FIGURE 7.

Recruitment of H1 to the regulatory region of pluripotency and differentiation genes. A, recruitment of total H1 in undifferentiated ES[4], ES-derived EBs at day 15 of differentiation and human fibroblasts (F). ChIP with a pan-H1 antibody (AE-4) was performed, and H1 occupancy at several promoters was interrogated with specific oligonucleotides by qPCR. Amplification of input DNA (representing 1% of immunoprecipitated DNA) was used for normalization. To allow proper comparison of the H1 occupancy between different cell lines, the data were normalized to the mean of H1 occupancy at two satellite regions (SATAB and SATXL) that may represent a highly invariable portion of the genome in terms of H1 occupancy. B, recruitment of overexpressed HA-tagged H1.0 in KiPS4F1, ES[4], and ES-derived EBs at day 15 of differentiation, keratinocytes (K) and fibroblasts (F). The cells were transduced with an HA-tagged H1.0 expressing lentivirus and submitted to ChIP analysis with an anti-HA antibody, and H1.0 occupancy at several gene promoters was interrogated as in A. To overcome potential differences in the expression of HA-H1.0 in each transduced cell line, the data were normalized to the mean of HA-H1.0 occupancy at two satellite regions (SATAB and SATXL). C, recruitment of H1 in undifferentiated and differentiated NT2 cells. ChIP with antibodies against total H1 (AE-4), H1X, or H1.2 was performed, and occupancy at the indicated promoters was interrogated with specific oligonucleotides by qPCR. Amplification of input DNA was used for normalization. D, H1 occupancy of NANOG and OCT4 promoters in breast cancer cells. T47D cells stably expressing HA-tagged H1.0, H1.2, or H1.4 were submitted to ChIP analysis with an anti-HA antibody, and H1 occupancy at several promoters was interrogated with specific oligonucleotides by qPCR. The data are normalized to amplified input DNA. The means and S.D. of at least two independent quantifications of representative experiments are shown throughout the figure.