Identification of a Distal Locus Enhancer Element That Controls Cell Type-Specific TNF and LTA Gene Expression in...

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• The human TNF/LT locus genes (TNF, LTA, LTB) exhibit cell type-specific expression patterns.
• Understanding the regulatory mechanisms governing these genes is crucial for immune response modulation.

Purpose of the Study:

• To investigate the role of a distal noncoding element (hHS-8) in controlling TNF and LTA gene expression in human T cells.
• To explore the potential of CRISPR/dead(d)Cas9 technology for modulating TNF and LTA transcription.

Main Methods:

• Analysis of NFAT binding site within hHS-8 in primary human CD4+ T cells.
• Assessing epigenetic modifications (H3K27 acetylation) and nuclease sensitivity.
• Utilizing CRISPR/dCas9 to target the hHS-8 NFAT site in CEM T cells.
• Measuring mRNA synthesis and RNA polymerase II recruitment.

Main Results:

• A conserved NFAT binding site in hHS-8 coordinates TNF and LTA expression in human T cells.
• T cell activation leads to increased hHS-8 and promoter activity, including enhancer RNA transcription.
• CRISPR/dCas9 targeting of the hHS-8 NFAT site significantly reduced TNF and LTA mRNA levels and RNA polymerase II recruitment.

Conclusions:

• The distal element hHS-8 plays a critical role in the inducible, cell type-specific gene expression of the human TNF/LT locus.
• CRISPR/dCas9 offers a potential strategy for therapeutic modulation of TNF and LTA transcription in human diseases.