Early Histone Research Articles

On the Hunt for the Histone Code

Our genome is not made of naked DNA, but a fiber (chromatin) composed of DNA and proteins packaged into our chromosomes. The basic building block of chromatin is the nucleosome, which has two copies of each of the proteins called histones (H2A, H2B, H3, and H4) wrapped by 146 base pairs of DNA. Regions of our genetic material are found between the more open (euchromatin) and more compact (heterochromatin) regions of the genome that can be variably accessible to the underlying genes. Furthermore, post-translational modifications (PTMs) on histones, such as on H3, are critical for regulating chromatin accessibility and gene expression. While site specific antibodies were the tool of choice for histone PTM analysis in the early days (pre-2000s), enter Don Hunt changing the histone PTM field forever. Don’s clever thinking brought new innovative mass spectrometry-based approaches to the epigenetics field. His lab’s effort led to the discovery of many new histone modifications and methods to facilitate the detection and quantification of histone PTMs, which are still considered state of the art in the proteomics field today. Due to Don’s pioneering work in this area, many labs have been able to jump into the epigenetics field and “Hunt” down their own histone targets. A walkthrough of those early histone years in the Hunt Lab are described by three of us who were fortunate enough to be at the right place, at the right time.

Assembly of continuous high-resolution draft genome sequence of Hemicentrotus pulcherrimus using long-read sequencing.

The update of the draft genome assembly of sea urchin, Hemicentrotus pulcherrimus, which is widely studied in East Asia as a model organism of early development, was performed using Oxford nanopore long-read sequencing. The updated assembly provided ~600-Mb genome sequences divided into 2,163 contigs with N50 = 516 kb. BUSCO completeness score and transcriptome model mapping ratio (TMMR) of the present assembly were obtained as 96.5% and 77.8%, respectively. These results were more continuous with higher resolution than those by the previous version of H. pulcherrimus draft genome, HpulGenome_v1, where the number of scaffolds = 16,251 with a total of ~100 Mb, N50 = 143 kb, BUSCO completeness score = 86.1%, and TMMR = 55.4%. The obtained genome contained 36,055 gene models that were consistent with those in other echinoderms. Additionally, two tandem repeat sequences of early histone gene locus containing 47 copies and 34 copies of all histone genes, and 185 of the homologous sequences of the interspecifically conserved region of the Ars insulator, ArsInsC, were obtained. These results provide further advance for genome-wide research of development, gene regulation, and intranuclear structural dynamics of multicellular organisms using H. pulcherrimus.

Hemorrhagic shock and tissue injury provoke distinct components of trauma-induced coagulopathy in a swine model.

Tissue injury (TI) and hemorrhagic shock (HS) are the major contributors to trauma-induced coagulopathy (TIC). However, the individual contributions of these insults are difficult to discern clinically because they typically coexist. TI has been reported to release procoagulants, while HS has been associated with bleeding. We developed a large animal model to isolate TI and HS and characterize their individual mechanistic pathways. We hypothesized that while TI and HS are both drivers of TIC, they provoke different pathways; specifically, TI reduces time to clotting, whereas, HS decreases clot strength stimulates hyperfibrinolysis. After induction of general anesthesia, 50kg male, Yorkshire swine underwent isolated TI (bilateral muscle cutdown of quadriceps, bilateral femur fractures) or isolated HS (controlled bleeding to a base excess target of -5mmol/l) and observed for 240min. Thrombelastography (TEG), calcium levels, thrombin activatable fibrinolysis inhibitor (TAFI), protein C, plasminogen activator inhibitor 1 (PAI-1), and plasminogen activator inhibitor 1/tissue-type plasminogen activator complex (PAI-1-tPA) were analyzed at pre-selected timepoints. Linear mixed models for repeated measures were used to compare results throughout the model. TI resulted in elevated histone release which peaked at 120min (p = 0.02), and this was associated with reduced time to clot formation (R time) by 240min (p = 0.006). HS decreased clot strength at time 30min (p = 0.003), with a significant decline in calcium (p = 0.001). At study completion, HS animals had elevated PAI-1 (p = 0.01) and PAI-1-tPA (p = 0.04), showing a trend toward hyperfibrinolysis, while TI animals had suppressed fibrinolysis. Protein C, TAFI and skeletal myosin were not different among the groups. Isolated injury in animal models can help elucidate the mechanistic pathways leading to TIC. Our results suggest that isolated TI leads to early histone release and a hypercoagulable state, with suppressed fibrinolysis. In contrast, HS promotes poor clot strength and hyperfibrinolysis resulting in hypocoagulability.

Poly(ADP-ribosyl)ation mediates early phase histone eviction at DNA lesions.

Nucleosomal histones are barriers to the DNA repair process particularly at DNA double-strand breaks (DSBs). However, the molecular mechanism by which these histone barriers are removed from the sites of DNA damage remains elusive. Here, we have generated a single specific inducible DSB in the cells and systematically examined the histone removal process at the DNA lesion. We found that histone removal occurred immediately following DNA damage and could extend up to a range of few kilobases from the lesion. To examine the molecular mechanism underlying DNA damage-induced histone removal, we screened histone modifications and found that histone ADP-ribosylation was associated with histone removal at DNA lesions. PARP inhibitor treatment suppressed the immediate histone eviction at DNA lesions. Moreover, we examined histone chaperones and found that the FACT complex recognized ADP-ribosylated histones and mediated the removal of histones in response to DNA damage. Taken together, our results reveal a pathway that regulates early histone barrier removal at DNA lesions. It may also explain the mechanism by which PARP inhibitor regulates early DNA damage repair.

Early Histone Deacetylase Inhibition Mitigates Ischemia/Reperfusion Brain Injury by Reducing Microglia Activation and Modulating Their Phenotype.

Histone deacetylase inhibitors (HDACi) are a promising therapeutic intervention for stroke. The involvement of the anti-inflammatory effects of HDACi in their neuroprotection has been reported, but the underlying mechanisms are still uncertain. Given the post-stroke inflammation is a time-dependent process, starting with acute and intense inflammation, and followed by a prolonged and mild one, we proposed whether target the early inflammatory response could achieve the neuroprotection of HDACi? To test this hypothesis, a single dose of suberoylanilide hydroxamic acid (SAHA) (50 mg/kg), a pan HDACi, was intraperitoneally (i.p.) injected immediately or 12 h after ischemia onset in a transient middle cerebral artery occlusion (tMCAO) mouse model. Compared with delayed injection, immediate SAHA treatment provided more protection, evidenced by smaller infarction volume, and a better outcome. This protection was accompanied by suppression of pro-inflammatory cytokines and reduction of activated microglia in the early stage of post-stroke inflammation. Moreover, SAHA treatment suppressed M1 cytokine expression (IL-6, TNF-α, and iNOS) while promoted the transcription of M2 cytokines (Arg-1 and IL-10) in LPS-challenged mouse microglia, and enhanced CD206 (M2 marker) but decreased CD86 (M1 markers) levels in microglia isolated from the ipsilateral hemisphere of MCAO mice. Collectively, our data suggested that the protection of SAHA on ischemic brain injury was closely associated with its inhibition on the early inflammatory response, and this inhibition was related to its reducing microglia activation and priming the activated microglia toward a more protective phenotype.

Early histone H4 acetylation during chromatin remodeling in equine spermatogenesis†

Chromatin remodeling during spermatogenesis culminates in the exchange of nucleosomes for transition proteins and protamines as an important part of spermatid development to give rise to healthy sperm. Comparative immunofluorescence analyses of equine and murine testis histological sections were used to characterize nucleoprotein exchange in the stallion. Histone H4 hyperacetylation is considered a key event of histone removal during the nucleoprotein transition to a protamine-based sperm chromatin structure. In the stallion, but not the mouse, H4 was already highly acetylated in lysine residues K5, K8, and K12 in round spermatids almost immediately after meiotic division. Time courses of transition protein 1 (TP1), protamine 1, H2A histone family member Z (H2AFZ), and testis-specific histone H2B variant (TH2B) expression in stallion spermatogenesis were similar to the mouse where protamine 1 and TP1 were only expressed in elongating spermatids much later in spermatid development. The additional acetylation of H4 in K16 position (H4K16ac) was detected during a brief phase of spermatid elongation in both species, concomitant with the phosphorylation of the noncanonical histone variant H2AFX resulting from DNA strand break-mediated DNA relaxation. The results suggest that H4K16 acetylation, which is dependent on DNA damage signaling, may be more important for nucleosome replacement in spermiogenesis than indicated by data obtained in rodents and highlight the value of the stallion as an alternative animal model for investigating human spermatogenesis. A revised classification system of the equine spermatogenic cycle for simplified comparison with the mouse is proposed to this end.

Dynamic changes in the interchromosomal interaction of early histone gene loci during development of sea urchin.

The nuclear positioning and chromatin dynamics of eukaryotic genes are closely related to the regulation of gene expression, but they have not been well examined during early development, which is accompanied by rapid cell cycle progression and dynamic changes in nuclear organization, such as nuclear size and chromatin constitution. In this study, we focused on the early development of the sea urchin Hemicentrotus pulcherrimus and performed three-dimensional fluorescence in situ hybridization of gene loci encoding early histones (one of the types of histone in sea urchin). There are two non-allelic early histone gene loci per sea urchin genome. We found that during the morula stage, when the early histone gene expression levels are at their maximum, interchromosomal interactions were often formed between the early histone gene loci on separate chromosomes and that the gene loci were directed to locate to more interior positions. Furthermore, these interactions were associated with the active transcription of the early histone genes. Thus, such dynamic interchromosomal interactions may contribute to the efficient synthesis of early histone mRNA during the morula stage of sea urchin development.

Glycolysis-Mediated Changes in Acetyl-CoA and Histone Acetylation Control the Early Differentiation of Embryonic Stem Cells

Loss of pluripotency is a gradual event whose initiating factors are largely unknown. Here we report the earliest metabolic changes induced during the first hours of differentiation. High-resolution NMR identified 44 metabolites and a distinct metabolic transition occurring during early differentiation. Metabolic and transcriptional analyses showed that pluripotent cells produced acetyl-CoA through glycolysis and rapidly lost this function during differentiation. Importantly, modulation of glycolysis blocked histone deacetylation and differentiation in human and mouse embryonic stem cells. Acetate, a precursor of acetyl-CoA, delayed differentiation and blocked early histone deacetylation in a dose-dependent manner. Inhibitors upstream of acetyl-CoA caused differentiation of pluripotent cells, while those downstream delayed differentiation. Our results show a metabolic switch causing a loss of histone acetylation and pluripotent state during the first hours of differentiation. Our data highlight the important role metabolism plays in pluripotency and suggest that a glycolytic switch controlling histone acetylation can release stem cells from pluripotency.

Establishment of regions of genomic activity during the Drosophila maternal to zygotic transition.

We describe the genome-wide distributions and temporal dynamics of nucleosomes and post-translational histone modifications throughout the maternal-to-zygotic transition in embryos of Drosophila melanogaster. At mitotic cycle 8, when few zygotic genes are being transcribed, embryonic chromatin is in a relatively simple state: there are few nucleosome free regions, undetectable levels of the histone methylation marks characteristic of mature chromatin, and low levels of histone acetylation at a relatively small number of loci. Histone acetylation increases by cycle 12, but it is not until cycle 14 that nucleosome free regions and domains of histone methylation become widespread. Early histone acetylation is strongly associated with regions that we have previously shown to be bound in early embryos by the maternally deposited transcription factor Zelda, suggesting that Zelda triggers a cascade of events, including the accumulation of specific histone modifications, that plays a role in the subsequent activation of these sequences.

The Compass-like Locus, Exclusive to the Ambulacrarians, Encodes a Chromatin Insulator Binding Protein in the Sea Urchin Embryo

Chromatin insulators are eukaryotic genome elements that upon binding of specific proteins display barrier and/or enhancer-blocking activity. Although several insulators have been described throughout various metazoans, much less is known about proteins that mediate their functions. This article deals with the identification and functional characterization in Paracentrotus lividus of COMPASS-like (CMPl), a novel echinoderm insulator binding protein. Phylogenetic analysis shows that the CMPl factor, encoded by the alternative spliced Cmp/Cmpl transcript, is the founder of a novel ambulacrarian-specific family of Homeodomain proteins containing the Compass domain. Specific association of CMPl with the boxB cis-element of the sns5 chromatin insulator is demonstrated by using a yeast one-hybrid system, and further corroborated by ChIP-qPCR and trans-activation assays in developing sea urchin embryos. The sns5 insulator lies within the early histone gene cluster, basically between the H2A enhancer and H1 promoter. To assess the functional role of CMPl within this locus, we challenged the activity of CMPl by two distinct experimental strategies. First we expressed in the developing embryo a chimeric protein, containing the DNA-binding domain of CMPl, which efficiently compete with the endogenous CMPl for the binding to the boxB sequence. Second, to titrate the embryonic CMPl protein, we microinjected an affinity-purified CMPl antibody. In both the experimental assays we congruently observed the loss of the enhancer-blocking function of sns5, as indicated by the specific increase of the H1 expression level. Furthermore, microinjection of the CMPl antiserum in combination with a synthetic mRNA encoding a forced repressor of the H2A enhancer-bound MBF1 factor restores the normal H1 mRNA abundance. Altogether, these results strongly support the conclusion that the recruitment of CMPl on sns5 is required for buffering the H1 promoter from the H2A enhancer activity, and this, in turn, accounts for the different level of accumulation of early linker and nucleosomal transcripts.

Early Histone Modifications in the Ventromedial Hypothalamus and Preoptic Area Following Oestradiol Administration

Expression of the primary female sex behaviour, lordosis, in laboratory animals depends on oestrogen-induced expression of progesterone receptor (PgR) within a defined cell group in the ventrolateral portion of the ventromedial nucleus of the hypothalamus (VMH). The minimal latency from oestradiol administration to lordosis is 18 h. During that time, ligand-bound oestrogen receptors (ER), members of a nuclear receptor superfamily, recruit transcriptional coregulators, which induce covalent modifications of histone proteins, thus leading to transcriptional activation or repression of target genes. The present study aimed to investigate the early molecular epigenetic events underlying oestrogen-regulated transcriptional activation of the Pgr gene in the VMH of female mice. Oestradiol (E₂) administration induced rapid and transient global histone modifications in the VMH of ovariectomised female mice. Histone H3 N-terminus phosphorylation (H3S10phK14Ac), acetylation (H3Ac) and methylation (H3K4me3) exhibited distinct temporal patterns facilitative to the induction of transcription. A transcriptional repressive (H3K9me3) modification showed a different temporal pattern. Collectively, this should create a permissive environment for the transcriptional activity necessary for lordosis, within 3-6 h after E₂ treatment. In the VMH, changes in the H3Ac and H3K4me3 levels of histone H3 were also detected at the promoter region of the Pgr gene within the same time window, although they were delayed in the preoptic area. Moreover, examination of histone modifications associated with the promoter of another ER-target gene, oxytocin receptor (Oxtr), revealed gene- and brain-region specific effects of E₂ treatment. In the VMH of female mice, E₂ treatment resulted in the recruitment of ERα to the oestrogen-response-elements-containing putative enhancer site of Pgr gene, approximately 200 kb upstream of the transcription start site, although it failed to increase ERα association with the more proximal promoter region. Finally, E₂ administration led to significant changes in the mRNA expression of several ER coregulators in a brain-region dependent manner. Taken together, these data indicate that, in the hypothalamus and preoptic area of female mice, early responses to E₂ treatment involve highly specific changes in chromatin structure, dependent on cell group, gene, histone modification studied, promoter/enhancer site and time following E₂.

Posttranslational modifications in histones underlie heat acclimation-mediated cytoprotective memory

We have demonstrated that heat acclimation (AC) causes selective, long-lasting, transcriptional changes in cytoprotective and chromatin remodeling-associated genes, which maintain their AC transcriptome profile, despite the loss of the AC phenotype (Tetievsky et al. Physiol Genomics 34: 78-87, 2008). We postulated that AC memory involves upstream epigenetic information, which predisposes to rapid reacclimation (ReAC) and cytoprotective memory. Here we tested the hypothesis that posttranslational histone modifications are linked to this process. Rats subjected to AC (34°C for 2 or 30 days), deacclimation (DeAC; 24°C, 30 days), and ReAC (34°C, 2 days), and untreated controls were used. Histone H4 lysine acetylation and histone H3 acetylation and phosphorylation in the heat shock element (HSE) of the promoters of heat shock protein-70 (hsp70) and -90 (hsp90) genes were examined. Histone acetyltransferase recruitment of TIP60 (60-kDa histone acetyltransferase-interactive protein), the catalytic subunit of NuH4, was used to validate acetylation. Heat shock factor-1 (HSF-1)-HSE binding to the hsp70 and hsp90 genes was measured to confirm HSF-1 binding to euchromatin. Our results indicate that, while histone H3Ser10 phosphorylation occurred during the AC 2-day phase, AC constitutively elevated histone H4 acetylation in the HSE of hsp70 and hsp90 promoters. HSF-1-HSE binding was detected in the hsp70 gene throughout AC-DeAC-ReAC. The hsp90 gene lacked HSF-1 binding during DeAC, but resumed a high binding level upon ReAC. HSP-90 is a critical cytoprotective protein, and the HSF-1-hsp90 binding profile matched levels of this protein. We conclude that, while early histone H3 phosphorylation is probably required for subsequent histone H4 acetylation, the constitutively acetylated histone H4 and the preserved euchromatin state throughout AC-DeAC-ReAC predispose to rapid cytoprotective acclimatory memory.

Electrophoretic patterns of spermatozoal nucleoproteins (NP) in fertile men and infertility patients and comparison with NP of somatic cells

During spermatogenesis in mammals the evolutionary early histones are replaced step by step by more species specific nucleoproteins (protamines). The protamines protect the genetic information of the male against mutagenic or other damaging influences and possibly play a role in male fertility. A method was presented to study the nucleoproteins (NP) of single human ejaculates. This procedure was checked concerning its usability and validity by bull spermatozoa. The NP of cryopreserved human spermatozoa of fertile men and of infertility patients were investigated and compared with the electrophoretic patterns of NP of somatic hepatocytes of rat and mouse. A total of 48 semen samples were studied. Ten of these samples were obtained from semen donors of proven fertility and 38 samples were collected from male partners of infertile couples. Photodensitometrically it could be distinguished between percentages of non-protamines (PNP) and percentages of protamines (PP). The PP varied intra-individually with a mean standard deviation of 34%, analogously to the alterations of classical semen parameters. The PP of fertile men did not significantly differ from the infertility patients with normal spermiogram parameters but showed a significantly higher value compared with the semen samples with pathological spermiograms. On average the PP of all infertile patients were significantly lower than the PP of fertile semen donors.

321. Sea Urchin sns Chromatin Insulator Prevents Silencing and Positional Effect Variegation of Oncoretroviral Vectors Transgene Expression in Murine Erythroid Cell Line

Silencing and position effect are considered significant obstacles to obtain a consistent level of transgene expression in viral gene therapy. Furthermore recent studies had shown that retroviruses tend to land on active genes with the potential consequence of insertional mutagenesis. The inclusion of elements, such as chromatin insulators, capable to insulate a gene from the surrounding chromatin effects at the integration site should improve both efficacy and safety of gene therapy vectors. We have previously characterized a 265 bp insulator element, termed sns, localized at the 3’ end of the early histone H2A gene of the sea urchin Paracentrotus lividus.

Functional characterization of the sea urchin sns chromatin insulator in erythroid cells

Chromatin insulators are regulatory elements that determine domains of genetic functions. We have previously described the characterization of a 265 bp insulator element, termed sns, localized at the 3′ end of the early histone H2A gene of the sea urchin Paracentrotus lividus. This sequence contains three cis-acting elements (Box A, Box B, and Box C + T) all needed for the enhancer-blocking activity in both sea urchin and human cells. The goal of this study was to further characterize the sea urchin sns insulator in the erythroid environment. We employed colony assays in human (K562) and mouse (MEL) erythroid cell lines. We tested the capability of sns to interfere with the communication between the 5′HS2 enhancer of the human β-globin LCR and the γ-globin promoter. We found that the sns sequence displays directional enhancer-blocking activity. By the use of antibodies against known DNA binding proteins, in electrophoretic mobility shift assays, we demonstrated the binding of the erythroid-specific GATA-1 and the ubiquitous Oct-1 and Sp1 transcription factors. These factors bind to Box A, Box B, and Box C + T, respectively, in both K562 and MEL nuclear extracts. These results may have significant implications for the conservation of insulator function in evolutionary distant organisms and may prove to be of practical benefit in gene transfer applications for erythroid disorders such as hemoglobinopathies and thalassemias.

Characterization of the grappa gene, the Drosophila histone H3 lysine 79 methyltransferase.

We have identified a novel gene named grappa (gpp) that is the Drosophila ortholog of the Saccharomyces cerevisiae gene Dot1, a histone methyltransferase that modifies the lysine (K)79 residue of histone H3. gpp is an essential gene identified in a genetic screen for dominant suppressors of pairing-dependent silencing, a Polycomb-group (Pc-G)-mediated silencing mechanism necessary for the maintenance phase of Bithorax complex (BX-C) expression. Surprisingly, gpp mutants not only exhibit Pc-G phenotypes, but also display phenotypes characteristic of trithorax-group mutants. Mutations in gpp also disrupt telomeric silencing but do not affect centric heterochromatin. These apparent contradictory phenotypes may result from loss of gpp activity in mutants at sites of both active and inactive chromatin domains. Unlike the early histone H3 K4 and K9 methylation patterns, the appearance of methylated K79 during embryogenesis coincides with the maintenance phase of BX-C expression, suggesting that there is a unique role for this chromatin modification in development.

Conservative segregation of maternally inherited CS histone variants in larval stages of sea urchin development.

Three sets of histone variants are coexisting in the embryo at larval stages of sea urchin's development: the maternally inherited cleavage stage variants (CS) expressed during the two initial cleavage divisions, the early histone variants, which are recruited into embryonic chromatin from middle cleavage stages until hatching and the late variants, that are fundamentally expressed from blastula stage onward. Since the expression of the CS histones is confined to the initial cleavage stages, these variants represent a very minor proportion of the histones present in the plutei larvae, whereas the late histone variants are predominant. To determine the position of these CS in the embryonic territories, we have immunolocalized the CS histone variants in plutei larvas harvested 72 h post-fertilization. In parallel, we have pulse labeled the DNA replicated during the initial cleavage cycle with bromodeoxyuridine (BrdU) and its position was further determined in the plutei larvas by immunofluorescence. We have found that the CS histone variants were segregated to specific territories in the plutei. The position in which the CS histone variants were found to be segregated was consistent with the position in which the DNA molecules that were replicated during the initial cleavage divisions were localized. These results strongly suggest that a specification of embryonic nuclei occurs at the initial cleavage divisions which is determined by a chromatin organized by CS histone variants.

The modulator is a constitutive enhancer of a developmentally regulated sea urchin histone H2A gene.

Going back to the late 1970s and early 1980s, we trace the Xenopus oocyte microinjection experiments that led to the emergence of the concept of "modulator". The finding that the modulator could transactivate transcription from far upstream and in either orientation suggested that a new genetic element, different from the classical prokaryotic promoter sequences, had been discovered. This particular enhancer transactivates transcription of the sea urchin early (alpha) histone H2A gene which is regulated in early sea urchin development. We summarise the data from sea urchin microinjection experiments that confirm and extend the results obtained with Xenopus oocytes. We conclude that the H2A enhancer is bipartite, is located approx. 100 bp upstream of the TATAAATA box in the H2A gene of two sea urchin species and enhances transcription when placed at a position far upstream or far downstream of the gene unless an insulator intervenes between enhancer and promoter. Evidence from microinjection experiments with sea urchin embryos suggests that the developmental control of H2A expression resides not with the enhancer, which is constitutively active, but with a striking chromatin structure with two positioned nucleosomes near the 3' end of the gene. Within this structure, there is an insulator element.

Developmentally-regulated interaction of a transcription factor complex containing CDP/cut with the early histone H3 gene promoter of the sea urchin Tetrapygus niger is associated with changes in chromatin structure and gene expression

During sea urchin embryogenesis the early histone genes are temporally expressed to accommodate the high demand for histone proteins during DNA replication at early cleavage stages of development. The early histone genes are transcriptionally active from the 16-cell stage, reaching a peak in expression at the 128-cell stage that gradually decreases until expression is completely inhibited at the late blastula stage. We are studying the gene regulatory mechanisms that control early histone gene expression in sea urchins to understand the interrelationships between chromatin remodeling and transcriptional activation during development. Here, we have investigated chromatin organization and transcription factor interactions by analyzing nuclease hypersensitivity and protein binding in the promoter region of the early histone H3 gene from the sea urchin Tetrapygus niger. We have found a DNase I hypersensitive domain centered at −90 in the early histone H3 gene promoter which is only detected in embryos at the 128-cell stage expressing high levels of early histone H3 mRNA. This hypersensitive site (−110 to −70) encompasses two regulatory elements (TnH3NFH3.1 and TnH3CCAAT). The −94 to −77 region of the histone H3 promoter is recognized by a transcription factor complex in nuclear extracts from 128-cell embryos. Methylation interference analysis and competition studies demonstrated a specific interaction at the CCAAT sequence. Using specific antibodies we find that the homeodomain transcription factor CDP/cut is the DNA-binding component of the complex interacting with the early histone H3 gene promoter in T. niger. Our results provide further evidence for the functional role of CDP/cut in developmental regulation of histone gene expression in phylogenetically diverse eukaryotic species.

Regulation of the sea urchin early H2A histone gene expression depends on the modulator element and on sequences located near the 3' end.

Transcription of the sea urchin early histone genes occurs transiently during early cleavage, reaching the maximum at the morula stage and declining to an undetectable level at the gastrula stage. To identify the regulatory elements responsible for the timing and the levels of transcription of the H2A gene, we used promoter binding studies in nuclear extracts and microinjection of a CAT transgene driven by the early H2A promoter. We found that morula and gastrula nuclear proteins produced indistinguishable DNase I footprint patterns on the H2A promoter. Two sites of interactions, centred on the modulator/enhancer and on the CCAAT box respectively, were detected. Deletion of the modulator or coinjection of an excess of modulator sequences severely affected the expression of two transgenes driven by the enhancer-less and modulator-containing H2A promoter. Finally, a DNA fragment containing 3' coding and post-H2A spacer sequences, where upon silencing three micrococcal nuclease hypersensitive sites were previously mapped, specifically repressed at the gastrula stage the expression of the transgene driven by the H2A promoter. These results indicate that the modulator is essential for the expression of early H2A gene and that sequences for downregulation are localized near the 3' end of the H2A gene.