A CTCF binding element regulates RA-induced early ESCs differentiation through orchestrating long-range chromatin interactions between its adjacent enhancers and HoxA
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ABSTRACT:
PROVIDER: PRJNA646489 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12229271_1.fastq.gz | Fastqsanger.gz | |||
| SRR12229271_2.fastq.gz | Fastqsanger.gz | |||
| SRR12229272_1.fastq.gz | Fastqsanger.gz | |||
| SRR12229272_2.fastq.gz | Fastqsanger.gz | |||
| SRR12229273_1.fastq.gz | Fastqsanger.gz |
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Project description:Proper Homeobox A (HoxA) cluster genes expression is essential for embryonic stem cells (ESCs) differentiation and individual development. However, the mechanisms controlling the precise spatiotemporal expression of HoxA cluster genes during early ESCs differentiation remain largely unknown. Here, we find a CTCF binding element (CBE+47kb) closest to the 3'-end of HoxA locus within a topologically associated domains (TAD) in ESCs. CRISPR-Cas9 mediated the CBE+47kb knockout significantly promotes expression of HoxA cluster genes and early ESCs differentiation induced by RA compared with wild type cells. In mechanism, we found that there was a significant differently long-range chromatin interactions between its adjacent enhancers and HoxA in CBE+47kb knockout cells through chromosome conformation capture assay (Capture-C), indicating that CBE+47kb can precisely organize interactions between its adjacent enhancers and HoxA chromatins. Furthermore, we also showed that its adjacent enhancers deletion shows significantly synthetic inhibition effect on HoxA genes expression, suggesting that these enhancers are required for RA-induced HoxA genes expression. Our study reveals that a new functional CBE+47 can regulate HoxA genes expression through orchestrating long-range chromatin interactions between its adjacent enhancers and HoxA, thus maintaining RA-induced early ESCs proper differentiation.
2020-07-16 | GSE154495 | GEO
Project description:To uncover the mechanism that Wnt signaling modulates RA-induced ESC differentiation, we conducted time course RNA-seq analysis during the differentiation. Transcriptome profiles of RA-induced and RA/CHIR-induced ESC differentiation were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000. The RNA-seq data revealed a significant decrease in mesoderm (one of the major source of vascular SMCs) markers and increase in PrE markers in CHIR treated samples, while other lineage markers showed no significant expression, implying a diverging to PrE fate by Wnt activation. Subsequent hierarchical clustering analysis also showed that the gene expression profiles of cells derived from RA induction alone clustered with adult SMCs, while cells derived from RA/CHIR induction gradually clustered to XEN cells with differentiation and was very close to XEN cells at day 16, suggesting that RA/CHIR-induced ESCs differentiated into PrE lineage. This study provided new insights into the importance of RA/Wnt crosstalk in the lineage differentiation of ESCs.
2019-12-10 | GSE123632 | GEO
Project description:Long noncoding RNAs (lncRNAs) have been implicated in controlling various aspects of embryonic stem cell (ESC) biology, although the functions of specific lncRNAs, and the molecular mechanisms through which they act, remain unclear. Here, we demonstrate discrete and opposing roles for the lncRNA transcript Haunt and its genomic locus in regulating the HOXA gene cluster during ESC differentiation. Reducing or enhancing Haunt expression, with minimal disruption of the Haunt locus, led to up- or down-regulation of HOXA genes, respectively. In contrast, increasingly large genomic deletions within the Haunt locus attenuated HOXA activation. The Haunt DNA locus contains potential enhancers of HOXA activation, whereas Haunt RNA acts to prevent aberrant HOXA expression. This work reveals a multi-faceted model of lncRNA-mediated transcriptional regulation of the HOXA cluster, with distinct roles for a lncRNA transcript and its genomic locus, while illustrating the power of rapid CRISPR/Cas9-based genome editing for assigning lncRNA functions. All RNA-seq(s) were designed to reveal the differentially expressed genes among different stages of ESCs differentiation, or differentially expressed genes between wild-type or Haunt or HOXA mutant cells during RA-induced differentiation. All ChIRP-Seq were used to reveal the DNA or RNA targets of Haunt before or after RA treatment.
2015-04-20 | E-GEOD-58514 | biostudies-arrayexpress
Project description:Next generation sequencing facilitates lineage identity of RA-induced and RA/CHIR-induced ESCs
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Project description:In the present study, we show that UTX plays an essential role in resolving and activating many retinoic acid (RA)-inducible bivalent genes during the RA-driven differentiation of mouse ESCs treated with a physiologically relevant RA concentration (0.2 μM). We showed that UTX loss and UTX knockdown interfered with the RA-induced differentiation of mouse ESCs. Therefore, our findings indicate that the UTX-mediated resolution and activation of many RA-inducible bivalent genes, including numerous Hoxa-d cluster genes, are required for RA-driven differentiation of mouse ESCs.
2016-02-05 | GSE76692 | GEO
Project description:Proper expression of Homeobox A cluster genes (HoxA) is essential for embryonic stem cell (ESC) differentiation and individual development. However, mechanisms controlling precise spatiotemporal expression of HoxA during early ESC differentiation remain poorly understood. Herein, we identified a functional CTCF-binding element (CBE+47) closest to the 3'-end of HoxA within the same topologically associated domain (TAD) in ESC. CRISPR-Cas9-mediated deletion of CBE+47 significantly upregulated HoxA expression and enhanced early ESC differentiation induced by retinoic acid (RA) relative to wild-type cells. Mechanistic analysis by chromosome conformation capture assay (Capture-C) revealed that CBE+47 deletion decreased interactions between adjacent enhancers, enabling formation of a relatively loose enhancer-enhancer interaction complex (EEIC), which overall increased interactions between that EEIC and central regions of HoxA chromatin. These findings indicate that CBE+47 organizes chromatin interactions between its adjacent enhancers and HoxA. Furthermore, deletion of those adjacent enhancers synergistically inhibited HoxA activation, suggesting that these enhancers serve as an EEIC required for RA-induced HoxA activation. Collectively, these results provide new insight into RA-induced HoxA expression during early ESC differentiation, also highlight precise regulatory roles of the CTCF-binding element in orchestrating high-order chromatin structure.
| S-EPMC7960549 | biostudies-literature
Project description:In the present study, we show that UTX plays an essential role in resolving and activating many retinoic acid (RA)-inducible bivalent genes during the RA-driven differentiation of mouse ESCs treated with a physiologically relevant RA concentration (0.2 μM). We showed that UTX loss and UTX knockdown interfered with the RA-induced differentiation of mouse ESCs. Therefore, our findings indicate that the UTX-mediated resolution and activation of many RA-inducible bivalent genes, including numerous Hoxa-d cluster genes, are required for RA-driven differentiation of mouse ESCs. ChIP-Seq data for H3K4me3 and H3K27me3 were generated along with input data for mouse stem cells with wild type or UTX-depleted genotype and with or without RA treatment.
2016-02-05 | E-GEOD-76692 | biostudies-arrayexpress
Project description:Differentiation is accompanied by extensive epigenomic reprogramming, leading to the repression of stemness factors and the transcriptional maintenance of activated lineage-specific genes. Here we used the mammalian Hoxa cluster of developmental genes as a model system to follow changes in DNA modification patterns during retinoic acid induced differentiation. We found the inactive cluster to be marked by defined patterns of 5-methylcytosine (5mC). Upon the induction of differentiation, the active anterior part of the cluster became increasingly enriched in 5-hydroxymethylcytosine (5hmC), following closely the colinear activation pattern of the gene array, which was paralleled by the reduction of 5mC. Depletion of the 5hmC generating dioxygenase Tet2 impaired the maintenance of Hoxa activity and partially restored 5mC levels. Our results indicate that gene specific 5mC-5hmC conversion by Tet2 is crucial for the maintenance of active chromatin states at lineage-specific loci. Genome wide DNA methylation profiling of uninduced and retinoic acid (RA) induced NTera2/D1 embryocarcinoma cells (NT2). Bisulphite converted DNA of uninduced, 7d RA and 14d RA treated NT2 cells were hybridised to the Illumina Infinium HumanMethylation450 Beadchip.
2012-05-02 | E-GEOD-32362 | biostudies-arrayexpress
Project description:Retinoic acid (RA) induces rapid differentiation of ESCs, partly by activating expression of the transcription factor Hoxa1, which regulates downstream target genes that promote ESCs differentiation. However, mechanisms of RA-induced Hoxa1 expression and ESCs early differentiation remain largely unknown. Here, we identify a distal enhancer interacting with the Hoxa1 locus through a long-range chromatin loop. Enhancer deletion significantly inhibited expression of RA-induced Hoxa1 and endoderm master control genes such as Gata4 and Gata6. Transcriptome analysis revealed that RA-induced early ESCs differentiation was blocked in Hoxa1 enhancer knockout cells, suggesting a requirement for the enhancer. Restoration of Hoxa1 expression partly rescued expression levels of ~40% of genes whose expression changed following enhancer deletion, and ~18% of promoters of those rescued genes were directly bound by Hoxa1. Our data show that a distal enhancer maintains Hoxa1 expression through long-range chromatin loop and that Hoxa1 directly regulates downstream target gene expression and then orchestrates RA-induced early differentiation of ESCs.
2018-12-24 | GSE124306 | GEO
Project description:Retinoic acid (RA) plays a pivotal role in different biological processes including inducing differentiation of embryonic stem cells (ESCs). We first knocked out one or both of RA receptors- Rarα and Rxrα to partially abolish the RA signaling, and then overexpressed one of them separately to hyper activation of RA signaling pathway. By transcriptome analysis, we show that RA signaling effects multi-lineage differentiation, particularly the endoderm, and identify RA signaling target genes, interesting, there are two family cluster genes- Hoxb and Cyp26. Chromosome immunoprecipitation (ChIP-seq) shows that RA induces novel proximal and distal enhancers around the cluster target genes and these enhancers are dependent of RA receptors(Rarα/Rxrα). 4C-seq reveals enhancer-enhancer and enhancer-promoter interactions in their regions and shows a decrease in the relative frequency of contact relative to WT after Rarα/Rxrα-knockout, suggesting Rarα/Rxrα participate in mediating chromatin interaction. We also identify that enhancers significantly maintain expression of RA-induced Hoxb and Cyp26 family genes and regulate multi-lineage marker genes. At the same time, we reveal that disrupting RA-response elements (RAREs) in the enhancer affects the expression of the target gene. Our study provides mechanistic insight into RA-induced early ESC differentiation to endoderm and potential regular regulation of downstream target genes.
2023-06-29 | GSE221992 | GEO