RESUMEN
AIMS: Here we report that chromatin, the complex and dynamic eukaryotic DNA packaging structure, is able to sense cellular redox changes. Histone H3, the only nucleosomal protein that possesses cysteine(s), can be modified by glutathione (GSH). RESULTS: Using Biotin labeled glutathione ethyl ester (BioGEE) treatment of nucleosomes in vitro, we show that GSH, the most abundant antioxidant in mammals, binds to histone H3. BioGEE treatment of NIH3T3 cells indicates that glutathionylation of H3 is maximal in fast proliferating cells, correlating well with enhanced levels of H3 glutathionylation in different tumor cell lines. Furthermore, glutathionylation of H3 in vivo decreases in livers from aged SAMP8 and C57BL/6J mice. We demonstrate biochemically and by mass spectrometry that histone variants H3.2/H3.3 are glutathionylated on their cysteine residue 110. Furthermore, circular dichroism, thermal denaturation of reconstituted nucleosomes, and molecular modeling indicate that glutathionylation of histone H3 produces structural changes affecting nucleosomal stability. INNOVATION: We characterize the implications of histone H3 glutathionylation in cell physiology and the modulation of core histone proteins structure affected by this modification. CONCLUSION: Histone H3 senses cellular redox changes through glutathionylation of Cys, which increases during cell proliferation and decreases during aging. Glutathionylation of histone H3 affects nucleosome stability structure leading to a more open chromatin structure.
Asunto(s)
Histonas/metabolismo , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional , S-Nitrosoglutatión/química , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular Tumoral , Proliferación Celular , Cisteína/química , Femenino , Histonas/química , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Células 3T3 NIH , Nucleosomas/química , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , S-Nitrosoglutatión/metabolismoRESUMEN
The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.
Asunto(s)
Empalme Alternativo , Histonas/genética , Histonas/fisiología , Nucleosomas/metabolismo , Adenosina Trifosfatasas/metabolismo , Línea Celular , Cromatina/metabolismo , ADN/metabolismo , Histona Acetiltransferasas/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Lisina Acetiltransferasa 5 , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de ProteínaRESUMEN
Nucleosomal incorporation of specialized histone variants is an important mechanism to generate different functional chromatin states. Here, we describe the identification and characterization of two novel primate-specific histone H3 variants, H3.X and H3.Y. Their messenger RNAs are found in certain human cell lines, in addition to several normal and malignant human tissues. In keeping with their primate specificity, H3.X and H3.Y are detected in different brain regions. Transgenic H3.X and H3.Y proteins are stably incorporated into chromatin in a similar fashion to the known H3 variants. Importantly, we demonstrate biochemically and by mass spectrometry that endogenous H3.Y protein exists in vivo, and that stress stimuli, such as starvation and cellular density, increase the abundance of H3.Y-expressing cells. Global transcriptome analysis revealed that knockdown of H3.Y affects cell growth and leads to changes in the expression of many genes involved in cell cycle control. Thus, H3.Y is a novel histone variant involved in the regulation of cellular responses to outside stimuli.
