Normal spermatogenesis in mice lacking the testis-specific linker histone H1t

H1 histones bind to linker DNA and nucleosome core particles and facilitate the folding of chromatin into a more compact structure. Mammals contain seven nonallelic subtypes of H1, including testis-specific subtype H1t, which varies considerably in primary sequence from the other H1 subtypes. H1t is found only in pachytene spermatocytes and early, haploid spermatids, constituting as much as 55% of the linker histone associated with chromatin in these cell types. To investigate the role of H1t in spermatogenesis, we disrupted the H1t gene by homologous recombination in mouse embryonic stem cells. Mice homozygous for the mutation and completely lacking H1t protein in their germ cells were fertile and showed no detectable defect in spermatogenesis. Chromatin from H1t-deficient germ cells had a normal ratio of H1 to nucleosomes, indicating that other H1 subtypes are deposited in chromatin in place of H1t and presumably compensate for most or all H1t functions. The results indicate that despite the unique primary structure and regulated synthesis of H1t, it is not essential for proper development of mature, functional sperm.     

质谱法分析大鼠精子发生过程中组蛋白H1变异体的多样性

目的为探究连接组蛋白H1在精子发生过程染色体重构中的功能,了解一共有多少种连接组蛋白H1参与各期生精细胞的染色体的构建。方法分离高纯度的SD大鼠的各期生精细胞,提取组蛋白,应用SDS-PAGE分离组蛋白的各组分,组蛋白（H1）经过蛋白酶（Glu-c和Arg-c）酶切,应用质谱进行检测。结果鉴定了组蛋白H1的体细胞亚型（H1.1-H1.5）和睾丸特异的连接组蛋白亚型（H1t）。组蛋白H1t分别表达在精原细胞,精母细胞和圆形精子细胞中。结论大鼠精子发生过程中,其主要连接组蛋白H1的种类是：H1.1-H1.5和H1t。     

Poly(ADP-ribose) metabolism is essential for proper nucleoprotein exchange during mouse spermiogenesis

Sperm chromatin is organized in a protamine-based, highly condensed form, which protects the paternal chromosome complement in transit, facilitates fertilization, and supports correct gene expression in the early embryo. Very few histones remain selectively associated with genes and defined regulatory sequences essential to embryonic development, while most of the genome becomes bound to protamine during spermiogenesis. Chromatin remodeling processes resulting in the dramatically different nuclear structure of sperm are poorly understood. This study shows that perturbation of poly(ADP-ribose) (PAR) metabolism, which is mediated by PAR polymerases and PAR glycohydrolase in response to naturally occurring endogenous DNA strand breaks during spermatogenesis, results in the abnormal retention of core histones and histone linker HIST1H1T (H1t) and H1-like linker protein HILS1 in mature sperm. Moreover, genetic or pharmacological alteration of PAR metabolism caused poor sperm chromatin quality and an abnormal nuclear structure in mice, thus reducing male fertility.     

Histone H1 depletion in mammals alters global chromatin structure but causes specific changes in gene regulation

Linker histone H1 plays an important role in chromatin folding in vitro. To study the role of H1 in vivo, mouse embryonic stem cells null for three H1 genes were derived and were found to have 50% of the normal level of H1. H1 depletion caused dramatic chromatin structure changes, including decreased global nucleosome spacing, reduced local chromatin compaction, and decreases in certain core histone modifications. Surprisingly, however, microarray analysis revealed that expression of only a small number of genes is affected. Many of the affected genes are imprinted or are on the X chromosome and are therefore normally regulated by DNA methylation. Although global DNA methylation is not changed, methylation of specific CpGs within the regulatory regions of some of the H1 regulated genes is reduced. These results indicate that linker histones can participate in epigenetic regulation of gene expression by contributing to the maintenance or establishment of specific DNA methylation patterns.     

Mouse Dnmt3a preferentially methylates linker DNA and is inhibited by histone H1

In mammals, DNA methylation is crucial for embryonic development and germ cell differentiation. The DNA methylation patterns are created by de novo-type DNA methyltransferases (Dnmts) 3a and 3b. Dnmt3a is crucial for global methylation, including that of imprinted genes in germ cells. In eukaryotic nuclei, genomic DNA is packaged into multinucleosomes with linker histone H1, which binds to core nucleosomes, simultaneously making contacts in the linker DNA that separates adjacent nucleosomes. In the present study, we prepared oligonucleosomes from HeLa nuclei with or without linker histone H1 and used them as a substrate for Dnmt3a. Removal of histone H1 enhanced the DNA methylation activity. Furthermore, Dnmt3a preferentially methylated the linker between the two nucleosome core regions of reconstituted dinucleosomes, and the binding of histone H1 inhibited the DNA methylation activity of Dnmt3a towards the linker DNA. Since an identical amount of histone H1 did not inhibit the activity towards naked DNA, the inhibitory effect of histone H1 was not on the Dnmt3a catalytic activity but on its preferential location in the linker DNA of the dinucleosomes. The central globular domain and C-terminal tail of the histone H1 molecule were indispensable for inhibition of the DNA methylation activity of Dnmt3a. We propose that the binding and release of histone H1 from the linker portion of chromatin may regulate the local DNA methylation of the genome by Dnmt3a, which is expressed ubiquitously in somatic cells in vivo.     

Histone H1 Subtypes Differentially Modulate Chromatin Condensation without Preventing ATP-Dependent Remodeling by SWI/SNF or NURF

Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.     

Binding of linker histones to the core nucleosome

Binding of chicken erythrocyte linker histones H1/H5 to the core nucleosome has been studied. Histones H1/H5 bind very efficiently to the isolated core nucleosome in vitro. The binding of linker histones to the core nucleosome is associated with aggregation of the particles. Approximately one molecule of linker histone binds per core nucleosome in the aggregates, irrespective of the concentration of the linker histones and the salt used. Histone H5 shows greater binding affinity to the core nucleosome as compared to H1. The carboxyl-terminal fragment of the linker histones binds strongly to the core nucleosome while the binding of the central globular domain is weak. Each core nucleosome is capable of binding two molecules of carboxyl-terminal fragment of linker histone. The core nucleosome containing one molecule of carboxyl-terminal fragment of linker histone requires higher salt concentration for aggregation while the core nucleosome containing two molecules of carboxyl-terminal fragment of linker histone can self-associate even at lower salt concentrations. On the basis of these results we are proposing a novel mechanism for the condensation of chromatin by linker histones and other related phenomena.     

Male mice lacking three germ cell expressed genes are fertile

In recent years, much knowledge about the functions of defined genes in spermatogenesis has been gained by making use of mouse transgenic and gene knockout models. Single null mutations in mouse genes encoding four male germ cell proteins, transition protein 2 (Tnp-2), proacrosin (Acr), histone H1.1 (H1.1), and histone H1t (H1t), have been generated and analyzed. Tnp-2 is believed to participate in the removal of the nuclear histones and initial condensation of the spermatid nucleus. Proacrosin is an acrosomal protease synthesized as a proenzyme and activated into acrosin during the acrosome reaction. The linker histone subtype H1.1 belongs to the group of main-type histones and is synthesized in somatic tissues and germ cells during the S-phase of the cell cycle. The histone gene H1t is expressed exclusively in spermatocytes and may have a function in establishing an open chromatin structure for the replacement of histones by transition proteins and protamines. Male mutant mice lacking any of these proteins show no apparent defects in spermatogenesis or fertility. To examine the synergistic effects of these proteins in spermatogenesis and during fertilization, two lines of triple null mice (Tnp-2-/-/Acr-/-/H1.1-/- and Tnp-2-/-/Acr-/-/H1t-/-) were established. Both lines are fertile and show normal sperm parameters, which clearly demonstrate the functional redundancy of these proteins in male mouse fertility. However, sperm only deficient for Acr (Acr-/-) are able to compete significantly with sperm from triple knockout mice Tnp-2-/-/Acr-/-/H1.1-/- (70.7% vs. 29.3%) but not with sperm from triple knockout mice Tnp-2-/-/Acr-/-/H1t-/- (53.6% vs. 46.4%). These results are consistent with a model that suggests that some sperm proteins play a role during sperm competition.     

Novel nucleosomal particles containing core histones and linker DNA but no histone H1

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these ‘proto-chromatosomes’ are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.     

Mouse oocytes and early embryos express multiple histone H1 subtypes

Oocytes and embryos of many species, including mammals, contain a unique linker (H1) histone, termed H1oo in mammals. It is uncertain, however, whether other H1 histones also contribute to the linker histone complement of these cells. Using immunofluorescence and radiolabeling, we have examined whether histone H10, which frequently accumulates in the chromatin of nondividing cells, and the somatic subtypes of H1 are present in mouse oocytes and early embryos. We report that oocytes and embryos contain mRNA encoding H10. A polymerase chain reaction-based test indicated that the poly(A) tail did not lengthen during meiotic maturation, although it did so beginning at the four-cell stage. Antibodies raised against histone H10 stained the nucleus of wild-type prophase-arrested oocytes but not of mice lacking the H10 gene. Following fertilization, H10 was detected in the nuclei of two-cell embryos and less strongly at the four-cell stage. No signal was detected in H10 -/- embryos. Radiolabeling revealed that species comigrating with the somatic H1 subtypes H1a and H1c were synthesized in maturing oocytes and in one- and two-cell embryos. Beginning at the four-cell stage in both wild-type and H10 -/- embryos, species comigrating with subtypes H1b, H1d, and H1e were additionally synthesized. These results establish that histone H10 constitutes a portion of the linker histone complement in oocytes and early embryos and that changes in the pattern of somatic H1 synthesis occur during early embryonic development. Taken together with previous results, these findings suggest that multiple H1 subtypes are present on oocyte chromatin and that following fertilization changes in the histone H1 complement accompany the establishment of regulated embryonic gene expression.     

Histone H1t is not replaced by H1.1 or H1.2 in pachytene spermatocytes or spermatids of H1t-deficient mice

The linker histone gene H1t is exclusively expressed in the mammalian testis. In former experiments we have shown that H1.1 and H1.2 histone gene expression is significantly enhanced in testis of adult H1t deficient mice. In this report we have quantified the mRNA of different H1 genes in 9-day- and 20-day-old wild type and H1t knock out mice. In addition, we have analysed the distribution of H1.1 and H1.2 protein by immunofluorescent staining in spread male germ cells. The aim of this work was to answer the question whether H1t can be replaced during spermatogenesis by H1.1 or H1.2. In our experiments we could not detect elevated levels of H1.1 or H1.2 in pachytene spermatocytes or haploid cells of H1t deficient testis. Therefore, in these cells, H1t seems not to be replaced by H1.1 or H1.2.