Figure 6.

CTCF is important for cell cycle progression. (A) Quantitative RT–PCR analysis in sorted DN, ISP and DP cell fractions from wild-type (WT) and Lck–Cre Ctcf^f/f mice. The DP fraction also contained CD4 SP cells. To obtain enough material, RNA was pooled from four WT and two Lck–Cre Ctcf^f/f mice. (B) Cell cycle status of DN, ISP and DP cells isolated from WT and Lck–Cre Ctcf^f/f mice. Cell cycle was analysed using 7-AAD, which measures DNA amount. A representative analysis is shown. Numbers indicate the percentage of cells in S/G2/M. In Lck–Cre Ctcf^f/f mice, this number is significantly reduced in ISP cells, showing that this population is hampered in the cell cycle. (C) Quantitative RT–PCR (left hand panel) and ChIP (right hand panel) analysis in wild-type and Ctcf^f/f MEFs, after treatment with Cre recombinase. mRNA expression in Cre-treated Ctcf^f/f MEFs (KO) is shown relative to wild type. Although residual Ctcf mRNA is present in the MEFs, p21 expression is increased. ChIP analysis was performed with anti-CTCF antibodies on four regions in the p21 gene. Potential CTCF-binding sites in the mouse p21 gene were chosen on the basis of a genome-wide analysis in human cells (Barski et al, 2007). An identical binding pattern was observed in wild-type MEFs, with relatively weak CTCF binding 2.3 kb upstream of the p21 promoter (up) and on the promoter (pr), and very strong binding on two adjacent regions within intron 1 (i-1a, i-1b). (D) Quantitative RT–PCR (left hand panels) and ChIP (right hand panel) analysis in wild-type thymocytes and T cells before (d0) and after (d3) 3 days of anti-CD3/CD28 stimulation in vitro. mRNA expression of p21 and Ctcf is shown relative to Hprt. ChIP analysis was performed with anti-CTCF antibodies on intron 1 of the p21 gene and, as positive control, on known binding sites in the c-Myc and Tcrα genes.