P-STAT1 mediates higher-order chromatin remodelling of the human MHC in response to IFN

JCS012328 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Time course of higher-order chromatin remodelling induced by IFNγ in HT1080 cells. The frequency with which the MHC class II gene HLA-DRA is present on an external chromatin loop, is shown for different times following the start of IFNγ treatment. The mean percentage of HLA-DRA signals on external chromatin loops is represented on the y-axis, with error bars showing the standard deviation. The different exposure times to IFNγ are shown on the x-axis. At least 200 chromosome territories were counted for each time point.

• Supplemental Figure S2 -

  Fig. S2. Expression of exogenous CIITA in STAT1-null U3A cells does not restore HLA-DRA expression or chromatin remodelling. (A) Western blot analysis ase performed in U3A cells, U3A cells stably transfected with CIITA (U3A/CIITA) and AHB B-lymphoblastoid cells, constitutively expressing CIITA. The upper section of the membrane was probed with anti-CIITA antibody, the lower section with anti-actin antibody as a loading control. (B) Reverse transcriptase RT-PCR shows no induction of HLA-DRA after expression of exogenous CIITA in U3A cells with and without 24 hours IFNγ treatment.

• Supplemental Figure S3 -

  Fig. S3. Map of the human MHC showing probes used for interphase distance measurements and regions analysed by ChIP. Probes used for distance measurements that are outside the MHC, RP11-239L20, RP11-373N24, BAC RP11-513I15, PAC RP1-50J22 at 27 Mb, 28.7 Mb, 34.3 Mb, and 36.4 Mb, respectively, are not shown here. The genomic distances (Mb) from the telomere on the short arm are given below the line. Vertical arrows show regions analysed by ChIP.

• Supplemental Figure S4 -

  Fig. S4. Exogenous BRG1 expression in SW13 cells restores the IFNγ response. (A) Reverse transcriptase RT-PCR analysis after re-expression of BRG1 in SW13 cells. SW13-SW13 cells infected with an empty vector; SW13-BRG1−SW13 cells infected with BRG1 vector; SW13-BRG1+IFNγ − SW13-BRG1 cells treated with IFNγ for 24 hours. (B) Western blot analysis was performed in B-lymphoblastoid cells AHB; BRG1/BRM deficient SW13 cells, stably infected with pBabe-hBRG1- IRESpuro (SW13/BRG1), and SW13 cells, infected with the empty vector. The upper section of the membrane was probed with anti-BRG1 antibody, the lower section with anti-actin antibody as a loading control.

• Supplemental Table S1 -

  Table S1. TFIIB binding to the genes across the MHC is independent of STAT1. (A) ChIP experiments were performed using TFIIB antibody in HT1080 and U3A cells. Immunoprecipitated DNA and serial dilutions of genomic DNA were subjected to real-time PCR reactions using SybrGreen Master Mix. The amount of immunoprecipitated DNA (ng) for each promoter was calculated using a genomic DNA standard curve. The average of at least 3 experiments is shown. (B) Primer sequences used for the ChIP assay. Positions of the genes in the MHC are shown in Supplemental Figure S5.

• Supplemental Table S2 -

  Table S2. Interphase distances between probe pairs from the MHC region on 6p21.3, the gene-poor region on 6p24 and from regions flanking the MHC ± standard error of the mean. The overall change in interphase distance is given in percent in the right-hand column.