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High-throughput functional characterization of combinations of transcriptional activators and repressors II

ABSTRACT: High-throughput functional characterization of combinations of transcriptional activators and repressors II

PROVIDER: PRJNA986924 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR25009940_1.fastq.gz	Fastqsanger.gz
	SRR25009940_2.fastq.gz	Fastqsanger.gz
	SRR25009941_1.fastq.gz	Fastqsanger.gz
	SRR25009941_2.fastq.gz	Fastqsanger.gz
	SRR25009942_1.fastq.gz	Fastqsanger.gz

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High-throughput functional characterization of combinations of transcriptional activators and repressors

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High-throughput functional characterization of combinations of transcriptional activators and repressors II

Project description:Despite growing knowledge of the functions of individual human transcriptional effector domains, much less is understood about how multiple effector domains within the same protein combine to regulate gene expression. Here, we measure transcriptional activity for 8,400 effector domain combinations by recruiting them to reporter genes in human cells. In our assay, weak and moderate activation domains synergize to drive strong gene expression, while combining strong activators often results in weaker activation. In contrast, repressors combine linearly and produce full gene silencing, and repressor domains often overpower activation domains. We use this information to build a synthetic transcription factor whose function can be tuned between repression and activation independent of recruitment to target genes by using a small molecule drug. Altogether, we outline the basic principles of how effector domains combine to regulate gene expression and demonstrate their value in building precise and flexible synthetic biology tools.

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Project description:Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy.

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Circular ANRIL isoforms switch from repressors to activators of p15/CDKN2B expression during RAF1 oncogene-induced senescence

Project description:Circular ANRIL isoforms switch from repressors to activators of p15/CDKN2B expression during RAF1 oncogene-induced senescence

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Circular ANRIL isoforms switch from repressors to activators of p15/CDKN2B expression during RAF1 oncogene-induced senescence

Project description:Long non-coding RNAs (ncRNAs) are major regulators of gene expression and cell fate. The INK4 locus encodes the tumour suppressor proteins p15INK4b, p16INK4a and p14ARF required for cell cycle arrest and whose expression increases during senescence. ANRIL is a ncRNA antisense to the p15 gene. In proliferative cells, ANRIL prevents senescence by repressing INK4 genes through the recruitment of Polycomb-group proteins. In models of replicative and RASval12 oncogene-induced senescence (OIS), the expression of ANRIL and Polycomb proteins decreases, thus allowing INK4 derepression. Here, we found in a model of RAF1 OIS that ANRIL expression rather increases, due in particular to an increased stability. This led us to search for circular ANRIL isoforms, as circular RNAs are rather stable species. We found that the expression of two circular ANRIL increases in several OIS models (RAF1, MEK1 and BRAF). In proliferative cells, they repress p15 expression, while in RAF1 OIS, they promote full induction of p15, p16 and p14ARF expression. Further analysis of one of these circular ANRIL shows that it interacts with Polycomb proteins and decreases EZH2 Polycomb protein localization and H3K27me3 at the p15 and p16 promoters, respectively. We propose that changes in the ratio between Polycomb proteins and circular ANRIL isoforms allow these isoforms to switch from repressors of p15 gene to activators of all INK4 genes in RAF1 OIS. Our data reveal that regulation of ANRIL expression depends on the senescence inducer and underline the importance of circular ANRIL in the regulation of INK4 gene expression and senescence.

2020-12-31 | GSE143957 | GEO

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