FIG. 6.

Expression of the C terminus of CAF-1 p150 in mouse fibroblasts disrupts the targeting by MBD1 of regions of heterochromatin without affecting the formation of heterochromatin complexes. NIH 3T3 cells grown on coverslips were cotransfected with the appropriate expression constructs and incubated with an anti-Flag MAb and an anti-HA polyclonal rabbit primary antibody. The cells were then washed and incubated with anti-mouse FITC-conjugated and anti-rabbit Cy5-conjugated secondary antibodies. Confocal images of the treated cells were taken to detect FITC (green) and Cy5 (red) immunofluorescence separately. The two images were merged to detect protein colocalization as in Fig. 4. (A) Flag-CAF-1 p150 C terminus and HA-MBD1 fail to target to heterochromatin foci in mouse cells. (B) Flag-CAF-1 p150 C terminus expression has no affect on the targeting of HA-MeCP2. (C) Expression of CAF-1 C terminus in mouse cells does not alter heterochromatin complexes at pericentromeric heterochromatin, as determined by normal HA-HP1α targeting.