Identification of CCR4 and other essential thyroidhormone receptor co-activators by modified yeastsynthetic genetic array analysisManjapra Govindana, Xianwang Mengb, Clyde L. Denisc, Paul Webbd, John D. Baxterd,1, and Paul G. Walfishb,1

aDepartment of Molecular Biology, Biochemistry and Pathology, Centre de Recherche L’Hotel-Dieu de Quebec, Universite Laval, QC, Canada G1R 2J6;bEndocrine Division, Mount Sinai Hospital, University of Toronto Medical School, Toronto, ON, Canada M5G 1X5; cDepartment of Biochemistryand Molecular Biology, University of New Hampshire, Durham, NH 03824; and dCenter for Diabetes Research, Methodist Hospital ResearchInstitute, Houston, TX 77030

Contributed by John D. Baxter, September 24, 2009 (sent for review November 8, 2007)

Identification of thyroid hormone receptor (TR) co-regulators hasenhanced our understanding of thyroid hormone (TH) action.However, it is likely that many other co-regulators remainedunidentified, and unbiased methods are required to discover theseproteins. We have previously demonstrated that the yeast Saccha-romyces cerevisiae is an excellent system in which to study TRaction, and that defined TR signaling complexes in a eukaryoticbackground devoid of complicating influences of mammalian cellco-regulators can be constructed and analyzed for endogenousyeast genes, many of which are conserved in mammals. Here, amodified synthetic genetic array analysis was performed by cross-ing a yeast strain that expressed TR�1 and the co-activator GRIP1/SRC2 with 384 yeast strains bearing deletions of known genes.Eight genes essential for TH action were isolated, of which 4 areconserved in mammals. Examination of one, the yeast CCR4 and itshuman homolog CCR4/NOT6 (hCCR4), confirmed that (i) trans-fected CCR4 potentiates a TH response in cultured cells moreefficiently than established TR co-activators and (ii) knockdown ofCCR4 expression strongly inhibited a TH response (>80%). THtreatment promoted rapid and sustained hCCR4 recruitment to theTH-responsive deiodinase 1 promoter and TR co-localizes withhCCR4 in the nucleus and interacts with hCCR4 in 2-hybrid andpull-down assays. These findings indicate that a modified yeastsynthetic genetic array strategy is a feasible method for unbiasedidentification of conserved genes essential for TR and other nuclearreceptor hormone functions in mammals.

nuclear receptor co-activators � conserved nuclear receptor signaling genes

Thyroid hormone (TH) receptors (TRs) are members of thenuclear receptor (NR) family (1, 2). TRs bind TH response

elements (TREs) located at variable distances from the tran-scription initiation site of genes with roles in growth, develop-ment, and metabolism. Unliganded TRs are transcriptionallyactive, either stimulating or inhibiting transcription. Hormonebinding (mostly triiodothyronine) generally reverses theseeffects; active repression by unliganded TRs becomes transcrip-tional enhancement and vice versa. These changes in transcrip-tional response are a consequence of hormone-dependent al-terations in the conformation of the receptor C-terminal ligandbinding domain, which result in release of cognate co-regulatorsand sequential recruitment of other co-regulator complexes (3).

TR co-activators have been identified on the basis of theirdirect interactions with TRs and other hormone-dependent NRs(4, 5). Among the best known are the steroid receptor co-activators SRC1, SRC2 (TIF2/GRIP1), and SRC3 (pCIP/RAC3/AIB1/ACTR), which are implicated in chromatin modification.SRC-1 and SRC-3 possess intrinsic histone acetyl-transferaseactivity and all SRCs bind auxiliary factors with histone acetyl-transferase activity, including p300, CREB-binding protein(CBP), and p300/CBP-associated factor P/CAF. Another cofac-tor, the 220-kDa TR associated protein (TRAP220/DRIP205),

is part of the mediator/SMCC complex, which contacts the basaltranscription machinery (6). Other co-activators with potentialroles in NR signaling include components of the CCR-NOTcomplexes (7). The well characterized CCR-NOT subunit iscalled carbon catabolite repressor 4 (CCR4) in yeast and NOT6in higher eukaryotes. CCR4/NOT6 (hCCR4) resembles theExoIII family of nuclease/phosphatases (8) and is implicated inmRNA deadenylation in the cytoplasm (9). Although identifi-cation of TR interacting cofactors has provided great insightsinto the mechanism of transcriptional activation, questions aboutthe identity, function, and interactions of many TR co-activatorsremain unanswered. Potential roles of cofactors with alternatecontact modes, or cofactors that lie completely downstream ofdirect TR contacts, are less clear. New unbiased techniques areneeded to systematically identify and categorize key TRco-activators.

We previously proposed that the yeast Saccharomyces cerevi-siae is an excellent system in which to study NR signaling (10).Yeast lack conventional NRs and SRCs, but contain homologsof key mammalian enzymes required for chromatin modificationand general transcription factors. Thus, it is possible to recon-struct defined NR signaling complexes in the absence of con-founding effects of other NR cofactors present in mammaliancells. We reconstituted TH-dependent gene activation at TRE-driven �-gal reporters in yeast that express TR, with and withoutthe heterodimer partner retinoid X receptor (RXR) and GRIP1/SRC2 (11–14), and used similar approaches to investigate TRinteractions with adenovirus E1A and truncated versions of theco-repressor N-CoR, co-activators that bind TRs in the typicalco-repressor mode (15, 16). Our previous studies showed thatSWI/SNF, GCN5, and ADA2 (SAGA) multi-component com-plexes are also required for activity of thyroid hormone action(13, 16). This represents proof of concept that yeast geneticanalysis can reveal cofactor requirements for TR signaling and,given strong homologies between yeast and mammalian genes,suggests that similar approaches could be used in a morecomprehensive manner to identify factors needed for TR actionthat are also important in mammalian cells.

In the current report, we used a synthetic genetic analysis(SGA) to test requirements for yeast genes in TR signaling.

Author contributions: M.G. and P.G.W. designed research; M.G., X.M., and P.G.W. per-formed research; M.G. and C.L.D. contributed new reagents/analytic tools; M.G., X.M., P.W.,J.D.B., and P.G.W. analyzed data; and M.G., X.M., P.W., J.D.B., and P.G.W. wrote the paper.

Presented in part at the 77th Annual Meeting of the American Thyroid Association,Phoenix, October 11–15, 2006.

Conflict of interest statement: J.D.B. is a consultant to and shareholder of Karo Bio AB,which has commercial interests in thyroid hormone mimetics.

1To whom correspondence may be addressed. E-mail: jbaxter918@aol.com, walfish@mshri.on.ca or pwalfish@mtsinai.on.ca.

This article contains supporting information online at www.pnas.org/cgi/content/full/0910134106/DCSupplemental.

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Standard SGA relies on the fact that �80% of the 6,200predicted yeast genes are non-essential and that strains bearingdeletions of these genes are viable (17). A modified version ofSGA was devised in which a yeast strain that contains aTRE-driven �-gal reporter and expresses TR�1 and GRIP1/SRC2 is crossed with strains that bear deletions of 384 differentgenes implicated in transcription or chromatin remodeling. Wefind that 8 of these genes are needed for optimal TR activity andpresent evidence that one, hCCR4, is also essential for TRsignaling in cultured HeLa cells. These observations indicatethat the proposed modified yeast SGA strategy is a feasiblemethod for the systematic identification of mammalian genesessential for NR function.

ResultsYeast SGA Screen. To test the possibility that SGA could revealnovel factors that are required for TR activity, we obtainedsample plates with yeast strains bearing individual deletions of384 genes. The genes included are listed in Table S1; the panelcorresponds to a portion of the complete ordered array of yeastmutant strains (17). It does not include genes that we havepreviously shown to be needed for TR signaling in yeast—ADA2, GCN5 and swi/snf 2, 3, and 6—but represents a rea-sonable fraction of known yeast genes and contains multiple

genes with potential roles in transcription and chromatin re-modeling.

The screen was performed in accordance with the previouslydescribed SGA procedures, which were modified as outlined inFig. 1A and described in Materials and Methods. A typical platescreen is illustrated in Fig. 1B. One hundred twenty-five cloneswere selected on the basis of suspected reductions in bluecoloration. Each was grown in liquid culture for direct �-galassays, and 31 clones with �60% loss of function in the test SGAstrain were selected for further study. We also verified require-ments for these genes by purchasing commercially available yeastdeletion mutants from the American Type Culture Collection(ATCC), which were prepared in a slightly different geneticbackground. Eight of the 31 selected clones continued to exhibit�60% loss of function by a �-gal assay.

Of the 8 identified genes (Table S2), 4 have known humanhomologs. These include CCR4/NOT6, the major component ofthe CCR4/NOT complex as previously described (7, 9). Inaddition, TR also exhibited strong requirements for SAC3 (18),a nuclear pore-associated protein; SHP1 (19), a protein involvedin ubiquitin dependent protein-degradation; and the SWI4 sub-unit of the swi/snf chromatin remodeling complex (20). Fourgenes that were needed for TR action were specific to yeastwithout a known human homolog: YBR175W, YJL175W, SRB2,

Fig. 1. SGA screening reveals yeast genes essential for hTR�1 action. (A)Strategy: MAT�:Y5563 yeast strain co-transformants that expressed hTR�1

and GRIP1 and contained a TRE-F2 � 1 �-gal reporter were crossed with anordered array of MATa gene deletion mutants. To select for growth of mutantmeiotic progeny expressing TR, GRIP1, and TRE-F2 � 1 �-gal reporter, theMATa meiotic progeny was transferred to medium that contains kanamycinand lacks leucine. (B) Yeast X-gal agarose overlay of SGA deletion mutants: thecultures from A were spotted an agarose plate (6% dimethylformamide, 0.1%SDS, 0.1 mg/mL X-Gal, 0.5% agarose, 10�7 M Triac or nil) grown overnight at30 °C. A blue color indicated that the deleted gene was not required for aTH-dependent activation of the TRE- reporter, whereas white indicated afailure of gene activation.

Fig. 2. Validation of yCCR4 as a gene needed for TR action in yeast. (A)Comparison of hTR�1 activation in WT and CCR4 deletion strains. Results of�-gal assays performed with commercial (ATCC) WT yeast strain YBR175WBY4741 (4741-WT) and CCR4 deletion mutant yeast strain YAL021C BY4741(4741-�yCCR4). This strain was transformed with a single copy of the chickenlysozyme (F2) TRE, hTR�1, GRIP1, or empty vector. Cultures were treated withvehicle (empty bars) or 10�7 M Triac (solid bars). �-Gal activities were expressedas Miller units per milligram protein. Data shown were pooled from 3 inde-pendent experiments and calculated as mean � SE. �-Gal assays were per-formed as described as outlined in Materials and Methods. (B) CCR4 replace-ment restores TH-dependent gene activation in the CCR4 deletion strain.Experimental procedures and �-gal assays were performed as described in Aexcept that the LexA plasmid was substituted for GRIP1/SRC2 with the sameselection marker restrictions.

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and LGE1. For further details on their known functional prop-erties, see Table S2.

Validation of yCCR4 as a TR Regulator. To validate our modifiedSGA method, we focused on one putative essential yeast TRinteracting protein with a known human homolog, namely CCR4(9, 21). We compared TR activity at the TRE driven reporter inthe WT 4741 strain versus the �yCCR4 deletion mutant gener-ated in the SGA screen in the absence and presence of GRIP1/SRC2 (Fig. 2A). In accordance with previous results, hTR�1exhibited moderate activity at the F2-driven �-gal reporter withL-triiodothyroacetic acid (Triac), and this was potentiated �5-fold by GRIP1/SRC2. TR activity was severely diminished(�90%) in the �yCCR4 mutant strain in the absence andpresence of GRIP1/SRC2. Exogenous yCCR4 (expressed as aLexA fusion) did not potentate TR activity in WT yeast but didrescue the signaling defect in the CCR4 deletion mutant strain(Fig. 2B). Thus, TR action shows a strong requirement foryCCR4, and this is not absolutely dependent on exogenousGRIP1/SRC2.

Other NRs also exhibited reduced activity in the �yCCR4deletion strain (Fig. S1 A–D). CCR4 was required for all 4 NRstested [TR, ER androgen receptor (AR), retinoic acid receptor(RAR), and RXR]. However, the magnitude of this effect wassmaller for ERs �60%), RXRs (�60–70%), and ARs (�50%)than for TRs (�90%). We conclude that yCCR4 is required foroptimal activity of exogenous NRs in yeast, but that this factorplays an especially prominent role in TR action.

hCCR4-NOT6 Is Essential for hTR�1 Function in Human Cells. Todetermine whether the human homolog of yCCR4 (hCCR4/NOT6) is required for TR signaling in mammalian cells, we

Fig. 3. hCCR4 is needed for hTR�1 action in mammalian cells. (A) hCCR4enhances hTR�1 TH-dependent gene activation. Co-transfection of increasingDNA concentrations of hCCR4 with 0.1 �g of hTR�1 progressively enhances

Fig. 4. Enhanced binding of hCCR4 to the DIO1 gene promoter. Shown arethe results of ChIP assays for HeLa cells transfected with hCCR4 and treatedwith vehicle (lane 1) or Triac (10�7 M) for different times: 0 min (lane 1), 5 min(lane 2), 10 min (lane 3), 60 min (lane 4), 120 min (lane 5), 360 min (lane 6), andovernight (lane 7). Following formaldehyde cross-linking, lysates were immu-noprecipitated with antibodies against hTR�1 or pre-immune serum as indi-cated. Primers specific for the DIO1 promoter and the coding region of GAPDHwere used to amplify the DNA associated with hTR�1 and hCCR4 in vivo.

TH-dependent hTR�1 reporter gene activation. (B) hCCR4 compared withother co-activators: co-transfection of TRE-DR4 � 3-LUC reporter with acomparable quantity of hCCR4 or CTR (control vector), GRIP1, SRC1, P/CAF,ADA2, and TIP60. Experiments and luciferase assays were performed as de-scribed in A. (C) Potency of co-activators on TH-dependent reporters withco-transfected hCCR4 as in B. Note that reporter activation by P/CAF, ADA2,and TIP60, but not GRIP1 or SRC1, was further enhanced by hCCR4. (D)Detection of impaired co-activator function in hCCR4 siRNA knockdown.Illustrated are the results comparing the luciferase reporter gene responsesfor WT HeLa cells (Left) compared with siRNA hCCR4-knockdown HeLa cells(Right) using standard techniques (see Materials and Methods). (E) Restora-tion of hCCR4 co-activator function by a WT replacement. hCCR4 siRNAknockdown HeLa cells were co-transfected with hTR�1, reporter TRE-DR4 �3-LUC, and increasing doses of hCCR4. Methods are otherwise similar to D.

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determined effects of hCCR4 transfection of TR�1 activity inHeLa cells. Fig. 3A shows that co-expression of hTR�1 withincreasing concentrations of hCCR4 potentiated TH response ata standard TRE-driven reporter (DR-4 � 3-LUC). This effectwas larger than effects obtained with GRIP1/SRC2 and com-parable to that obtained with several other TR co-activators(SRC1, P/CAF, ADA2, TIP60; Fig. 3B). Interestingly, hCCR4did not synergize with SRC1 or GRIP1/SRC2 in TR co-activation; GRIP1/SRC2 failed to potentiate TR activity in thepresence of exogenous hCCR4 and SRC1 weakly suppressed TRactivity in these conditions (Fig. 3C). By contrast, P/CAF,ADA2, and TIP60 continued to enhance TR activity in thepresence of exogenous hCCR4.

We next examined effects of hCCR4 knockdown on TRaction. We verified that parental HeLa cells express hCCR4transcripts by Northern blot and RT-PCR (Fig. S2 A and B). Wecreated a retrovirus that expresses an siRNA specific for hCCR4and obtained stable HeLa cell lines that express the hCCR4siRNA or contain a control retrovirus vector. The absence ofhCCR4 expression in the hCCR4 siRNA expressing cell line wasconfirmed by RT-PCR (Fig. S2 C and D). Transfection analysisrevealed that TR�1 action was severely curtailed in this back-ground but not in the control cell line (Fig. 3D). Moreover, thisdeficit was rescued by exogenous hCCR4; a transfected hCCR4expression vector enhanced response �8-fold (Fig. 3E) andrestored TR signaling to levels comparable to those with TR andtransfected hCCR4 in control HeLa cells. Co-expression oftransfected TR and hCCR4 did not affect expression levels of theother protein (Fig. S2E). Thus, TR activity shows a very strongrequirement for hCCR4. Similar observations were obtained inHeLa cells for ER and AR, although the requirement for hCCR4appeared strongest with TRs (Fig. S2 F–I).

TR Interacts with hCCR4 in Vivo and in Vitro. To determine whetherTR� recruits hCCR4 to a target promoter in cultured cells, weperformed ChIP analysis on extracts from a HeLa cell derivativethat stably expresses hTR�1 fused to a tandem affinity purifi-cation (tap) tag (Fig. 4).

As expected, ChIP analysis confirmed that hTR�1 is recruitedto the TRE in the human deiodinase 1 (DIO1) promoter in theabsence of ligand, and also revealed that TR� interactions weremodestly elevated at several times after TH treatment (Fig. 4).TH treatment also promoted very rapid recruitment of hCCR4to the target DIO1 promoter; hCCR4 was detected at the DIO1TRE within 5 min of TH treatment (Fig. 4). hCCR4 wasmaintained for several hours after TH induction, unlike SRCsand TRAP220, which tend to cycle on and off TR-regulated

promoters. Thus, we conclude that hCCR4 is rapidly and stablyrecruited to a TR�1 target promoter after TH treatment.

TR/hCCR4 interactions were also observed in immunofluo-rescence co-localization experiments. Studies performed inHeLa cells in the absence of co-expressed hCCR4 and hTR�1revealed weak endogenous signal for hCCR4 in the cytoplasmand nucleus and no signal for TR�1 in the absence or presenceof TH (Fig. S2 J). Compartmental localization of hCCR4 wasunchanged in the presence or absence of TH. By contrast, strongsignals were detected in the presence of co-expressed hCCR4and hTR�1 (Fig. 5). Detection of hCCR4 in the nuclear andcytoplasmic compartments is consistent with roles in transcrip-tional response and cytoplasmic mRNA modification (9, 21).This suggests that TRs interact with hCCR4 in living cells andthat TH alters the distribution of TR/hCCR4 complexes betweennucleus and cytoplasm.

Additionally, the binding mode by which TR interacts directlywith hCCR4 was also studied by using mammalian 2-hybrid (Fig.S2K) and GST pull-down studies (Fig. S2L). These studiesdemonstrated that TR binds directly to hCCR4 through itshormone binding domain, and these interactions are enhancedby TH. Moreover, further mutational studies have shown thatTH-dependent reduction in binding for the hCCR4200–558 mu-tant is likely localized in part to its N-terminal 100–200 aasequences—a leucine-rich repeat domain (Fig. S2L).

DiscussionIn the current report, we have confirmed, by using a modifiedyeast SGA analysis method, that one of the 4 genes with a knownhuman homolog, i.e., hCCR4, is an important TR co-activator inmammalian cells. Transfected hCCR4 in HeLa cells potentiatesTR action with a magnitude equal to or better than in previousstudies with defined TR cofactors such as SRC1 and SRC2/GRIP1 (22). The overall magnitude of deleting hCCR4 is similarto that obtained in mouse embryo fibroblasts with a targeteddeletion of the TRAP220 gene (23), suggesting that hCCR4plays a role in TH action that is as important as other establishedTR co-regulators. CHIP analysis reveals rapid recruitment ofhCCR4 to the promoter of an endogenous TR target gene(DIO1) in response to TH. As reported here, the fact that themajor site of TR/CCR4 co-localization changes from the cyto-plasm to the nucleus upon TH treatment leads us to suspect arole for hCCR4 in trafficking of TRs between different cellularcompartments. All of these findings suggest that hCCR4 plays avery important role in TR signaling that is distinct from otherknown TR co-activators.

Our analysis also suggests that hCCR4 plays a major role notonly in TR action but also in other hormone-dependent NRs; weobserved requirements for yCCR4 in ER, AR, RXR, and RARactivity in reconstituted transcription activation assays in yeastcells (Fig. S1), irrespective of the presence or absence ofGRIP1/SRC2, and in HeLa cells (Fig. S2 F–I). Subsequent to ourearlier report (24) on the important role of hCCR4 as a NRco-activator, other workers reached a similar conclusion on itseffects on TR� and other NRs by amplifying the activity of NRligand binding domain in the absence of direct contacts (25).Effects of hCCR4 on TR� in this study (25) did not appear asrobust (�3-fold for TR� compared with as high as 30-fold forTR�1 in our work), and siRNA knockdown had an even moremodest effect on NR action (i.e., 50% reduction vs. �10-fold forTR�1 in our experiments). Moreover, we demonstrated directTR binding to hCCR4 in three ways: GST pull-downs, immu-nofluorescent nuclear co-localization experiments in HeLa cells,and mammalian 2-hybrid assays. Association of hCCR4 with TRin vivo was further suggested by ChIP assays. The reasons fordifferences between the previously published results (25) andour studies are not clear, but could be related to the fact that theother group (25) used a shorter hCCR4 cDNA than the full-

Fig. 5. hCCR4 co-localizes with hTR�1. Co-transfection of hCCR4 and hTR�1in HeLa cells whereas only control empty vectors were transfected as control(Fig. S2J). The cells were treated with vehicle (�TH; Upper) or 10�6 M Triac(�TH; Lower). The green and red colors show localization of CCR4 and hTR�1,respectively. The merged image shows co-localization of CCR4 and hTR�1.Blue and white arrows indicate the cytoplasm and nucleus, respectively.

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length cDNA used in our experiments. Nevertheless, their study(25) supports our observations that hCCR4 is an important TRco-activator and a general NR co-regulator.

In conclusion, it is encouraging that the yeast SGA approachdescribed herein has identified at least one TR co-factor thatis important for TR activity in cultured cells and appears todisplay unique activities and binding modes. Studies usingsimilar methodology to that described in the current report forCCR4 are in progress to validate the other genes with humanhomologs identified in the SGA analysis. Thus, of 384 yeastgenes analyzed, eight (2%) were of major importance (i.e.,�60% loss of function; Table S2) for TR action. Our resultstherefore validate the feasibility of applying an SGA strategyfor systematic identification of conserved yeast genes withhuman homologs essential for TR and other hormone-dependent NRs.

Materials and MethodsModified Yeast SGAs. The SGA array was a gift from C. Boone (Toronto, ON,Canada). The yeast strain MATa:Y5563 (MATa, lys1D his3D1 leu2D0 ura3D0met15D0 LYS2�, can1D::MFA1pr-HIS3) was used as a mating strain. Usingthe available selection markers of LEU2 (hTR�1), NATR (GRIP1), and URA3(TRE-F2x1 �-gal reporter), YEp56-hTR�1 (LEU2), pRS426N-GRIP1 (NATR;nourseothricin; clonNAT; Werner BioAgents), and the pC2-TRE-F2 � 1-�-gal reporter (URA3) were transformed into the MATa:Y5563 yeast strain,and these strains were crossed with a MATa strain carrying gene deletionslinked to the kanamycin resistance marker (17). The MATa meiotic progenywere transferred to medium that contains kanamycin and lacks leucine toselect for growth of double-mutant meiotic progeny expressing hTR�1,GRIP1, and the TRE-F2x1 �-gal reporter in a background containing thegene deletion of choice. Mutations that affect hTR�1/TH-dependent �-galactivity were detected by a standard yeast X-gal agarose overlay assay withor without TH (Fig. 1A).

Yeast clones that had reduced blue color were retested by selecting themfrom a replicate plate and growing them in culture for standard liquid �-galassay. For clones with an impaired TH-dependent response, commercial (i.e.,ATCC) WT yeast strain (4741-WT) were compared with deletion mutants in the4741 background. All experiments had suitable controls for determiningspecific transcriptional effects for hTR�1 and GRIP1/SRC2 as previously de-scribed (11, 26).

Yeast Reporter Assays. Yeast transformants were grown in minimal medium,YEp56-hTR�1, pRS423-GRIP1/SRC2, and the �-gal reporter plasmids TRE-F2x1and �-gal assays were performed as described previously (13). LexA-yCCR4(HIS3) replacement was constructed as a LexA-CCR4 fusion plasmid in whichfull-length yeast CCR4 (residues 1–837) have been fused in-frame with LexA atthe EcoRI-BamHI polylinker site of pLexA1–87, a 2-�m-based plasmid describedpreviously (15, 16).

Expression, Interaction, and Silencing Vectors. The hTR�1 was cloned intoBamH1/EcoR1 sites of pcDNA3.1 HisC, pHIS6-CBP-SBP, pGEX 2TK, and pMvectors. hCCR4 (CCR4/NOT6, AB033020) was cloned using appropriate primersfrom MegaMan Transcriptome Library (Stratagene) and cloned into BamHIsite of pcDNA 3.1 HisC and pVP16 vectors. All plasmids were analyzed byrestriction digestion and sequenced to confirm the inserted cDNAs. ThehCCR4/NOT6 cDNA sequence was analyzed using siRNA converter program(Ambion), and siRNA sequences between 47% and 50% GC content werechosen for optimal knockdown effects. Two sets of oligonucleotides withflanking 5�ApaI and 3�EcoR1 were synthesized, annealed, and ligated intopMSCvneo-SIL vector with unique ApaI and EcoR1 sites.

Transfections and Reporter and Mammalian 2-Hybrid Assays. HeLa cells weretransferred to 6-well plates and grown to 70% confluence, and cells weretransfected with DR4-LUC, hTR�1, and/or vector alone with other factors orcarrier DNA and pCMV-�-Gal (up to 0.3 �g of total plasmid DNA) in MEMsupplemented with 10% charcoal-treated serum. Twelve hours later, cellswere washed with PBS solution and supplemented with hormone or vehicle inhormone-free MEM-charcoal treated FBS medium. Cells were harvested afterovernight incubation and whole cell extracts were prepared by 3 cycles offreezing and thawing in cell extraction buffer containing complete proteaseinhibitor mixture (Roche). �-Gal activities were determined in 10-�L aliquotsto normalize the transfection efficiency. Samples containing 10 U of �-galactivity were used for determining luciferase activity using a commercial

system (Promega). Two-hybrid protein-protein interaction assays were alsoperformed in HeLa, with a reporter plasmid (pGL4.14-G5-Luc) containing 5GAL4 binding elements up-stream of the thymidine kinase promoter regionfused to the luciferase reporter gene. Total light emission during the initial20 s of the reaction was measured with a luminometer (Lumat LB 9501;Berthold).

hCCR4 siRNA Knockdown. Purified plasmids were used for transfection ofPT67 cells (Becton-Dickinson) for viral packaging and to select transfectantswith G418 resistance. Six colonies were chosen for developing the viralsupernatants. Identical experiments were performed using vector withoutthe insertion of duplex oligonucleotides as control. The viral supernatantswere used for the infection of HeLa cells and selected using G418. Trans-formants were tested for absence of the selected gene by RT-PCR withappropriate primers, by Northern blot and transcription activation func-tion of hTR�1. Replacement of the hCCR4 siRNA knockdown in HeLa cellswas performed by transient co-transfection of pcDNA3.1-HisC-CCR4 ex-pression vector. Appropriate loss of transcriptional CCR4 expression in thesiRNA knockdown was monitored by comparison to WT cell lines and cellstransformed with control virus supernatants.

CHIP Assay. HeLa cells were engineered to stably express TAP-tagged hTRb1.pTAP-TR�1 plasmid was used to transfect exponentially growing HeLa cells bycalcium phosphate precipitation. Transfectants were selected using G418 andcolonies were collected after several weeks. hTR� was verified by dot blot andimmunoprecipitation using rabbit polyclonal antibodies raised against GST-hTR�1 and peroxidase coupled goat anti-rabbit IgGs. One HeLa cell cloneexpressing high levels of TAP-hTR�1 was selected for ChIP. The hTR�1 express-ing HeLa cells were transfected with an hCCR4 expression vector. WT HeLa cellswere used as a negative control. These cells were treated with vehicle or 10�7

M Triac for 5, 10, 60, 120, and 360 min, and overnight, and the chromatin wascross-linked using formaldehyde for 10 min at room temperature. ChIP assayswere performed using a ChIP-IT Express kit from Active Motif. One hundredfifty milligrams of total protein determined by Bio-Rad reagent and 2 mL ofpolyclonal antibodies were raised against GST-hTRb1. DNA from both IP inputand IP bound fractions were amplified by PCR using pairs of DIO1 primers andthe coding region of the GAPDH gene as internal control. The input DIO1 PCRproduct was measured in an aliquot of the cross-linked chromatin without IPusing 0.5% of material used for hTR�1 ChIP or 1% for CCR4 ChIP. PCR productswere analyzed by agarose gel electrophoresis and ethidium bromide staining.ChIP was repeated at least 3 times using 3 different chromatin preparationswith identical results. The primer sequences for the human iodothyronineDIO1 promoter were identical to a previous report (27). The human GAPDHgene primers were used as controls.

Immunofluorescence and Microscopy. HeLa cells were seeded on glass cover-slips and grown for 24 h and maintained in 5% CO2 at 37°C to 70% confluence.They were transfected with hCCR4 and/or hTR�1 or carrier DNA (up to 0.3 �gof total plasmid DNA) in MEM supplemented with 10% charcoal-treatedserum. Twelve hours later, cells were washed with PBS solution and supple-mented with hormone or vehicle in hormone-free MEM-charcoal treated FBSmedium for 24 h. For immunofluorescence staining, cells were fixed for 20 minat room temperature in 4% paraformaldehyde in PBS solution, permeabilizedin methanol for 2 min, and washed 4 times with PBS solution and probed withprimary and secondary antibodies. Primary rabbit polyclonal anti-hCCR4 (1:10;produced by our laboratory) and mouse monoclonal anti-hTR�1 (1:100; SantaCruz Biotechnology) antibodies were used. Secondary antibodies were FITC-labeled anti-rabbit (1:00) and TRITC-labeled anti-mouse (1:100) antibodies(Santa Cruz Biotechnology). Imaging was performed using a Leica 2.00 build0871 confocal laser system.

Pull-Down Assays. GST-hTR�1, GST-hTR�1 DNA binding domain, or GST-hTR�1-hormone binding domain was expressed in Escherichia coli BL21 cellsand prepared by standard methods. Full-length or deletional mutants of[35S]methionine-labeled factors were synthesized by in vitro transcription andtranslation (TNT; Promega). Equivalent amounts of GST or GST-fusion proteinwere used for in vitro binding assays as previously described. hCCR4, hCCR4100–

558, or hCCR4200–558 was cloned into pcDNA1 AR for transcription and transla-tion to produce 35S-Met-labeled hCCR4s.

ACKNOWLEDGMENTS. We acknowledge the excellent technical assistance ofMr. Y.-F. Yang and Ms. R. Wang, and the collaborative assistance and advicefor SGA experiments of Drs. C. Boone and N. Krogan of the Banting and BestDepartment of Medical Research, University of Toronto. This work was sup-ported in part by a Diana Meltzer Abramsky Research Fellowship from the

19858 � www.pnas.org�cgi�doi�10.1073�pnas.0910134106 Govindan et al.

Thyroid Foundation of Canada (to X.M.); Canadian Institutes of Health Oper-ational Grant MOP-49448 (to P.G.W.); the Mount Sinai Hospital Foundation ofToronto and Department of Medicine Research Funds; the Julius Kuhl and

Temmy Latner/Dynacare Family Foundations; the Joseph and Mildred Son-shine Family Centre for Head and Neck Diseases (to P.G.W.); and NationalInstitutes of Health Grants GM 78078 and DK51281 (to C.L.D and J.D.B.).

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