Preparative two-step purification of recombinant H1.0 linker histone and its domains
نویسندگان
چکیده
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 subtypes 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.
منابع مشابه
Linker histone H1.0 interacts with an extensive network of proteins found in the nucleolus
The H1 linker histones are abundant chromatin-associated DNA-binding proteins. Recent evidence suggests that linker histones also may function through protein-protein interactions. To gain a better understanding of the scope of linker histone involvement in protein-protein interactions, we used a proteomics approach to identify H1-binding proteins in human nuclear extracts. Full-length H1.0 and...
متن کاملThe linker histone H1.0 generates epigenetic and functional intratumor heterogeneity.
Tumors comprise functionally diverse subpopulations of cells with distinct proliferative potential. Here, we show that dynamic epigenetic states defined by the linker histone H1.0 determine which cells within a tumor can sustain the long-term cancer growth. Numerous cancer types exhibit high inter- and intratumor heterogeneity of H1.0, with H1.0 levels correlating with tumor differentiation sta...
متن کاملHistone H1 Variants Are Differentially Expressed and Incorporated into Chromatin during Differentiation and Reprogramming to Pluripotency Running Title: H1 Variant Content in Pluripotent and Differentiated Cells
There are seven linker histone variants in human somatic cells (H1.0 to H1.5, and H1X), their prevalence varying as a function of cell type and differentiation stage, suggesting that the different variants may have distinct roles. We have revisited this notion by using new methodologies to study pluripotency and differentiation, including the in vitro differentiation of human embryonic stem (ES...
متن کاملMapping of six somatic linker histone H1 variants in human breast cancer cells uncovers specific features of H1.2
Seven linker histone H1 variants are present in human somatic cells with distinct prevalence across cell types. Despite being key structural components of chromatin, it is not known whether the different variants have specific roles in the regulation of nuclear processes or are differentially distributed throughout the genome. Using variant-specific antibodies to H1 and hemagglutinin (HA)-tagge...
متن کاملHistone H1 variants are differentially expressed and incorporated into chromatin during differentiation and reprogramming to pluripotency.
There are seven linker histone variants in human somatic cells (H1.0 to H1.5 and H1X), and their prevalence varies as a function of cell type and differentiation stage, suggesting that the different variants may have distinct roles. We have revisited this notion by using new methodologies to study pluripotency and differentiation, including the in vitro differentiation of human embryonic stem (...
متن کامل