scholarly journals Histone ChIP-Seq identifies differential enhancer usage during chondrogenesis as critical for defining cell-type specificity

2019 ◽  
Author(s):  
Kathleen Cheung ◽  
Matthew J. Barter ◽  
Julia Falk ◽  
Carole Proctor ◽  
Louise N. Reynard ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factor ◽  
Human Mesenchymal Stem Cells ◽  
Cell Types ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Epigenetic Mechanisms ◽  
Enhancer Activity ◽  
Chromatin States

AbstractEpigenetic mechanisms are known to regulate gene expression during chondrogenesis. In this study, we have characterised the epigenome during in vitro differentiation of human mesenchymal stem cells (hMSCs) into chondrocytes. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) was used to assess a range of N-terminal post-transcriptional modifications (marks) to histone H3 lysines (H3K4me3, H3K4me1, H3K27ac, H3K27me3 and H3K36me3) in both hMSCs and differentiated chondrocytes. Chromatin states were characterised using histone ChIP-seq and cis-regulatory elements were identified in chondrocytes. Chondrocyte enhancers were associated with chondrogenesis related gene ontology (GO) terms. In silico analysis and integration of DNA methylation data with chondrogenesis chromatin states revealed that enhancers marked by histone marks H3K4me1 and H3K27ac were de-methylated during in vitro chondrogenesis. Similarity analysis between hMSC and chondrocyte chromatin states defined in this study with epigenomes of cell-types defined by the Roadmap Epigenomics project revealed that enhancers are more distinct between cell-types compared to other chromatin states. Motif analysis revealed that the transcription factor SOX9 is enriched in chondrocyte enhancers. Luciferase reporter assays confirmed that chondrocyte enhancers characterised in this study exhibited enhancer activity which may be modulated by inducing DNA methylation and SOX9 overexpression. Altogether, these integrated data illustrate the cross-talk between different epigenetic mechanisms during chondrocyte differentiation.SummaryHuman mesenchymal stem cells are able to differentiate into chondrocytes, the cell type found in cartilage, making them an accessible system to study gene regulation during this process. Epigenetic mechanisms such as histone modifications and DNA methylation together with transcription factor binding play a role in activating and repressing gene expression. In this study, we investigated the genome-wide histone modification changes during chondrocyte differentiation. Integration of this data with DNA methylation and SOX9 transcription factor ChIP-seq revealed epigenetic changes at gene enhancer elements. Regions of the genome that transition from non-enhancers to enhancers in chondrocytes are enriched for SOX9 transcription factor binding sites. Luciferase reporter assays revealed that enhancer activity may be modulated by manipulating DNA methylation and SOX9 expression. This study has defined important regulatory elements in chondrocytes which could serve as targets for future mechanistic studies.

scholarly journals 4277 Functional consequences of the juvenile idiopathic arthritis risk variant at 1q24.3

2020 ◽  
Vol 4 (s1) ◽  
pp. 95-96
Author(s):  
Halima Moncrieffe ◽  
Katelyn Dunn ◽  
Yongbo Huang ◽  
Xiaoting Chen ◽  
Carl D. Langefeld ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Juvenile Idiopathic Arthritis ◽  
Transcription Factor Binding ◽  
Luciferase Reporter ◽  
Common Disease ◽  
Reference Allele ◽  
Enhancer Activity ◽  
Risk Variant ◽  
Factor Binding

OBJECTIVES/GOALS: Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatologic disease childhood and a cause of pain and potential disability. JIA has a strong genetic component and no known cure. The goal of this study is to evaluate allele-dependent effects of a novel JIA risk variant at 1q24.3. METHODS/STUDY POPULATION: JIA patients meeting criteria for the two most common disease subtypes (oligoarticular and RF neg polyarthritis) were genotyped using the Immunochip, an Illumina array with dense coverage of the HLA region and 186 other loci previously reported in autoimmune diseases. Phase I association findings (Hinks, 2013) and Phase II analysis (unpublished) of an expanded cohort (4,271 JIA and 14,390 controls) identified new risk loci, including rs78037977 at 1q24.3. We prioritized rs78037977 and predicted possible impacted mechanisms based on Bayesian predictions of attributable risk, the surrounding chromatin landscape, and transcription factor binding data. A luciferase reporter assay was used to assess allele-dependent enhancer activity. RESULTS/ANTICIPATED RESULTS: rs78037977 is located between FASLG and TNFSF18 at chromosome 1q24.3 is associated with JIA (p = 6.3x10−09), and explains 94% of the posterior probability at this locus; no other SNPs in linkage disequilibrium (r2>0.6). The chromatin landscape around rs78037977 contains H3K4Me1 and H3K27Ac marks, which are indicative of enhancer activity. Further, >160 transcription factors have chromatin immunoprecipitation followed by sequencing (ChIP-seq) peaks overlapping rs78037977 in various cellular contexts. In luciferase reporter assays, the region around rs78037977 containing the reference A allele had ~2-fold increased enhancer activity compared to the non-reference allele. DISCUSSION/SIGNIFICANCE OF IMPACT: This work provides in vitro evidence to support allele-dependent enhancer activity of a novel JIA-risk variant at 1q24.3. Our ongoing work investigates the effect of the DNA-containing region of rs78037977 on gene expression and differential transcription factor binding at rs78037977.

scholarly journals CpG traffic lights are markers of regulatory regions in humans

2016 ◽  
Author(s):  
Abdullah M. Khamis ◽  
Anna V. Lioznova ◽  
Artem V. Artemov ◽  
Vasily Ramensky ◽  
Vladimir B. Bajic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Regulation Of Gene Expression ◽  
Cell Types ◽  
Regulatory Elements ◽  
Cpg Methylation ◽  
Population Heterogeneity ◽  
Gene Body Methylation ◽  
Enhancer Activity ◽  
Traffic Lights

AbstractDNA methylation is involved in regulation of gene expression. Although modern methods profile DNA methylation at single CpG sites, methylation levels are usually averaged over genomic regions in the downstream analyses. In this study we demonstrate that single CpG methylation can serve as a more accurate predictor of gene expression compared to average promoter / gene body methylation. CpG positions with significant correlation between methylation and expression of a gene nearby (named CpG traffic lights) are evolutionary conserved and enriched for exact TSS positions and active enhancers. Among all promoter types, CpG traffic lights are especially enriched in poised promoters. Genes that harbor CpG traffic lights are associated with development and signal transduction. Methylation levels of individual CpG traffic lights vary between cell types dramatically with the increased frequency of intermediate methylation levels, indicating cell population heterogeneity in CpG methylation levels. Being in line with the concept of the inherited stochastic epigenetic variation, methylation of such CpG positions might contribute to transcriptional regulation. Alternatively, one can hypothesize that traffic lights are markers of absent gene expression resulting from inactivation of their regulatory elements. The CpG traffic lights provide a promising insight into mechanisms of enhancer activity and gene regulation linking methylation of single CpG to expression.

scholarly journals cis-Acting Elements within the Candida albicans ERG11 Promoter Mediate the Azole Response through Transcription Factor Upc2p

2007 ◽  
Vol 6 (12) ◽  
pp. 2231-2239 ◽  
Author(s):  
Brian G. Oliver ◽  
Jia L. Song ◽  
Jake H. Choiniere ◽  
Theodore C. White
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Regulatory Elements ◽  
Antifungal Drugs ◽  
Start Codon ◽  
Luciferase Reporter ◽  
Ergosterol Biosynthesis ◽  
Luciferase Reporter Gene ◽  
Enhancer Element

ABSTRACT The azole antifungal drugs are used to treat infections caused by Candida albicans and other fungi. These drugs interfere with the biosynthesis of ergosterol, the major sterol in fungal cells, by inhibiting an ergosterol biosynthetic enzyme, lanosterol 14 α-demethylase, encoded by the ERG11 gene. In vitro, these drugs as well as other ergosterol biosynthesis inhibitors increase ERG11 mRNA expression by activation of the ERG11 promoter. The signal for this activation most likely is the depletion of ergosterol, the end product of the pathway. To identify cis-acting regulatory elements that mediate this activation, ERG11 promoter fragments have been fused to the luciferase reporter gene from Renilla reniformis. Promoter deletions and linker scan mutations localized the region important for azole induction to a segment from bp −224 to −251 upstream of the start codon, specifically two 7-bp sequences separated by 13 bp. These sequences form an imperfect inverted repeat. The region is recognized by the transcription factor Upc2p and functions as an enhancer of transcription, as it can be placed upstream of a heterologous promoter in either direction, resulting in the azole induction of that promoter. The promoter constructs are not azole inducible in the upc2/upc2 homozygous deletion, demonstrating that Upc2p controls the azole induction of ERG11. These results identify an azole-responsive enhancer element (ARE) in the ERG11 promoter that is controlled by the Upc2p transcription factor. No other ARE is present in the promoter. Thus, this ARE and Upc2p are necessary and sufficient for azole induction of ERG11.

scholarly journals Long Noncoding RNA LncPGCR mediated by TCF7L2 regulates chicken PGCs formation

2020 ◽  
Author(s):  
Jingyi Jiang ◽  
Qisheng Zuo ◽  
Shaoze Cheng ◽  
Xia Yuan ◽  
Jing Jin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factor ◽  
Histone Acetylation ◽  
Noncoding Rna ◽  
Signal Pathway ◽  
Luciferase Reporter ◽  
Over Expression ◽  
Phosphorylation Level

Abstract Background: Several of the thousands of long noncoding RNAs (lncRNAs) have been functionally characterized, yet its specific function and molecular mechanism in the formation of chicken primordial germ cells (PGCs) remains poorly understood. In the present study, we aim to investigate the role of LncPGCR (LncRNA PGCs Regulator) in PGCs formation.Methods: LncPGCR, Cvh, Nanog, C-kit, gga-mir-6577-5p and Btrc expressions were detected by qRT-PCR. The percentage of PGCs cells was detected by flow cytometry, immunocytochemistry, and PAS staining. The interaction of histone acetylation, DNA methylation, transcription factor TCF7L2, and LncPGCR was confirmed by Luciferase reporter assay. The interaction of GAPDH and LncPGCR was measured by RNA pull-down, RIP, and Western blot.Results: We observed the increased expression of LncPGCR in PGCs. It is mainly expressed in the cytoplasm and encodes small peptides. Moreover, over-expression of LncPGCR could promote PGCs formation in vitro and in vivo. Besides, we first reported that histone acetylation, DNA methylation, and transcription factor TCF7L2 can regulate LncPGCR expression in PGCs. In addition, LncPGCR activates WNT signaling pathways to promote PGCs formation by adsorbing gga-mir-6577-5p, relieving its inhibitory effect on target gene Btrc. Meanwhile, LncPGCR contributed to PGCs formation by increasing the phosphorylation level of GAPDH to activate the TGF-β signal pathway.Conclusion: LncPGCR over-expression promoted ESCs differentiation into PGCs through the potential LncPGCR/miR-6577-5p/BTRC pathway or increasing the phosphorylation level of GAPDH to activate the TGF-β signal pathway.

scholarly journals Epigenetic Modifications During Sex Change Repress Gonadotropin Stimulation of Cyp19a1a in a Teleost Ricefield Eel (Monopterus albus)

Endocrinology ◽  
2013 ◽  
Vol 154 (8) ◽  
pp. 2881-2890 ◽  
Author(s):  
Yang Zhang ◽  
Shen Zhang ◽  
Zhixin Liu ◽  
Lihong Zhang ◽  
Weimin Zhang
Keyword(s):  
Dna Methylation ◽  
Binding Protein ◽  
Sex Change ◽  
Gonadal Development ◽  
Proximal Region ◽  
Histone Deacetylation ◽  
Epigenetic Mechanisms ◽  
Gonadal Differentiation ◽  
Stimulation Of

Abstract In vertebrates, cytochrome P450 aromatase, encoded by cyp19a1, converts androgens to estrogens and plays important roles in gonadal differentiation and development. The present study examines whether epigenetic mechanisms are involved in cyp19a1a expression and subsequent gonadal development in the hermaphroditic ricefield eel. The expression of the ricefield eel cyp19a1a was stimulated by gonadotropin via the cAMP pathway in the ovary but not the ovotestis or testis. The CpG within the cAMP response element (CRE) of the cyp19a1a promoter was hypermethylated in the ovotestis and testis compared with the ovary. The methylation levels of CpG sites around CRE in the distal region (region II) and around steroidogenic factor 1/adrenal 4 binding protein sites and TATA box in the proximal region (region I) were inversely correlated with cyp19a1a expression during the natural sex change from female to male. In vitro DNA methylation decreased the basal and forskolin-induced activities of cyp19a1a promoter. Chromatin immunoprecipitation assays indicated that histone 3 (Lys9) in both regions I and II of the cyp19a1a promoter were deacetylated and trimethylated in the testis, and in contrast to the ovary, phosphorylated CRE-binding protein failed to bind to these regions. Lastly, the DNA methylation inhibitor 5-aza-2′-deoxycytidine reversed the natural sex change of ricefield eels. These results suggested that epigenetic mechanisms involving DNA methylation and histone deacetylation and methylation may abrogate the stimulation of cyp19a1a by gonadotropins in a male-specific fashion. This may be a mechanism widely used to drive natural sex change in teleosts as well as gonadal differentiation in other vertebrates.

Abstract 1384: Osteogenic Transcription Factor Runx2 Regulates Expression of RANKL during VSMC Calcification

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Chang Hyun Byon ◽  
Jay McDonald ◽  
Yabing Chen
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Molecular Mechanism ◽  
Luciferase Reporter ◽  
Rankl Expression ◽  
Reporter System ◽  
Mobility Shift ◽  
Atherosclerotic Lesions ◽  
Flanking Region

The expression of receptor activator of nuclear factor κ B (RANKL) is up-regulated in calcified atherosclerotic lesions, whereas it is frequently undetectable in normal vessels. The underlying molecular mechanism of increased expression of RANKL in calcified vessels is not known. We have previously demonstrated that oxidative stress induces calcification of vascular smooth muscle cells (VSMC) in vitro . Therefore, we determined whether oxidative stress regulates RANKL expression in VSMC and the underlying molecular mechanism. Consistent with previous observations in vivo , we found that the expression of RANKL in VSMC isolated from mouse. However, hydrogen peroxide (H 2 O 2 ), which induces VSMC calcification, induced a 33-fold increase in the transcripts of RANKL as determined by real-time PCR. Increased expression of RANKL protein was further confirmed by ELISA. Using flow cytometry, we demonstrated that membrane-bound RANKL was increased by oxidative stress. To characterize the molecular mechanism underlying H 2 O 2 -induced RANKL expression, we employed the luciferase reporter system with a series of deletion mutants of the RANKL 5′-flanking region. The H 2 O 2 responsive region is located between −200 to −400 in the 5′-flanking region of RANKL gene. Analyses of the sequence of this region identified multiple binding sites for the key osteogenic transcription factor, Runx2, which we previously reported to be an essential regulator of VSMC calcification. Electrophoretic mobility shift analyses demonstrated increased binding of Runx2 on the RANKL promoter sequence in nuclear extracts from VSMC exposed to H 2 O 2 . To further determine the role of Runx2 in regulating RANKL expression, we generated stable Runx2 knockdown VSMC with the use of lentivirus-carrying shRNA for Runx2 gene. H 2 O 2 -induced RANKL expression was abrogated in VSMC with Runx2 knockdown. In addition, adenovirus-mediated overexpression of Runx2 in VSMC induced the expression of RANKL. In summary, we have demonstrated that H 2 O 2 induces the expression of RANKL in VSMC, which is regulated by the osteogenic transcription factor Runx2. These observations provide novel molecular insights into the regulation of RANKL and its role on the pathogenesis of calcified atherosclerotic lesions.

Regulation of the twist target gene tinman by modular cis-regulatory elements during early mesoderm development

Development ◽  
1997 ◽  
Vol 124 (24) ◽  
pp. 4971-4982 ◽  
Author(s):  
Z. Yin ◽  
X.L. Xu ◽  
M. Frasch
Keyword(s):  
Homeobox Gene ◽  
Regulatory Elements ◽  
Bhlh Protein ◽  
Expression Domain ◽  
Enhancer Activity ◽  
Enhancer Element ◽  
Mesoderm Development ◽  
Visceral Mesoderm

The Drosophila tinman homeobox gene has a major role in early mesoderm patterning and determines the formation of visceral mesoderm, heart progenitors, specific somatic muscle precursors and glia-like mesodermal cells. These functions of tinman are reflected in its dynamic pattern of expression, which is characterized by initial widespread expression in the trunk mesoderm, then refinement to a broad dorsal mesodermal domain, and finally restricted expression in heart progenitors. Here we show that each of these phases of expression is driven by a discrete enhancer element, the first being active in the early mesoderm, the second in the dorsal mesoderm and the third in cardioblasts. We provide evidence that the early-active enhancer element is a direct target of twist, a gene encoding a basic helix-loop-helix (bHLH) protein, which is necessary for tinman activation. This 180 bp enhancer includes three E-box sequences which bind Twist protein in vitro and are essential for enhancer activity in vivo. Ectodermal misexpression of twist causes ectopic activation of this enhancer in ectodermal cells, indicating that twist is the only mesoderm-specific activator of early tinman expression. We further show that the 180 bp enhancer also includes negatively acting sequences. Binding of Even-skipped to these sequences appears to reduce twist-dependent activation in a periodic fashion, thus producing a striped tinman pattern in the early mesoderm. In addition, these sequences prevent activation of tinman by twist in a defined portion of the head mesoderm that gives rise to hemocytes. We find that this repression requires the function of buttonhead, a head-patterning gene, and that buttonhead is necessary for normal activation of the hematopoietic differentiation gene serpent in the same area. Together, our results show that tinman is controlled by an array of discrete enhancer elements that are activated successively by differential genetic inputs, as well as by closely linked activator and repressor binding sites within an early-acting enhancer, which restrict twist activity to specific areas within the twist expression domain.

scholarly journals Architectural Epigenetics: Mitotic Retention of Mammalian Transcriptional Regulatory Information

2010 ◽  
Vol 30 (20) ◽  
pp. 4758-4766 ◽  
Author(s):  
Sayyed K. Zaidi ◽  
Daniel W. Young ◽  
Martin Montecino ◽  
Jane B. Lian ◽  
Janet L. Stein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Transcription Factor ◽  
Epigenetic Mechanisms ◽  
Cellular Phenotype ◽  
Transcriptional Regulatory ◽  
Regulatory Information ◽  
Cellular Identity ◽  
Epigenetic Signatures ◽  
Transcription Factor Occupancy

ABSTRACT Epigenetic regulatory information must be retained during mammalian cell division to sustain phenotype-specific and physiologically responsive gene expression in the progeny cells. Histone modifications, DNA methylation, and RNA-mediated silencing are well-defined epigenetic mechanisms that control the cellular phenotype by regulating gene expression. Recent results suggest that the mitotic retention of nuclease hypersensitivity, selective histone marks, as well as the lineage-specific transcription factor occupancy of promoter elements contribute to the epigenetic control of sustained cellular identity in progeny cells. We propose that these mitotic epigenetic signatures collectively constitute architectural epigenetics, a novel and essential mechanism that conveys regulatory information to sustain the control of phenotype and proliferation in progeny cells by bookmarking genes for activation or suppression.

scholarly journals The disruption of circadian clockwork in differentiating cells from rat reproductive tissues as identified by in vitro real-time monitoring system

2007 ◽  
Vol 193 (3) ◽  
pp. 413-420 ◽  
Author(s):  
Pei-Jian He ◽  
Masami Hirata ◽  
Nobuhiko Yamauchi ◽  
Seiichi Hashimoto ◽  
Masa-aki Hattori
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Monitoring System ◽  
Cell Types ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Peripheral Tissues ◽  
Reproductive Tissues ◽  
Clock Gene Expression

The circadian clock, regulating hormonal secretion and metabolisms in accordance with the environmental light–dark cycle, resides in almost all peripheral tissues as well as in the superchiasmatic nucleus. Clock gene expression has been found to be noncyclic during spermatogenesis and the differentiation of thymocytes. However, currently little is known about how cell differentiation could affect circadian clockwork. We performed this study using the in vitro real-time oscillation monitoring system to examine the clockwork in several types of differentiating cells originated from reproductive tissues of transgenic rats (constructed with Period gene 2 (Per2) promoter-destabilized luciferase reporter gene). After treatment with dexamethasone (DXM), persistent oscillation of Per2 expression was observed in both gonadotropin-induced and pregnant ovarian luteal cells, proliferative uterine stromal cells (USCs), and nondifferentiating testicular interstitial cells, with a cyclic period of ~24 h. In contrast to these cell types, only one cycle of oscillation was sustained in granulosa cells undergoing differentiation. Additionally, Per2 oscillation was irregular in USCs undergoing decidualization induced by medroxyprogesterone acetate plus N6, 2-O-dibutyryl adenosine 3′:5′-cyclic monophosphate. Furthermore, no oscillation of Per2 expression was evoked by DXM in Leydig cells and thymocytes. In conclusion, the present study characterized the oscillation of Per2 gene expression in several types of ovarian, uterine, and testicular cells, and it is strongly suggested that circadian clockwork is affected during cellular differentiation.

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