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D Doenecke

Publications and source records attributed to D Doenecke.

At least 37 records · Page 2Linked to original sources
PubMed1998

cAMP/phorbol ester response element is involved in transcriptional regulation of the human replacement histone gene H3.3B.

The human histone H3.3B gene belongs to the group of replacement histone genes, which are up-regulated during differentiation of cells. Here we provide evidence that a cAMP response element/PMA response element (CRE/TRE) located in the proximal promoter contributes to the expression of the H3.3B gene. (1) Band shift and supershift analysis demonstrated the binding of AP-1 and transcription factors of the CRE-binding protein/activating-transcription-factor family to the H3.3B CRE/TRE. (2) Treatment of HeLa cells with PMA led to a 4-fold increase in H3. 3B mRNA levels within 2 h, whereas transcription of the cell cycle-dependent H3 histone genes remained constant. In contrast with PMA, cAMP did not affect H3.3B transcription. (3) PMA treatment of cells transiently transfected with H3.3B promoter constructs linked to a luciferase gene caused a 4-5-fold increase in reporter gene activity, whereas mutation of the CRE/TRE element abolished the PMA response. These results demonstrate that activation of the protein kinase C pathway by PMA results in an early up-regulation of H3.3B gene expression via the CRE/TRE element. Furthermore treatment with PMA apparently leads to differential induction of H3 histone subtype genes and this in turn can result in a remodelling of chromatin structure of cells before or during differentiation processes.

Activating Transcription Factors↗
PubMed1998

Testicular expression of the mouse histone H1.1 gene.

The replication-dependent H1 histone subtype H1.1 is the predominant H1 histone subtype in germ cell chromatin during early spermatogenesis. H1.1 gene transcription was assigned by in situ hybridization with H1.1-specific RNA probes to the basal layer of germ cells within the seminiferous tubule, but not to the somatic cells of the testis. In situ hybridization and immunohistochemistry with testis from 5-, 9-, and 20-day-old mice showed that H1.1 mRNA and protein was already present in spermatogonia of the prepuberal testis. In testis from 20-day-old and adult mice, the H1.1 mRNA remained confined to the most peripheral layer of germ cells. In contrast to the H1.1 mRNA, the H1.1 protein persisted in the germ cell chromatin with decreasing concentration throughout meiosis and in postmeiotic cells. These data demonstrate that in situ hybridization with H1.1 mRNA-specific probes can serve to identify germ cells with ongoing DNA replication. Comparison of results obtained from in situ hybridization and immunocytochemistry indicates that the period of H1.1 gene transcription is restricted to the proliferative phase in which cells undergo successive divisions, whereas the H1.1 protein is a component of the proliferative, meiotic, and spermiogenic phases.

Animals↗
PubMed1998

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

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.

Animals↗
PubMed1998

Expression of mRNA and protein of nucleoproteins during human spermiogenesis.

The most important event determining the nuclear status of sperm cells is the replacement of histones by protamines, which are the basic nuclear proteins of mature spermatozoa. A first step in this exchange is the displacement of histones by transition proteins (TP). Our study demonstrates, for the first time, the sequential expression of the testis-specific histone (H1t) and the transition proteins (TP1 and TP2) during normal human spermatogenesis. H1t mRNA could only be detected in the cytoplasm of mid and late pachytene spermatocytes. Concomitant with the onset of H1t transcription, the H1t protein appeared in the nuclei of pachytene spermatocytes and remain as a nuclear protein constituent up to step 5 spermatids. While transition protein 1 gene TNP-1 mRNA was present in spermatids from step 2 to early step 4, the TP1 protein occured, with temporal delay, in the nuclei of step 3 and step 4 spermatids. The TP2 protein was observed in the nuclei of spermatids from step 1 to step 5. The transition protein 2 gene TNP-2 mRNA was only detected by reverse transcription-polymerase chain reaction, but not on paraffin sections. These data demonstrate a strong temporal association between H1t gene transcription and synthesis of the H1t protein. Since the TP1 protein appeared with temporal delay we can assume that the corresponding TNP-1 mRNA is translationally delayed.

Blotting, Western↗
PubMed1997

Transcriptional regulation of the human replacement histone gene H3.3B.

In contrast to the cell-cycle-dependent histone genes, replacement histone genes are transcribed independently of DNA replication and their expression is upregulated during differentiation. We have investigated the transcriptional regulation of the recently characterized human replacement histone gene H3.3B. Using reporter gene assays of promoter-luciferase gene-constructs, we show that promoter activity largely depends on an intact Oct and CRE/TRE element within the proximal 145 bp of the promoter. DNase I footprinting revealed binding of proteins to a 40-bp region covering these two elements. Band shift experiments identified binding proteins as Oct-1 and factors of the CREB/ATF and AP-1 family, respectively. The unexpected transcriptional regulation of this replacement histone gene is discussed.

Base Sequence↗
PubMed1997

Characterization of the two H1(zero)-encoding genes from Xenopus laevis.

We have analyzed the promoter and the coding sequences of the two homologous histone H1(zero)-encoding genes from Xenopus laevis, here termed H1(zero)-1 and H1(zero)-2. Both genes encode proteins of 193 amino acids and differ at just 16 amino-acid residues. Putative regulatory sequences identified in the promoter region are the same and are highly conserved. However, significant differences exist in the 5' untranslated regions (UTR) of the transcribed sequences of these two genes, such as several deletions in the 5'-UTR of the H1(zero)-2 gene in comparison with the H1(zero)-1 gene 5'-UTR. The 3'-UTR is a short sequence of about 200 bp which is unexpected compared with the long 3'-UTR of mammalian H1(zero) mRNA, but it is in the same size range as in avian H5 mRNA. Thus, the main differences between these two genes are observed in sequences potentially involved in the regulation of the H1(zero) gene expression such as the 5'-UTR. The two genes are expressed during embryogenesis and in several adult tissues. We discuss these findings in terms of the evolution of histone H1(zero) genes in vertebrates and the appearance of histone H5 in avian species.

Amino Acid Sequence↗
PubMed1997

Transgenic mice transcribing the human H1 zero histone gene exhibit a normal phenotype.

The linker histone H1degree accumulates in terminally differentiating cells and replaces other members of the H1 histone family, even in the absence of cell division. To study the role of H1degree in vivo, we have created two lines of transgenic mice with either the human H1degree promoter (HH minigene) or the mouse metallothionein T promoter (MH minigene) upstream of the human H1degree gene. Mice bearing the minigenes HH or MH overexpress human H1degree mRNA at 10-20-fold higher levels than in normal mice in a constitutive or metal-inducible manner. In contrast to this increase in mRNA content, which was studied in liver, kidney and brain, no significant changes in the relative proportions of the H1 protein subtypes, including H1degree were observed. Transgenic mice exhibited normal anatomic phenotypes, growth rates and reproduction rates. Thus, our results suggest a posttranscriptional and/or translational mechanism that compensates the unbalanced linker-histone expression in different tissues.

Animals↗
PubMed1997

Nuclear transport of H1 histones meets the criteria of a nuclear localization signal-mediated process.

We have studied the nuclear transport of H1 histones using the digitonin permeabilization assay system in order to establish the transport requirements for H1 translocation to the nucleus. Using HeLa cells and fluorescence-labeled calf thymus H1, we show that the H1 nuclear transport in permeabilized cells requires the addition of cytoplasmic extract. Furthermore, it can be blocked by energy depletion and by chilling or by addition of wheat germ agglutinin or by nonhydrolyzable GTP analogs. Thus, the import of H1 histones follows the criteria established for nuclear import mediated by nuclear localization signals (NLS). The distribution of basic amino acids in average H1 sequences, however, does not allow the assignment of a specific element as a classical NLS.

Biological Transport↗
PubMed1997

Human histone gene organization: nonregular arrangement within a large cluster.

We have previously located the genes of the five human main type H1 genes and the gene encoding the testicular subtype H1t to the region 21.1 to 22.2 on the short arm of chromosome 6. To investigate the organization of the histone genes in this region, we isolated two YACs from a human YAC library by PCR screening with primers specific for histone H1.1. This screen revealed two YAC clones, YAC Y23 (corresponding to ICRFy901D1223) contains an insert of about 480 kb, whereas the smaller YAC 4A (corresponding to ICRFy900C104) spans about 340 kb and is completely covered by YAC Y23. We have subcloned the YAC inserts in cosmids, determined the linear orientation of the cosmids by cosmid walking, and constructed a restriction map of the entire region by mapping the individual cosmids using partial digests and hybridization with labeled oligonucleotides complementary to the cos site of the vector. Hybridization analysis, subcloning, restriction mapping, and sequencing revealed that most of the previously isolated phage and cosmid clones containing histone genes are part of this YAC including the clones containing the four human main type H1 histone genes H1.1 to H1.4, the H1t gene, and core histone genes. Thirty-five histone genes map within 260 kb of the YAC Y23 insert. All newly identified histone genes were sequenced, and the sequences were deposited with the EMBL nucleotide sequence database. The histone H1.5 gene is not part of this region, and we therefore conclude that the H1.5 gene and the associated core histone genes form a separate subcluster within this chromosomal region.

Chromosome Mapping↗
PubMed1997

Characterization of the H1.5 gene completes the set of human H1 subtype genes.

The H1 histone family in mammals contains at least seven subtypes. In the past we have isolated six of the seven genes encoding these isoforms. To complete the set of the human H1 histone genes, we have designed two PCR primers deduced from a partially published sequence of the remaining histone H1 gene [Carozzi et al. (1984) Science 224, 1115-1118] and from a consensus sequence which we have derived from the conserved region of human histone H1 genes. Using these primers we have amplified a 417-bp DNA fragment from total human DNA. This fragment was used for screening a human phage genomic library. Two overlapping clones were isolated. The region contains a set of 5 genes representing each of the five histone classes. In continuation of our numbering of human H1 genes, we have named this H1 gene H1.5. This gene encodes a protein almost identical to the previously published protein sequence designated H1a [Ohe et al. (1986) J. Biochem. 100, 359-368]; since the changes are in a region of some uncertainty of the peptide sequencing, we conclude that the newly isolated gene codes for the H1a protein. The structures of the flanking regions of the genes except the H2B gene are typical for histone genes. They include: (1) a CCAAT element in the promotor region, (2) a TATA box and (3) a palindromic termination element. The H2B sequence shows no typical regulatory elements and no complete ORF, therefore we consider it as a pseudogene. The expression of the H1.5 gene was examined in several cell lines.

Amino Acid Sequence↗
PubMed1997

Histone gene expression and chromatin structure during spermatogenesis.

The chromatin of male germ cells is restructured throughout spermatogenesis. Analysis of differential histone protein patterns at specific stages of spermatogenesis may contribute towards an understanding of the changes in chromatin structure and function during this differentiation process. The most striking changes in histone patterns occur at the stage of pachytene spermatocytes when most of the linker H1 histones are replaced by the testis specific subtype H1t. In addition, replacement of core histone subtypes is observed at this stage. These structural changes precede the reorganization of chromatin at haploid stages when histones are replaced first by transition proteins and then by protamines.

Animals↗
PubMed1997

Histones: genetic diversity and tissue-specific gene expression.

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, H3 and 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 degree, and H1t. The subtypes H1.2 and H1.4 are found in most somatic cell nuclei, whereas H1 degree 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.

Animals↗
PubMed1997

The human histone gene cluster at the D6S105 locus.

The sequences and organization of the histone genes in the histone gene cluster at the chromosomal marker D6S105 have been determined by analyzing the Centre d'Etude du Polymorphisme Humain yeast artificial chromosome (YAC) 964f1. The insert of the YAC was subcloned in cosmids. In the established contig of the histone-gene-containing cosmids, 16 histone genes and 2 pseudogenes were identified: one H1 gene (H1.5), five H2A genes, four H2B genes and one pseudogene of H2B, three H3 genes, and three H4 genes plus one H4 pseudogene. The cluster extends about 80 kb with a nonordered arrangement of the histone genes. The dinucleotide repeat polymorphic marker D6S105 was localized at the telomeric end of this histone gene cluster. Almost all human histone genes isolated until now have been localized within this histone gene cluster and within the previously described region of histone genes, about 2 Mb telomeric of the newly described cluster or in a small group of histone genes on chromosome 1. We therefore conclude that the data presented here complete the set of human histone genes. This now allows the general organization of the human histone gene complement to be outlined on the basis of a compilation of all known histone gene clusters and solitary histone genes.

Chromosome Mapping↗
PubMed1997

Differential expression of the murine histone genes H3.3A and H3.3B.

The histone family of proteins is subdivided into two major groups: the main type histones, which are synthesized in coordination with DNA replication during the S-phase of the cell cycle, and the replacement histones, which can be synthesized in the absence of DNA replication substituting main type histone isoforms. Accumulation of replacement histone variants has been observed in several terminally differentiated tissues that have stopped cell division. The replacement subtype of the H3 class is termed H3.3. This protein is encoded by two different genes (H3.3A and H3.3B) that both code for the same amino acid sequence, but differ in nucleotide sequences and gene organization. This has been shown for human and avian H3.3A and H3.3B genes and for a murine H3.3B cDNA. In an attempt to define patterns of replacement histone H3.3 gene expression during male germ cell differentiation, we have constructed mouse testicular cDNA libraries and have isolated cDNAs corresponding to the murine H3.3A and H3.3B genes. Using probes specific for these two different genes we show by RNase protection analysis and by nonradioactive in situ hybridization with testis sections that H3.3A mRNA is present in pre- and postmeiotic cells, whereas expression of the H3.3B gene is essentially restricted to cells of the meiotic prophase.

Amino Acid Sequence↗
PubMed1997

Varied expression patterns of human H1 histone genes in different cell lines.

Five main type H1 histones have been described in man (H1.1-H1.5) in addition to the testis specific type H1t and the replacement subtype H1 degrees, which is found mainly in highly differentiated cells. We have isolated this whole complement of H1 genes and have studied the expression of the seven human H1 subtype genes in several cell lines. The RNAase protection assay was used to discriminate between the very similar transcripts derived from the seven H1 subtype genes. With the exception of H1.2 and H1.4, we found substantial differences between the H1 mRNA levels in the different cell lines tested. No H1.1 mRNA was detected in most of the cell lines and just a low level of H1.1 mRNA was found in human testis. In contrast to the differential patterns of the other subtypes, H1.2 and H1.4 were in all cells expressed at a high level, indicating a basal function compared with the other H1 histones. Because differences in the timing of H1 protein subtype synthesis have been reported, we have analyzed the kinetics of accumulation of H1 subtypes in synchronized HeLa cells and observed that all H1 subtypes examined (H1 degrees, H1.2-H1.5) were expressed in a replication-dependent manner. The analysis showed a differential rise of mRNA levels during S-phase, from four-fold (H1 degrees) to 15-fold (H1.5). Our results may point at a specific function of each subtype and suggest that expression of the H1 histone subtype genes depends on common S-phase-depent factors as well as on individual regulatory systems. Thus, the data presented here provide a basis for further analysis of the regulation and function of the complex H1 gene and protein family.

Cell Line↗
PubMed1997

Testis-specific expression of the mouse histone gene H1t is regulated by several promoter elements.

The testis-specific histone gene H1t is expressed only in mammalian testis at the stage of pachytene spermatocytes. The tissue-specific regulation of the mouse H1t gene was examined in mouse testicular primary culture cells with gene constructs consisting of H1t promoter elements fused to the chloramphenicol acetyltransferase or the firefly luciferase reporter gene. Our experiments demonstrate that expression of the mouse H1t gene is enhanced by a conserved H1 histone gene-specific TG box 452 base pairs upstream of the transcription start site. The transcription of the H1t gene appears to be reduced by sequences between -1999 and -1506. No regulatory effect could be shown for the H1 box in the expression of the mouse H1t gene. Binding of nuclear protein extracted from mouse testis to these consensus elements was shown by electrophoretic mobility-shift assays with mouse testicular nuclear proteins and labeled oligonucleotides containing the upstream TG box sequences or the two testis-specific elements of the H1t gene.

Animals↗
PubMed1997

Physical mapping of two histone gene clusters on human chromosome 6p22.1-22.2.

Histones are basic proteins which are responsible for the assembly and maintenance of the nucleosomal structure within the chromosomal fiber in eukaryotes. Two clusters of these genes have previously been mapped to the region 6p21.1-p22.2. We describe here a radiation hybrid map, a long range restriction map and a YAC contig covering and linking these two clusters and giving the precise localisation with respect to the HLA complex. The large cluster contains five H1 histone genes in the 6p22.2 region, the smaller only one, H1F5 (H1.5), in 6p22.1. In both clusters, each H1 locus is accompanied by several core histone genes. The large cluster has additionally been covered by a sequence ready PAC contig and three probably unrelated genes (TRMI2, BTN and SSADH) have been accurately localized within the 6p22.2-p22.1 region.

Chromosomes, Human, Pair 6↗