Project description:It remains unclear how ESCs rapidly establish enhancer regulatory elements to activate lineage-specific genes at the earliest differentiation stages. Previous studies suggest that some tissue-specific enhancers are pre-marked in ESCs with permissive features that support their competence to induce gene expression in response to the binding of lineage transcription factors (TFs). However, the absence of functional assays to identify chromatin regions with the competence to activate gene expression upon TF binding has impeded to better define any pre-marking features and their role on the transcriptional competence of the chromatin. To address this, we leveraged a CRISPR-activation(a) system to systematically interrogate the presence of chromatin regions able to induce the expression of 5 lineage-specific genes that are inactive in human ESCs (hESCs). Multiple CRISPRa screens discovered 42 regions, which we termed Competent Chromatin Regions (CCRs), that can induce the expression of the 5 lineage genes upon CRISPRa targeting. ChIP seq analyses showed that CCRs do not represent primed or active enhancers, since they are not enriched in H3K4me1 or H3K27ac histone modifications. Instead, CCRs exhibit a significant enrichment in POU sequence motifs, along with higher levels of binding by OCT4 and other pluripotent TFs. CCRs have an enriched presence of several repressive and activating chromatin-associated factors, including the DNA demethylation factors TET1 and QSER1 and various HDAC enzymes, which regulate their transcriptional competence. Although CCRs are exclusively found at the topologically associated domains shared with their related genes, we found they do not require an enrichment in pre-established chromatin contacts to exert their function. To assess the extent of our findings, we simulated the earliest stages of lineage differentiation by inducing the expression of the lineage TF FOXA2 in undifferentiated hESCs and we found it preferentially binds to the regions predicted as CCRs. Our results suggest that the factors enriched at CCRs facilitate the binding of lineage TFs and enable ESCs to promptly activate the expression of lineage genes. This study advances our understanding of pluripotency regulation in ESCs by identifying a novel type of regulatory regions that supports their developmental plasticity.

Project description:It remains unclear how ESCs rapidly establish enhancer regulatory elements to activate lineage-specific genes at the earliest differentiation stages. Previous studies suggest that some tissue-specific enhancers are pre-marked in ESCs with permissive features that support their competence to induce gene expression in response to the binding of lineage transcription factors (TFs). However, the absence of functional assays to identify chromatin regions with the competence to activate gene expression upon TF binding has impeded to better define any pre-marking features and their role on the transcriptional competence of the chromatin. To address this, we leveraged a CRISPR-activation(a) system to systematically interrogate the presence of chromatin regions able to induce the expression of 5 lineage-specific genes that are inactive in human ESCs (hESCs). Multiple CRISPRa screens discovered 42 regions, which we termed Competent Chromatin Regions (CCRs), that can induce the expression of the 5 lineage genes upon CRISPRa targeting. ChIP seq analyses showed that CCRs do not represent primed or active enhancers, since they are not enriched in H3K4me1 or H3K27ac histone modifications. Instead, CCRs are characterized by the enriched occupancy of OCT4, NANOG and demethylation factors TET1 and QSER1, which prevent DNA hypermethylation. Although CCRs are exclusively found at the topologically associated domains shared with their related genes, we found they do not require an enrichment in pre-established chromatin contacts to exert their function. To assess the extent of our findings, we simulated the earliest stages of lineage differentiation by inducing the expression of the lineage TF FOXA2 in undifferentiated hESCs and we found it preferentially binds to the regions predicted as CCRs. Our results suggest that the factors enriched at CCRs facilitate the binding of lineage TFs and enable ESCs to promptly activate the expression of lineage genes. This study advances our understanding of pluripotency regulation in ESCs by identifying a novel type of regulatory regions that supports their developmental plasticity.

Project description:It remains unclear how ESCs rapidly establish enhancer regulatory elements to activate lineage-specific genes at the earliest differentiation stages. Previous studies suggest that some tissue-specific enhancers are pre-marked in ESCs with permissive features that support their competence to induce gene expression in response to the binding of lineage transcription factors (TFs). However, the absence of functional assays to identify chromatin regions with the competence to activate gene expression upon TF binding has impeded to better define any pre-marking features and their role on the transcriptional competence of the chromatin. To address this, we leveraged a CRISPR-activation(a) system to systematically interrogate the presence of chromatin regions able to induce the expression of 5 lineage-specific genes that are inactive in human ESCs (hESCs). Multiple CRISPRa screens discovered 42 regions, which we termed Competent Chromatin Regions (CCRs), that can induce the expression of the 5 lineage genes upon CRISPRa targeting. ChIP seq analyses showed that CCRs do not represent primed or active enhancers, since they are not enriched in H3K4me1 or H3K27ac histone modifications. Instead, CCRs exhibit a significant enrichment in POU sequence motifs, along with higher levels of binding by OCT4 and other pluripotent TFs. CCRs have an enriched presence of several repressive and activating chromatin-associated factors, including the DNA demethylation factors TET1 and QSER1 and various HDAC enzymes, which regulate their transcriptional competence. Although CCRs are exclusively found at the topologically associated domains shared with their related genes, we found they do not require an enrichment in pre-established chromatin contacts to exert their function. To assess the extent of our findings, we simulated the earliest stages of lineage differentiation by inducing the expression of the lineage TF FOXA2 in undifferentiated hESCs and we found it preferentially binds to the regions predicted as CCRs. Our results suggest that the factors enriched at CCRs facilitate the binding of lineage TFs and enable ESCs to promptly activate the expression of lineage genes. This study advances our understanding of pluripotency regulation in ESCs by identifying a novel type of regulatory regions that supports their developmental plasticity.

Project description:Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs). When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. Here we ask whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, we first compare two different approaches to detect open chromatin in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. We find that both methods reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, we screened for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and identified 3778 regions that become (over-)activated during tumor development. Next, we applied motif discovery to search for candidate transcription factors that could bind these regions and identified AP-1 and Stat92E as key regulators. We validated the importance of Stat92E in the development of the tumors by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally we tested if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs, and observed that similar chromatin changes indeed occurred. Finally, we determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, we show that FAIRE-seq and ATAC-seq based open chromatin profiling, combined with motif discovery, is a straightforward approach to identify functional genomic regulatory regions, master regulators, and gene regulatory networks controlling complex in vivo processes. FAIRE-Seq in Drosophila wild type eye-antennal imaginal discs (2 wt strains); ATAC-Seq in Drosophila wild type eye-antennal imaginal discs (3 wt strains) ; FAIRE-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila eye discs with Unpaired over-expression (2 biological replicates); CTCF ChIP-seq in Drosophila eye discs; ChIP-seq input in Drosophila eye discs

Project description:Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs). When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. Here we ask whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, we first compare two different approaches to detect open chromatin in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. We find that both methods reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, we screened for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and identified 3778 regions that become (over-)activated during tumor development. Next, we applied motif discovery to search for candidate transcription factors that could bind these regions and identified AP-1 and Stat92E as key regulators. We validated the importance of Stat92E in the development of the tumors by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally we tested if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs, and observed that similar chromatin changes indeed occurred. Finally, we determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, we show that FAIRE-seq and ATAC-seq based open chromatin profiling, combined with motif discovery, is a straightforward approach to identify functional genomic regulatory regions, master regulators, and gene regulatory networks controlling complex in vivo processes.

			Action	DRS
	SRR24756048_1.fastq.gz	Fastqsanger.gz
	SRR24756048_2.fastq.gz	Fastqsanger.gz
	SRR24756049.fastq.gz	Fastqsanger.gz
	SRR24756050.fastq.gz	Fastqsanger.gz
	SRR24756051.fastq.gz	Fastqsanger.gz

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Discovery of Competent Chromatin Regions in Human Embryonic Stem Cells

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