Analysis of TGFβ1 and IL-10 transcriptional regulation in CTCL cells by chromatin immunoprecipitation

Methods Mol Biol. 2014:1172:329-41. doi: 10.1007/978-1-4939-0928-5_30.

Abstract

The immunosuppressive cytokines transforming growth factor β1 (TGFβ1) and interleukin-10 (IL-10) regulate a variety of biological processes including differentiation, proliferation, tissue repair, tumorigenesis, inflammation, and host defense. Aberrant expression of TGFβ1 and IL-10 has been associated with many types of autoimmune and inflammatory disorders, as well as with many types of cancer and leukemia. Patients with cutaneous T cell lymphoma (CTCL) have high levels of malignant CD4+ T cells expressing IL-10 and TGFβ1 that suppress the immune system and diminish the antitumor responses. The transcriptional regulation of TGFβ1 and IL-10 expression is orchestrated by several transcription factors, including NFκB. However, while the transcriptional regulation of pro-inflammatory and anti-apoptotic genes by NFκB has been studied extensively, much less is known about the NFκB regulation of immunosuppressive genes. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the transcriptional regulation of TGFβ1 and IL-10 by measuring recruitment of NFκB p65, p50, c-Rel, Rel-B, and p52 subunits to TGFβ1 and IL-10 promoters in human CTCL Hut-78 cells.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • CD4-Positive T-Lymphocytes / metabolism*
  • CD4-Positive T-Lymphocytes / pathology
  • Chromatin Immunoprecipitation
  • Humans
  • Interleukin-10 / genetics*
  • Interleukin-10 / metabolism
  • Lymphoma, T-Cell, Cutaneous / genetics*
  • Lymphoma, T-Cell, Cutaneous / metabolism
  • Lymphoma, T-Cell, Cutaneous / pathology
  • NF-kappa B p50 Subunit / genetics
  • NF-kappa B p50 Subunit / metabolism
  • NF-kappa B p52 Subunit / genetics
  • NF-kappa B p52 Subunit / metabolism
  • Promoter Regions, Genetic
  • Proto-Oncogene Proteins c-rel / genetics
  • Proto-Oncogene Proteins c-rel / metabolism
  • Real-Time Polymerase Chain Reaction
  • Signal Transduction
  • Transcription Factor RelA / genetics
  • Transcription Factor RelA / metabolism
  • Transcription Factor RelB / genetics
  • Transcription Factor RelB / metabolism
  • Transcription, Genetic
  • Transforming Growth Factor beta1 / genetics*
  • Transforming Growth Factor beta1 / metabolism
  • Tumor Cells, Cultured

Substances

  • IL10 protein, human
  • NF-kappa B p50 Subunit
  • NF-kappa B p52 Subunit
  • Proto-Oncogene Proteins c-rel
  • RELA protein, human
  • RELB protein, human
  • Transcription Factor RelA
  • Transforming Growth Factor beta1
  • Interleukin-10
  • Transcription Factor RelB