Cis Regulatory Element Scan by Tiling-deletion and Sequencing
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ABSTRACT:
PROVIDER: PRJNA320305 | ENA |
REPOSITORIES: ENA
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Project description:Here we report a highly scalable strategy for unbiased discovery and functional characterization of cis regulatory elements in the genome. These results demonstrate the utility of our cis element discovery strategy and reveal the dual function of gene promoters: they not only initiate transcription of their own genes, but could also control transcription of nearby genes.
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Project description:Millions of cis-regulatory elements are predicted to be present in the human genome, but direct evidence for their biological function is scarce. Here we report a high-throughput method, cis-regulatory element scan by tiling-deletion and sequencing (CREST-seq), for the unbiased discovery and functional assessment of cis-regulatory sequences in the genome. We used it to interrogate the 2-Mb POU5F1 locus in human embryonic stem cells, and identified 45 cis-regulatory elements. A majority of these elements have active chromatin marks, DNase hypersensitivity, and occupancy by multiple transcription factors, which confirms the utility of chromatin signatures in cis-element mapping. Notably, 17 of them are previously annotated promoters of functionally unrelated genes, and like typical enhancers, they form extensive spatial contacts with the POU5F1 promoter. These results point to the commonality of enhancer-like promoters in the human genome.
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Project description:Transcriptome analysis of effect of Lockd knockout on cells Many long non-coding (lnc) RNAs are reported to regulate gene expression and protein functions. However, the proportion of lncRNAs with biological activities among the thousands expressed in mammalian cells is controversial. We studied Lockd (Downstream of p27), a 434 bp polyadenylated lncRNA originating 4 kb 3â to the Cdkn1b gene. Heterozygous and homozygous deletion of the 25 kb Lockd locus reduced Cdkn1b transcription by approximately 35 and 70% respectively in a mouse erythroid cell line. In contrast, homozygous insertion of a polyadenylation cassette 80 bp downstream of the Lockd transcription start site reduced the entire lncRNA transcript level by > 90%, but had no effect on Cdkn1b transcription. The 5â region of the Lockd gene contains a DNase hypersensitive site, binds numerous transcription factors (TFs), and physically associates with the Cdkn1b promoter in chromosomal conformation capture (NG Capture-C) studies. Thus, the Lockd gene positively regulates Cdkn1b transcription through an enhancer-like cis element and not via the lncRNA transcript. These findings demonstrate that the biological functions of a lncRNA cannot be inferred simply from phenotypes that arise after deleting the corresponding genomic locus. We analyzed mouse G1E erythroid cell line clones with Control Lockd (C - 3 replicates) and with Lockd deletion with CRISPR (KO - 4 replicates) using Mouse Gene 2.0 ST Array platform (transcript version). Array data was processed by RMA algorithm.
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