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First published online 28 August 2007
doi: 10.1242/jcs.012328

Journal of Cell Science 120, 3262-3270 (2007)
Published by The Company of Biologists 2007
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P-STAT1 mediates higher-order chromatin remodelling of the human MHC in response to IFN{gamma}

Rossitza Christova1,2,*, Tania Jones1,2,*, Pei-Jun Wu1,*, Andreas Bolzer1, Ana P. Costa-Pereira3,4, Diane Watling3,4, Ian M. Kerr3 and Denise Sheer1,2,{ddagger}

1 Human Cytogenetics Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK
2 Institute of Cell and Molecular Science, Queen Mary's School of Medicine and Dentistry, 4 Newark St, London, E1 2AT, UK
3 Biochemical Regulatory Mechanisms Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK
4 Imperial College London, Faculty of Medicine, Department of Oncology/SORA, Hammersmith Hospital, Du Cane Road, London, W21 ONN, UK


Figure 1
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  		Fig. 1. P-STAT1 is essential for the induction of external chromatin loops carrying the MHC locus in response to 24-hour IFN{gamma} treatment. FISH signals for HLA-DRA are detected in red and for the CT6 in green. (A) IFN{gamma} induction of external chromatin loops carrying the HLA-DRA gene. (B) No IFN{gamma} induction of external chromatin loops carrying the HLA-DRA gene in this STAT1-null cell line. U3A cells are tetraploid and contain four copies of chromosome 6. (C) IFN{gamma} induction of external chromatin loops carrying the HLA-DRA gene in U3A cells complemented with STAT1. (D) No IFN{gamma} induction of external chromatin loops carrying the HLA-DRA gene in U3A cells complemented with a STAT1 point mutant (Y701F) that cannot be phosphorylated by JAKs.

 

Figure 2
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  		Fig. 2. ChIP analysis of transcription factor recruitment upon IFN{gamma} induction. HT1080 and STAT1-null U3A cells were treated with 200 IU/ml IFN{gamma} at the times shown, and crosslinked with formaldehyde. Immunoprecipitated DNA and serial dilutions of genomic DNA were then subjected to quantitative RT-PCR. The fold enrichment was calculated relative to the non-induced levels. ChIP experiments were performed using antibodies recognizing P-STAT1, RNAP II, BRG1 and acetylated Histone H3. The mean values of at least three experiments are shown, together with standard error bars.

 

Figure 3
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  		Fig. 3. Analysis of the role of BRG1 and BRM in large-scale chromatin remodelling of the MHC. SW13 cells (BRG1-BRM-negative) were treated with IFN{gamma} for 24 hours where indicated. HLA-DRA is detected in red and the CT6 in green. (A) SW13 cells without IFN{gamma}. (B) SW13 cells treated with IFN{gamma}. No external chromatin loops are observed. (C) SW13 cells, complemented with BRG1 and treated with IFN{gamma}. External chromatin loops carrying the MHC are clearly observed.

 

Figure 4
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  		Fig. 4. Chromatin remodelling can be induced in MRC5 fibroblasts by IFN{gamma} and sodium butyrate (SB). (A) FISH image showing HLA-DRA (red) extending from the painted CT6 (green) in MRC5 cells after treatment with SB for 1 hour. (B) Comparison of external chromatin loops for the MHC and the 6p24 region in untreated control cells, in cells treated with IFN{gamma} for 24 hours and in cells treated with SB for 1 hour (n>=100). A significant increase in the percentage of external chromatin loops was observed for all three MHC regions in IFN{gamma} and SB-treated fibroblasts compared with untreated cells. (C) External chromatin loops carrying HLA-DRA induced by SB treatment over a 1-hour time course. (D) Comparison of external chromatin loops induced by IFN{gamma} over a 24-hour time course in control cells (without IFN{gamma}) and cells treated with SB for the last 1 hour of the IFN{gamma} treatment. The mean percentage of signals on external chromatin loops is represented on the y-axis, error bars show the standard deviation. The value at the 0-minute (0') time point for MRC5+SB+IFN{gamma} is high because the cells have been exposed to SB for 1 hour.

 

Figure 5
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  		Fig. 5. Chromatin in the MHC becomes decondensed by IFN{gamma} treatment. Relationship between mean square interphase distance and genomic separation (Mb) of the probe pairs within the MHC. Black diamonds: control cells without IFN{gamma}. Grey squares: cells treated with IFN{gamma} for 24 h. The slope of each regression line is indicated by y, r is the correlation coefficient. (A-C) Distances were measured between probe pairs, regardless of whether they were (A) on an external loop or within the CT6, (B) located within the CT6 or, (C) located on an external loop.

 

Figure 6
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  		Fig. 6. Model for chromatin changes during activation of the MHC by IFN{gamma}. The MHC is located within the CT6 before induction. GAS elements ({diamond}) and CIITA-binding sites (bullet) are shown. Binding of P-STAT1 ({diamondsuit}) leads to the release of the entire MHC, which loops outside the CT6 domain. After 2-4 hours the locus becomes hyperacetylated and accessible to transcription factors. CIITA (Figure 6) is synthesised and binds to the HLA genes 4-6 hours after IFN{gamma} induction to activate transcription.

 

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© The Company of Biologists Ltd 2007