HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

Area of Science:

• Genomics
• Epigenetics
• Molecular Biology

Background:

• Topologically associating domains (TADs) are crucial for regulating DNA element interactions.
• CCCTC-binding factor (CTCF) regulates HOX gene expression, impacting development and disease.
• The role of HOX loci-associated CTCF boundaries in chromatin organization and gene expression is not well understood.

Purpose of the Study:

• To investigate how CTCF boundaries at HOX loci influence chromatin organization and HOX gene expression.
• To identify specific CTCF binding sites critical for maintaining oncogenic chromatin domains and HOX gene expression patterns.

Main Methods:

• Generation of a pooled sgRNA library targeting CTCF binding sites in HOXA/B/C/D loci.
• CRISPR-Cas9 genetic screening to assess the impact of CTCF disruption.
• Analysis of TAD formation and HOX gene expression changes.

Main Results:

• Disruption of the CTCF binding site between HOXA7/HOXA9 genes (CBS7/9) significantly altered TAD formation.
• This specific CTCF site was identified as a critical regulator of oncogenic chromatin domains.
• The CBS7/9 site is important for maintaining ectopic HOX gene expression in MLL-rearranged acute myeloid leukemia (AML).

Conclusions:

• sgRNA library screening provides insights into CTCF-mediated genome organization at specific gene loci.
• CTCF boundaries play a key role in regulating HOX gene expression and chromatin structure.
• This approach facilitates functional characterization of genetic regulatory elements in biological processes.