CIITA is silenced by epigenetic mechanisms that prevent the recruitment of transactivating factors in rhabdomyosarcoma cells

Abstract

Rhabdomyosarcomas (RMS) are highly malignant pediatric sarcomas. We have discovered that the gene encoding the major histocompatibilty complex class II transactivator, CIITA, is silenced in cells representing both major subtypes of RMS. Silencing of CIITA prevents the IFN-γ inducible expression of MHC class II genes in these cells. Overexpression of CIITA in these cells can restore MHC expression. We have found that IFN-γ signaling is intact in these cells, but pSTAT1 and IRF1 do not bind to the CIITA PIV promoter. The CIITA promoter is not hypermethylated in RD (ERMS) cells but does show a modestly enhanced methylation status in SJRH30 (ARMS) cells. We have found that histone acetylation, which normally increases on the CIITA PIV promoter following IFN-γ treatment, is blocked in both types of RMS cells. In RD cells, treatment with a histone deacetylase inhibitor (TSA) reverses the silencing of CIITA. In SJRH30 cells, treatment with DNA methyltransferase inhibitors and TSA cooperatively restores CIITA expression. Surprisingly, we have also shown that the expression of two components of the immunoproteasome, which are embedded in the class II locus, is stimulated by IFN-γ in certain RMS cells in the absence of stimulation by CIITA. CIITA overexpression can also activate the expression of these genes, indicating that the immunoproteasome genes LMP2 and LMP7 can be activated by both CIITA dependent and CIITA independent pathways.

Figure 1. IFN-γ does not stimulate CIITA and MHC class I and II gene expression in RMS cells

A. IFN-γ does not stimulate CIITA expression in RMS cells. The indicated cell lines were stimulated with 500 U IFN-γ for 24 hours (+). Untreated samples are indicated with a negative sign. Gene expression was assayed for CIITA. B. IFN-γ does not stimulate MHC class II expression in RMS cells. Cell lines were stimulated as in A. Gene expression was assayed for HLA-DRA for human samples and H2-Ea for murine samples. C. IFN-γ does not stimulate MHC class I expression in RMS cells. Cells were stimulated as in A. Gene expression was assayed for HLA-B for human samples and H2-K1 for murine samples.

Figure 2. MHC expression can be restored by CIITA over expression

A. CIITA over expression restores MHC class I and II expression. Stable cell lines over expressing CIITA were assayed for the expression of CIITA, HLA-DRA or HLA-B. For A., error bars reflect standard deviation from the mean from the replicated samples. B. MHC class I and II presentation is restored by over expression of CIITA. SJRH30 cells with and without CIITA were assayed for cell surface expression of HLA-A,B,C and HLA-DR with or without IFN-γ stimulation by FACS analysis. The black curves represent the control cells and the grey curve represents the CIITA overexpression cells.

Figure 3. IFN-γ can stimulate pSTAT1 in RMS cells, but pSTAT1 does not bind DNA

A. The IFN-γ receptor is expressed in RMS cells. Gene expression was assayed with primers against IFNGR and represented as expression levels relative to the levels found in HeLa cells normalized to HPRT expression levels. Error bars reflect standard deviation from the mean from the replicated samples. B. STAT1 is present in RMS cells. Protein extracts from the indicated cells lines were used for western blot analysis. Blot was probed with anti-STAT1 and anti-GAPDH antibodies. C. STAT1 is phosphorylated in response to IFN-γ in RMS cells. Indicated cell lines were stimulated with 500 u IFN-γ for 24 hours and harvested for protein and used for western blot analysis. Blot was probed with anti-phospho STAT and anti-GAPDH antibodies. D. Indicated cells were cross linked and used for ChIP assays with antibodies against phospho-STAT1 and IgG and primers against the pIV promoter of CIITA. Data shown are fold change between signal detected at the CIITA PIV promoter for the pSTAT1 antibody minus the signal detected for IgG. Standard error from the mean was calculated from replicate [Δ][Δ]Ct values. A region of Chr19 was used to normalize the fold enrichments for the individual promoters. E. IRF1 is induced by IFN-γ in ARMS cell, but not ERMS cells. Cells were stimulated with IFN-γ or vehicle control for 24 hours and gene expression was monitored with primers against IRF1. F. IRF1 does not bind the PIV promoter of CIITA. ChIP assays on cells treated as in E. with antibodies against IRF1 and primers against the PIV promoter of CIITA are shown.

Figure 4. The CIITA promoter is not hypermethylated but IFN-γ stimulated histone acetylation and RNAPII recruitment are blocked in RMS cells

A. Schematic representation of the CIITA PIV region. CpG nucleotides are indicated in bold and numbered. Known transcription factor binding sites are boxed and labeled. B. Bisulfite sequencing of the template strand of genomic DNA from RD, SJRH30 and HeLa cells. Numbers refer to the CpG nucleotide numbers in A. At least ten individual CIITA-PIV amplicons were examined in each case. Each sequenced amplicon is represented by a horizontal row of boxes. Methylated CpGs are indicated by black boxes and unmethylated CpGs are indicated by white boxes. C. ChIP assays on IFN-γ stimulated and unstimulated HeLa cells with antibodies against RNAPII, histone H3 9,18 acetylation, histone H3 and IgG. Data shown are fold change between signal detected at the CIITA PIV promoter for each antibody minus the signal detected for IgG. A gene desert region of Chr19 was used to normalize the data. D. As in C., except RD cells were used. E. As in C., except SJRH30 cells were used.

Figure 5. Inhibition of histone deacetylases activates CIITA and class II expression in RD cells and partially reactivates CIITA in SJRH30 cells

A. Histone deacetylase inhibitors stimulate CIITA expression in RD cells. Cells were treated with the histone deacetylase inhibitor TSA or with 5-aza-2'-deoxycytidine. 5-aza-2'-deoxycytidine was used at 1uM, 2uM and 5uM as indicated on the graph. Gene expression of CIITA was assayed by qRT-PCR. Data are shown as fold stimulation of treated samples vs. untreated samples. B. Histone deacetylase inhibitors and methyltransferase inhibitors cooperatively activate CIITA expression in SJRH30 cells. Data as in panel A, except SJRH30 cells were used. C. TSA and 5-aza-2'-deoxycytidine treatment increases the level of H3Ac and induces the binding of pSTAT1. ChIP assays were performed on SJRH30 cells treated with IFN-γ or vehicle control with antibodies against H3K9,18Ac and pSTAT1 and primers against the pIV promoter of CIITA.

Figure 6. Immunoproteasome components are stimulated by IFN-γ and CIITA in certain RMS cells

A. Schematic representation of the locus containing LMP2 and LMP7. Shown is the mouse locus. B. Immunoproteasome components are stimulated by IFN-γ in many RMS cell lines. RD2, U48484 and HeLa cells do not significantly activate these genes in response to IFN-γ. Cell lines were stimulated with 100 u/ml IFN-γ and gene expression assayed for LMP2 and LMP7. Transcript levels were determined by qRTPCR and normalized to the mRNA levels of HPRT. Numbers indicate induction factors relative to untreated samples. C. Overexpression of CIITA in RMS lines also induces LMP2 and LMP7. Cell line overexpressing CIITA were assayed for expression of LMP2 and LMP7. Transcript levels were determined by qRT-PCR and normalized to the mRNA levels of HPRT. Numbers indicate induction factors relative to vector only samples.