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Non-coding CRISPR interference screen to identify regulatory elements of Xist in TX1072 XX SP107 mESCs at the onset of random X inactivation

ABSTRACT: Developmental genes are controlled by complex cis-regulatory landscapes that integrate multiple signals to ensure the correct spatio-temporal expression pattern. To investigate the underlying regulatory principles, we use the Xist locus as a model, which encodes the master regulator of X-chromosome inactivation. Xist is upregulated at the primed pluripotent state in a female-specific manner, thus integrating developmental cues and X-dosage information. It remains poorly understood how these signals are decoded by the ~800kb genomic region that controls Xist. While a series of repressive cis-regulatory elements have been identified, the distal enhancers that activate Xist transcription remain largely unknown. Here we use an inducible CRISPRi system in a non-coding CRISPR screen to profile 138 candidate regulatory elements on their effect on Xist upregulation during early differentiation of mouse Embryonic Stem Cells (mESCs).

ORGANISM(S): synthetic construct Mus musculus

PROVIDER: GSE167352 | GEO | 2021/03/04

REPOSITORIES: GEO

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PolyA-tail enriched RNA-seq in differentiating TX1072 XX mESCs

Project description:Developmental genes are controlled by complex cis-regulatory landscapes that integrate multiple signals to ensure the correct spatio-temporal expression pattern. To investigate the underlying regulatory principles, we use the Xist locus as a model, which encodes the master regulator of X-chromosome inactivation. Xist is upregulated at the primed pluripotent state in a female-specific manner, thus integrating developmental cues and X-dosage information. It remains poorly understood how these signals are decoded by the ~800kb genomic region that controls Xist. While a series of repressive cis-regulatory elements have been identified, the distal enhancers that activate Xist transcription remain largely unknown. Here we use polyA-tail enriched RNA-seq within the wildtype TX1072 XX cell line after two days of differentiation.

2021-03-04 | GSE167354 | GEO

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Project description:Developmental genes are controlled by complex cis-regulatory landscapes that integrate multiple signals to ensure the correct spatio-temporal expression pattern. To investigate the underlying regulatory principles, we use the Xist locus as a model, which encodes the master regulator of X-chromosome inactivation. Xist is upregulated at the primed pluripotent state in a female-specific manner, thus integrating developmental cues and X-dosage information. It remains poorly understood how these signals are decoded by the ~800kb genomic region that controls Xist. While a series of repressive cis-regulatory elements have been identified, the distal enhancers that activate Xist transcription remain largely unknown. Here we use TT-seq and RNA-seq to profile nascent and processed RNA transcription within the X inactivation center at the onset of random X-chromosome inactivation using an endogenous cell model of the inactive (TX 1072 XXΔXic) and active (TX 1072 XO) X chromosome.

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Project description:Developmental genes are controlled by complex cis-regulatory landscapes that integrate multiple signals to ensure the correct spatio-temporal expression pattern. To investigate the underlying regulatory principles, we use the Xist locus as a model, which encodes the master regulator of X-chromosome inactivation. Xist is upregulated at the primed pluripotent state in a female-specific manner, thus integrating developmental cues and X-dosage information. It remains poorly understood how these signals are decoded by the ~800kb genomic region that controls Xist. While a series of repressive cis-regulatory elements have been identified, the distal enhancers that activate Xist transcription remain largely unknown. Here we use capture Hi-C to profile chromatin contacts within the X inactivation center at the onset of random X-chromosome inactivation using an endogenous cell model of the inactive (TX 1072 XXΔXic) and active (TX 1072 XO) X chromosome.

2021-03-04 | GSE167351 | GEO

CUT&Tag on H3K4me3, H3K27ac, H3K4me1, H3K9me3, H3K27me3, H3K36me3 and H2AK119ub in TX1072 XXΔXic and XO mESCs during differentiation

Project description:Developmental genes are controlled by complex cis-regulatory landscapes that integrate multiple signals to ensure the correct spatio-temporal expression pattern. To investigate the underlying regulatory principles, we use the Xist locus as a model, which encodes the master regulator of X-chromosome inactivation. Xist is upregulated at the primed pluripotent state in a female-specific manner, thus integrating developmental cues and X-dosage information. It remains poorly understood how these signals are decoded by the ~800kb genomic region that controls Xist. While a series of repressive cis-regulatory elements have been identified, the distal enhancers that activate Xist transcription remain largely unknown. Here we use CUT&Tag on several histone modifications to profile the chromatin landscape of the X inactivation center at the onset of random X-chromosome inactivation using an endogenous cell model of the inactive (TX 1072 XXΔXic) and active (TX 1072 XO) X chromosome.

2021-03-04 | GSE167353 | GEO

A conserved noncoding locus modulates random monoallelic Xist expression across a TAD boundary

Project description:Topologically associating domains (TADs) are thought to restrict the action of distal cis-regulatory elements, such as enhancers, to target gene promoters within the same TAD. Here we discover that a cis-regulatory element of Xist is located almost 200kb away from its promoter and across a TAD boundary. This element, the promoter of a noncoding RNA, Linx, opposes Xist up-regulation in cis and influences the choice of X chromosome to be inactivated. Even though Linx lies in the same TAD as Xist’s antisense Tsix locus, we show that the repressive action of Linx on Xist is independent of Tsix. Furthermore, this Xist regulatory action is independent of Linx transcript or of its transcription. Finally, we demonstrate that Linx cis-regulatory repressive element is well conserved in mammals, unlike Tsix, suggesting that it represents an ancestral mechanism for random monoallelic Xist expression. Our study uncovers a novel regulatory axis for X-chromosome inactivation and a new class of cis-regulatory effects that rely on TAD partitioning to modulate developmental decisions.

2019-12-01 | GSE124596 | GEO

Distal and proximal cis-regulatory elements sense X-dosage and developmental state at the Xist locus

Project description:Developmental genes such as Xist, the master regulator of X-chromosome inactivation (XCI), are controlled by complex cis-regulatory landscapes, which decode multiple signals to establish specific spatio-temporal expression patterns. Xist integrates information on X-chromosomal dosage and developmental stage to trigger XCI at the primed pluripotent state in females only. Through a pooled CRISPR interference screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements of Xist during the onset of random XCI. By quantifying how enhancer activity is modulated by X-dosage and differentiation, we find that X-dosage controls the promoter-proximal region in a binary switch-like manner. By contrast, differentiation cues activate a series of distal elements and bring them into closer spatial proximity of the Xist promoter. The strongest distal element is part of an enhancer cluster ~200 kb upstream of the Xist gene which is associated with a previously unannotated Xist-enhancing regulatory transcript, we named Xert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specific Xist upregulation at the correct developmental time. Our study is the first step to disentangle how multiple, functionally distinct regulatory regions interact to generate complex expression patterns in mammals.

2021-03-04 | GSE167358 | GEO

Non-coding CRISPR interference screen to identify regulatory elements of Xist in TX1072 XX SP107 mESCs at the onset of random X inactivation

Project description:Non-coding CRISPR interference screen to identify regulatory elements of Xist in TX1072 XX SP107 mESCs at the onset of random X inactivation

| PRJNA704374 | ENA

RNA-seq in TX1072 XO mESCs during differentiation

Project description:As the master regulator of X-chromosome inactivation (XCI), the Xist RNA is expressed nearly ubiquitously in female mice. Xist is only absent in the germ line and at the pluripotent state. Xist is generally assumed to be expressed “by default” in females, while being actively repressed in the few tissues where it is silent. Whether activating mechanisms also contribute remained largely unknown. Through a pooled CRISPR screen we identify the GATA family of transcription factors as potent direct activators of Xist. We describe a GATA-responsive regulatory element (RE79), located ~100 kb upstream of the Xist promoter. In cell lines derived from the two extraembryonic lineages, XEN and TS cells, where imprinted Xist expression is maintained, RE79 is bound by different sets of GATA factors expressed in those tissues. Here we use RNA-seq on differentiating XO mouse embryonic stem cells as a reference for gene expression during early differentiation.

2022-01-26 | GSE194016 | GEO

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