The Relationship Between Long-Range Chromatin Occupancy and Polymerization of the Drosophila ETS Family Transcriptional Repressor Yan
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INVESTIGATION The Relationship Between Long-Range Chromatin Occupancy and Polymerization of the Drosophila ETS Family Transcriptional Repressor Yan Jemma L. Webber,*,1 Jie Zhang,*,†,1 Lauren Cote,* Pavithra Vivekanand,*,2 Xiaochun Ni,‡,§ Jie Zhou,§ Nicolas Nègre,**,3 Richard W. Carthew,†† Kevin P. White,‡,§,** and Ilaria Rebay*,†,4 *Ben May Department for Cancer Research, †Committee on Cancer Biology, ‡Department of Ecology and Evolution, §Department of Human Genetics, and **Institute for Genomics and Systems Biology, University of Chicago, Chicago, Illinois 60637, and ††Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208 ABSTRACT ETS family transcription factors are evolutionarily conserved downstream effectors of Ras/MAPK signaling with critical roles in development and cancer. In Drosophila, the ETS repressor Yan regulates cell proliferation and differentiation in a variety of tissues; however, the mechanisms of Yan-mediated repression are not well understood and only a few direct target genes have been identified. Yan, like its human ortholog TEL1, self-associates through an N-terminal sterile a-motif (SAM), leading to speculation that Yan/TEL1 polymers may spread along chromatin to form large repressive domains. To test this hypothesis, we created a monomeric form of Yan by recombineering a point mutation that blocks SAM-mediated self-association into the yan genomic locus and compared its genome-wide chromatin occupancy profile to that of endogenous wild-type Yan. Consistent with the spreading model predictions, wild-type Yan-bound regions span multiple kilobases. Extended occupancy patterns appear most prominent at genes encoding crucial developmental regulators and signaling molecules and are highly conserved between Drosophila melanogaster and D. virilis, suggest- ing functional relevance. Surprisingly, although occupancy is reduced, the Yan monomer still makes extensive multikilobase contacts with chromatin, with an overall pattern similar to that of wild-type Yan. Despite its near-normal chromatin recruitment, the repressive function of the Yan monomer is significantly impaired, as evidenced by elevated target gene expression and failure to rescue a yan null mutation. Together our data argue that SAM-mediated polymerization contributes to the functional output of the active Yan repressive complexes that assemble across extended stretches of chromatin, but does not directly mediate recruitment to DNA or chromatin spreading. YNAMIC regulation of gene expression during develop- et al. 2010, 2011; Bulger and Groudine 2011; Dunipace Dment requires the combined and coordinated action of et al. 2011; He et al. 2011). However, the molecular deter- transcriptional activators and repressors across multiple cis- minants underlying long-range transcriptional regulation re- regulatory modules (CRMs). Research over the last decade main poorly understood. has led to a growing appreciation of the existence and im- One long-standing hypothesis of transcriptional repres- portance of both short-range linear and long-range three- sion is that the biochemical ability of a factor to polymerize dimensional chromatin interactions to overall regulation of might drive spreading of repressive complexes along the gene expression (Hong et al. 2008; Frankel et al. 2010; Perry chromatin, thereby providing a mechanism of long-range repression (Courey and Jia 2001; Roseman et al. 2001). Copyright © 2013 by the Genetics Society of America doi: 10.1534/genetics.112.146647 Well-studied examples include multiple Polycomb Group Manuscript received October 10, 2012; accepted for publication November 6, 2012 (PcG) corepressors and the ETS family transcriptional Supporting information is available online at http://www.genetics.org/lookup/suppl/ doi:10.1534/genetics.112.146647/-/DC1. repressors TEL1 (ETV6) and Yan, all of which carry a strong 1 These authors contributed equally to this work. oligomerization domain termed the sterile a-motif (SAM) 2Present address: Biology Department, Dickinson College, Carlisle, PA 17013. 3Present address: Institut National de la Recherche Agronomique, UMR1333, (Kim et al. 2001, 2002, 2005; Tran et al. 2002; Qiao et al. Université de Montpellier II, Place Eugène Bataillon, 34095 Montpellier, France. 2004; Qiao and Bowie 2005). In vitro, the isolated SAM 4Corresponding author: Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637. E-mail: [email protected] domains from these proteins form helical, head-to-tail Genetics, Vol. 193, 633–649 February 2013 633 polymers whose overall structural homology suggests a com- model in which SAM-mediated polymerization of Yan does mon mode of function. In vivo, mutations that disrupt SAM- not provide the primary chromatin recruitment mechanism, mediated self-association have been shown to reduce or but is instead required for the function, stability, and/or ablate repression activity of both the PcG and the ETS pro- maintenance of long-range repressive complexes. Given that teins in a variety of cultured cell and transgenic overexpres- SAM-mediated chromatin spreading has also been proposed sion assays (Roseman et al. 2001; Song et al. 2005; Zhang to underlie the extensive chromatin interactions and long- et al. 2010; Robinson et al. 2012). Genome-wide occupancy range repression of PcG proteins, our findings may have analyses of two polymerization-competent PcG proteins in broader implications with respect to the relationship be- Drosophila, Polycomb (Pc) and Polyhomeotic (Ph), have tween polymerization, long-range chromatin occupancy, shown that chromatin occupancy “spreads” over regions rang- and transcriptional repression. ing from several to hundreds of kilobases (Negre et al. 2006; Schwartz et al. 2006; Tolhuis et al. 2006). Comparable stud- Materials and Methods ies have not been performed yet for either human TEL1 or Drosophila Yan, and although it is widely inferred, it has not Fly strains, genotypic selection of yan mutant embryos, been demonstrated that SAM-mediated oligomerization and rescue experiments drives the long-range PcG chromatin occupancy patterns. For chromatin immunoprecipitation (ChIP)–quantitative (q) Here we have focused on the ETS family repressor Yan PCR (ChIP-qPCR) verification of the ChIP-chip results, 400 that acts downstream of receptor tyrosine kinase signaling in stage-11 GFP-negative yan null embryos were hand selected Drosophila to orchestrate a proper balance between prolif- from the cross yanER433/CyO, twist-Gal4, UAS-GFP (CTG)· eration and differentiation in a variety of tissues. Thus yanE833/CTG. For YanV105R ChIP (800 embryos per repli- depending on context, loss of yan leads to overproliferation cate) and rescue experiments, GFP-negative stage-11 em- fi or inappropriate cell fate speci cation, while overexpression bryos were selected from the cross yanER433/CTG; YanV105R · of a constitutively active form can block the induction of yanE833/CTG; YanV105R. Controls were similarly selected a variety of neural, epithelial, and mesodermal cell fates from the cross yanER433/CTG; YanWT · yanE833/CTG; YanWT. (Rebay and Rubin 1995; Rogge et al. 1995; Halfon et al. For the genetic rescue experiments, embryos that hatched 2000; Hsu and Schulz 2000). In-depth investigation of were counted as rescued to larval stage. Embryos that did fi a small number of direct transcriptional targets identi ed not hatch were hand removed from their chorion and vitel- from genetic studies has led to the suggestion that Yan func- line membrane, incubated in a 1:1 solution of glycerol:acetic tions as a short-range passive repressor that competes with acid overnight, mounted in Hoyers:lactic acid, and dried at the ETS family activator Pointed (Pnt) for access to GGA(A/ 65° for 18 hr before imaging on a wide-field microscope. T) ETS consensus-binding motifs within specific cis-regula- Chromatin immunoprecipitation tory enhancers (Klambt 1993; Scholz et al. 1993; O’Neill et al. 1994). Competition between Yan and Pnt is regulated w1118 embryos were collected; aged to stage 5–7 (2h10– by MAPK activation, which attenuates Yan-mediated repres- 3h10) or stage 11 (5h20–7h20, D. melanogaster;10–12 hr sion while stimulating Pnt-mediated activation (Gabay et al. at room temperature, D. virilis); dechorionated in 50% 1996). These regulatory interactions have been proposed to bleach; cross-linked in 10 ml of cross-linking solution (50 provide a bistable switch that must be flipped for a cell to mM HEPES, pH 7.6, 1 mM EDTA, 0.5 mM EGTA, 100 mM commit to a fate (Graham et al. 2010). NaCl, 1.8% formaldehyde) and 30 ml of n-Heptane for To test the model that Yan self-association through its 15 min; washed in Stop Solution (PBS, 0.1% Triton X-100, SAM domain can induce spreading of repression complexes 125 mM Glysine), PBS/T (PBS, 0.1% Triton X-100), and over extended stretches of chromatin and to gain further Wash Solution (10 mM HEPES, 10 mM EDTA, 0.5 mM insight into Yan-mediated regulation of gene expression EGTA, 0.25% Triton X-100); homogenized in ChIP lysis during development, we compared the global chromatin buffer [50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 1 mM occupancy profile of endogenous wild-type Yan to that of EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, prote- a recombineered genomic transgene carrying a missense ase inhibitor tablet (Roche)]; and chromatin sonicated to mutation in the SAM domain that restricts the Yan protein to a final size of 500 bp, using a Fisher Scientific (Pittsburgh) a monomeric form. Consistent with