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CYP19A1 encodes aromatase, which converts testosterone to estrogen, and is induced during placental maturation. To elucidate the molecular mechanism underlying this function, histone methylation was analyzed using the placental cytotrophoblast cell line, JEG3. Treatment of JEG3 cells with 3-deazaneplanocin A, an inhibitor of several methyltransferases, resulted in increased CYP19A1 expression, accompanied by removal of the repressive mark H3K27me3 from the CYP19A1 promoter. However, this increase was not observed in cells treated with GSK126, another specific inhibitor for H3K27me3 methylation. Expression of TFAP2C, which encodes AP-2γ, a transcription factor that regulates CYP19A1, was also elevated on 3-deazaneplanocin A treatment. Interestingly, TFAP2C messenger RNA (mRNA) was readily degraded in JEG3 cells but protected from degradation in the presence of 3-deazaneplanocin A. TFAP2C mRNA contained N6-methyladenosines, which were reduced on drug treatment. These observations indicate that the TFAP2C mRNA undergoes adenosine methylation and rapid degradation, whereas 3-deazaneplanocin A suppresses methylation, resulting in an increase in AP-2γ levels. We conclude that the increase in AP-2γ expression via stabilization of the TFAP2C mRNA is likely to underlie the increased CYP19A1 expression., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The 'open' and 'compact' regions of chromatin are considered to be regions of active and silent transcription, respectively. However, individual genes produce transcripts at different levels, suggesting that transcription output does not depend on the simple open-compact conversion of chromatin, but on structural variations in chromatin itself, which so far have remained elusive. In this study, weakly crosslinked chromatin was subjected to sedimentation velocity centrifugation, which fractionated the chromatin according to its degree of compaction. Open chromatin remained in upper fractions, while compact chromatin sedimented to lower fractions depending on the level of nucleosome assembly. Although nucleosomes were evenly detected in all fractions, histone H1 was more highly enriched in the lower fractions. H1 was found to self-associate and crosslinked to histone H3, suggesting that H1 bound to H3 interacts with another H1 in an adjacent nucleosome to form compact chromatin. Genome-wide analyses revealed that nearly the entire genome consists of compact chromatin without differences in compaction between repeat and non-repeat sequences; however, active transcription start sites (TSSs) were rarely found in compact chromatin. Considering the inverse correlation between chromatin compaction and RNA polymerase binding at TSSs, it appears that local states of chromatin compaction determine transcription levels., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Human CYP19 spans a region of chromosome 15 of approximately 130 kb and encodes aromatase, an enzyme required for estrogen synthesis. In the human granulosa cell-line KGN, there are seven open chromatin regions within the CYP19 locus. In this study, we demonstrate that two of these regions ~40 kb upstream and ~15 kb downstream of the CYP19 promoter are cohesin-loading sites, physically interacting with the promoter to negatively and positively regulate transcription, respectively. These observations suggest that CYP19 expression is controlled by a balance between the upstream silencer and downstream enhancer. When cohesin is depleted, CYP19 expression is elevated since the silencer is 2.5-fold further from the promoter than the enhancer and most likely depends on cohesin-mediated tethering to influence expression., (© 2021 The Authors. Published by Elsevier B.V.)

Abp1 is a fission yeast CENP-B homologue that contributes to centromere function, silencing at pericentromeric heterochromatin and silencing of retrotransposons. We identified the sfh1 gene, encoding a core subunit of the fission yeast chromatin remodeling complex RSC as an Abp1-interacting protein. Because sfh1 is essential for growth, we isolated temperature-sensitive sfh1 mutants. These mutants showed defects in centromere functions, reflected by sensitivity to an inhibitor of spindle formation and minichromosome instability. Sfh1 localized at both kinetochore and pericentromeric heterochromatin regions. Although sfh1 mutations had minor effect on silencing at these regions, they decreased the levels of cohesin on centromeric heterochromatin. Sfh1 also localized at a retrotransposon, Tf2, in a partly Abp1-dependent manner, and assisted in silencing of Tf2 by Abp1 probably in the same pathway as a histone chaperon, HIRA, which is also known to involve in Tf2 repression. Furthermore, sfh1 mutants were sensitive to several DNA-damaging treatments (HU, MMS, UV and X-ray). Increase in spontaneous foci of Rad22, a recombination Mediator protein Rad52 homologue, in sfh1 mutant suggests that RSC functions in homologous recombination repair of double-stranded break downstream of the Rad22 recruitment. These results indicate that RSC plays multiple roles in the maintenance of genome integrity., (© 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence. The use of this adapter in LM-PCR reduced the impairment of PCR by substrate DNA with a high GC content, compared with the use of traditional LM-PCR adapters. This result suggested that our method has the potential to contribute to reduction of the amplification bias that is caused by an intrinsic property of the sequence context in substrate DNA. A DNA preparation obtained from a chromatin immunoprecipitation assay using pulldown of a specific form of histone H3 was successfully amplified using the modified LM-PCR, and the amplified products could be used as probes in a fluorescence in situ hybridization analysis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

The human CYP19 gene encodes aromatase, which converts androgens to estrogens. CYP19 mRNA variants are transcribed mainly from three promoters. Quantitative RT-PCR was used to measure the relative amounts of each of the three transcripts and determine the on/off state of the promoters. While some of the promoters were silent, CYP19 mRNA production differed among the other promoters, whose estimated transcription levels were 0.001% to 0.1% of that of the TUBB control gene. To investigate the structural aspects of chromatin that were responsible for this wide range of activity of the CYP19 promoters, we used a fractionation protocol, designated SEVENS, which sequentially separates densely packed nucleosomes from dispersed nucleosomes. The fractional distribution of each inactive promoter showed a similar pattern to that of the repressed reference loci; the inactive regions were distributed toward lower fractions, in which closed chromatin comprising packed nucleosomes was enriched. In contrast, active CYP19 promoters were raised toward upper fractions, including dispersed nucleosomes in open chromatin. Importantly, these active promoters were moderately enriched in the upper fractions as compared to active reference loci, such as the TUBB promoter; the proportion of open chromatin appeared to be positively correlated to the promoter strength. These results, together with ectopic transcription accompanied by an increase in the proportion of open chromatin in cells treated with an H3K27me inhibitor, indicate that CYP19 mRNA could be transcribed from a promoter in which chromatin is shifted toward an open state in the equilibrium between closed and open chromatin.

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL), a malignancy of CD4(+) T cells whose etiology is thought to be associated with the viral trans-activator Tax. We have shown recently that Tax can drastically upregulate the expression of p27(Kip1) and p21(CIP1/WAF1) through protein stabilization and mRNA trans-activation and stabilization, respectively. The Tax-induced surge in p21(CIP1/WAF1) and p27(Kip1) begins in S phase and results in cellular senescence. Importantly, HeLa and SupT1 T cells infected by HTLV-1 also arrest in senescence, thus challenging the notion that HTLV-1 infection causes cell proliferation. Here we use time-lapse microscopy to investigate the effect of Tax on cell cycle progression in two reporter cell lines, HeLa/18x21-EGFP and HeLa-FUCCI, that express enhanced green fluorescent protein (EGFP) under the control of 18 copies of the Tax-responsive 21-bp repeat element and fluorescent ubiquitin cell cycle indicators, respectively. Tax-expressing HeLa cells exhibit elongated or stalled cell cycle phases. Many of them bypass mitosis and become single senescent cells as evidenced by the expression of senescence-associated β-galactosidase. Such cells have twice the normal equivalent of cellular contents and hence are enlarged, with exaggerated nuclei. Interestingly, nocodazole treatment revealed a small variant population of HeLa/18x21-EGFP cells that could progress into mitosis normally with high levels of Tax expression, suggesting that genetic or epigenetic changes that prevent Tax-induced senescence can occur spontaneously at a detectable frequency.

It has been demonstrated that the mammalian Ad4BP/SF-1 (NR5A1) gene is regulated precisely in sex, tissue, and developmental stage specific manners. To clarify the complex transcriptional regulation, we investigated in the present study whether the gene transcription is regulated by multiple promoters accompanied by noncoding first exons. Novel first exons (Io and Ig) were identified downstream of the already identified exon Ia. Nucleotide sequences revealed that Ia and Ig exons were well conserved, whereas Io exon was less conserved among the mouse, rat, and human genes. Interestingly, the splice donor of the mouse and human Io and human Ig exons do not satisfy the consensus sequence. Transcripts containing Ia, Io, and Ig were detected in all rat tissues examined, while the transcript containing Io was undetectable in the corresponding tissues of mice. The lack of exon Io usage in the mouse was confirmed by transient transfection assays with cultured cells. Quantitative RT-PCR analysis revealed that the transcript containing Ig exon was the main product in the pituitary but significantly less in the spleen, suggesting that the regulation of Ad4BP/SF-1 gene transcription in the pituitary and spleen is distinct from that of other tissues. The above findings, together with the structural abnormality at the splice donor site, suggest that acquisition of the multiple first exons enables the Ad4BP/SF-1 gene to be regulated differentially in different animal species and in different tissues in the same animal., (Copyright 2000 Academic Press.)

Acute promyelocytic leukemia (APL) has been characterized by 15;17 chromosomal translocation, which involves the retinoic acid receptor alpha (RARA) gene on chromosome 17 and the PML gene on chromosome 15. An extremely restricted region (ERR) of 50 bps within the second intron of the RARA gene was identified as the cluster region of breakpoints by sequencing analyses. ERR was tested by in vitro transfection-recombination assay, and was shown to be the recombination hot spot. In this study, presence of DNA binding proteins to the 148 bps DNA fragment which contains ERR was confirmed by gel-mobility shift analysis in the nuclear extract of NIH3T3 cells and human leukemia cell lines. Furthermore, in vitro study with the mouse sarcoma cell lines using the recombination reporter plasmid containing ERR showed that ERR might be involved in the homologous recombination in addition to the illegitimate recombination. The DNA binding proteins specific to ERR might play an important role in chromosome translocation.

ELP is a transcription factor belonging to the nuclear receptor superfamily. The consensus binding sequence for ELP contains a half site of the nuclear receptor recognition element. We demonstrated previously that ELP1, the repressor type isoform of ELP, competes for binding with the retinoic acid receptor and represses retinoic acid-induced transactivation. In this study, competitive repression by ELP1 was investigated for several other nuclear receptors. As in the case of the retinoic acid receptor, binding of vitamin D receptor, thyroid hormone receptor, and estrogen receptor could be competed by ELP1, resulting in repression of their ligand-dependent transactivation. Interestingly, the activator-type ELP isoforms were capable of repressing retinoic acid-induced transactivation through binding to the retinoic acid receptor binding element. These data suggest that competition for target DNA binding is a general mechanism of transcriptional repression by ELP isoforms.

The Rad51 protein, which is a homologue of the bacterial RecA protein, is involved in mitotic and meiotic recombination and in repair of double-strand breaks of DNA in yeast. The Rad51 homologue is conserved from yeast to human. In this study, the Rad51 protein was shown to be induced in peripheral blood lymphocytes (PBLs) 36 h after phytohemagglutinin (PHA) stimulation. Immunofluorescence study revealed that the distribution of the Rad51 protein in the nucleus was not uniform and focus-like staining was observed. Formation of the Rad51 foci was induced at 36 h after treatment of the cells with PHA. Twenty five percent of the cells had the foci at this time and the number of cells with foci declined thereafter. Cell cycle study using laser microscope by double staining method suggested that the appearance of the Rad51 nuclear foci was S phase specific. Furthermore, double staining study for the Rad51 protein and incorporated BrdU confirmed S phase specific appearance of the Rad51 nuclear foci. Formation of the Rad51 nuclear foci in PHA-stimulated lymphocytes might be involved in DNA recombination or DNA repair in S phase. The roles of RAD51 foci in S-phase will be discussed.

The chromatin structure of the ELP gene was analyzed by DNase I hypersensitivity. A strong DNase I hypersensitive site (Y1DH) was identified at the exon 1 region of the ELP gene in steroidogenic Y1 cells. A strong DNase 1 hypersensitive site (ECDH2) downstream of the exon 8 of the ELP gene and a weak DNase 1 hypersensitive site (ECDH1) upstream of the exon 2 of the ELP gene was identified in undifferentiated EC cells. These differences of the DNase I hypersensitive sites may be related to the differential expression of ELP isoforms in Y1 cells and EC cells. In addition, the gel shift assay and DMS protection assay revealed that ELP binds to the ECDH2 region.

Instability of microsatellite sequences are frequently found in human tumors. In addition, minisatellite sequences, another group of highly unstable sequences, serve as sensitive markers of genetic instability. We have studied minisatellite instability in methylcholanthrene-induced mouse sarcomas. These sarcomas frequently carry the amplified c-myc gene. Seven sarcomas without the amplification and seven others with the amplification were selected randomly. Regardless of the state of the c-myc gene amplification, these sarcomas exhibited a varying degree of transplantability in syngeneic mice. The hypervariable mouse minisatellite locus Ms6hm was found to be highly unstable, specifically among sarcomas with the amplified c-myc gene. However, chromosome instability, as analyzed by micronucleus assay, was observed similarly for two groups of sarcomas. In addition, transversion of G to C and A to T was detected at the K-ras gene in four of the seven sarcomas with the amplified c-myc gene, and these mutations are thought to be induced directly by methylcholanthrene. Thus, concomitant occurrence was observed for three seemingly unrelated mutations, amplification of the c-myc locus, point mutation of the K-ras gene, and instability at the hypervariable mouse minisatellite locus. The present study indicates a possible involvement of K-ras mutation and c-myc amplification in induction of genetic instability in methylcholanthrene-induced mouse sarcomas.

Analysis was made on the genomic structure, functions, and expression of the mouse ELP gene, which codes for the embryonal long terminal repeat binding protein. Extensive screening of the cDNA library of embryonal carcinoma cells (EC cells) identified four isoforms of ELP: ELP1 (the original ELP isolate), ELP2, ELP3, and Ad4BP/SF1. Analysis of the genomic sequences revealed that these ELP isoforms were generated by alternative promoter usage and differential splicing. The mRNAs of isoforms initiated at four transcription start sites distributed on three exons. Sequence analysis of the four isoforms identified three polypeptides. The N-terminal portion of ELP1 and ELP2 was longer than ELP3, and Ad4BP/SF1 by 77 aa. The DNA-binding domain and region II were shared by all four isoforms. The C-terminal portion shared by ELP2, ELP3, and Ad4BP/SF1 was 131 aa in length, and that specific to ELP1 was 57 aa in length. The ELP3 and Ad4BP/SF1 isoforms were identical for the coding sequence, but the two differed at the 5' noncoding region. Region II and III domains of nuclear receptors were thought to be involved in ligand-binding and transcriptional activation. ELP1, which lacked region III, functioned as a repressor. The isoforms carrying intact region II and region III functioned as transactivators. Expression of the four isoforms was studied in mouse tissues and in tissue culture cells by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Complex patterns of expression of these isoforms were observed in various tissues. All four ELP isoforms were expressed only in EC cells.

The mechanism of repression of transcription by ELP, the embryonal long terminal repeat binding protein, was investigated. ELP represses the Moloney murine leukemia virus long terminal repeat by binding to a site which overlaps with a sequence element for retinoic acid receptor binding. This suggests possible competition of ELP with retinoic acid receptor for the same sequence elements. Oligonucleotides corresponding to ELP and/or retinoic acid receptor binding elements were placed upstream of the SV40 promoter and their effect on gene expression was analyzed by CAT assay. Elements which have affinity to both ELP and retinoic acid receptor were activated by retinoic acid receptor and these activations were repressed by ELP. An ELP binding element without affinity to retinoic acid receptor was insensitive to both activation by retinoic acid receptor and repression by ELP. Furthermore, cellular ELP binding elements and the Moloney leukemia virus long terminal repeat were activated by retinoic acid. These data suggest that one of the mechanism of transcriptional repression by ELP is competition for binding sites with transactivators such as retinoic acid receptors.

Acute promyelocytic leukemia (APL) has been characterized by 15;17 chromosomal translocation, which involves the retinoic acid receptor alpha (RARA) gene on chromosome 17 and the PML gene on chromosome 15. The breakpoints have been mapped to three cluster regions in the PML gene, and to RARA gene intron 2. We have examined the distribution of breakpoints within RARA gene intron 2. An extremely restricted region (ERR) of 50 bp within RARA gene intron 2 was identified as the cluster region of breakpoints by polymerase chain reaction and sequence analysis of DNA from APL patients. To study experimentally the mechanism involved in the translocation, ERR was tested in NIH3T3 cells by in vitro transfection-recombination assay, in which target sequences were placed either downstream of the SV40 promoter or upstream of the neo gene. Cells were conferred resistance to G418 only when the promoter was fused to the neo gene by recombination of two target sequences during transfection. The molecular junctions were analysed in five clones, and all of them were shown to be confined within a 20 bp region in a 148 bp DNA fragment containing ERR. This suggests that ERR might be the illegitimate recombination hot spot in mammalian cells.

A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the beta-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the beta-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.