Linker histone variant H1t is closely associated with repressed repeat-element chromatin domains in pachytene spermatocytes

摘要

Generation and validation of specificity of H1t-specific antibodies. The tripartite structure of linker histones—the linker histones possess three major domains: N-terminal domain, globular domain, and the C-terminal domain. The DNA-binding motifs like SPKK that are present in the somatic linker histone H1.2 have been underlined using blue lines. Since the C-terminal of H1t protein is highly divergent in comparison with other variants, we used 112–207 amino acid residues as protein fragment for the generation of H1t-specific antibodies in rabbits. Western blotting of H1t and H1.2 antibodies against perchloric acid extracts prepared from P20 mouse testicular cells. The anti-H1t and anti-H1.2 antibodies showed reactivity to the specific linker histones, as seen in the blot images on the left, where bands corresponding to molecular weights of H1t and H1.2 were obtained. The blots on the right indicate the immunoblotting performed with the H1t and H1.2 antibodies after preincubation with the recombinant H1t C-terminal protein fragment, before their addition to the blot. Ponceau and Coomassie-stained images are given for reference. Immunoblotting using H1t and H1.2 antibodies against 0.4 N H SO acid extracted histones prepared from P20 mouse testicular cells. The blots on the left show the western blotting data performed using H1t and H1.2 antibodies against the acid extracted histones. The blots on the right indicate the western blotting data performed using H1t and H1.2 after preincubation with the recombinant H1t C-terminal protein fragment. Ponceau and Coomassie-stained images are given for reference