Whole genome functional characterization of RE1 silencers using a modified massively parallel reporter assay BRB-seq
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ABSTRACT:
PROVIDER: PRJNA874716 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR21296608_1.fastq.gz | Fastqsanger.gz | |||
| SRR21296608_2.fastq.gz | Fastqsanger.gz |
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Project description:Both upregulation and downregulation by cis-regulatory elements help establish precise gene expression. Our understanding of how elements repress transcriptional activity is far more limited than activating elements. To address this gap, we characterized RE1, a group of transcriptional silencers bound by REST, on a genome-wide scale using an optimized massively parallel reporter assay (MPRAduo). MPRAduo empirically defined a minimal binding strength of REST required by silencer (REST m-value), above which multiple cofactors colocalize and act to directly silence transcription. We identified 1,500 human variants that alter RE1 silencing and found their effect sizes are predictable when they overlap with REST binding sites above the m-value. In addition, we demonstrate that non-canonical REST binding motifs exhibit silencer function only if they precisely align two half sites with specific spacer length. Our results show mechanistic insights into RE1 silencer which allows us to predict its activity and effect of variants on RE1, providing a paradigm for performing genome-wide functional characterization transcription factors binding sites.
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Project description:Both upregulation and downregulation by cis-regulatory elements help establish precise gene expression. Our understanding of how elements repress transcriptional activity is far more limited than activating elements. To address this gap, we characterized RE1, a group of transcriptional silencers bound by REST, on a genome-wide scale using an optimized massively parallel reporter assay (MPRAduo). MPRAduo empirically defined a minimal binding strength of REST required by silencer (REST m-value), above which multiple cofactors colocalize and act to directly silence transcription. We identified 1,500 human variants that alter RE1 silencing and found their effect sizes are predictable when they overlap with REST binding sites above the m-value. In addition, we demonstrate that non-canonical REST binding motifs exhibit silencer function only if they precisely align two half sites with specific spacer length. Our results show mechanistic insights into RE1 silencer which allows us to predict its activity and effect of variants on RE1, providing a paradigm for performing genome-wide functional characterization transcription factors binding sites.
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Project description:Whole genome functional characterization of RE1 silencers using a modified massively parallel reporter assay
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Project description:Enhancers and silencers often depend on the same transcription factors (TFs) and are imperfectly distinguished from each other by genomic assays of TF binding or chromatin state. To identify sequence features that define enhancers and silencers, we assayed massively parallel reporter libraries of genomic sequences targeted by the photoreceptor TF CRX and found instances of enhancer, silencer, or no activity. Both enhancers and silencers contain more TF motifs than inactive sequences, but enhancers contain motifs from a more diverse collection of TFs. We developed a measure of information content that describes the number and diversity of motifs in a sequence and found that, while both enhancers and silencers depend on CRX motifs, enhancers have higher information content. Our results indicate that enhancers contain motifs for a diverse but degenerate collection of TFs, while silencers depend on a smaller and less diverse collection of TFs.
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Project description:Silencers, the yin to enhancers' yang, play a pivotal role in fine-tuning gene expression throughout the genome. However, despite their recognized importance, comprehensive identification of these regulatory elements in the genome is still in its early stages. We developed a method called Ss-STARR-seq to directly determine the activity of silencers in the whole genome. In this study, we applied Ss-STARR-seq to human cell lines K562, LNCaP, and 293T, and identified 134,171, 137,753, and 125,307 silencers on a genome-wide scale, respectively, these silencers function in various cells in a cell-specific manner. Silencers exhibited a substantial enrichment of transcriptional-inhibitory motifs, including REST, and demonstrated overlap with the binding sites of repressor transcription factors within the endogenous environment. Interestingly, H3K27me3 did not reflect silencer activity but facilitated the silencer's inhibitory role on gene expression. Additionally, the silencer did not have any significant histone markers at the genome-wide level. Our findings unveil that aspect-silencers not only transition into enhancers throughout diverse cell lines but also achieve functional conversion with insulators. Regarding to biological effects, knockout experiments underscored the functional redundancy and specificity of silencers in regulating gene expression and cell proliferation. In summary, this study pioneers the elucidation of the genome-wide silencer landscape in human cells, delineates their global regulatory features, and identifies specific silencers influencing cancer cell proliferation.
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Project description:The majority of the human genome does not encode proteins. Many of these noncoding regions contain important regulatory sequences that control gene expression. To date, most studies have focused on activators such as enhancers, but regions that repress gene expression⎯silencers⎯have not been systematically studied. We have developed a system that identifies silencer regions in a genome-wide fashion based on silencer-mediated transcriptional repression of caspase 9. We found that silencers are widely distributed and may function in a tissue-specific fashion. These silencers harbor unique epigenetic signatures and are associated with specific transcription factors. Silencers also act at multiple genes, and at the level of chromosomal domains and long-range interactions. Deletion of silencer regions linked to the drug transporter genes ABCC2 and ABCG2 caused chemo-resistance. Overall, our study demonstrates that tissue-specific silencing is widespread throughout the human genome and likely contributes significantly to the regulation of gene expression and human biology.
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