Figure 8.

H3K9me3 and H3K9me2 double knockdown inhibits peripheral targeting of endogenous human β-globin locus (a) Schematic of an ∼2.5-Mbp region showing human β-globin locus and its flanking LAD regions (Lamin B1 DamID, human fibroblast Tig3 cells, hg19 assembly; University of California, Santa Cruz, Genome Browser). DNA FISH BAC probes shown below the map as black lines are labeled red for HBB and green for LAD sequences. chr, chromosome. (b–e) Representative DNA FISH images showing classes of HBB (red) and adjacent LAD localization (green). Gray, DAPI. (b) HBB and LAD both interior. (c–c″″) HBB and LAD both peripheral. (d and e) Polarized orientation with HBB locus at the periphery and LAD extended into the interior (d) or LAD locus attached at one end to the periphery but extended such that HBB is in the interior (e). (f–i) statistics for these different localization classes (b–e). Suv39H (H3K9me3 [me3] knockdown [KD]) knockdown or G9a (H3K9me2 [me2] knockdown) inhibition did not change the localization of HBB; however, double knockdown of H3K9me3 and H3K9me2 significantly reduces the preferential peripheral localization of HBB. (e and i) The polarized orientation with the flanking LAD attached peripherally at its distal end suggests existence of a third tethering mechanism, independent of Suv39H and G9a (Fig. 10 a). n > 100; data shown are pooled from at least three independent experiments. Bars, 2 µm. Insets are at 2×. Statistical significance: *, P < 0.05.