Knockdown of Histone H1.5 leads to a loss in centromeric transcription
Mammalian linker histone proteins (Histone H1) comprise a group of 11 non-allelic variants which have key roles in modulating chromatin organization. Subtype specific genomic distribution can contribute to a fine- tuned regulation of gene expression and chromatin architecture. In this study, we report that the variant histone H1.5 localizes to human centromeres and has a functional role in regulation of centromeric noncoding transcription. We used immunofluorescence to visualize the localization of the H1.5 to the centromere in astrocytes and performed chromatin immunoprecipitation which revealed an interaction of H1.5 with centromeric-protein A (CENP-A) nucleosomes in vivo in human cells. In vitro reconstitution of chromatin demonstrates that H1.5 can robustly bind to both CENP-A mono-nucleosomes and arrays of nucleosomes. Functionally, depletion of H1.5 from cells results in the loss of centromeric satellite transcription and leads to accumulation of mitotic defects. These data point to a hitherto unreported role for H1.5 in regulation of mitotic integrity in human cells. Overall design: SVGp12 cells were subjected to histone H1 subtype knockdown using siH1.5/siH1.0/siScramble smartpool  oligos. After 72hours , cells were harvested for total RNA- extraction.