RESUMO
The recent discovery of the cancer-associated E76K mutation in histone H2B (H2BE76-to-K) in several types of cancers revealed a new class of oncohistone. H2BE76K weakens the stability of histone octamers, alters gene expression, and promotes colony formation. However, the mechanism linking the H2BE76K mutation to cancer development remains largely unknown. In this study, we knock in the H2BE76K mutation in MDA-MB-231 breast cancer cells using CRISPR/Cas9 and show that the E76K mutant histone H2B preferentially localizes to genic regions. Interestingly, genes upregulated in the H2BE76K mutant cells are enriched for the E76K mutant H2B and are involved in cell adhesion and proliferation pathways. We focused on one H2BE76K target gene, ADAM19 (a disintegrin and metalloproteinase-domain-containing protein 19), a gene highly expressed in various human cancers including breast invasive carcinoma, and demonstrate that H2BE76K directly promotes ADAM19 transcription by facilitating efficient transcription along the gene body. ADAM19 depletion reduced the colony formation ability of the H2BE76K mutant cells, whereas wild-type MDA-MB-231 cells overexpressing ADAM19 mimics the colony formation phenotype of the H2BE76K mutant cells. Collectively, our data demonstrate the mechanism by which H2BE76K deregulates the expression of genes that control oncogenic properties through a combined effect of its specific genomic localization and nucleosome destabilization effect.
Assuntos
Proteínas ADAM/genética , Neoplasias da Mama/genética , Histonas/genética , Proteínas ADAM/metabolismo , Neoplasias da Mama/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Feminino , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Histonas/metabolismo , Humanos , Mutação/genética , Nucleossomos , Oncogenes/genética , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Canonical histones (H2A, H2B, H3, and H4) are present in all eukaryotes where they package genomic DNA and participate in numerous cellular processes, such as transcription regulation and DNA repair. In addition to the canonical histones, there are many histone variants, which have different amino acid sequences, possess tissue-specific expression profiles, and function distinctly from the canonical counterparts. A number of histone variants, including both core histones (H2A/H2B/H3/H4) and linker histones (H1/H5), have been identified to date. Htz1 (H2A.Z) and CENP-A (CenH3) are present from yeasts to mammals, and H3.3 is present from Tetrahymena to humans. In addition to the prevalent variants, others like H3.4 (H3t), H2A.Bbd, and TH2B, as well as several H1 variants, are found to be specific to mammals. Among them, H2BFWT, H3.5, H3.X, H3.Y, and H4G are unique to primates (or Hominidae). In this review, we focus on localization and function of primate- or hominidae-specific histone variants.
Assuntos
Histonas/classificação , Primatas/genética , Primatas/fisiologia , Sequência de Aminoácidos , Animais , Encéfalo , Neoplasias da Mama , Nucléolo Celular , DNA , Regulação da Expressão Gênica , Histonas/genética , Humanos , Mamíferos , FilogeniaRESUMO
RecQL5, a mammalian RecQ family protein, is involved in the regulation of transcription elongation, DNA damage response, and DNA replication. Here, we identified and characterized an alternative splicing isoform of RECQL5 (RECQL5ß1), which contains 17 additional amino acid residues within the RECQL5 KIX domain when compared with the canonical isoform (RECQL5ß). RECQL5ß1 had a markedly decreased binding affinity to RNA polymerase II (Pol II) and poorly competed with the transcription elongation factor TFIIS for binding to Pol II. As a result, this isoform has a weaker activity for repression of transcription elongation. In contrast, we discovered that RECQL5ß1 could bind stronger to MRE11, which is a primary sensor of DNA double-strand breaks (DSBs). Furthermore, we found that RECQL5ß1 promoted DNA repair in the RECQL5ß1 rescue cells. These results suggest that RECQL5ß mainly functions as a transcription repressor, while the newly discovered RECQL5ß1 has a specialized role in DNA damage response. Taken together, our data suggest a cellular-functional specialization for each KIX splicing isoform in the cell.
Assuntos
Reparo do DNA , Proteína Homóloga a MRE11/metabolismo , RNA Polimerase II/metabolismo , RecQ Helicases/metabolismo , Transcrição Gênica , Linhagem Celular , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Ligação Proteica , Isoformas de Proteínas , Fatores de Elongação da Transcrição/metabolismoAssuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Histonas/genética , Neoplasias/genética , Proliferação de Células/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , Histonas/ultraestrutura , Humanos , Mutação/genética , Neoplasias/patologia , Conformação de Ácido Nucleico , Nucleossomos/genética , Oncogenes/genética , FenótipoRESUMO
Histone variants, present in various cell types and tissues, are known to exhibit different functions. For example, histone H3.3 and H2A.Z are both involved in gene expression regulation, whereas H2A.X is a specific variant that responds to DNA double-strand breaks. In this study, we characterized H4G, a novel hominidae-specific histone H4 variant. We found that H4G is expressed in a variety of human cell lines and exhibit tumor-stage dependent overexpression in tissues from breast cancer patients. We found that H4G localized primarily to the nucleoli of the cell nucleus. This localization was controlled by the interaction of the alpha-helix 3 of the histone fold motif with a histone chaperone, nucleophosmin 1. In addition, we found that modulating H4G expression affects rRNA expression levels, protein synthesis rates and cell-cycle progression. Our data suggest that H4G expression alters nucleolar chromatin in a way that enhances rDNA transcription in breast cancer tissues.
Assuntos
Neoplasias da Mama/genética , DNA Ribossômico/genética , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Proteínas Nucleares/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , DNA Ribossômico/química , DNA Ribossômico/metabolismo , Feminino , Gorilla gorilla , Histonas/química , Histonas/metabolismo , Humanos , Camundongos , Camundongos Knockout , Estadiamento de Neoplasias , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleofosmina , Pan troglodytes , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Background Hypertrophic cardiomyopathy (HCM) is a serious disorder and one of the leading causes of mortality worldwide. HCM is characterized as left ventricular hypertrophy in the absence of any other loading conditions. In previous studies, mutations in at least 50 genes have been identified in HCM patients. Methods In this research, the genetic lesion of an HCM patient was identified by whole exome sequencing. Real-time polymerase chain reaction (PCR), immunofluorescence and Western blot were used to analyze the effects of the identified mutation. Results According to whole exome sequencing, we identified a de novo mutation (c.814T>C/p.F272L) of SET and MYND domain containing histone methyltransferase 1 (SMYD1) in a Chinese patient with HCM exhibiting syncope. We then generated HIS-SMYD1-pcDNA3.1+ (WT and c.814T>C/p.F272L) plasmids for transfection into AC16 cells to functionalize the mutation. The immunofluorescence experiments indicated that this mutation may block the SMYD1 protein from entering the nucleus. Both Western blot and real-time PCR revealed that, compared with cells transfected with WT plasmids, the expression of HCM-associated genes such as ß-myosin heavy chains, SMYD1 chaperones (HSP90) and downstream targets including TGF-ß were all disrupted in cells transfected with the mutant plasmid. Previous studies have demonstrated that SMYD1 plays a crucial role in sarcomere organization and heart development. Conclusions This novel mutation (c.814T>C/p.F272L) may be the first identified disease-causing mutation of SMYD1 in HCM patients worldwide. Our research expands the spectrum of HCM-causing genes and contributes to genetic counseling for HCM patients.