A genetic approach to the functions of specific H1 linker histones in mammalian spermatogenesis

Abstract

Histones are small, basic proteins found in abundance in most eukaryotic cells. The H1 histones bind the linker DNA between nucleosome core particles and facilitate the packaging of chromatin into the 30-nm fiber and higher order structures.;To investigate the role of the testis-specific linker Histone H1t in spermatogenesis, the H1t gene was disrupted by homologous recombination in mouse embryonic stem (ES) cells that were then used to generate mice that transmitted the disrupted H1t allele. Analysis of H1s in testis extracts from H1t(-/-) male mice showed that they were completely lacking H1t. These mice are fertile and produce normal numbers of mature sperm. The results indicate that despite the unique properties and expression pattern of H1t it is not essential for spermatogenesis.;Analysis of male germ cells from H1t -/- mice showed that the somatic H1a subtype predominated in chromatin. To determine the effect of creating a larger linker histone deficiency in chromatin than that established by the disruption of H1t alone, we disrupted the H1a gene in ES cells also containing a disrupted H1t allele. Targeting of the H1a gene in ES cells occurred both in cis and in trans to the modified H1t allele. Both strains of mutant mice are viable, appear normal, and have normal body weights and testis weights. Both strains also are fertile and have normal numbers of mature sperm.;HPLC analyses showed that the H1 to nucleosome ratio is the same in H1a(-/-) as in wildtype littermates. HPLC data also showed that the H1d and H1e subtypes preferentially substitute for the loss of H1a. The percentage of H1t in total H1s was not altered. This result indicates that H1t is able to replace H1c, H1d, and H1e, as effectively as it normally replaces H1a, Thus H1a, is not required for the H1stone protein transition from somatic H1s to H1t.;Analyses of germ cell chromatin in the H1at double mutants showed that the H1 to nucleosome ratio decreased by 25%, indicating that the mice cannot fully compensate for loss of 70% of their H1. The observed 25% decrease in the H1 to nucleosome ratio in the total germ cells of the double mutant implies that pachytene spermatocytes in the double mutant must have an H1 to nucleosome ratio 50% that of normal. Nevertheless spermatogenesis is normal in these mice. These results indicate that both H1a and H1t are not essential for normal spermatogenesis, suggesting that H1s may be partially functionally redundant, at least in spermatogenesis. (Abstract shortened by UMI.).