Recruitment of the Mammalian Histone-modifying EMSY Complex to Target Genes Is Regulated by ZNF131

Radhika A Varier,Enrique Carrillo De Santa Pau,Petra Van Der Groep,Rik G.H Lindeboom,Filomena Matarese,Anneloes Mensinga,Arne H Smits,Raghu Ram Edupuganti,Marijke P Baltissen,Pascal W.T.C Jansen,Natalie Ter Hoeve,Danny R Van Weely,Ina Poser,Paul J Van Diest,Hendrik G Stunnenberg,Michiel Vermeulen

doi:10.1074/jbc.m115.701227

Abstract

Recent work from others and us revealed interactions between the Sin3/HDAC complex, the H3K4me3 demethylase KDM5A, GATAD1, and EMSY. Here, we characterize the EMSY/KDM5A/SIN3B complex in detail by quantitative interaction proteomics and ChIP-sequencing. We identify a novel substoichiometric interactor of the complex, transcription factor ZNF131, which recruits EMSY to a large number of active, H3K4me3 marked promoters. Interestingly, using an EMSY knock-out line and subsequent rescue experiments, we show that EMSY is in most cases positively correlated with transcriptional activity of its target genes and stimulates cell proliferation. Finally, by immunohistochemical staining of primary breast tissue microarrays we find that EMSY/KDM5A/SIN3B complex subunits are frequently overexpressed in primary breast cancer cases in a correlative manner. Taken together, these data open venues for exploring the possibility that sporadic breast cancer patients with EMSY amplification might benefit from epigenetic combination therapy targeting both the KDM5A demethylase and histone deacetylases.

Highlights

Recent work from others and us revealed interactions between the Sin3/HDAC complex, the H3K4me3 demethylase KDM5A, GATAD1, and EMSY
By immunohistochemical staining of primary breast tissue microarrays we find that EMSY/KDM5A/SIN3B complex subunits are frequently overexpressed in primary breast cancer cases in a correlative manner
We identify a novel interactor of the EMSY complex, ZNF131, which recruits EMSY to a subset of its genome-wide targets

Summary

Experimental Procedures

Generating Stable Cell Lines and Cell Culture—To generate mammalian cells expressing proteins of the EMSY/KDM5A complex at near endogenous levels bacterial artificial chromosomes containing EMSY, GATAD1, PHF12, or KDM5A, respectively were obtained from the BACPAC Resources Center and a GFP cassette was inserted as a C-terminal in-frame fusion using BAC recombineering (9). HeLa or MCF7 cells were transfected with the recombineered BAC using Lipofectamine 2000 or PEImax (for EMSY) and selected with 400 ␮g/ml geneticin (G418). Short hairpin knockdown of ZNF131 in the EMSY GFP expressing HeLa cells was mediated by lentiviral transduction. Re-expression of EMSYGFP in the EMSY CRISPR knock-out lines was performed using an EMSY-GFP (EX-E2426-M29) from Genecoepia (EX-NEGM29 was the empty control vector). Nuclear extracts obtained from GFP-tagged protein-expressing and wild-type cells were subjected to GFP-affinity pull-downs. EMSY knock-out cells were stably transfected (PEI transfection reagent, G418 selection) with an EMSY-GFP construct or an empty vector control (Fig. 4, D and E). The wells were scanned to obtain representative images, and the number of colonies in each well was counted This was expressed as % colonies survival over the initial number of cells seeded. Beads containing immobilized DNA were incubated with GSTlysates expressing the recombinant ZNF131 domains in a total volume of 600 ␮l of protein binding buffer

A Wild type cells

WDR83 DHPS

Results

Discussion