H4 Gene Transcription Research Articles

In vivo occupancy of histone gene proximal promoter elements reflects gene copy number‐dependent titratable transactivation factors and cross‐species compatibility of regulatory sequences

To assess systematically the structural and functional aspects of histone gene transcription within a chromosomal context, we stably integrated an extensive set of human histone H4 gene constructs into mouse C127 cells. Levels of expression were determined by S1 nuclease protection assays for multiple mouse monoclonal cell lines containing these human H4 genes. For each cell line, we quantitated the number of integrated human H4 genes by Southern blot analysis. The results indicate that the expression of the human H4 gene is in part copy number dependent at low gene dosages. However, the level of expression varies among different cell lines containing similar numbers of copies of the same H4 gene construct. This result suggests that position-dependent chromosomal integration effects contribute to H4 gene transcription, consistent with the roles of long-range gene organization and nuclear architecture in gene regulation. At high copy number, the level of human H4 gene expression per copy decreased, and endogenous mouse H4 mRNA levels were also reduced. Furthermore, in vivo occupancy at the human H4 gene immediate 5' regulatory elements, as defined by genomic fingerprinting, showed copy number-dependent protein/DNA interactions. Hence, human and mouse H4 genes compete for titratable transcription factors in a cellular environment. Taken together, these results indicate cross-species compatibility and suggest limited representation in vivo of the factors involved in regulating histone H4 gene transcription.

Transcription of histone H4, H3, and H1 cell cycle genes: promoter factor HiNF-D contains CDC2, cyclin A, and an RB-related protein.

Cell cycle-controlled human histone genes are coordinately expressed during S phase, and transcriptional regulation involves a series of trans-acting factors (HiNFs). The proliferation-specific factor HiNF-D interacts with multiple recognition motifs in histone H4, H3, and H1 promoters. Using gel shift immunoassays, we show that CDC2, cyclin A, and an RB-related protein are ubiquitous subunits of HiNF-D binding activity isolated from several cell types. HiNF-D levels in vivo are sensitive to okadaic acid and staurosporine, indicating that HiNF-D activity and/or assembly is influenced by phosphorylation status. Thus, HiNF-D appears to be a multicomponent phosphoprotein that participates in coordinate control of multiple histone H4, H3, and H1 genes during the cell cycle. The presence of cell cycle mediators in the HiNF-D complex suggests linkage between transcriptional control of histones, enzymes involved in DNA synthesis, and the onset of DNA replication during the G1/S phase transition.

Delineation of a human histone H4 cell cycle element in vivo: the master switch for H4 gene transcription.

Histone gene expression is cell cycle regulated at the transcriptional and the post-transcriptional levels. Upon entry into S phase, histone gene transcription is stimulated 2- to 5-fold and peaks within 1-3 hr of the initiation of DNA synthesis. We have delineated the proximal promoter element responsible for cell cycle-dependent transcription of a human histone H4 gene in vivo. Our results indicate that H4 cell cycle-dependent transcriptional regulation is mediated by an 11-base-pair element, the cell cycle element (5'-CTTTCG-GTTTT-3'), that resides in the in vivo protein-DNA interaction site, site II (nucleotides -64 to -24). The H4 cell cycle element functions as a master switch for expression of the FO108 human histone H4 gene in vivo; mutations within the H4 cell cycle element drastically reduce the level of expression as well as abrogate cell cycle-regulated transcription. Furthermore, these mutations result in a loss of binding in vitro of the cognate nuclear factor HiNF-M. In vivo competition analysis indicates that the cell cycle element mediates specific competition for a DNA-binding factor, presumably HiNF-M, that is a rate-limiting step in transcription of this H4 gene.

Downregulation of histone H4 gene transcription during postnatal development in transgenic mice and at the onset of differentiation in transgenically derived calvarial osteoblast cultures.

In vivo regulation of cell cycle dependent human histone gene expression was examined in transgenic mice using a fusion construct containing 6.5 kB of a human H4 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Transcriptional control of histone gene expression, as a function of proliferative activity, was determined. We established the relationship between DNA replication dependent H4 mRNA levels (Northern blot analysis) and H4 promoter activity (CAT assay) during postnatal development in a broad spectrum of tissues. In most tissues sampled in adult animals, the cellular representation of H4 gene transcripts declined in parallel with promoter activity. This result is consistent with transcriptional control of H4 gene expression at the cessation of proliferation. Interestingly, while H4 mRNA was detectable at very low levels post-proliferatively in brain, promoter activity persisted in adult brain, where most of the cells are terminally differentiated. This dissociation between histone gene promoter activity and histone mRNA accumulation points to the possibility of post-transcriptional regulation of histone gene expression in brain. Cultures of osteoblasts were prepared from calvaria of transgenic mice carrying the H4 promoter/CAT reporter construct. In contrast to the brain, in these bone-derived cells, we established by immunohistochemistry that the transition to the quiescent, differentiated state is associated with a transcriptionally mediated downregulation of histone gene expression at the single cell level.

UHF-1, a factor required for maximal transcription of early and late sea urchin histone H4 genes: analysis of promoter-binding sites.

A protein, denoted UHF-1, was found to bind upstream of the transcriptional start site of both the early and late H4 (EH4 and LH4) histone genes of the sea urchin Strongylocentrotus purpuratus. A nuclear extract from hatching blastulae contained proteins that bind to EH4 and LH4 promoter fragments in a band shift assay and produced sharp DNase I footprints upstream of the EH4 gene (from -133 to -106) and the LH4 gene (from -94 to -66). DNase I footprinting performed in the presence of EH4 and LH4 promoter competitor DNAs indicated that UHF-1 binds more strongly to the EH4 site. A sequence match of 11 of 13 nucleotides was found within the two footprinted regions: [sequence: see text]. Methylation interference and footprinting experiments showed that UHF-1 bound to the two sites somewhat differently. DNA-protein UV cross-linking studies indicated that UHF-1 has an electrophoretic mobility on sodium dodecyl sulfate-acrylamide gels of approximately 85 kDa and suggested that additional proteins, specific to each promoter, bind to each site. In vitro and in vivo assays were used to demonstrate that the UHF-1-binding site is essential for maximal transcription of the H4 genes. Deletion of the EH4 footprinted region resulted in a 3-fold decrease in transcription in a nuclear extract and a 2.6-fold decrease in expression in morulae from templates that had been injected into eggs. In the latter case, deletion of the binding site did not grossly disrupt the temporal program of expression from the injected EH4 genes. LH4 templates containing a 10-bp deletion in the consensus region or base substitutions in the footprinted region were transcribed at 14 to 58% of the level of the wild-type LH4 template. UHF-1 is therefore essential for maximal expression of the early and late H4 genes.

UHF-1, a Factor Required for Maximal Transcription of Early and Late Sea Urchin Histone H4 Genes: Analysis of Promoter-Binding Sites

A protein, denoted UHF-1, was found to bind upstream of the transcriptional start site of both the early and late H4 (EH4 and LH4) histone genes of the sea urchin Strongylocentrotus purpuratus. A nuclear extract from hatching blastulae contained proteins that bind to EH4 and LH4 promoter fragments in a band shift assay and produced sharp DNase I footprints upstream of the EH4 gene (from -133 to -106) and the LH4 gene (from -94 to -66). DNase I footprinting performed in the presence of EH4 and LH4 promoter competitor DNAs indicated that UHF-1 binds more strongly to the EH4 site. A sequence match of 11 of 13 nucleotides was found within the two footprinted regions: [sequence: see text]. Methylation interference and footprinting experiments showed that UHF-1 bound to the two sites somewhat differently. DNA-protein UV cross-linking studies indicated that UHF-1 has an electrophoretic mobility on sodium dodecyl sulfate-acrylamide gels of approximately 85 kDa and suggested that additional proteins, specific to each promoter, bind to each site. In vitro and in vivo assays were used to demonstrate that the UHF-1-binding site is essential for maximal transcription of the H4 genes. Deletion of the EH4 footprinted region resulted in a 3-fold decrease in transcription in a nuclear extract and a 2.6-fold decrease in expression in morulae from templates that had been injected into eggs. In the latter case, deletion of the binding site did not grossly disrupt the temporal program of expression from the injected EH4 genes. LH4 templates containing a 10-bp deletion in the consensus region or base substitutions in the footprinted region were transcribed at 14 to 58% of the level of the wild-type LH4 template. UHF-1 is therefore essential for maximal expression of the early and late H4 genes.

Down-regulation of histone H3 and H4 gene transcription in differentiated L6 myotubes

The core histone mRNA levels in terminally differentiated L6 myotubes decrease to less than 5% of the amount present in proliferating myoblasts, in parallel with the cessation of DNA synthesis (Bird, R.C., Jacobs, F.A., Stein, G., Stein, J. and Sells, B.H. (1985) Biochim. Biophys. Acta 824, 209–217). The role of gene transcription in the down-shift of histone mRNA levels was assessed using a cell-free system. The level of transcription from the differentiation-independent adenovirus major late promoter was directly related to the RNA polymerase II activity of myoblast and myotube nuclear extracts. In addition, both extracts actively transcribed the histone H4 gene template containing only the 5′ proximal promoter region (−210 bp). In contrast, inclusion of the distal-proximal promoter region (−410 to −210 bp) in the template resulted in a 60% decrease in transcription by the myotube extract. A similar down-shift in transcription of the histone H3 gene template (containing 900 bp 5′ of the initiation site) by myotube nuclear extracts was also observed. The decrease in histone mRNA levels in myotubes may therefore be controlled in part by a transcriptional mechanism involving a negative regulatory factor.

Histone H4 mRNA is stored as a small cytoplasmic RNP during the G 2 phase in Physarum polycephalum

In Physarum polycephalum the triggering of histone H4 gene transcription occurs in G2 phase. The rate of synthesis of histone H4 mRNA was measured by in vivo pulse-labeling experiments. We show that it begins to increase in mid-G2. During the second part of G2 it increases approximately 20 fold over its minimum value and reaches a maximum at the end of G2. After entry of the cells in S, histone H4 gene transcription rate begins to decrease and reaches a minimum value in early G2. The histone H4 mRNA which accumulates in G2 is not translated immediately into proteins but is stored in an inactive form until the beginning of the next S phase. Immediately after its transcription the H4 mRNA is transported to the cytoplasm where it is stored and stabilized as an inactive mRNP complex. This was shown by fractionation of cytoplasmic RNP in sucrose gradients and blot hybridization of subcellular fractions.

Level of histone H4 mRNA in Xenopus laevis embryonic cells cultured in the absence of cell adhesion

The amount of histone H4 mRNA per embryo was followed during early development of Xenopus laevis by Northern blot analyses using a cloned histone H4 cDNA as the probe. The H4 mRNA content was nearly constant until the blastula stage, increased greatly at the gastrula stage and then decreased at the neurula stage. Experiments with actinomycin D suggested that most H4 mRNA molecules detected at the late gastrula and neurula stages were maintained depending on new transcription of H4 genes during these stages. To see if the H4 mRNA level is affected by cell adhesion, we prepared dissociated cells and measured H4 mRNA content under conditions that inhibit cellular reaggregation. It was found that the amount of H4 mRNA per embryo in dissociated and reaggregation-inhibited cells was nearly equal to that of the control embryo at the neurula stage. Therefore, we conclude that the synthetic activity of histone H4 mRNA is not dependent on the cellular adhesion during development.

Transcription of alpha-tubulin and histone H4 genes begins at the same point in the Physarum cell cycle.

In naturally synchronous plasmodia of Physarum polycephalum, both tubulin and histone gene transcription define periodic cell cycle-regulated events. Using a slot-blot hybridization assay and Northern blot analysis, we have demonstrated that a major peak of accumulation of both alpha-tubulin and histone H4 transcripts occurs in late G2 phase. Nuclear transcription assays indicate that both genes are transcriptionally activated at the same point in the cell cycle: mid G2 phase. While the rate of tubulin gene transcription drops sharply at the M/S-phase boundary, the rate of histone gene transcription remains high through most of S phase. We conclude that the cell cycle regulation of tubulin expression occurs primarily at the level of transcription, while histone regulation involves both transcriptional and posttranscriptional controls. It is possible that the periodic expression of both histone and tubulin genes is triggered by a common cell cycle regulatory mechanism.