Role of mammalian H1 linker histones studied by gene inactivation

Abstract

Mice contain seven H1 subtypes (H1a--e, H1t and H10) that differ in amino acid sequence and expression during development. Previous studies showed that mice develop normally when any one of several of these H1 genes is inactivated homozygously. In each of the single H1 null mice, other H1s compensate for the lost subtype, resulting in a normal stoichiometry of total H1 to nucleosomes. All the H1 genes, except H10, are tightly linked and interspersed with other genes. To further investigate the in vivo role of H1 histones in chromatin structure, gene expression and mouse development, I have generated a series of compound H1 knock-out (KO) mice by sequential targeting of three H1 genes in ES cells. Mice lacking H1c, H1d and H1e, generated by three rounds of gene inactivation in ES cells, die at embryonic day E9.5--E10.5, demonstrating that H1 histones are essential for mouse development. H1c/H1e double KO mice are viable and fertile, although they display subtle growth retardation and slightly shorter life span. H1d KO mice develop normally. H1c/H1d/H1e (-,+/-,-/-,-,-) mice (5(-) mice) have a very low birth rate and they are severely growth retarded and exhibited reduced fertility. H10/H1c/H1e triple KO mice were also generated. They exhibit lower birth rate and mild growth retardation. Examination of the H1/nucleosome ratio by HPLC analysis of chromatin proteins showed a 47% reduction in H1c/H1d/H1e triple KO embryos, a 40% reduction in several tissues of 5(-) mice, a 20--40% reduction in H1 0/H1c/H1e triple KO mice, and a normal total H1 content in H1c/H1e double KO mice. The fact that any single H1 KO strain and several double KO mice develop normally indicates that it is not any individual H1, but rather the total amount of H1, i.e. the H1/nucleosome ratio, which is important for proper development. These experiments also show that a marked reduction in H1 content in some tissues is compatible with postnatal life.;Chromatin analyses in tissues from compound H1 KO animals with a substantial reduction in total H1 content shows a decrease in global nucleosome spacing. The extent of the decrease in nucleosome spacing correlates with the extent of the decrease in the H1/nucleosome ratio. These results provide the first in vivo evidence that linker histones affect nucleosome packaging.;To investigate the in vivo role of the differentiation specific H1 0 linker histone in gene expression, a subtractive cloning strategy was used and three genes that are activated and one gene that is repressed in H10 single KO mice were identified. The results indicate that H10 does indeed regulate specific gene expression.