Saturating mutagenesis of the GADD45A intron 3 enhancer using STARRseq
Ontology highlight
ABSTRACT:
ORGANISM(S): synthetic construct Homo sapiens
PROVIDER: GSE226617 | GEO | 2023/03/12
REPOSITORIES: GEO
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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 miRNA microarray to identify miRNAs and signals pathways that regulated by Gadd45a.
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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.
2014-04-22 | GSE56943 | GEO
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. The dataset includes RRBS anlysis of 2 WT ES cell samples and 2 Gadd45a/b DKO ES cell samples.
2015-05-07 | E-GEOD-62496 | biostudies-arrayexpress
Project description:GADD45a is a nuclear protein involved in the regulation of cell growth, apoptosis and DNA repair. We previously found that GADD45a physically and functionally interacts with TET1 to mediate active DNA demethylation and is recruited by ING1 to its target sites. In this study, we identified GADD45a bound genomic loci in mouse embryonic fibroblasts (MEFs) via ChIP-Sequencing.
2018-06-11 | GSE99602 | GEO
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. Genes expression of MEFs was measured at day8 in reprogramming. Three samples were set: MEFs infected with SKO plus Flag, MEFs infected with SKO plus Gadd45a and MEFs infected with SKO plus G39A which is a negative mutant of Gadd45a.
2014-04-22 | E-GEOD-56943 | biostudies-arrayexpress
Project description:Gadd45a can enhance somatic cell reprogramming significantly. To explore the roles of Gadd45a playing in reprogramming, we performed miRNA microarray to identify miRNAs and signals pathways that regulated by Gadd45a. miRNAs expression of MEFs was measured at day8 in reprogramming. Four samples were set: MEFs infected with SKO plus Flag, MEFs infected with SKO plus Gadd45a, MEFs infected with SKOM plus Flag and MEFs infected with SKOM+Ga.
2014-04-22 | E-GEOD-56942 | biostudies-arrayexpress
Project description:Gadd45a is a stress-induced protein that causes skeletal muscle atrophy. The goal of these studies was to determine the effects of Gadd45a overexpression on mRNA levels in mouse skeletal muscle. For additional details see Ebert et al, Stress-Induced Skeletal Muscle Gadd45a Expression Reprograms Myonuclei and Causes Muscle Atrophy. JBC epub. June 12, 2012.
2012-09-15 | GSE39196 | GEO
Project description:Gadd45a is a stress-induced protein that causes skeletal muscle atrophy. The goal of these studies was to determine the effects of Gadd45a overexpression on mRNA levels in mouse skeletal muscle. For additional details see Ebert et al, Stress-Induced Skeletal Muscle Gadd45a Expression Reprograms Myonuclei and Causes Muscle Atrophy. JBC epub. June 12, 2012. Tibialis anterior (TA) muscles from muscle-specfic ATF4 knockout mice (ATF4 mKO) were transfected with either 20 mg empty plasmid (pcDNA3) (left TA) or 20 mg pCMV-FLAG-Gadd45a (right TA) and harvested 7 days later. mRNA levels in Gadd45a-transfected muscles were normalized to levels in control transfected muscles.
2012-09-15 | E-GEOD-39196 | biostudies-arrayexpress