scholarly journals CRISPRi screens reveal a DNA methylation-mediated 3D genome dependent causal mechanism in prostate cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Musaddeque Ahmed ◽  
Fraser Soares ◽  
Ji-Han Xia ◽  
Yue Yang ◽  
Jing Li ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Dna Methylation ◽  
Cell Proliferation ◽  
Cell Line ◽  
Regulatory Elements ◽  
Ctcf Binding ◽  
Ctcf Binding Site ◽  
Causal Mechanism ◽  
3D Genome ◽  
Risk Snps

AbstractProstate cancer (PCa) risk-associated SNPs are enriched in noncoding cis-regulatory elements (rCREs), yet their modi operandi and clinical impact remain elusive. Here, we perform CRISPRi screens of 260 rCREs in PCa cell lines. We find that rCREs harboring high risk SNPs are more essential for cell proliferation and H3K27ac occupancy is a strong indicator of essentiality. We also show that cell-line-specific essential rCREs are enriched in the 8q24.21 region, with the rs11986220-containing rCRE regulating MYC and PVT1 expression, cell proliferation and tumorigenesis in a cell-line-specific manner, depending on DNA methylation-orchestrated occupancy of a CTCF binding site in between this rCRE and the MYC promoter. We demonstrate that CTCF deposition at this site as measured by DNA methylation level is highly variable in prostate specimens, and observe the MYC eQTL in the 8q24.21 locus in individuals with low CTCF binding. Together our findings highlight a causal mechanism synergistically driven by a risk SNP and DNA methylation-mediated 3D genome architecture, advocating for the integration of genetics and epigenetics in assessing risks conferred by genetic predispositions.

scholarly journals Exploring the Effects of Genetic Variation on Gene Regulation in Cancer in the Context of 3D Genome Structure

2021 ◽  
Author(s):  
Noha Osman ◽  
Abd-El-Monsif Shawky ◽  
Michal Brylinski
Keyword(s):  
High Risk ◽  
Genetic Variants ◽  
Large Scale ◽  
Target Genes ◽  
Genome Structure ◽  
Regulatory Elements ◽  
3D Genome ◽  
Risk Snps ◽  
Prostate Cancers ◽  
3D Genome Structure

Abstract Background: Numerous genome-wide association studies (GWAS) conducted to date revealed genetic variants associated with various diseases, including breast and prostate cancers. Despite the availability of these large-scale data, relatively few variants have been functionally characterized, mainly because the majority of single-nucleotide polymorphisms (SNPs) map to the non-coding regions of the human genome. The functional characterization of these non-coding variants and the identification of their target genes remain challenging.Results: In this communication, we explore the potential functional mechanisms of non-coding SNPs by integrating GWAS with the high-resolution chromosome conformation capture (Hi-C) data for breast and prostate cancers. We show that more genetic variants map to regulatory elements through the 3D genome structure than the 1D linear genome lacking physical chromatin interactions. Importantly, the association of enhancers, transcription factors, and their target genes with breast and prostate cancers tends to be higher when these regulatory elements are mapped to high-risk SNPs through spatial interactions compared to simply using a linear proximity. Finally, we demonstrate that topologically associating domains (TADs) carrying high-risk SNPs also contain gene regulatory elements whose association with cancer is generally higher than those belonging to control TADs containing no high-risk variants.Conclusions: Our results suggest that many SNPs may contribute to the cancer development by affecting the expression of certain tumor-related genes through long-range chromatin interactions with gene regulatory elements. Integrating large-scale genetic datasets with the 3D genome structure offers an attractive and unique approach to systematically investigate the functional mechanisms of genetic variants in disease risk and progression.

scholarly journals Determining cellular CTCF and cohesin abundances to constrain 3D genome models

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Claudia Cattoglio ◽  
Iryna Pustova ◽  
Nike Walther ◽  
Jaclyn J Ho ◽  
Merle Hantsche-Grininger ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Site Occupancy ◽  
Cellular Protein ◽  
Protein Quantification ◽  
Ctcf Binding ◽  
Ctcf Binding Site ◽  
3D Genome ◽  
Quantitative Measurements ◽  
Dna Loop ◽  
Predictive Understanding

Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved. Here, we report the quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells. Extending our previous imaging studies (Hansen et al., 2017), we estimate bounds on the density of putatively DNA loop-extruding cohesin complexes and CTCF binding site occupancy. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) suggest the presence of cohesin dimers and/or oligomers. Finally, based on our cell lines with accurately measured protein abundances, we report a method to conveniently determine the number of molecules of any Halo-tagged protein in the cell. We anticipate that our results and the established tool for measuring cellular protein abundances will advance a more quantitative understanding of 3D genome organization, and facilitate protein quantification, key to comprehend diverse biological processes.

210 DNA METHYLATION AND HYDROXYMETHYLATION ANALYSIS IN A MODEL OF OOCYTE DIFFERENTIAL DEVELOPMENTAL COMPETENCE IN SHEEP

2015 ◽  
Vol 27 (1) ◽  
pp. 195
Author(s):  
L. Masala ◽  
D. Bebbere ◽  
G. P. Burrai ◽  
F. Ariu ◽  
L. Bogliolo ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Embryo Development ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Ctcf Binding ◽  
Ctcf Binding Site ◽  
Global Methylation ◽  
Tet Genes ◽  
Lower Expression

DNA methylation is an important epigenetic mark that plays a role in gene regulation by the addition of a methyl group to CpG islands in the DNA. Despite being relatively stable in somatic cells, DNA methylation is subject to reprogramming during embryo development and gametogenesis. The aim of this work was to evaluate different aspects of DNA methylation in relation to oocyte quality in the ovine species. A model of differential developmental competence consisting in ovine oocytes and in vitro produced (IVP) blastocysts derived from adult (AD) and prepubertal (PR) donors, was used. The methylation was analysed in terms of: expression of a panel of genes involved in DNA methylation [DNA methyltransferases (DNMTs)] and demethylation [ten-eleven translocation dioxygenases (TET)] in oocytes and blastocysts; global methylation and hydroxymethylation by direct immunofluorescence; locus-specific methylation of 2 imprinted genes by pyrosequencing. Gene relative quantification was performed by RNA reverse transcription followed by real-time PCR. Pools of 10 immature (GV) and in vitro-matured (MII) oocytes and (IVP) blastocysts derived from AD and PR donors (4 replicates per class) were analysed. Lower expression of TET1, TET2, and TET3 was observed in PR GV oocytes (ANOVA; P < 0.05), while no significant differences were found for the enzymes involved in methylation (DNMT1, DNMT3A, DNMT3B; ANOVA; P > 0.05). The levels of all the genes studied showed no significant differences in embryos at blastocyst stage (ANOVA; P > 0.05). Methylation and hydroxymethylation immunostaining were performed in GV and MII oocytes using anti-5-methylcytosine mouse mAb and 5-hydroxymethylcytosine rabbit pAB. High levels of DNA methylation were observed in both AD and PR GV and MII oocytes, while hydroxymethylation immunopositivity was scattered evident throughout the gamete chromatin. Pyrosequencing of bisulfite converted DNA was used to determine the methylation status within differentially methylated regions (DMR) of maternally imprinted H19 (CTCF binding site IV; 11 CpG sites) and paternally imprinted IGF2R (17CpG sites within intron 2). No differences were observed between classes of oocytes for each gene (pools of 40 oocytes per replicate, 3 replicates per class; ANOVA; P > 0.05). Our work shows no differences in the expression of the enzymes involved in methylation, in accordance with the results of global and locus specific methylation analysis. Conversely, we observed lower expression of the TET genes in PR GV oocytes (ANOVA; P > 0.05). TET1, TET2, and TET3, whose expression has never been studied in ovine, generate 5-hydroxymethlcytosine (5hmC) by oxidation of 5-methylcytosine (5mC), and are involved in active DNA demethylation during early embryo development. Our observation of lower expression of the TET genes in lower competence PR GV oocytes suggests that epigenetic mechanisms may affect oocyte quality and paves the way to better understand methylation dynamics during sheep pre-implantation development.

scholarly journals miR-100-5p downregulates mTOR to suppress the proliferation, migration and invasion of prostate cancer cells

2020 ◽  
Author(s):  
Su-Liang Li ◽  
Yun Ye ◽  
Jian-Jun Wang
Keyword(s):  
Prostate Cancer ◽  
Cell Proliferation ◽  
Cell Line ◽  
Expression Analysis ◽  
Immunohistochemical Staining ◽  
Regulatory Mechanism ◽  
Differential Expression Analysis ◽  
Migration And Invasion ◽  
Lncap Cells ◽  
Qrt Pcr

AbstractBackgroundPrevious studies have shown that miR-100-5p expression is abnormal in prostate cancer. However, the role and regulatory mechanism of miR-100-5p requires further investigation. Thus, the aim of this study was to observe the effects of miR-100-5p on the proliferation, migration and invasion of prostate cancer (PCa) cells and to explore the potential related regulatory mechanism.MethodsDifferential miRNA expression analysis was performed using next-generation sequencing (NGS) in the PCa cell line LNCaP and the normal prostatic epithelial cell line RWPE-1. The expression levels of miR-100-5pwere detected using real-time fluorescence quantitative PCR (qRT-PCR). LNCaP cells were transfected with NC-mimics or miR-100-5p mimics by using liposome transfection. Moreover, the CCK-8 proliferation assay, cell scratch assay and Transwell assay were used to detect the effects of miR-100-5p on cell proliferation, migration and invasion. In addition, the potential target gene of miR-100-5p was predicted, and the influence of miR-100-5p on the expression of mTOR mRNA by qRT-PCR and the expression of mTOR protein was detected by western blot and immunohistochemical staining.ResultsDifferential expression analysis of high-throughput sequencing data showed low expression of miR-100-5p in the PCa cell line LNCaP. It was further confirmed by qRT-PCR that the expression of miR-100-5p in LNCaP cells was significantly lower than that in RWPE-1 cells (P<0.01). miR-100-5p expression in LNCaP cells was markedly upregulated after transfection with miR-100-5p mimics (P<0.01), while cell proliferation, migration and invasion capacities were clearly reduced (P<0.01), and mTOR mRNA and protein expression was also substantially lowered (P<0.01). Finally, we further confirmed by immunohistochemical staining that miR-100-5p regulated the expression of mTOR.ConclusionmiR-100-5p is expressed at low levels in LNCaP cells, and it can suppress LNCaP cell proliferation, migration and invasion, the mechanism of which is related to downregulating the expression of mTOR.

scholarly journals Transposable elements strongly contribute to cell-specific and species-specific looping diversity in mammalian genomes

2019 ◽  
Author(s):  
Adam G Diehl ◽  
Ningxin Ouyang ◽  
Alan P Boyle
Keyword(s):  
Transposable Elements ◽  
Large Fraction ◽  
Population Level ◽  
Cell Types ◽  
Ctcf Binding ◽  
Ctcf Binding Site ◽  
3D Genome ◽  
Chromatin Looping ◽  
Mammalian Genomes ◽  
Species Specific

AbstractBackgroundChromatin looping is exceedingly important to gene regulation and a host of other nuclear processes. Many recent insights into 3D chromatin structure across species and cell types have contributed to our understanding of the principles governing chromatin looping. However, 3D genome evolution and how it relates to Mendelian selection remain largely unexplored. CTCF, an insulator protein found at most loop anchors, has been described as the “master weaver” of mammalian genomes, and variations in CTCF occupancy are known to influence looping divergence. A large fraction of mammalian CTCF binding sites fall within transposable elements (TEs) but their contributions to looping variation are unknown. Here we investigated the effect of TE-driven CTCF binding site expansions on chromatin looping in human and mouse.ResultsTEs have broadly contributed to CTCF binding and loop boundary specification, primarily forming variable loops across species and cell types and contributing nearly 1/3 of species-specific and cell-specific loops.ConclusionsOur results demonstrate that TE activity is a major source of looping variability across species and cell types. Thus, TE-mediated CTCF expansions explain a large fraction of population-level looping variation and may play a role in adaptive evolution.

scholarly journals Determining cellular CTCF and cohesin abundances to constrain 3D genome models

2018 ◽  
Author(s):  
Claudia Cattoglio ◽  
Iryna Pustova ◽  
Nike Walther ◽  
Jaclyn J. Ho ◽  
Merle Hantsche-Grininger ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Site Occupancy ◽  
Cellular Protein ◽  
Protein Quantification ◽  
Ctcf Binding ◽  
Ctcf Binding Site ◽  
3D Genome ◽  
Quantitative Measurements ◽  
Dna Loop ◽  
Predictive Understanding

Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved. Here we report the quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells. Extending our previous imaging studies (Hansen et al., 2017), we estimate bounds on the density of putatively DNA loop-extruding cohesin complexes and CTCF binding site occupancy. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) suggest the presence of cohesin dimers and/or oligomers. Finally, based on our cell lines with accurately measured protein abundances, we report a method to conveniently determine the number of molecules of any Halo-tagged protein in the cell. We anticipate that our results and the established tool for measuring cellular protein abundances will advance a more quantitative understanding of 3D genome organization, and facilitate protein quantification, key to comprehend diverse biological processes.

scholarly journals Hypermethylation of human DNA: Fine-tuning transcription associated with development

2017 ◽  
Author(s):  
Carl Baribault ◽  
Kenneth C. Ehrlich ◽  
V. K. Chaithanya Ponnaluri ◽  
Sriharsa Pradhan ◽  
Michelle Lacey ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Regulatory Elements ◽  
Fine Tuning ◽  
Ctcf Binding ◽  
Specific Gene ◽  
Dna Hypermethylation ◽  
Specific Expression ◽  
Lncrna Gene ◽  
Tissue Specific ◽  
Tissue Specific Expression

AbstractTissue-specific gene transcription can be affected by DNA methylation in ways that are difficult to discern from studies focused on genome-wide analyses of differentially methylated regions (DMRs). We studied 95 genes in detail using available epigenetic and transcription databases to detect and elucidate less obvious associations between development-linked hypermethylated DMRs in myoblasts (Mb) and cell-and tissue-specific expression. Many of these genes encode developmental transcription factors and display DNA hypermethylation also in skeletal muscle (SkM) and a few heterologous samples (e.g., aorta, mammary epithelial cells, or brain) among the 38 types of human cell cultures or tissues examined. Most of the DMRs overlapped transcription regulatory elements, including canonical, alternative, or cryptic promoters; enhancers; CTCF binding sites; and long-noncoding RNA (lncRNA) gene regions. Among the prominent relationships between DMRs and expression was promoter-region hypermethylation accompanying repression in Mb but not in many other repressed samples (26 genes). Another surprising relationship was down-modulated (but not silenced) expression in Mb associated with DNA hypermethylation at cryptic enhancers in Mb although such methylation was absent in both non-expressing samples and highly expressing samples (24 genes). The tissue-specificity of DNA hypermethylation can be explained for many of the genes by their roles in prenatal development or by the tissue-specific expression of neighboring genes. Besides elucidating developmental epigenetics, our study provides insights into the roles of abnormal DNA methylation in disease, e.g., cancer, Duchenne muscular dystrophy, and congenital heart malformations.

scholarly journals Exploring the effects of genetic variation on gene regulation in cancer in the context of 3D genome structure

2020 ◽  
Author(s):  
Noha Osman ◽  
Michal Brylinski
Keyword(s):  
High Risk ◽  
Genetic Variants ◽  
Large Scale ◽  
Target Genes ◽  
Genome Structure ◽  
Regulatory Elements ◽  
3D Genome ◽  
Risk Snps ◽  
Prostate Cancers ◽  
3D Genome Structure

AbstractNumerous genome-wide association studies (GWAS) conducted to date revealed genetic variants associated with various diseases, including breast and prostate cancers. Despite the availability of these large-scale data, relatively few variants have been functionally characterized, mainly because the majority of single-nucleotide polymorphisms (SNPs) map to the non-coding regions of the human genome. The functional characterization of these non-coding variants and the identification of their target genes remain challenging. In this communication, we explore the potential functional mechanisms of non-coding SNPs by integrating GWAS with the high-resolution chromosome conformation capture (Hi-C) data for breast and prostate cancers. We show that more genetic variants map to regulatory elements through the 3D genome structure than the 1D linear genome lacking physical chromatin interactions. Importantly, the association of enhancers, transcription factors, and their target genes with breast and prostate cancers tends to be higher when these regulatory elements are mapped to high-risk SNPs through spatial interactions compared to simply using a linear proximity. Finally, we demonstrate that topologically associating domains (TADs) carrying high-risk SNPs also contain gene regulatory elements whose association with cancer is generally higher than those belonging to control TADs containing no high-risk variants. Our results suggest that many SNPs may contribute to the cancer development by affecting the expression of certain tumor-related genes through long-range chromatin interactions with gene regulatory elements. Integrating large-scale genetic datasets with the 3D genome structure offers an attractive and unique approach to systematically investigate the functional mechanisms of genetic variants in disease risk and progression.

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