Regulatory role of regulatory factor for box (RFX5) complex and class II transactivator (CIITA) in the transcription repression of the collagen alpha2(I) gene
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Type I collagen, which consists of two alpha1 chains [α1(I), COL1A1 ] and one alpha2 chain [α2(I), COL1A2 ], is the most abundant member of the collagen family. Interferon-gamma (IFN-γ), which is both an important pro-inflammatory and a anti fibrotic cytokine, coordinately suppresses both α1(I) and α2(I) production primarily at the transcriptional level. Previous work identified a regulatory factor for X-box (RFX) binding site in the first exon of the collagen α2(I) gene. RFX5 complex, consisting of three proteins (RFX5, RFXB, and RFXAP), as well as class II transactivator (CIITA), activates major histocompatibility complex class II (MHC II) transcription during IFN-γ stimulation. This thesis demonstrates that these factors mediate repression of collagen gene transcription in response to IFN-γ. All three RFX5 complex proteins were required for maximum repression of COL1A2 promoter activity. Mutations in regions of RFX5 proteins important for complex formation either reversed repression of collagen transcription or activated collagen transcription, presumably due to dominant negative effects. CIITA, recruited to the collagen transcription start site by RFX5, repressed collagen gene transcription mainly through its acidic and proline-serine-threonine rich domains. Repression of the collagen promoter by CIITA was enhanced in the presence of the RFX5 complex. Inhibition of IFN-γ induced expression of CIITA by RNA interference (RNAi) led to partial alleviation of collagen repression and MHC II activation. IFN-γ decreased collagen transcription at the same time that it increased the expression of RFX5/CIITA complex. In addition, IFN-γ increased the expression of RFXB, but decreased the expression of an RFXB splice variant, RFXBSV. RFXBSV reversed collagen repression by IFN-γ. Both RFX5 and CIITA were present in a large repressor/co-repressor complex containing histone deacetylase (HDAC) and Sin3 proteins. IFN-γ promoted the recruitment of RFX5/CIITA complex as well as HDAC2/Sin3B to the collagen transcription start site, which resulted in the deacetylation of histories surrounding this site. IFN-γ also blocked the occupancy of RNA polymerase II (Pol II) on the collagen transcription start site in conjunction with the increase in CIITA binding. Taken together, these data identify the RFX5/CIITA complex as a repressor of collagen gene transcription.
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