Saturating mutagenesis of the GADD45A intron 3 enhancer using STARRseq
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ABSTRACT:
PROVIDER: PRJNA940815 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23697876_1.fastq.gz | Fastqsanger.gz | |||
| SRR23697876_2.fastq.gz | Fastqsanger.gz | |||
| SRR23697877_1.fastq.gz | Fastqsanger.gz | |||
| SRR23697877_2.fastq.gz | Fastqsanger.gz | |||
| SRR23697878_1.fastq.gz | Fastqsanger.gz |
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Project description:Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.
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Project description:Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA demethylation in vertebrates. However, it remained unclear how they contribute to the demethylation process. Here, we demonstrate that Gadd45a promotes active DNA demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet) -initiated oxidative demethylation. The connection of Gadd45a with oxidative demethylation is evidenced by the enhanced activation of a methylated reporter gene in HEK293T cells expressing Gadd45a in combination with catalytically active TDG and Tet. Gadd45a interacts with TDG physically and increases the removal of 5fC and 5caC from genomic and transfected plasmid DNA by TDG. Knockout of both Gadd45a and Gadd45b from mouse ES cells leads to hypermethylation of specific genomic loci most of which are also targets of TDG and show 5fC enrichment in TDG-deficient cells. These observations indicate that the demethylation effect of Gadd45a is mediated by TDG activity. This finding thus unites Gadd45a with the recently defined Tet-initiated demethylation pathway.
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Project description:Gadd45a can enhance somatic cell reprogramming significantly. To explore the roles of Gadd45a playing in reprogramming, we performed whole genome microarray to identify genes and signals pathways that regulated by Gadd45a.
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Project description:Regulatory elements (REs) consist of enhancers and promoters that occupy a significant portion of the non-coding genome and control gene expression programs either in –cis or in –trans. Putative REs have been identified largely based on their regulatory features (co-occupancy of ESC-specific transcription factors, enhancer histone marks and DNase hypersensitivity) in mouse embryonic stem cells (mESCs). However, less has been established regarding their regulatory functions in their native context. We deployed cis- and trans-regulatory elements scanning through saturating mutagenesis and sequencing (ctSCAN-SMS) to target elements within the ~12kb cis-region of the Oct4 gene locus, as well as genome-wide 2,613 high-confidence trans-REs (TREs), in mESCs. The ctSCAN-SMS identified 10 CREs and 12 TREs, as novel candidate REs of the Oct4 gene in mESCs. Furthermore, deletion of these REs confirmed that the majority of the CREs and TREs are functionally active, and involved in regulating Oct4 gene expression. Additionally, a subset of the functional CREs and TREs physically interact with the Oct4 promoter to varying degrees through intra- and inter-chromosomal interactions, respectively. Comparative genomics analysis reveals that functional CREs are more conserved in terms of their regulatory sequence conservation between mouse and primates (including humans) than TREs. Notably, a few active CREs are devoid of canonical regulatory features. Taken together, our work demonstrates the reliability and robustness of ctSCAN-SMS screening to identify critical REs, and probe their roles in the regulation of transcriptional output of a target gene (in this case Oct4).
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