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Requirement for intron structures in activating the Cd8a locus.

Hisashi Wada1, Nighat Yasmin1, Kiyokazu Kakugawa2

  • 1Laboratory for Transcriptional Regulation, RIKEN Center for Integrative Medical Sciences, 230-0045 Yokohama, Japan.

Proceedings of the National Academy of Sciences of the United States of America
|March 14, 2018
PubMed
Summary

Coreceptor reversal in T cell development involves CD8a gene reactivation. Intron sequences are crucial for this stage-specific gene expression, influencing T cell lineage commitment.

Keywords:
Cd8 geneDNA methylationT cell developmentintron-mediated enhancement

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Area of Science:

  • Immunology
  • Molecular Biology
  • Developmental Biology

Background:

  • During T cell differentiation, CD4+CD8+ double-positive (DP) thymocytes become CD4-CD8+ single-positive (CD8SP) cells, a process involving the termination and reinitiation of Cd8a gene transcription (coreceptor reversal).
  • While a transcriptional enhancer in the Cd8a gene is known, the precise molecular mechanisms governing Cd8a gene reactivation remain unclear.

Purpose of the Study:

  • To elucidate the molecular mechanisms underlying the stage-specific reactivation of the Cd8a gene during T cell differentiation.
  • To investigate the role of intron sequences and DNA methylation in regulating Cd8a gene expression in thymocytes.

Main Methods:

  • Generation of a human CD2 (hCD2) reporter transgene inserted into the first exon of the Cd8a gene locus.
  • Analysis of hCD2 expression in DP and mature CD8+ T cells under various conditions, including the presence or absence of intron sequences and polyadenylation signals.
  • Assessment of DNA methylation status at the Cd8a reporter locus in relation to gene expression.

Main Results:

  • hCD2 reporter expression from the modified Cd8a locus required intron incorporation into the hCD2 transcript.
  • Polyadenylation signals downstream of hCD2 cDNA inhibited expression in mature CD8+ T cells, while DP thymocyte expression mimicked endogenous Cd8a expression.
  • Restoration of hCD2 expression in mature CD8+ T cells was achieved by incorporating endogenous or heterologous intron structures, indicating intron-mediated enhancement.
  • Impaired stage-specific DNA demethylation was observed in non-expressing reporter alleles.
  • Even intron sequences lacking splicing signals restored hCD2 expression, suggesting a role beyond canonical splicing.

Conclusions:

  • Intron-mediated enhancement plays a critical role in the stage-specific reactivation of the Cd8a locus during T cell development.
  • Intron sequences, independent of canonical splicing, are essential for efficient Cd8a gene expression in mature CD8+ T cells.
  • The findings provide new insights into the complex regulation of coreceptor gene expression during T cell lineage commitment.