Birgit Drabent

Histones: genetic diversity and tissue-specific gene expression.

Abstract Histones are the major protein constituents of the chromatin of eukaryotic cell nuclei. This group of basic proteins is extremely conserved throughout evolution and includes five classes termed H1, H2A, H2B, H3and H4. In mammals, each of these classes except H4 is subdivided into several subtypes. The most divergent class of histones is the H1 protein family, which consists of seven different subtypes, termed H1.1–H1.5, H1°, and H1t. The subtypes H1.2 and H1.4 are found in most somatic cell nuclei, whereas H1° is found in several differentiated tissues, and H1t is restricted to mammalian testicular cells. Similarly, core histone subtypes replacing the major forms of H2A, H2B or H3 have been described. Biochemical analysis of protein and RNA from different tissues and cell lines demonstrates varied patterns of expression of individual histone subtype genes. Moreover, antibodies against specific histone subtypes and in situ hybridization with subtype-specific probes indicate that the expression of histone subtype genes is in several cases modulated in a tissue-specific manner. This is particularly evident at the different stages of spermatogenesis when chromatin undergoes substantial reorganization, which finally results in the highly condensed state of chromatin of the mature sperm head.

Spermatogenesis proceeds normally in mice without linker histone H1t.

Abstract. The histone gene H1t is expressed exclusively in pachytene spermatocytes of the testis. In this report we have eliminated the single copy H1t gene by homologous recombination from the mouse genome to analyse the function of the H1t protein during spermatogenesis. Mice homozygous for the mutated H1t gene locus developed normally and showed no anatomic abnormalities until the adult stage. In addition, H1t-deficient mice were fertile and reproduced as wild-type mice. The process of spermatogenesis and the testicular morphology remained unchanged in the absence of H1t. RNase protection analysis demonstrated that H1.1, H1.2 and H1.4 histone gene expression is enhanced during spermatogenesis in H1t-deficient mice.

EXPRESSION OF THE MOUSE TESTICULAR HISTONE GENE H1T DURING SPERMATOGENESIS

The testicular H1 histone variant, H1t, is synthesized during spermatogenesis in mammalian male germ cells. In situ hybridization and immunohistochemical techniques were used to assign the expression of either the H1t mRNA or the H1t protein to specific cell stages of spermatogenesis. Our results show the presence of the H1t mRNA only in the late and mid-pachytene stages, whereas the protein occurs first in pachytene spermatocytes, and persists until later stages from round up to elongated spermatids.

Isolation and characterization of two human H1 histone genes within clusters of core histone genes

Two human H1 histone genes, termed H1.3 and H1.4, were isolated from two cosmid clones. The H1.4 gene is associated with an H2B gene, whereas genes coding for all four core histones are located in the vicinity of the H1.3 gene. This cluster arrangement was found both in the two cosmid clones and on overlapping bacteriophage clones isolated from an EMBL3 library. In continuation of our previous analysis of two human H1 genes, this analysis raises the number of completely sequenced H1 histone genes within clusters of core histone genes to four.

Structure and expression of the mouse testicular H1 histone gene (H1t).

A mouse genomic library was screened with a human testicular H1 (H1t) gene fragment. One phage containing the testis specific mouse H1t histone gene and its flanking regions was isolated. Northern blot analysis showed that the mouse H1t gene is expressed only in mouse testis at the stage of pachytene spermatocytes and that the H1t mRNA is not polyadenylated. This mouse H1t gene encodes a protein which differs from the somatic mouse H1 proteins, but is similar to the known H1t proteins from rat, and man.

Isolation of two murine H1 histone genes and chromosomal mapping of the H1 gene complement

The mammalian H1 histone gene complement consists of at least seven H1 protein isoforms. These include five S-phase-dependent H1 protein subtypes and two more distantly related proteins, which are expressed upon terminal differentiation (H10) or during the pachytene stage of spermatogenesis (H1t). In the past, three replication-dependent murine H1 genes plus the H10 and H1t genes have been isolated and characterized. In this report, we describe the sequences of two more H1 genes, and we show that all five murine replication-dependent H1 genes and the H1t gene map to the region A2-3 on Chromosome (Chr) 13. This is in agreement with our previous finding that the human H1 histone gene complement maps to 6p21.3, which corresponds to the A2-3 region on the murine Chr 13. Previous reports have shown that the replication-independent H10 genes map to syntenic regions on Chrs 22 (human H10) and 15 (murine H10).

Expression of the mouse histone gene H1t begins at premeiotic stages of spermatogenesis

Abstract The gene encoding H1t, a testicular variant of histone H1, is expressed in mammals during spermatogenesis. Northern blot and in situ hybridization has detected H1t mRNA only at the stage of pachytene spermatocytes. We have extended this analysis to more sensitive approaches and demonstrate, by RNase protection and electron-microscopic in situ hybridization, that H1t mRNA is detectable even in spermatogonia. Just a faint H1t band is seen in Western blots of nuclear protein from 9-day-old mice. This indicates that the H1t gene is expressed at premeiotic stages, albeit at a low level. In contrast to H1t mRNA, the H1t protein has not been detected in spermatogonia by electron microscopy after immunogold staining.

Expression of murine H1 histone genes during postnatal development

Murine genes encoding the seven H1 histone isoforms H1.1-H1.5, H1(o) and H1t have been isolated and sequenced. We have established expression patterns of these genes in several tissues during postnatal development. For that analysis, RNase protection assay rather than Northern blot hybridization was used, since the sequences of these genes are highly similar and would cross-hybridize under Northern blot conditions. Expression patterns of H1.1 to H1.5 and H1(o) were determined in tissues of animals at days 5, 9 and 20 after birth and of adult mice. In addition, RNA was analyzed in three mouse cell lines (NIH3T3, P19, TM4). Transcription of the subtype genes H1.2 and H1.4 was found in all tissues and cell lines studied. The most varied expression patterns were obtained with the H1.1 subtype. H1.1 mRNA was found at high concentrations in thymus and spleen throughout development and in testis beginning with a low expression in 5-day-old animals and increasing levels in testis RNA from 9- and 20-day-old and adult mice. H1(o) mRNA was found primarily in highly differentiated tissues with concentrations decreasing from 5-day-old to adult animals.

Male Mice Lacking Three Germ Cell Expressed Genes Are Fertile

Abstract 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.

Mytilus edulis Core Histone Genes Are Organized in Two Clusters Devoid of Linker Histone Genes

Comparison of histone gene cluster arrangements in several species has revealed a broad spectrum of histone gene patterns. To elucidate the core histone gene organization in a mollusk, we have analyzed a Mytilus edulis genomic library and have isolated eight phage clones containing core histone genes. Analysis of insert DNA revealed that the core histone genes are arranged as regular gene repeats of all four core histones. The repeats do not contain linker histone genes. The clones are distributed into two groups of dissimilar repeated units with a common size of about 5.6 kb. The genes of each core histone class in the distinct repeats encode identical histone proteins and have comparable gene arrangements in the two repeat units. However, the intergenic sequences differ significantly. The core histone genes are organized as large clusters of about 100 repeats each. Previously, we have shown that the linker histone genes in M. edulis are also organized in a cluster of repeats of solitary H1 genes. Hence, this is the first case of a separate, clustered organization of both core and linker histone genes, repectively.