Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs

Abstract

Heterochromatin protein 1 (HP1a) has conserved roles in gene silencing and heterochromatin and is also implicated in transcription, DNA replication, and repair. Here we identify chromatin-associated protein and RNA interactions of HP1a by BioTAP-XL mass spectrometry and sequencing from Drosophila S2 cells, embryos, larvae, and adults. Our results reveal an extensive list of known and novel HP1a-interacting proteins, of which we selected three for validation. A strong novel interactor, dADD1 (Drosophila ADD1) (CG8290), is highly enriched in heterochromatin, harbors an ADD domain similar to human ATRX, displays selective binding to H3K9me2 and H3K9me3, and is a classic genetic suppressor of position-effect variegation. Unexpectedly, a second hit, HIPP1 (HP1 and insulator partner protein-1) (CG3680), is strongly connected to CP190-related complexes localized at putative insulator sequences throughout the genome in addition to its colocalization with HP1a in heterochromatin. A third interactor, the histone methyltransferase MES-4, is also enriched in heterochromatin. In addition to these protein-protein interactions, we found that HP1a selectively associated with a broad set of RNAs transcribed from repetitive regions. We propose that this rich network of previously undiscovered interactions will define how HP1a complexes perform their diverse functions in cells and developing organisms.

Correspondence ofDrosophilapolycomb group proteins with broad H3K27me3 silent domains

“…15 The crosslinking-coupled tandem affinity purification strategy not only improves preservation of bait protein-protein interactions, but also allows the analysis of target DNA in parallel. [16][17][18] We found that Sex comb on midleg (Scm), previously known as a substoichiometric component of PRC1, 3,19 is the only protein strongly enriched with both complexes. 15 Surprisingly, we also found through BioTAP-XL DNA sequencing that Pc, E(z) and Scm show a broad distribution on silent chromatin and overlap significantly with the broad domains of H3K27me3, though they also still show sharp peaks on PREs.…”

“…This demonstrates that BioTAP-XL profiles reflect the distribution of each specific bait protein, rather than any shared affinity of the epitope tag. 17 To probe the colocalization pattern of H3K27me3 and Polycomb proteins with respect to topologically associating domains (TADs) genome-wide, we compared the enrichment of H3K27me3 and Polycomb proteins around the topological boundaries from Hi-C data in embryos ( Fig. 2A).…”

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

“…Previously, our group identified MSL3 associated proteins using large-scale amounts (5). To determine the proteins enriched by the pulldown, we first normalized the total peptide counts for each protein (Dataset S1) to the protein length and to the total number of peptides identified across all proteins in the LC-MS experiment (10) before comparing the pulldown with the input.…”

BioTAP‐XL: Cross‐linking/Tandem Affinity Purification to Study DNA Targets, RNA, and Protein Components of Chromatin‐Associated Complexes

“…In each case, previously known protein subunits as well as novel, yet functionally relevant interactors were identified (Alekseyenko et al, 2014a, Alekseyenko et al, 2014b). Similarly, the DNA targeting of the tagged bait was resolved, and agreed strongly with previous experiments.…”