RESUMO
The structure of the nucleosome, the basic building block of the chromatin fiber, plays a key role in epigenetic regulatory processes that affect DNA-dependent processes in the context of chromatin. Members of the HMGN family of proteins bind specifically to nucleosomes and affect chromatin structure and function, including transcription and DNA repair. To better understand the mechanisms by which HMGN 1 and 2 alter chromatin, we analyzed their effect on the organization of histone tails and linker histone H1 in nucleosomes. We find that HMGNs counteract linker histone (H1)-dependent stabilization of higher order 'tertiary' chromatin structures but do not alter the intrinsic ability of nucleosome arrays to undergo salt-induced compaction and self-association. Surprisingly, HMGNs do not displace H1s from nucleosomes; rather these proteins bind nucleosomes simultaneously with H1s without disturbing specific contacts between the H1 globular domain and nucleosomal DNA. However, HMGNs do alter the nucleosome-dependent condensation of the linker histone C-terminal domain, which is critical for stabilizing higher-order chromatin structures. Moreover, HMGNs affect the interactions of the core histone tail domains with nucleosomal DNA, redirecting the tails to more interior positions within the nucleosome. Our studies provide new insights into the molecular mechanisms whereby HMGNs affect chromatin structure.
Assuntos
DNA/química , Proteína HMGN1/química , Proteína HMGN2/química , Histonas/química , Nucleossomos/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Galinhas , DNA/genética , DNA/metabolismo , Expressão Gênica , Proteína HMGN1/genética , Proteína HMGN1/metabolismo , Proteína HMGN2/genética , Proteína HMGN2/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Conformação de Ácido Nucleico , Nucleossomos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
An interplay between the nucleosome binding proteins H1 and HMGN is known to affect chromatin dynamics, but the biological significance of this interplay is still not clear. We find that during embryonic stem cell differentiation loss of HMGNs leads to down regulation of genes involved in neural differentiation, and that the transcription factor OLIG2 is a central node in the affected pathway. Loss of HMGNs affects the expression of OLIG2 as well as that of OLIG1, two transcription factors that are crucial for oligodendrocyte lineage specification and nerve myelination. Loss of HMGNs increases the chromatin binding of histone H1, thereby recruiting the histone methyltransferase EZH2 and elevating H3K27me3 levels, thus conferring a repressive epigenetic signature at Olig1&2 sites. Embryonic stem cells lacking HMGNs show reduced ability to differentiate towards the oligodendrocyte lineage, and mice lacking HMGNs show reduced oligodendrocyte count and decreased spinal cord myelination, and display related neurological phenotypes. Thus, the presence of HMGN proteins is required for proper expression of neural differentiation genes during embryonic stem cell differentiation. Specifically, we demonstrate that the dynamic interplay between HMGNs and H1 in chromatin epigenetically regulates the expression of OLIG1&2, thereby affecting oligodendrocyte development and myelination, and mouse behavior.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Epigênese Genética , Proteínas HMGN/fisiologia , Histonas/metabolismo , Proteínas do Tecido Nervoso/genética , Oligodendroglia/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Proteína HMGN1/genética , Proteína HMGN1/fisiologia , Proteína HMGN2/genética , Proteína HMGN2/fisiologia , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição 2 de OligodendrócitosRESUMO
DNase I hypersensitive sites (DHSs) are a hallmark of chromatin regions containing regulatory DNA such as enhancers and promoters; however, the factors affecting the establishment and maintenance of these sites are not fully understood. We now show that HMGN1 and HMGN2, nucleosome-binding proteins that are ubiquitously expressed in vertebrate cells, maintain the DHS landscape of mouse embryonic fibroblasts (MEFs) synergistically. Loss of one of these HMGN variants led to a compensatory increase of binding of the remaining variant. Genome-wide mapping of the DHSs in Hmgn1(-/-), Hmgn2(-/-), and Hmgn1(-/-)n2(-/-) MEFs reveals that loss of both, but not a single HMGN variant, leads to significant remodeling of the DHS landscape, especially at enhancer regions marked by H3K4me1 and H3K27ac. Loss of HMGN variants affects the induced expression of stress-responsive genes in MEFs, the transcription profiles of several mouse tissues, and leads to altered phenotypes that are not seen in mice lacking only one variant. We conclude that the compensatory binding of HMGN variants to chromatin maintains the DHS landscape, and the transcription fidelity and is necessary to retain wild-type phenotypes. Our study provides insight into mechanisms that maintain regulatory sites in chromatin and into functional compensation among nucleosome binding architectural proteins.
Assuntos
Sítios de Ligação , Desoxirribonuclease I/metabolismo , Elementos Facilitadores Genéticos , Proteínas HMGN/metabolismo , Animais , Linhagem Celular , Cromatina/metabolismo , Análise por Conglomerados , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Proteínas HMGN/genética , Proteína HMGN1/genética , Proteína HMGN1/metabolismo , Proteína HMGN2/genética , Proteína HMGN2/metabolismo , Humanos , Camundongos , Camundongos Knockout , Nucleossomos/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas , Estresse Fisiológico/genéticaRESUMO
Thyroid cancer originates from genetic and epigenetic changes that alter gene expression and cellular signaling pathways. Here, we report that altered expression of the nucleosome-binding protein HMGN4 potentiates thyroid tumorigenesis. Bioinformatics analyses reveal increased HMGN4 expression in thyroid cancer. We find that upregulation of HMGN4 expression in mouse and human cells, and in the thyroid of transgenic mice, alters the cellular transcription profile, downregulates the expression of the tumor suppressors Atm, Atrx and Brca2, and elevates the levels of the DNA damage marker γH2AX. Mouse and human cells overexpressing HMGN4 show increased tumorigenicity as measured by colony formation, by tumor generation in nude mice, and by the formation of preneoplastic lesions in the thyroid of transgenic mice. Our study identifies a novel epigenetic factor that potentiates thyroid oncogenesis and raises the possibility that HMGN4 may serve as an additional diagnostic marker, or therapeutic target in certain thyroid cancers.
Assuntos
Carcinogênese/genética , Transformação Celular Neoplásica/genética , Expressão Gênica/genética , Proteínas HMGN/genética , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Animais , Biomarcadores Tumorais/genética , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/genética , Regulação para Baixo/genética , Epigênese Genética/genética , Humanos , Camundongos , Camundongos Nus , Camundongos Transgênicos , Transdução de Sinais/genética , Transcrição Gênica/genética , Regulação para Cima/genéticaRESUMO
The dynamic interaction of nucleosome binding proteins with their chromatin targets is an important element in regulating the structure and function of chromatin. Histone H1 variants and High Mobility Group (HMG) proteins are ubiquitously expressed in all vertebrate cells, bind dynamically to chromatin, and are known to affect chromatin condensation and the ability of regulatory factors to access their genomic binding sites. Here, we review the studies that focus on the interactions between H1 and HMGs and highlight the functional consequences of the interplay between these architectural chromatin binding proteins. H1 and HMG proteins are mobile molecules that bind to nucleosomes as members of a dynamic protein network. All HMGs compete with H1 for chromatin binding sites, in a dose dependent fashion, but each HMG family has specific effects on the interaction of H1 with chromatin. The interplay between H1 and HMGs affects chromatin organization and plays a role in epigenetic regulation.
Assuntos
Cromatina/química , Proteínas de Grupo de Alta Mobilidade/fisiologia , Histonas/fisiologia , Animais , Proteínas de Transporte/fisiologia , HumanosRESUMO
Structural changes in specific chromatin domains are essential to the orderly progression of numerous nuclear processes, including transcription. We report that the nuclear protein NSBP1 (HMGN5), a recently discovered member of the HMGN nucleosome-binding protein family, is specifically targeted by its C-terminal domain to nucleosomes in euchromatin. We find that the interaction of NSBP1 with nucleosomes alters the compaction of cellular chromatin and that in living cells, NSBP1 interacts with linker histones. We demonstrate that the negatively charged C-terminal domain of NSBP1 interacts with the positively charged C-terminal domain of H5 and that NSBP1 counteracts the linker histone-mediated compaction of a nucleosomal array. Dysregulation of the cellular levels of NSBP1 alters the transcription level of numerous genes. We suggest that mouse NSBP1 is an architectural protein that binds preferentially to euchromatin and modulates the fidelity of the cellular transcription profile by counteracting the chromatin-condensing activity of linker histones.
Assuntos
Montagem e Desmontagem da Cromatina , Eucromatina/metabolismo , Proteínas HMGN/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Transativadores/metabolismo , Transcrição Gênica , Animais , Sítios de Ligação , Linhagem Celular Transformada , Eucromatina/química , Perfilação da Expressão Gênica , Proteínas HMGN/química , Proteínas HMGN/genética , Histonas/química , Lisina , Metilação , Camundongos , Microscopia Confocal , Modelos Moleculares , Células NIH 3T3 , Conformação de Ácido Nucleico , Conformação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/química , Transativadores/genética , TransfecçãoRESUMO
HMGNs are nucleosome-binding proteins that alter the pattern of histone modifications and modulate the binding of linker histones to chromatin. The HMGN3 family member exists as two splice forms, HMGN3a which is full-length and HMGN3b which lacks the C-terminal RD (regulatory domain). In the present study, we have used the Glyt1 (glycine transporter 1) gene as a model system to investigate where HMGN proteins are bound across the locus in vivo, and to study how the two HMGN3 splice variants affect histone modifications and gene expression. We demonstrate that HMGN1, HMGN2, HMGN3a and HMGN3b are bound across the Glyt1 gene locus and surrounding regions, and are not enriched more highly at the promoter or putative enhancer. We conclude that the peaks of H3K4me3 (trimethylated Lys(4) of histone H3) and H3K9ac (acetylated Lys(9) of histone H3) at the active Glyt1a promoter do not play a major role in recruiting HMGN proteins. HMGN3a/b binding leads to increased H3K14 (Lys(14) of histone H3) acetylation and stimulates Glyt1a expression, but does not alter the levels of H3K4me3 or H3K9ac enrichment. Acetylation assays show that HMGN3a stimulates the ability of PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] to acetylate nucleosomal H3 in vitro, whereas HMGN3b does not. We propose a model where HMGN3a/b-stimulated H3K14 acetylation across the bodies of large genes such as Glyt1 can lead to more efficient transcription elongation and increased mRNA production.
Assuntos
Cromatina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/genética , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Proteínas HMGN/metabolismo , Histonas/metabolismo , Acetilação , Animais , Sítios de Ligação , Linhagem Celular , Proteínas HMGN/genética , Histonas/genética , Camundongos , TransfecçãoRESUMO
BACKGROUND: Nucleosomal binding proteins, HMGN, is a family of chromatin architectural proteins that are expressed in all vertebrate nuclei. Although previous studies have discovered that HMGN proteins have important roles in gene regulation and chromatin accessibility, whether and how HMGN proteins affect higher order chromatin status remains unknown. RESULTS: We examined the roles that HMGN1 and HMGN2 proteins play in higher order chromatin structures in three different cell types. We interrogated data generated in situ, using several techniques, including Hi-C, Promoter Capture Hi-C, ChIP-seq, and ChIP-MS. Our results show that HMGN proteins occupy the A compartment in the 3D nucleus space. In particular, HMGN proteins occupy genomic regions involved in cell-type-specific long-range promoter-enhancer interactions. Interestingly, depletion of HMGN proteins in the three different cell types does not cause structural changes in higher order chromatin, i.e., in topologically associated domains (TADs) and in A/B compartment scores. Using ChIP-seq combined with mass spectrometry, we discovered protein partners that are directly associated with or neighbors of HMGNs on nucleosomes. CONCLUSIONS: We determined how HMGN chromatin architectural proteins are positioned within a 3D nucleus space, including the identification of their binding partners in mononucleosomes. Our research indicates that HMGN proteins localize to active chromatin compartments but do not have major effects on 3D higher order chromatin structure and that their binding to chromatin is not dependent on specific protein partners.
Assuntos
Cromatina , Proteínas HMGN , Epigênese Genética , Proteínas HMGN/química , Proteínas HMGN/genética , Proteínas HMGN/metabolismo , Nucleossomos , Ligação ProteicaRESUMO
Nucleosomes containing acetylated H3K27 are a major epigenetic mark of active chromatin and identify cell-type specific chromatin regulatory regions which serve as binding sites for transcription factors. Here we show that the ubiquitous nucleosome binding proteins HMGN1 and HMGN2 bind preferentially to H3K27ac nucleosomes at cell-type specific chromatin regulatory regions. HMGNs bind directly to the acetylated nucleosome; the H3K27ac residue and linker DNA facilitate the preferential binding of HMGNs to the modified nucleosomes. Loss of HMGNs increases the levels of H3K27me3 and the histone H1 occupancy at enhancers and promoters and alters the interaction of transcription factors with chromatin. These experiments indicate that the H3K27ac epigenetic mark enhances the interaction of architectural protein with chromatin regulatory sites and identify determinants that facilitate the localization of HMGN proteins at regulatory sites to modulate cell-type specific gene expression.
Assuntos
Proteínas HMGN , Nucleossomos , Cromatina/genética , Proteínas HMGN/química , Proteínas HMGN/genética , Proteínas HMGN/metabolismo , Nucleossomos/genética , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
High mobility group nucleosome-binding (HMGN) proteins are architectural non-histone chromosomal proteins that bind to nucleosomes and modulate the structure and function of chromatin. The interaction of HMGN proteins with nucleosomes is dynamic and the proteins compete with the linker histone H1 chromatin-binding sites. HMGNs reduce the H1-mediated compaction of the chromatin fiber and facilitate the targeting of regulatory factors to chromatin. They modulate the cellular epigenetic profile, affect gene expression and impact the biological processes such as development and the cellular response to environmental and hormonal signals. Here we review the role of HMGN in chromatin structure, the link between HMGN proteins and histone modifications, and discuss the consequence of this link on nuclear processes and cellular phenotype.
Assuntos
Cromatina/química , Cromatina/metabolismo , Proteínas HMGN/metabolismo , Sequência de Aminoácidos , Animais , Proteínas HMGN/química , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Moldes Genéticos , Transcrição GênicaRESUMO
The dynamic nature of the chromatin epigenetic landscape plays a key role in the establishment and maintenance of cell identity, yet the factors that affect the dynamics of the epigenome are not fully known. Here we find that the ubiquitous nucleosome binding proteins HMGN1 and HMGN2 preferentially colocalize with epigenetic marks of active chromatin, and with cell-type specific enhancers. Loss of HMGNs enhances the rate of OSKM induced reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs), and the ASCL1 induced conversion of fibroblast into neurons. During transcription factor induced reprogramming to pluripotency, loss of HMGNs accelerates the erasure of the MEF-specific epigenetic landscape and the establishment of an iPSCs-specific chromatin landscape, without affecting the pluripotency potential and the differentiation potential of the reprogrammed cells. Thus, HMGN proteins modulate the plasticity of the chromatin epigenetic landscape thereby stabilizing, rather than determining cell identity.
Assuntos
Membrana Celular/metabolismo , Fibroblastos/metabolismo , Proteína HMGN1/metabolismo , Proteína HMGN2/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Reprogramação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Embrião de Mamíferos/citologia , Epigênese Genética , Fibroblastos/citologia , Células HEK293 , Proteína HMGN1/genética , Proteína HMGN2/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos Knockout , Camundongos Nus , Ligação ProteicaRESUMO
We report that loss of HMGN1, a nucleosome-binding protein that alters the compaction of the chromatin fiber, increases the cellular sensitivity to ionizing radiation and the tumor burden of mice. The mortality and tumor burden of ionizing radiation-treated Hmgn1-/- mice is higher than that of their Hmgn1+/+ littermates. Hmgn1-/- fibroblasts have an altered G2-M checkpoint activation and are hypersensitive to ionizing radiation. The ionizing radiation hypersensitivity and the aberrant G2-M checkpoint activation of Hmgn1-/- fibroblasts can be reverted by transfections with plasmids expressing wild-type HMGN1, but not with plasmids expressing mutant HMGN proteins that do not bind to chromatin. Transformed Hmgn1-/- fibroblasts grow in soft agar and produce tumors in nude mice with a significantly higher efficiency than Hmgn1+/+ fibroblasts, suggesting that loss of HMGN1 protein disrupts cellular events controlling proliferation and growth. Hmgn1-/- mice have a higher incidence of multiple malignant tumors and metastases than their Hmgn1+/+ littermates. We suggest that HMGN1 optimizes the cellular response to ionizing radiation and to other tumorigenic events; therefore, loss of this protein increases the tumor burden in mice.
Assuntos
Transformação Celular Neoplásica/efeitos da radiação , Proteína HMGN1/deficiência , Neoplasias Induzidas por Radiação/metabolismo , Tolerância a Radiação/fisiologia , Animais , Divisão Celular/efeitos da radiação , Transformação Celular Neoplásica/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Fase G2/efeitos da radiação , Proteína HMGN1/metabolismo , Proteína HMGN1/fisiologia , Masculino , Camundongos , Camundongos Nus , Neoplasias Induzidas por Radiação/patologiaRESUMO
HMGN1 is a nuclear protein that binds to nucleosomes and alters the accessibility of regulatory factors to their chromatin targets. To elucidate its biological function and identify specific HMGN1 target genes, we generated Hmgn1-/- mice. DNA microarray analysis of Hmgn1+/+ and Hmgn1-/- embryonic fibroblasts identified N-cadherin as a potential HMGN1 gene target. RT-PCR and western blot analysis confirmed a linkage between HMGN1 expression and N-cadherin levels. In both transformed and primary mouse embryonic fibroblasts (MEFs), HMGN1 acted as negative regulator of N-cadherin expression. Likewise, the N-cadherin levels in early embryos of Hmgn1-/- mice were higher than those of their Hmgn1+/+ littermates. Loss of HMGN1 increased the adhesiveness, motility and aggregation potential of Hmgn1-/- MEFs, a phenotype consistent with increased levels of N-cadherin protein. Re-expression of wild-type HMGN1, but not of the mutant HMGN1 protein that does not bind to chromatin, in Hmgn1-/- MEFs, decreased the levels of N-cadherin and restored the Hmgn1+/+ phenotype. These studies demonstrate a role for HMGN1 in the regulation of specific gene expression. We suggest that in MEFs, and during early mouse development, the interaction of HMGN1 with chromatin down-regulates the expression of N-cadherin.
Assuntos
Caderinas/metabolismo , Cromossomos/química , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteína HMGN1/metabolismo , Animais , Western Blotting , Adesão Celular , Moléculas de Adesão Celular , Linhagem Celular Transformada , Movimento Celular , Células Cultivadas , Cromatina/metabolismo , Regulação para Baixo , Embrião de Mamíferos , Fibroblastos/citologia , Marcação de Genes , Proteína HMGN1/genética , Camundongos , Camundongos Knockout , Mutação , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta Catenina/metabolismoRESUMO
UNLABELLED: We report that HMGN1, a nucleosome-binding protein that affects chromatin structure and function, affects the growth of N-nitrosodiethylamine (DEN)-induced liver tumors. Following a single DEN injection at 2 weeks of age, Hmgn1(tm1/tm1) mice, lacking the nucleosome-binding domain of HMGN1, had earlier signs of liver tumorigenesis than their Hmgn1(+/+) littermates. Detailed gene expression profiling revealed significant differences between DEN-injected and control saline-injected mice, but only minor differences between the injected Hmgn1(tm1/tm1) mice and their Hmgn1(+/+) littermates. Pathway analysis revealed that the most significant process affected by loss of HMGN1 involves the lipid/sterol metabolic pathway. Our study indicates that in mice, loss of HMGN1 leads to transcription changes that accelerate the progression of DEN-induced hepatocarcinogenesis, without affecting the type of tumors or the final total tumor burden of these mice. IMPLICATIONS: Loss of HMGN1 leads to accelerated progression of DEN-induced hepatocarcinogenesis in mice.
Assuntos
Transformação Celular Neoplásica/genética , Dietilnitrosamina/farmacologia , Proteína HMGN1/genética , Metabolismo dos Lipídeos/genética , Neoplasias Hepáticas/genética , Animais , Cromatina/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fígado/patologia , Neoplasias Hepáticas/induzido quimicamente , Camundongos , Camundongos Knockout , Carga TumoralRESUMO
Proliferating cell nuclear antigen (PCNA) is a ubiquitous protein that interacts with multiple partners and regulates nuclear activities, including chromatin assembly, histone modifications, replication, and DNA damage repair. The role of specific partners in regulating PCNA activities is not fully understood. Here we identify the nucleosome binding protein HMGN1 as a new PCNA-interacting protein that enhances the binding of PCNA to chromatin but not to purified DNA. Two tetrapeptides in the conservative domain of HMGN1 contain amino acids necessary for the binding of HMGN1 to PCNA. Deletion of both tetrapeptides abolishes the HMGN1-PCNA interaction. PCNA preferentially binds to the linker DNA adjacent to an HMGN-containing nucleosome. In living cells, loss of HMGN1 decreases the rate of PCNA recruitment to damaged DNA sites. Our study identifies a new factor that facilitates the interaction of PCNA with chromatin and provides insights into mechanisms whereby nucleosome binding architectural proteins affect the cellular phenotype.
Assuntos
Cromatina/metabolismo , Proteína HMGN1/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Transformada , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Humanos , Camundongos , Nucleossomos/metabolismo , Oligopeptídeos , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Alarmins are endogenous mediators capable of promoting the recruitment and activation of antigen-presenting cells (APCs), including dendritic cells (DCs), that can potentially alert host defense against danger signals. However, the relevance of alarmins to the induction of adaptive immune responses remains to be demonstrated. In this study, we report the identification of HMGN1 (high-mobility group nucleosome-binding protein 1) as a novel alarmin and demonstrate that it contributes to the induction of antigen-specific immune responses. HMGN1 induced DC maturation via TLR4 (Toll-like receptor 4), recruitment of APCs at sites of injection, and activation of NF-κB and multiple mitogen-activated protein kinases in DCs. HMGN1 promoted antigen-specific immune response upon co-administration with antigens, and Hmgn1(-/-) mice developed greatly reduced antigen-specific antibody and T cell responses when immunized with antigens in the presence of lipopolysaccharide (LPS). The impaired ability of Hmgn1(-/-) mice to mount antigen-specific immune responses was accompanied by both deficient DC recruitment at sites of immunization and reduced production of inflammatory cytokines. Bone marrow chimera experiments revealed that HMGN1 derived from nonleukocytes was critical for the induction of antigen-specific antibody and T cell responses. Thus, extracellular HMGN1 acts as a novel alarmin critical for LPS-induced development of innate and adaptive immune responses.
Assuntos
Proteína HMGN1/metabolismo , Imunidade , Lipopolissacarídeos/imunologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Antígenos/imunologia , Diferenciação Celular , Linhagem Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Feminino , Células HEK293 , Proteína HMGN1/genética , Proteína HMGN1/imunologia , Humanos , Imunidade/genética , Imunidade Inata/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/metabolismo , Fenótipo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismoRESUMO
The HMGN family is a family of nucleosome-binding architectural proteins that affect the structure and function of chromatin in vertebrates. We report that the HMGN5 variant, encoded by a gene located on chromosome X, is a rapidly evolving protein with an acidic C-terminal domain that differs among vertebrate species. We found that the intranuclear organization and nucleosome interactions of human HMGN5 are distinct from those of mouse HMGN5 and that the C-terminal region of the protein is the main determinant of the chromatin interaction properties. Despite their apparent differences, both mouse and human HMGN5 proteins interact with histone H1, reduce its chromatin residence time, and can induce large-scale chromatin decompaction in living cells. Analysis of HMGN5 mutants suggests that distinct domains in HMGN5 affect specific steps in the interaction of H1 with chromatin. Elevated levels of either human or mouse HMGN5 affect the transcription of numerous genes, most in a variant-specific manner. Our study identifies HMGN5 as a rapidly evolving vertebrate nuclear protein with species-specific properties. HMGN5 has a highly disordered structure, binds dynamically to nucleosome core particles, modulates the binding of H1 to chromatin, reduces the compaction of the chromatin fiber, and affects transcription.
Assuntos
Sequência Conservada , Proteínas HMGN/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatina/metabolismo , Cromossomos Humanos X/genética , Expressão Gênica , Perfilação da Expressão Gênica , Proteínas HMGN/genética , Histonas/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , RNA Interferente Pequeno/genética , Transativadores/genéticaAssuntos
Bioquímica/métodos , Proteínas HMGN/química , Proteínas HMGN/isolamento & purificação , Animais , Cátions , Núcleo Celular/metabolismo , Cromatina/química , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Células HeLa , Humanos , Nuclease do Micrococo/metabolismo , Reação em Cadeia da Polimerase , Testes de Precipitina , Ligação Proteica , Proteínas Recombinantes/química , Fatores de TempoAssuntos
Bioquímica/métodos , Cromatina/química , Proteínas HMGN/química , Nucleossomos/metabolismo , Animais , Southern Blotting , Cromatina/metabolismo , Cromossomos/ultraestrutura , Relação Dose-Resposta a Droga , Proteínas HMGN/metabolismo , Histonas/química , Nuclease do Micrococo/metabolismo , Microscopia Eletrônica , Nucleossomos/ultraestrutura , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
The nucleosome-binding protein HMGN1 affects the structure and function of chromatin; however, its role in regulating specific gene expression in living cells is not fully understood. Here we use embryonic fibroblasts from Hmgn1(+/+) and Hmgn1(-/-) mice to examine the effect of HMGN1 on the heat shock-induced transcriptional activation of Hsp70, a well characterized gene known to undergo a rapid chromatin re-structuring during transcriptional activation. We find that loss of HMGN1 decreases the levels of Hsp70 transcripts at the early stages of heat shock. HMGN1 enhances the rate of heat shockinduced changes in the Hsp70 chromatin but does not affect the chromatin structure before induction, an indication that it does not predispose the gene to rapid activation. Heat shock elevates the levels of H3K14 acetylation in the Hsp70 chromatin of wild type cells more efficiently than in the chromatin of Hmgn1(-/-) cells, whereas treatment with histone deacetylase inhibitors abrogates the effects of HMGN1 on the heat shock response. We suggest that HMGN1 enhances the rate of heat shock-induced H3K14 acetylation in the Hsp70 promoter, thereby enhancing the rate of chromatin remodeling and the subsequent transcription during the early rounds of Hsp70 activation when the gene is still associated with histones in a nucleosomal conformation.