Smith, E. M.; Khalizeva, E.; Haug, S.; Cheeseman, I. M.

The heterochromatin protein 1 (HP1) protein family maintains heterochromatin by reading H3K9 methylation and recruiting diverse chromatin effectors. Multiple effectors compete for the same binding surface on HP1, requiring cells to regulate the ability of HP1 to engage the right effectors across differing conditions. Here, we show that mammalian cells produce multiple N-terminally extended isoforms of HP1{beta} from conserved, in-frame near-cognate start codons in the CBX1 5' UTR. Each of these isoforms retains the localization to H3K9me3 marks. However, the extended HP1{beta} isoforms shift its binding preference away from proteins with a conserved PxVxL motif and toward the G9a/GLP complex. Structural predictions suggest that the extension blocks the PxVxL interaction surface. Indeed, we find that an extended HP1{beta} isoform fails to bind a model PxVxL-containing substrate in vitro. Together, our data reveal an alternative HP1{beta} isoform that reshapes its effector binding preferences, providing an additional regulatory layer for heterochromatin organization.