Heterochromatic 3D genome organization is directed by HP1a and H3K9-dependent and independent mechanisms[CUT&RUN]
Whether and how histone post-translational modifications and the proteins that bind them drive 3D genome organization remains unanswered. Here, we evaluate the contribution of H3K9-methylated constitutive heterochromatin to 3D genome organization in Drosophila tissues. We find that the predominant organizational feature of wildtype tissues is segregation of euchromatic chromosome arms from heterochromatic pericentromeres. Reciprocal perturbation of HP1a•H3K9me binding, using a point mutation in the HP1a chromodomain or replacement of the replication-dependent histone H3 with H3K9R mutant histones, revealed that HP1a binding to methylated H3K9 in constitutive heterochromatin is required to limit contact frequency between pericentromeres and chromosome arms and regulate the distance between arm and pericentromeric regions. Surprisingly, self-association of pericentromeric regions is largely preserved despite loss of H3K9 methylation and HP1a occupancy. Thus, the HP1a•H3K9 interaction contributes to, but does not solely drive, segregation of euchromatin and heterochromatin inside the nucleus. Overall design: Comparative analysis of CUT&RUN data collected from HP1a chromdomain mutant wing imaginal discs and their controls.