Preparative two-step purification of recombinant H1.0 linker histone and its domains

H1 linker histones are small basic proteins that have a key role in the formation and maintenance of higher-order chromatin structures. Additionally, many examples have shown that linker histones play an important role in gene regulation, modulated by their various sub-types and posttranslational modifications. Obtaining high amounts of very pure linker histones, especially for efficient antibody production, remains a demanding and challenging procedure. Here we present an easy and fast method to purify human linker histone H1.0 overexpressed in Escherichia coli, as well as its domains: N-terminal/globular domain and C-terminal intrinsically disordered domain. This purification protocol relies on a simple affinity chromatography step followed by cation exchange due to the highly basic properties of histone proteins. Therefore, this protocol can also be applied to other linker histones. Highly pure proteins in amounts sufficient for most biochemical experiments can be obtained. The functional quality of purified H1.0 histone and its domains has been confirmed by pull-down, gel-mobility shift assays and the nuclear import assay

List of primers used in this study.

<p>List of primers used in this study.</p

Purification of <i>H</i>. <i>sapiens</i> H1.0 linker histone.

<p>(A) SDS-PAGE analysis of expression and Ni-NTA purification of GST-H1.0-His6 (~55 kDa). (B) SDS-PAGE analysis of GST-H1.0-His6 before and after cleavage treatment with HRV 3C protease. Two prominent lower molecular bands can be observed, GST (~26 kDa) and H1.0-His6 (runs ~32 kDa). (C) Chromatogram and SDS-PAGE gel analysis of cation exchange chromatography. Ft, unbound fraction containing GST; PM, protein marker PageRuler Unstained Protein Ladder; 10–18, protein fractions containing full-length H1.</p

Gel filtration analysis of purified full-length H1.0 linker histone and its domains H1g and H1-C.

<p>Peak fractions were analysed by SDS-PAGE. PM, protein marker PageRuler Unstained Protein Ladder, Thermo Scientific.</p

Domain architecture of recombinant <i>H</i>. <i>sapiens</i> H1.0 linker histone constructs.

<p>Full-length H1.0 was cloned into the pGEX-4T-1 vector with a GST fusion at the N-terminus and 6xHis-tag at the C-terminus. Globular H1.0 (H1g) and C-terminal H1.0 (H1-C) were generated by iPCR from full-length H1.0 with an N-terminal 6xHis-SUMO-HRV 3C site fusion in the pETDuet-1 vector.</p

Purified recombinant H1.0 and its domains are functional.

<p>(A) SDS—PAGE analysis of purified proteins used for <i>in vitro</i> assays. Protein samples were analysed using 17% SDS—PAGE gels. Proteins were visualized by staining with SimplyBlue SafeStain. PageRuler Unstained Protein Ladder, Thermo Scientific, was used as protein marker. (B) Native PAGE of complex formation between histone chaperone Nap1 and H1 histone or its domains. H2AH2B histone dimer was used as a control for Nap1 binding. A histone chaperone:histone complex migrates slower compared to the histone chaperone alone. Several slower migrating bands can be observed in native polyacrylamide gels, indicative of the formation of higher order association states, typical for Nap1 chaperone. (C) Native PAGE of complex formation between DNA and H1 histone or its domains. H2AH2B histone dimer was used as a control for DNA binding. DNA:histone complex migrates slower compared to DNA. The complexes were visualized by SyberGold. (D) GST pull-down assays using GST-H1.0 and NCP. The proteins were visualized by Simply Blue SafeStain.</p

Nuclear import assay of recombinant H1.0 in digitonin-permeabilized HeLa cells reconstituted with the import factors.

<p>H1.0 import is dependent on the presence of both Importin 7 and Importin β. Linker histone import is blocked if WGA is added to the reaction. Histone H1.0 alone or with only one importin were used as a negative control. On the upper panels, cell nuclei stained with DAPI are shown. On the lower panels, cells with NT-495 labelled H1 are shown.</p

Disordered region of H3K9 methyltransferase Clr4 binds the nucleosome and contributes to its activity

Heterochromatin is a distinctive chromatin structure that is essential for chromosome segregation, genome stability and regulation of gene expression. H3K9 methylation (H3K9me), a hallmark of heterochromatin, is deposited by the Su(var)3-9 family of proteins; however, the mechanism by which H3K9 methyltransferases bind and methylate the nucleosome is poorly understood. In this work we determined the interaction of Clr4, the fission yeast H3K9 methyltransferase, with nucleosomes using nuclear magnetic resonance, biochemical and genetic assays. Our study shows that the Clr4 chromodomain binds the H3K9me3 tail and that both, the chromodomain and the disordered region connecting the chromodomain and the SET domain, bind the nucleosome core. We show that interaction of the disordered region with the nucleosome core is independent of H3K9me and contributes to H3K9me in vitro and in vivo. Moreover, we show that those interactions with the nucleosome core are contributing to de novo deposition of H3K9me and to establishment of heterochromatin.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Proposal for Guidelines for the Treatment of Vitiligo in Croatia

Vitiligo, an acquired depigmentation disorder, because of its impressive clinical presentation has a large impact on psychosocial life of patients. Although the exact etiopathogenesis still remains uncertain, several therapeutic options are available for treatment of this condition. Unfortunately, patients are often confronted with difficulties regarding to receiving suitable therapy. Because of the fact that vitiligo is not contagious and not life-threatening disease, physicians usually do not recognize patients’ problems and consider vitiligo as only a cosmetic problem, which should be treated only by camouflage and sun protection products. On the other hand, because of the lack of the accurate information for patients, a widely open market for different kind of alternative questionable therapies occurs so patients are often experimenting with different types of unproven medications. The need for widely accepted consensus concerning vitiligo treatment and establishment of the therapeutic guidelines exists worldwide. Our aim was to introduce for the first time vitiligo therapy guidelines in Republic of Croatia, based on the evidence-based accepted vitiligo therapy world recommendations and our experience. We present a review of therapy for vitiligo regarding to various vitiligo types and severity of lesions as well adequate therapeutic options. Also, our intention is to improve social component of patient’s life through rising awareness of this condition which affects over 35 million people worldwide

Proposal for Guidelines for the Treatment of Vitiligo in Croatia

Abstract: Vitiligo, an acquired depigmentation disorder, because of its impressive clinical presentation has a large impact on psychosocial life of patients. Although the exact etiopathogenesis still remains uncertain, several therapeutic options are available for treatment of this condition. Unfortunately, patients are often confronted with difficulties regarding to receiving suitable therapy. Because of the fact that vitiligo is not contagious and not life-threatening disease, physicians usually do not recognize patients’ problems and consider vitiligo as only a cosmetic problem, which should be treated only by camouflage and sun protection products. On the other hand, because of the lack of the accurate information for patients, a widely open market for different kind of alternative questionable therapies occurs so patients are often experimenting with different types of unproven medications. The need for widely accepted consensus concerning vitiligo treatment and establishment of the therapeutic guidelines exists worldwide. Our aim was to introduce for the first time vitiligo therapy guidelines in Republic of Croatia, based on the evidence-based accepted vitiligo therapy world recommendations and our experience. We present a review of therapy for vitiligo regarding to various vitiligo types and severity of lesions as well adequate therapeutic options. Also, our intention is to improve social component of patient’s life through rising awareness of this condition which affects over 35 million people worldwide