Sequence Requirements for Combinatorial Recognition of Histone H3 by the MRG15 and Pf1 Subunits of the Rpd3S/Sin3S Corepressor Complex

Abstract

The transcriptional output at a genomic locus in eukaryotes is determined, in part, by the pattern of histone modifications that are read and interpreted by key effector proteins. The histone deacetylase activity of the evolutionarily-conserved Rpd3S/Sin3S complex is crucial for suppressing aberrant transcription from cryptic start sites within intragenic regions of actively transcribed genes. Precise targeting of the complex relies on the chromatin binding activities of the MRG15 and Pf1 subunits. Whereas the molecular target of the MRG15 chromodomain (CD) has been suggested to be H3K36me2/3, the precise molecular target of the Pf1 plant homeodomain 1 (PHD1) has remained elusive. Here we show that Pf1 PHD1 binds preferentially to the unmodified extreme N-terminus of histone H3 (H3K4me0) but not to H3K4me2/3, which are enriched in the promoter and 5′ regions of genes. Unlike previously characterized CD and PHD domains that bind to their targets with micromolar affinity, both MRG15 CD and Pf1 PHD1 bind to their targets with >100 μM affinity, offering an explanation for why both MRG15 CD and Pf1 PHD1 domains are required to target the Rpd3S/Sin3S complex to chromatin. Our results also suggest that bivalency, rather than cooperativity, is the operative mechanism by which Pf1 and MRG15 combine to engage H3 in a biologically significant manner. Finally, the studies reveal an unanticipated role of Pf1 PHD1 in engaging the MRG15 MRG domain, albeit in a Pf1 MRG-binding domain (MBD)-dependent manner, implying a key role for the MRG15 MRG-Pf1 MBD interaction in chromatin targeting of the Rpd3S/Sin3S complex.

Combinatorial Histone Readout by the Dual Plant Homeodomain (PHD) Fingers of Rco1 Mediates Rpd3S Chromatin Recruitment and the Maintenance of Transcriptional Fidelity

“…1F, we consistently found that both PHD1 and PHD2 preferentially bound to the N-terminal region (residues 1-20) of the H3 tail. This result is consistent with previous analyses of PHD1 (22,23). Interestingly, we also found that trimethlyation of K4 (H3K4me3) decreases the ability of both PHD1 and PHD2 to bind N-terminal H3 1-20 peptides, suggesting that these domains bind to the extreme N terminus of H3 and are affected by N-terminal post-translational modifications.…”

“…Finally, we note that although a previous survey of yeast PHD domains was performed using solution peptide pulldowns (22), our studies differ in regards to the ability of PHD1 and PHD2 to bind H3K36me3, a result that is also true for the characterized PHD1 domain of the human Rco1 counterpart, Pf1 (23). We note that PHD1 and PHD2 expression and maintaining their stability in vitro was found to be extremely difficult, and further, that binding and washing conditions greatly impacted weak interactions and nonspecific binding.…”

MRG15-mediated tethering of PALB2 to unperturbed chromatin protects active genes from genotoxic stress

“…S1 D and E) (19,20). This prompted us to investigate whether H3K36me3 was required for MRG15-mediated PALB2 chromatin association.…”

The Chromatin-Associated Phf12 Protein Maintains Nucleolar Integrity and Prevents Premature Cellular Senescence

“…To identify the molecular basis underlying the impact of Pf1 on nucleolar structure and function, we aimed to identify novel Pf1-interacting proteins. To do so, we generated Flag-tagged constructs of wild-type Pf1 and two Pf1 variants with point mutations unable to interact either with MRG15 or with histone H3 (the Pf1 F210A and Pf1 D57N mutants, respectively [5,31]). We then stably expressed the three constructs in HeLa S3 cells, extracted nuclear proteins, immunoprecipitated protein complexes containing the Pf1 construct using anti-Flag beads, and analyzed the protein composition of the precipitated complexes by liquid chromatography-tandem mass spectrometry (LC-MS/MS).…”