Transcription factor C/EBPß induces genome-wide H3K27ac and upregulates gene expression during decidualization of human endometrial stromal cells.

We previously reported that H3K27 acetylation (H3K27ac) increases throughout the genome during decidualization of human endometrial stromal cells (ESCs). However, its mechanisms have not been clarified. We also reported that C/EBPß acts as a pioneer factor initiating chromatin remodeling by increasing H3K27ac of IGFBP-1 and PRL promoters. Therefore, C/EBPß may be involved in the genome-wide increase of H3K27ac during decidualization. In this study, we investigated whether C/EBPß causes genome-wide H3K27ac modifications and regulates gene expressions during decidualization. cAMP was used to induce decidualization. Three types of cells (control cells, cAMP-treated cells, and cAMP-treated + C/EBPß-knockdowned cells by siRNA) were generated. Of 4190 genes that were upregulated by cAMP, C/EBPß knockdown inhibited these upregulation in 2239 genes (53.4%), indicating that they are under the regulation of C/EBPß. cAMP increased H3K27ac in 1272 of the 2239 genes. C/EBPß knockdown abolished the increase of H3K27ac in almost all genes (1263 genes, 99.3%), suggesting that C/EBPß can upregulate gene expression by increasing H3K27ac. To investigate how C/EBPß regulates H3K27ac throughout the genome, we tested the hypothesis that C/EBPß binds to its binding regions and recruits cofactors with histone acetyltransferase activities. To do this, we collated our ChIP-sequence data with public ChIP-sequence database of transcription factors, and found that p300 is the most likely cofactor that binds to the H3K27ac-increased-regions with C/EBPß. ChIP-qPCR of several genes confirmed that C/EBPß binds to the target regions, recruits p300, and increases H3K27ac. Our genome-wide analysis revealed that C/EBPß induces H3K27ac throughout the genome and upregulates gene expressions during decidualization by recruiting p300 to the promoters.

FABP7 Regulates Acetyl-CoA Metabolism Through the Interaction with ACLY in the Nucleus of Astrocytes.

Fatty acid binding protein 7 (FABP7) is an intracellular fatty acid chaperon that is highly expressed in astrocytes, oligodendrocyte-precursor cells, and malignant glioma. Previously, we reported that FABP7 regulates the response to extracellular stimuli by controlling the expression of caveolin-1, an important component of lipid raft. Here, we explored the detailed mechanisms underlying FABP7 regulation of caveolin-1 expression using primary cultured FABP7-KO astrocytes as a model of loss of function and NIH-3T3 cells as a model of gain of function. We discovered that FABP7 interacts with ATP-citrate lyase (ACLY) and is important for acetyl-CoA metabolism in the nucleus. This interaction leads to epigenetic regulation of several genes, including caveolin-1. Our novel findings suggest that FABP7-ACLY modulation of nuclear acetyl-CoA has more influence on histone acetylation than cytoplasmic acetyl-CoA. The changes to histone structure may modify caveolae-related cell activity in astrocytes and tumors, including malignant glioma.

Glucose regulates the histone acetylation of gene promoters in decidualizing stromal cells.

Decidualization stimuli activate the insulin signaling pathway and increase the glucose uptake in human endometrial stromal cells (ESCs). The inductions of prolactin (PRL) and IGF-binding protein-1 (IGFBP1), specific markers of decidualization, were inhibited by incubating ESCs under low glucose concentrations. These results suggested that decidualization stimuli activate the insulin signaling pathway, which contributes to decidualization through the increase of glucose uptake. Here, we investigated the mechanisms by which glucose regulates decidualization. ESCs were incubated with cAMP to induce decidualization. We examined whether low glucose affects the expression levels of transcription factors that induce decidualization. Forkhead box O1 (FOXO1) expression was significantly suppressed under low glucose conditions. Knockdown of FOXO1 by siRNA inhibited the expression levels of PRL and IGFBP1 during decidualization. Taken together, our results showed that low glucose inhibits decidualization by decreasing FOXO1 expression. We also examined the levels of histone H3K27 acetylation (H3K27ac), which is related to active transcription, of the promoter regions of FOXO1, PRL and IGFBP1 by ChIP assay. The H3K27ac levels of these promoter regions were increased by decidualization under normal glucose conditions, but not under low glucose conditions. Thus, our results show that glucose is indispensable for decidualization by activating the histone modification status of the promoters of PRL, IGFBP1 and FOXO1.

The distal upstream region of insulin-like growth factor-binding protein-1 enhances its expression in endometrial stromal cells during decidualization.

We have previously shown that decidualization of human endometrial stromal cells (ESCs) causes a genome-wide increase in the levels of acetylation of histone-H3 Lys-27 (H3K27ac). We also reported that the distal gene regions, more than 3 kb up- or downstream of gene transcription start sites have increased H3K27ac levels. Insulin-like growth factor-binding protein-1 (IGFBP-1) is a specific decidualization marker and has increased H3K27ac levels in its distal upstream region (-4701 to -7501 bp). Here, using a luciferase reporter gene construct containing this IGFBP-1 upstream region, we tested the hypothesis that it is an IGFBP-1 enhancer. To induce decidualization, we incubated ESCs with cAMP and found that cAMP increased luciferase expression, indicating that decidualization increased the transcriptional activity from the IGFBP-1 upstream region. Furthermore, CRISPR/Cas9-mediated deletion of this region in HepG2 cells significantly reduced IGFBP-1 expression, confirming its role as an IGFBP-1 enhancer. A ChIP assay revealed that cAMP increased the recruitment of the transcriptional regulators CCAAT enhancer-binding protein ß (C/EBPß), forkhead box O1 (FOXO1), and p300 to the IGFBP-1 enhancer in ESCs. Of note, C/EBPß knockdown inhibited the stimulatory effects of cAMP on the levels of H3K27ac, chromatin opening, and p300 recruitment at the IGFBP-1 enhancer. These results indicate that the region -4701 to -7501 bp upstream of IGFBP-1 functions as an enhancer for IGFBP-1 expression in ESCs undergoing decidualization, that C/EBPß and FOXO1 bind to the enhancer region to up-regulate IGFBP-1 expression, and that C/EBPß induces H3K27ac by recruiting p300 to the IGFBP-1 enhancer.

Identification of uterine leiomyoma-specific marker genes based on DNA methylation and their clinical application.

Differential diagnosis of uterine leiomyomas and leiomyosarcomas is needed to determine whether the uterus can be retained. Therefore, biomarkers for uterine leiomyomas, and reliable and objective diagnostic methods have been desired besides the pathological diagnosis. In the present study, we identified 12 genes specific to uterine leiomyomas based on DNA methylation. Using these marker genes specific to uterine leiomyomas, we established a hierarchical clustering system based on the DNA methylation level of the marker genes, which could completely differentiate between uterine leiomyomas and normal myometrium. Furthermore, our hierarchical clustering system completely discriminated uterine cancers and differentiated between uterine leiomyosarcomas and leiomyomas with more than 70% accuracy. In conclusion, this study identified DNA methylation-based marker genes specific to uterine leiomyomas, and our hierarchical clustering system using these marker genes was useful for differential diagnosis of uterine leiomyomas and leiomyosarcomas.

Identification of consensus motifs associated with mitotic recombination and clinical characteristics in patients with paternal uniparental isodisomy of chromosome 11.

Uniparental disomy (UPD) is defined as the inheritance of both homologs of a given genomic region from only one parent. The majority of UPD includes an entire chromosome. However, the extent of UPD is sometimes limited to a subchromosomal region (segmental UPD). Mosaic paternal UPD (pUPD) of chromosome 11 is found in approximately 20% of patients with Beckwith-Wiedemann syndrome (BWS) and almost all pUPDs are segmental isodisomic pUPDs resulting from mitotic recombination at an early embryonic stage. A mechanism initiating a DNA double strand break (DSB) within 11p has been predicted to lead to segmental pUPD. However, no consensus motif has yet been found. Here, we analyzed 32 BWS patients with pUPD by SNP array and searched for consensus motifs. We identified four consensus motifs frequently appearing within breakpoint regions of segmental pUPD. These motifs were found in another nine BWS patients with pUPD. In addition, the seven motifs found in meiotic recombination hot spots could not be found within pUPD breakpoint regions. Histone H3 lysine 4 trimethylation, a marker of DSB initiation, could not be found either. These findings suggest that the mechanism(s) of mitotic recombination leading to segmental pUPD are different from that of meiotic recombination. Furthermore, we found seven patients with paternal uniparental diploidy (PUD) mosaicism. Comparison of clinical features between segmental pUPDs and PUDs showed that developmental disability and cardiac abnormalities were additional characteristic features of PUD mosaicism, along with high risk of tumor development. We also found that macroglossia was characteristic of segmental pUPD mosaicism.

Tissue-Specific Expression of Estrogen Receptor 1 Is Regulated by DNA Methylation in a T-DMR.

The mechanism controlling tissue-specific expression of estrogen receptor 1 (ESR1) is unclear. In other genes, DNA methylation of a region called the tissue-dependent and differentially methylated region (T-DMR) has been associated with tissue-specific gene expression. This study investigated whether human ESR1 has a T-DMR and whether DNA methylation of the T-DMR regulates its expression. ESR1 expression was tissue-specific, being high in the endometrium and mammary gland and low/nil in the placenta and skin. Therefore, DNA methylation profiles of the promoter of ESR1 were analyzed in these tissues and in breast cancer tissues. In all of the normal tissues, the proximal promoter regions were unmethylated. On the other hand, the distal regions (T-DMR) were unmethylated in the endometrium and mammary gland, but were moderately methylated and hypermethylated in the placenta and skin, respectively. T-DMR-methylated reporter assay was performed to examine whether DNA methylation at the T-DMR suppresses ESR1 transcription. T-DMR, but not the promoter region, had transcriptional activities and DNA methylation of the T-DMR suppressed ESR1 transcription. Early growth response protein 1 was shown to be a possible transcription factor to bind the T-DMR and up-regulate ESR1 expression. ESR1 has several upstream exons, and each upstream exon, Exon-A/Exon-B/Exon-C, had its own T-DMR. In some breast cancer cases and breast cancer cell lines, ESR1 expression was not regulated by DNA methylation at T-DMR as it is in normal tissues. In conclusion, ESR1 has a T-DMR. DNA methylation status at the T-DMR is involved in tissue-specific ESR1 expression in normal tissues but not always in breast cancer.

Retinoic acid has the potential to suppress endometriosis development.

BACKGROUND: Despite endometriosis is common estrogen dependent disease afflicting women in reproductive age, the pathogenesis has not been fully elucidated. Retinoic acid has various functions in cells as biologic modulator, and aberrant retinoid metabolism seems to be involved in the lesions of endometriosis. In order to evaluate the potential of all-trans retinoic acid (ATRA) for therapeutic treatment, a transcriptome analysis and estradiol measurements in cultured endometriotic cells and tissues were conducted. METHODS: The mRNA expression levels in ATRA-treated endometriotic stromal cells (ESC) isolated from ovarian endometrial cysts (OEC) were investigated. Estradiol production in OEC tissues was also investigated. RESULTS: In the isolated ESC culture supplemented with ATRA for four days, total RNA was extracted followed by a transcriptome analysis using GeneChip. Forty-nine genes were upregulated and four genes were down-regulated by the ATRA treatment. Many upregulated genes were associated with the negative regulation of cellular proliferation. In addition, ATRA treatment decreased the mRNA expression of 17-beta-dehydrogenase 2 (HSD17B2) which converts estradiol into estrone in a dose-dependent manner, and the ELISA measurements indicated that estradiol production in the OEC tissue was inhibited by ATRA treatment. CONCLUSIONS: Retinoic acid has the potential to suppress endometriosis development.

Fibroadenoma in Beckwith-Wiedemann syndrome with paternal uniparental disomy of chromosome 11p15.5.

Herein is described a case of breast fibroadenomas in a 16-year-old girl with Beckwith-Wiedemann syndrome (BWS) and uniparental disomy (UPD) of chromosome 11p15.5. She was clinically diagnosed with BWS and direct closure was performed for an omphalocele at birth. Subtotal and 90% pancreatectomy were performed for nesidioblastosis at the ages 2 months and 8 years, respectively. Bilateral multiple breast fibroadenomas were noted at the age of 16 and 17 years. In this case, paternal UPD of chromosome 11p15.5 was identified on microsatellite marker analysis. The relevant imprinted chromosomal region in BWS is 11p15.5, and UPD of chromosome 11p15 is a risk factor for BWS-associated tumorigenicity. Chromosome 11p15.5 consists of imprinting domains of IGF2, the expression of which is associated with the tumorigenesis of various breast cancers. This case suggests that fibroadenomas occurred in association with BWS.

Genome-wide analysis of histone modifications in human endometrial stromal cells.

Dramatic changes of gene expressions occur in human endometrial stromal cells (ESCs) during decidualization. The changes in gene expression are associated with changes of chromatin structure, which are regulated by histone modifications. Here we investigated genome-wide changes in histone modifications associated with decidualization in human ESCs using chromatin immunoprecipitation combined with next-generation sequencing. ESCs were incubated with estradiol and medroxyprogesterone acetate for 14 days to induce decidualization. The chromatin immunoprecipitation-sequence data showed that induction of decidualization increased H3K27ac and H3K4me3 signals in many genomic regions but decreased in only a few regions. Most of the H3K27ac-increased regions (80%) and half of the H3K4me3-increased regions were located in the distal promoter regions (more than 3 kb upstream or downstream of the transcription start site). RNA sequence showed that induction of decidualization up-regulated 881 genes, 223 of which had H3K27ac- or H3K4me3-increased regions in the proximal and distal promoter regions. Induction of decidualization increased the mRNA levels of these genes more than it increased the mRNA levels of genes without H3K27ac- or H3K4me3-increased regions. Pathway analysis revealed that up-regulated genes with the H3K27ac- or H3K4me3-increased regions were associated with the insulin signaling, which may be involved in glucose uptake that is necessary for ESCs to undergo decidualization. These results show that histone modification statuses on a genome-wide basis change in human ESCs during decidualization. The main changes of histone modifications are increases of H3K27ac and H3K4me3 in both the proximal and distal promoter regions, which are involved in the up-regulation of gene expression that occurs during decidualization.

Differences in distribution of single nucleotide polymorphisms among intracellular pattern recognition receptors in pigs.

Pathogens localized extracellularly or incorporated into endosomes are recognized mainly by Toll-like receptors, whereas pathogens and pathogen-derived molecules that invade into the cytoplasm of host cells typically are recognized by intracellular pattern recognition receptors (PRRs), such as retinoic acid-inducible gene (RIG)-like helicases (RLHs) and nucleotide-binding oligmerization domain (NOD)-like receptors (NLRs). RIG-I and melanoma differentiation-associated gene 5 (MDA5), which belong to the RLH family, recognize viral genomic RNA, whereas NOD2, a member of the NLR family, responds to microbial peptidoglycans. These receptors may play an important role in pig opportunistic infectious diseases, such as pneumonia and diarrhea, which markedly impair livestock productivity, such that polymorphisms of these receptor genes are potential targets of pig breeding to increase disease resistance. Here, we report single nucleotide polymorphisms (SNPs) in porcine DDX58, IFIH1, and NOD2, which encode RIG-I, MDA5, and NOD2, respectively. Interestingly, compared with DDX58 and IFIH1, NOD2 abounded in nonsynonymous SNPs both throughout the coding sequence and in sequences encoding domains important for ligand recognition, such as helicase domains for RIG-I and MDA5 and leucine-rich repeats in NOD2. These differences in the distribution of SNPs in intracellular PRRs may parallel the diversity of their ligands, which include nucleic acids and peptidoglycans.