Figure 1. Developmentally regulated cohesin and CTCF binding at IFNG.

a) ChIP and genomic tiling array data for the mouse B3 pre-B cell line¹⁰ 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 rodents²⁰.

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).