Cohesins form chromosomal cis-interactions at the developmentally regulated IFNG locus

Abstract

Cohesin-mediated sister chromatid cohesion is essential for chromosome segregation and post-replicative DNA repair. In addition, evidence from model organisms and from human genetics suggests that cohesin is involved in the control of gene expression. This non-canonical role has recently been rationalized by the findings that mammalian cohesin complexes are recruited to a subset of DNase I hypersensitive sites and to conserved noncoding sequences by the DNA-binding protein CTCF. CTCF functions at insulators (which control interactions between enhancers and promoters) and at boundary elements (which demarcate regions of distinct chromatin structure), and cohesin contributes to its enhancer-blocking activity. The underlying mechanisms remain unknown, and the full spectrum of cohesin functions remains to be determined. Here we show that cohesin forms the topological and mechanistic basis for cell-type-specific long-range chromosomal interactions in cis at the developmentally regulated cytokine locus IFNG. Hence, the ability of cohesin to constrain chromosome topology is used not only for the purpose of sister chromatid cohesion, but also to dynamically define the spatial conformation of specific loci. This new aspect of cohesin function is probably important for normal development and disease.

Figure 1. Developmentally regulated cohesin and CTCF binding at IFNG

a) ChIP and genomic tiling array data for the mouse B3 pre-B cell line show log2 enrichment of the cohesin subunit Rad21 (black) and CTCF (gray) at Ifng. Schematic representation of the mouse Ifng region (m chr.10) and the human IFNG region (h Chr.12), a flash indicates genomic rearrangement at Il26, a pseudogene in rodents. b) ChIP and real time PCR mapping of Rad21 (top) and CTCF (bottom) at human IFNG in 293T cells (white), non-polarized CD4 T cells (grey), CCR5⁺ Th1 (red) and CRTh2⁺ Th2 (black) effector memory cells. Star symbols indicate statistical significance (Student’s T test) *** p<0.005, ** p<0.05, * p<0.05. Primer positions are indicated relative to the IFNG transcription start site. Human/mouse identity at the −63 kb site is 59% over 291bp surrounding a canonical CTCF consensus motif. Two evolutionary conserved regions (ECR, www.dcode.org) are found at +1.5 kb (70.2% and 69.1% identity over 104 and 217bp) and three at +119 kb (70.0%, 71.0% and 62.2 and 69,1% identity over 120, 100 and 45bp). Positive control sites at Chr11:118283kb, Chr11:118333kb, CD8 Cluster I and ZPF54 were included in all experiments (supplementary figure 1) and data are normalized to Chr11:118283kb (mean±SD, n=4). c) Isolation (left) of human effector memory CCR5⁺ Th1 and CRTh2⁺ Th2 cells and cytokine expression assessed by intracellular staining and flow cytometry (middle) or real time RT-PCR analysis before (contr.) and after activation with plate bound anti-CD3 and anti-CD28 (activ., right).

Figure 2. Cell type-specific long-range chromosomal interactions at IFNG are based on CTCF/cohesin sites

a) Chromosome conformation capture (3C) and Taqman PCR were used to determine interactions between IFNG +1.5 kb (beacon) and primers (black arrowheads) placed within the indicated HindIII fragments (shaded blue) in non-polarized CD4 T cells (grey), CCR5⁺ Th1 (red) and CRTh2⁺ Th2 (black) cells (supplementary figure 2 and table 1). Crosslinking efficiencies are expressed relative to +119 kb in CCR5 cells and normalised to mouse Acta2 (see supplementary methods and supplementary figure 2) or to human ACTA2 with similar results (supplementary figure 3a). Cohesin sites and the approximate positions of the IFNG and IL26 genes are indicated (mean±SD, n=5 for CCR5 and CRTh2, n=4 for non-polarized CD4 T cells). *** p<0.005 (Student’s T test, Th1 vs. non-polarized and Th1 vs. Th2). b) 3D-FISH to determine nuclear diameters of CCR5⁺ Th1 (red, 9.4+/−1.1 μm, n=50) and CRTh2⁺ Th2 cells (black, 9.5+/−1.5 μm, n=50) and distances between IFNG alleles in individual CCR5 (red, 4.5+/−1.6 μm, n=50, minimum 1.5 μm) and CRTh2 cells (black, 5.0+/−1.9 μm, n=50, minimum 1.1 μm). c) Long range interactions at IFNG in CCR5⁺ Th1 cells occur prior to DNA replication. 3C analysis of IFNG in total CCR5⁺ Th1 cells (red) compared to CCR5⁺ Th1 cells in the G1 phase of the cell cycle (orange). Data are presented as in a), (mean±SE, n=2).

Figure 3. Long-range chromosomal interactions at IFNG require cohesin

a) CCR5⁺ Th1 cells were transfected with control (red) or Rad21 (light blue) siRNA oligonucleotides. Long-range interactions at IFNG were assessed by 3C (mean±SE, n=4 for −63, −38 and +119kb, mean±SE, n=2 for −98, −28, +42, +78 and +142kb). Data are expressed relative to +119 kb in control siRNA treated CCR5⁺ cells after normalisation to mouse Acta2 in admixed mouse chromatin, which also served to control for technical aspects of the 3C methodology (supplementary figure 2). Normalisation to human ACTA2 gave similar results (supplementary figure 3b). *** p<0.005, ** p<0.05 (Student’s T test). The expression of CTCF and Rad21 was assessed by immunoblotting 72 hours after transfection of CCR5⁺ Th1 cells with Rad21 or control siRNA. Lamin is a control. b) ChIP for Rad21 (top) and CTCF (bottom) occupancy of IFNG 72 hours after transfection of CCR5⁺ Th1 cells with control (red) or Rad21 siRNA (light blue). Primer positions are indicated in kb (mean± SE, n=2). c) Real time RT-PCR analysis of CCR5⁺ Th1 cells 72 hours after transfection with Rad21 siRNA or control siRNA. Rad21 and CTCF are shown as knockdown controls (top panel, mean± SD, n=5). Levels of IFNG are shown one week (basal, mean± SD, n=5) or 4 hours after T cell activation (induced, mean± SD, n=4) with anti-CD3 and anti-CD28. IL2 is shown as an activation control (mean± SD, n=3) and IGF2 as known cohesin target (mean± SD, n=3). Data are normalised to GAPDH, HPRT and TBP and shown relative to control siRNA transfected CCR5 cells (middle panels). ELISA for basal (mean± SD, n=4) and activation-induced (mean± SD, n=4) IFNγ protein secretion. Data are shown relative to control siRNA CCR5 cells (bottom panels).