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Cooperative Binding of Transcription Regulators02:13

Cooperative Binding of Transcription Regulators

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Transcriptional regulators bind to specific cis-regulatory sequences in the DNA to regulate gene transcription. These cis-regulatory sequences are very short, usually less than ten nucleotide pairs in length. The short length means that there is a high probability of the exact same sequence randomly occurring throughout the genome.  Since regulators can also bind to groups of similar sequences, this further increases the chances of random binding. Transcriptional regulators form...
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Combinatorial gene control is the synergistic action of several transcriptional factors to regulate the expression of a single gene. The absence of one or more of these factors may lead to a significant difference in the level of gene expression or repression.
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Tissue-specific transcription factors contribute to diverse cellular functions in mammals. For example, the gene for beta globin, a major component of hemoglobin, is present in all cells of the body. However, it is only expressed in red blood cells because the transcription factors that can bind to the promoter sequences of the beta globin gene are only expressed in these cells. Tissue-specific transcription factors also ensure that mutations in these factors may impair only the function of...
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Gene transcription is regulated by the synergistic action of several proteins that form a complex at a gene regulatory site. This is observed in eukaryotes, where the regulation of gene expression is a complex process. Regulatory proteins in eukaryotes can broadly be classified into two types – regulators that bind directly to specific DNA sequences and co-regulators that associate with regulatory proteins but cannot directly bind to the DNA. These co-regulators are further divided into...
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Rational gRNA design based on transcription factor binding data.

David Bergenholm1, Yasaman Dabirian1, Raphael Ferreira1

  • 1Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.

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|October 29, 2021
PubMed
Summary
This summary is machine-generated.

Integrating transcription factor binding data improves CRISPR gene regulation. Targeting dCas9-VPR near motifs upregulates genes, while binding motifs causes downregulation, suggesting steric competition.

Keywords:
CRISPRaSaccharomyces cerevisiaegRNA designglycolytic promoterstranscription factor binding

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Area of Science:

  • Molecular Biology
  • Gene Regulation
  • Biotechnology

Background:

  • The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is a key tool in genome engineering.
  • Endonuclease-deficient Cas9 (dCas9) fused to effector domains enables programmable gene regulation.
  • Understanding the interplay between transcription factor (TF) binding and dCas9 binding is crucial for optimizing gene regulation strategies.

Purpose of the Study:

  • To investigate the influence of Saccharomyces cerevisiae transcription factor binding sites on the activity of dCas9 fused to the VPR activator domain.
  • To determine how targeting dCas9-VPR to TF binding motifs affects gene expression in central carbon metabolism genes.
  • To assess the utility of TF binding data in designing guide RNAs for CRISPR interference and activation.

Main Methods:

  • Utilized Saccharomyces cerevisiae transcription factor binding data.
  • Targeted dCas9-VPR to specific binding sites of Gcr1-Gcr2 and Tye7 transcription factors in gene promoters.
  • Analyzed gene expression changes in response to dCas9-VPR targeting relative to TF binding motifs.

Main Results:

  • Upregulation of gene expression was observed when dCas9-VPR was targeted adjacent to a TF binding motif.
  • Downregulation or no significant change in gene expression occurred when dCas9-VPR bound directly on a TF motif, indicating steric hindrance.
  • TF binding data proved valuable for predicting dCas9-VPR activity and designing effective guide RNAs.

Conclusions:

  • The positioning of dCas9 relative to TF binding sites significantly impacts gene expression outcomes.
  • Steric competition between dCas9 and endogenous TFs influences gene regulation.
  • Integrating TF binding information enhances the design and efficacy of CRISPR-based gene regulation tools.