is a primate-specific, distant H3 variant. It is evolutionarily derived from H3.3, and may function in transcription regulation. However, the mechanism by which H3.Y regulates transcription has not been elucidated. In the present study, we determined the crystal structure of the H3.Y nucleosome, and found that many H3.Y-specific residues are located on the entry/exit sites of the nucleosome. Biochemical analyses revealed that the DNA ends of the H3.Y nucleosome were more flexible than those of the H3.3 nucleosome, although the H3.Y nucleosome was stable in vitro and in vivo Interestingly, the linker histone H1, which compacts nucleosomal DNA, appears to bind to the H3.Y nucleosome less efficiently, as compared to the H3.3 nucleosome. These characteristics of the H3.Y nucleosome are also conserved in the H3.Y/H3.3 heterotypic nucleosome, which may be the predominant form in cells. In human cells, H3.Y preferentially accumulated around transcription start sites (TSSs). Taken together, H3.Y-containing nucleosomes around transcription start sites may form relaxed chromatin that allows transcription factor access, to regulate the transcription status of specific genes.
Assuntos
Cromatina/genética , Histonas/genética , Nucleossomos/genética , Transcrição Gênica , DNA/genética , Histonas/metabolismo , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
Non-allelic histone variants are considered as epigenetic factors that regulate genomic DNA functions in eukaryotic chromosomes. In this study, we identified three new human histone H3 variants (named H3.6, H3.7, and H3.8), which were previously annotated as pseudogenes. H3.6 and H3.8 conserve the H3.3-specific amino acid residues, but H3.7 shares the specific amino acid residues with H3.1. We successfully reconstituted the nucleosome containing H3.6 in vitro and determined its crystal structure. In the H3.6 nucleosome, the H3.6-specific Val62 residue hydrophobically contacts the cognate H4 molecule, but its contact area is smaller than that of the corresponding H3.3 Ile62 residue. The thermal stability assay revealed that the H3.6 nucleosome is substantially unstable, as compared to the H3.3 nucleosome. Interestingly, mutational analysis demonstrated that the H3.6 Val62 residue is fully responsible for the H3.6 nucleosome instability, probably because of the weakened hydrophobic interaction with H4. We also reconstituted the nucleosome containing H3.8, but its thermal stability was quite low. In contrast, purified H3.7 failed to form nucleosomes in vitro. The identification and characterization of these novel human histone H3 variants provide important new insights into understanding the epigenetic regulation of the human genome.
Assuntos
Histonas/química , Isoformas de Proteínas/química , Cromatina/metabolismo , Cristalografia por Raios X , Histonas/genética , Histonas/metabolismo , Humanos , Conformação ProteicaRESUMO
CENP-A and CENP-B are major components of centromeric chromatin. CENP-A is the histone H3 variant, which forms the centromere-specific nucleosome. CENP-B specifically binds to the CENP-B box DNA sequence on the centromere-specific repetitive DNA. In the present study, we found that the CENP-A nucleosome more stably retains human CENP-B than the H3.1 nucleosome in vitro. Specifically, CENP-B forms a stable complex with the CENP-A nucleosome, when the CENP-B box sequence is located at the proximal edge of the nucleosome. Surprisingly, the CENP-B binding was weaker when the CENP-B box sequence was located in the distal linker region of the nucleosome. This difference in CENP-B binding, depending on the CENP-B box location, was not observed with the H3.1 nucleosome. Consistently, we found that the DNA-binding domain of CENP-B specifically interacted with the CENP-A-H4 complex, but not with the H3.1-H4 complex, in vitro. These results suggested that CENP-B forms a more stable complex with the CENP-A nucleosome through specific interactions with CENP-A, if the CENP-B box is located proximal to the CENP-A nucleosome. Our in vivo assay also revealed that CENP-B binding in the vicinity of the CENP-A nucleosome substantially stabilizes the CENP-A nucleosome on alphoid DNA in human cells.
Assuntos
Autoantígenos/metabolismo , Proteína B de Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Nucleossomos/metabolismo , Autoantígenos/química , Linhagem Celular Tumoral , Centrômero/química , Proteína Centromérica A , Proteínas Cromossômicas não Histona/química , DNA/química , DNA/metabolismo , Histonas/metabolismo , Humanos , Domínios e Motivos de Interação entre ProteínasRESUMO
The crotonylation of histones is an important post-translational modification, and epigenetically functions in the regulation of genomic DNA activity. The histone modifications in the structured "histone-fold" domains are considered to have an especially important impact on the nucleosome structure and dynamics. In the present study, we reconstituted the human nucleosome containing histone H3.2 crotonylated at the Lys122 residue, and determined its crystal structure at 2.56 Å resolution. We found that the crotonylation of the H3 Lys122 residue does not affect the overall nucleosome structure, but locally impedes the formation of the water-mediated hydrogen bond with the DNA backbone. Consistently, thermal stability assays revealed that the H3 Lys122 crotonylation, as well as the H3 Lys122 acetylation, clearly reduced the histone-DNA association.
Assuntos
Histonas/química , Histonas/ultraestrutura , Lisina/química , Modelos Moleculares , Nucleossomos/química , Nucleossomos/ultraestrutura , Cristalografia , Modelos Químicos , Conformação ProteicaRESUMO
The cyclobutane pyrimidine dimer (CPD) is induced in genomic DNA by ultraviolet (UV) light. In mammals, this photolesion is primarily induced within nucleosomal DNA, and repaired exclusively by the nucleotide excision repair (NER) pathway. However, the mechanism by which the CPD is accommodated within the nucleosome has remained unknown. We now report the crystal structure of a nucleosome containing CPDs. In the nucleosome, the CPD induces only limited local backbone distortion, and the affected bases are accommodated within the duplex. Interestingly, one of the affected thymine bases is located within 3.0 Å from the undamaged complementary adenine base, suggesting the formation of complementary hydrogen bonds in the nucleosome. We also found that UV-DDB, which binds the CPD at the initial stage of the NER pathway, also efficiently binds to the nucleosomal CPD. These results provide important structural and biochemical information for understanding how the CPD is accommodated and recognized in chromatin.
Assuntos
DNA/ultraestrutura , Nucleossomos/efeitos da radiação , Nucleossomos/ultraestrutura , Dímeros de Pirimidina/química , Dímeros de Pirimidina/efeitos da radiação , Raios Ultravioleta , Sítios de Ligação , DNA/química , DNA/efeitos da radiação , Ligação de Hidrogênio , Conformação Molecular/efeitos da radiação , Conformação de Ácido Nucleico , Ligação ProteicaRESUMO
The centromere is a specific genomic region upon which the kinetochore is formed to attach to spindle microtubules for faithful chromosome segregation. To distinguish this chromosomal region from other genomic loci, the centromere contains a specific chromatin structure including specialized nucleosomes containing the histone H3 variant CENP-A. In addition to CENP-A nucleosomes, we have found that centromeres contain a nucleosome-like structure comprised of the histone-fold CENP-T-W-S-X complex. However, it is unclear how the CENP-T-W-S-X complex associates with centromere chromatin. Here, we demonstrate that the CENP-T-W-S-X complex binds preferentially to â¼ 100 bp of linker DNA rather than nucleosome-bound DNA. In addition, we find that the CENP-T-W-S-X complex primarily binds to DNA as a (CENP-T-W-S-X)2 structure. Interestingly, in contrast to canonical nucleosomes that negatively supercoil DNA, the CENP-T-W-S-X complex induces positive DNA supercoils. We found that the DNA-binding regions in CENP-T or CENP-W, but not CENP-S or CENP-X, are required for this positive supercoiling activity and the kinetochore targeting of the CENP-T-W-S-X complex. In summary, our work reveals the structural features and properties of the CENP-T-W-S-X complex for its localization to centromeres.
Assuntos
Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Super-Helicoidal/metabolismo , Animais , Linhagem Celular , Galinhas , Proteínas Cromossômicas não Histona/química , DNA/metabolismo , Cinetocoros/metabolismo , Nucleossomos/metabolismoRESUMO
Nucleosomes are extremely stable histone-DNA complexes that form the building blocks of chromatin, which accommodates genomic DNA within the nucleus. The dynamic properties of chromatin play essential roles in regulating genomic DNA functions, such as DNA replication, recombination, repair, and transcription. Histones are the protein components of nucleosomes, and their diverse modifications and variants increase the versatility of nucleosome structures and their dynamics in chromatin. Therefore, a technique to evaluate the physical properties of nucleosomes would facilitate functional studies of the various nucleosomes. In this report, we describe a convenient assay for evaluating the thermal stability of nucleosomes in vitro.
Assuntos
Histonas/química , Microscopia de Fluorescência/métodos , Nucleossomos/química , Histonas/metabolismo , Modelos Químicos , Modelos Moleculares , Nucleossomos/metabolismo , TemperaturaRESUMO
The Epstein-Barr virus (EBV) genome is episomally maintained in latently infected cells. The viral protein EBNA1 is a bridging molecule that tethers EBV episomes to host mitotic chromosomes as well as to interphase chromatin. EBNA1 localizes to cellular chromosomes (chromatin) via its chromosome binding domains (CBDs), which are rich in glycine and arginine residues. However, the molecular mechanism by which the CBDs of EBNA1 attach to cellular chromatin is still under debate. Mutation analyses revealed that stepwise substitution of arginine residues within the CBD1 (amino acids 40-54) and CBD2 (amino acids 328-377) regions with alanines progressively impaired chromosome binding activity of EBNA1. The complete arginine-to-alanine substitutions within the CBD1 and -2 regions abolished the ability of EBNA1 to stably maintain EBV-derived oriP plasmids in dividing cells. Importantly, replacing the same arginines with lysines had minimal effect, if any, on chromosome binding of EBNA1 as well as on its ability to stably maintain oriP plasmids. Furthermore, a glycine-arginine-rich peptide derived from the CBD1 region bound to reconstituted nucleosome core particles in vitro, as did a glycine-lysine rich peptide, whereas a glycine-alanine rich peptide did not. These results support the idea that the chromosome binding of EBNA1 is mediated by electrostatic interactions between the basic amino acids within the CBDs and negatively charged cellular chromatin.
Assuntos
Aminoácidos Básicos/metabolismo , Cromatina/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/química , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Plasmídeos/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Cromossomos Humanos/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Nucleossomos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The histone H2A variant H2A.W occupies transposons and thus prevents access to them in Arabidopsis thaliana. H2A.W is deposited by the chromatin remodeler DDM1, which also promotes the accessibility of chromatin writers to heterochromatin by an unknown mechanism. To shed light on this question, we solve the cryo-EM structures of nucleosomes containing H2A and H2A.W, and the DDM1-H2A.W nucleosome complex. These structures show that the DNA end flexibility of the H2A nucleosome is higher than that of the H2A.W nucleosome. In the DDM1-H2A.W nucleosome complex, DDM1 binds to the N-terminal tail of H4 and the nucleosomal DNA and increases the DNA end flexibility of H2A.W nucleosomes. Based on these biochemical and structural results, we propose that DDM1 counters the low accessibility caused by nucleosomes containing H2A.W to enable the maintenance of repressive epigenetic marks on transposons and prevent their activity.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Montagem e Desmontagem da Cromatina , Microscopia Crioeletrônica , Histonas , Nucleossomos , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/química , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Nucleossomos/química , Histonas/metabolismo , Histonas/genética , Histonas/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ligação Proteica , Modelos Moleculares , DNA de Plantas/metabolismo , DNA de Plantas/genéticaRESUMO
The histone H2A.Z variant is widely conserved among eukaryotes. Two isoforms, H2A.Z.1 and H2A.Z.2, have been identified in vertebrates and may have distinct functions in cell growth and gene expression. However, no structural differences between H2A.Z.1 and H2A.Z.2 have been reported. In the present study, the crystal structures of nucleosomes containing human H2A.Z.1 and H2A.Z.2 were determined. The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical. This structural polymorphism may have been induced by a substitution that evolutionally occurred at the position of amino acid 38 and by the flexible nature of the L1 loops of H2A.Z.1 and H2A.Z.2. It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2. A mutational analysis revealed that the amino-acid difference at position 38 is at least partially responsible for the distinctive dynamics of H2A.Z.1 and H2A.Z.2. These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.