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HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries
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CTCF Binding Polarity Determines Chromatin Looping.

Elzo de Wit1, Erica S M Vos1, Sjoerd J B Holwerda1

  • 1Hubrecht Institute-KNAW and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands.

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Summary
This summary is machine-generated.

CCCTC-binding factor (CTCF) polarity dictates chromatin loop formation. CTCF binding orientation is crucial for higher-order chromatin structure, influencing gene regulation.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Epigenetics

Background:

  • CCCTC-binding factor (CTCF) is a key architectural protein.
  • CTCF plays a critical role in organizing the three-dimensional (3D) genome.
  • The orientation of CTCF binding sites is hypothesized to influence chromatin loop formation.

Purpose of the Study:

  • To investigate the role of CTCF binding site orientation in chromatin loop formation.
  • To experimentally validate the hypothesis that convergent CTCF binding sites drive loop formation.
  • To understand the functional significance of CTCF polarity in 3D genome organization.

Main Methods:

  • High-resolution 4C-seq was used to assay 3D genomic contact profiles at CTCF binding sites.
  • CRISPR/Cas9 genome editing was employed to delete core CTCF binding sites in specific loci.
  • CTCF and cohesin recruitment were assessed following genetic manipulation and re-insertion experiments.

Main Results:

  • Deletion of CTCF binding sites resulted in loss of CTCF and cohesin recruitment.
  • Chromatin loops involving distal, convergent CTCF sites were disrupted or destabilized upon CTCF site deletion.
  • Re-insertion of CTCF sites with reversed orientation restored protein recruitment but not loop formation.

Conclusions:

  • CTCF binding polarity is a critical functional determinant in the formation of chromatin loops.
  • The orientation of CTCF binding sites, not just their presence, is essential for establishing higher-order chromatin structure.
  • CTCF polarity directly influences the recruitment of cohesin and the stability of genomic interactions.