cbfa2t3-znf652 corepressor complex regulates transcription of

CBFA2T3-ZNF652 Corepressor Complex RegulatesTranscription of the E-box Gene HEB*

Received for publication, November 7, 2007, and in revised form, April 4, 2008 Published, JBC Papers in Press, May 2, 2008, DOI 10.1074/jbc.M709136200

Raman Kumar‡1, Kelly M. Cheney‡, Ross McKirdy‡, Paul M. Neilsen‡, Renee B. Schulz‡, Jaclyn Lee‡, Juliane Cohen‡,Grant W. Booker§, and David F. Callen‡

From the ‡Breast Cancer Genetics Group, Dame Roma Mitchell Cancer Research Laboratories, Discipline of Medicine, University ofAdelaide and Hanson Institute, Institute of Medical and Veterinary Science, Adelaide, South Australia 5000, Australia and the§School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia 5005, Australia

Transcriptional repression plays a critical role in develop-ment and homeostasis. The ETO family represents a group ofhighly conserved and ubiquitously expressed transcriptionalregulatory proteins that are components of a diverse range ofmultiprotein repressor complexes. ETO proteins function astranscriptional repressors by interacting with a number of tran-scription factors that bind to their cognate consensus DNAbinding sequences within the promoters of target genes. Wepreviously reported that the classical C2H2 zinc finger DNA-binding protein, ZNF652, specifically and functionally interactswith the ETO protein CBFA2T3 and has a role in the suppres-sion of breast oncogenesis. Here we report the identificationand validation of the ZNF652 consensus DNA bindingsequence. Our results show that the E-box gene HEB is adirect target of CBFA2T3-ZNF652-mediated transcriptionalrepression. The CBFA2T3-ZNF652 complex regulates HEBexpression by binding to a single ZNF652 response elementlocated within the promoter sequence of HEB. This study alsoshows that the NHR3 and NHR4 domains of CBFA2T3 interactwith a conserved proline-rich region locatedwithin theC termi-nus of ZNF652. Our results, together with previous reports,indicate that HEB has a complex relationship with CBFA2T3;CBFA2T3 interacts with ZNF652 to repress HEB expression,and in addition CBFA2T3 interacts with the HEB protein toinhibit its activator function. These findings suggest thatCBFA2T3-ZNF652-mediated HEB regulation may play animportant role in hematopoiesis and myogenesis.

Gene regulation occurs through the balanced activity of tran-scriptional activators and repressors and is critical for theordered development and maintenance of homeostasis. Inher-ited or acquired defects in transcription factor structure andfunction can result in irreversible alterations in this balance.The ensuing aberrant expression of target genes can lead todevelopmental abnormalities or to the initiation and promo-

tion of cancer (1). Spatial and temporal gene regulation by tran-scriptional activation has been intensely investigated, whereasthe critical role of transcriptional repression in developmentand disease has only been recently recognized (2).CBFA2T1 (RUNX1T1, MTG8) together with CBFA2T2

(MTGR1) and CBFA2T3 (MTG16) form a group of ubiqui-tously expressed transcriptional regulatory proteins called the“ETO” family. The ETO proteins have modular structures andare characterized by the presence of four regions, NHR1 toNHR4, so named because of their homology to the Drosophilanervy protein (3). ETO proteins are primarily nuclear-localizedand are capable of oligomerization and interaction with otherproteins through their NHR2 domains (4–6). The NHR1 (alsocalled eTAFH)domain ofCBFA2T1has been shown to bind therepression domain I of nuclear receptor co-repressor N-CoR invitro and repress transcription in a reporter assay (7). NHR1also mediates ETO interactions with a conserved activationdomain of the E-box proteins HEB and E2A and inhibits theirtransactivation functions (8). Therefore, the NHR1 motif caninterchange negative and positive coregulatory proteins to con-trol transcription (9). The second domain, NHR2, is describedas a hydrophobic heptad repeat domain and is required forhomo- and heterotetramerization of the ETO proteins (6). Thetetramerization of the ETO proteins is not essential for inter-action with the corepressors N-CoR, SMRT, mSin3A, andHDAC1–3. However, NHR2-mediated tetramerization of theAML1-CBFA2T1 fusion protein contributes to the develop-ment of leukemia in t(8:21) translocation carriers (6). The thirddomain in CBFA2T1, NHR3, has been shown to interact withthe regulatory subunit of type II cyclic AMP-dependent proteinkinase (PKA RII�) in lymphocytes (10). The last of thesedomains, NHR4, also called the MYND (Myeloid-Nervy-DEAF-1) domain, contains two nonclassical zinc fingers and, inconjunction with NHR1 and NHR3, interacts with the core-

* This work was supported by Grant 207703 from the National Health andMedical Research Council of Australia, the Youth Breast Cancer Associa-tion, and the Susan G. Komen Breast Cancer Foundation. The costs of pub-lication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed: Breast Cancer GeneticsGroup, Dame Roma Mitchell Cancer Research Laboratories, Hanson Inst.,IMVS, Frome Rd., Adelaide, South Australia 5000, Australia. Fax: 61-8-8222-3217; E-mail: [email protected].

2 The abbreviations used are: NHR, nervy homology region; AML, acute mylel-oid leukemia; BSA, bovine serum albumin; ChIP, chromatin immunopre-cipitation; CHO, Chinese hamster ovary; dsDNA, double-stranded DNA;DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; Gfi-1,growth factor independence 1; HA, hemagglutinin; HDAC, histonedeacetylase; HEB, HeLa E-box-binding protein; HEK, human embryonic kid-ney; HRP, horseradish peroxidase; HuT78, human T-lymphoblastoid; Luc,luciferase; MBP, maltose-binding protein; MT, mutant; MYND, Myeloid-Nervy-DEAF-1; N-CoR, nuclear receptor co-repressor; RE, response ele-ment; SCL, stem cell leukemia; siRNA, small interfering RNA; SMRT, silenc-ing mediator of retinoic acid and thyroid hormone receptor; TK, thymidinekinase; WT, wild type; ZNF652, zinc finger protein 652.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 27, pp. 19026 –19038, July 4, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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pressor N-CoR (7). Additionally, the NHR4 domain ofCBFA2T1 also directly interacts with SMRT (5, 11), althoughthe significance of this interaction in AML1-CBFA2T1-in-duced leukemogenic activity has not been completely resolved(12, 13). Consistent with the role of the ETO proteins in tran-scriptional repression, NHR2, NHR3, and NHR4 also interactwith various members of the HDAC family.CBFA2T1 is themost studied of the ETOproteins because of

its involvement in the non-random t(8;21) translocations thatare frequently associated with acute myeloid leukemia (AML).The t(8;21) in AML generates an in-frame fusion of the codingsequences of the N-terminal zinc finger DNA-binding domainof AML1 (also called RUNX1) with almost the entire CBFA2T1encoding region, leading to a chimeric AML1-CBFA2T1 pro-tein (14). AML1normally functions as an activator of transcrip-tion, but when fused with CBFA2T1, it acts as a repressor. Theresulting changes in gene expression are thought to be the basisfor the development of leukemia. The ETOproteinCBFA2T3 isalso involved in a recurrent t(16;21) translocation with AML1and is associatedwith therapy-relatedmyeloidmalignancy (15).Gene knock-out studies in mice have been utilized to deter-

mine the normal cellular role of the ETO family of proteins,although the observed phenotypes may be influenced by func-tional complementation between the ETO family members.Studies of knock-out mice show that Cbfa2t1 plays a criticalrole in gut development (16), whereas Cbfa2t2 is required formaintenance of the secretory cell lineage in the small intestine(17).Further clues regarding the normal function of the ETO pro-

teins have been obtained through their interacting proteins.CBFA2T1 regulates early adipogenesis by inhibiting transcrip-tional activity of C/EBP�, a member of the C/EBP (CCAAT/enhancer-binding protein) family of leucine zipper transcrip-tion factors (18). Both CBFA2T1 and CBFA2T3 have beenshown to interact with the corepressor ATN1 (19). An expan-sion of a polyglutamine repeat within theATN1 protein is asso-ciated with the neurodegenerative disease dentatorubral-pal-lidoluysian atrophy (DRPLA) (19). As a component ofmultiprotein complexes, CBFA2T3 has been shown to coordi-nate cellular proliferation and differentiation during erythro-poiesis (20, 21).CBFA2T3 is located in the 16q24.3 loss-of-heterozygosity

region in breast cancer, and our functional studies are consist-entwith a role of CBFA2T3 as a breast cancer tumor suppressor(22, 23). We have previously shown that loss, or reduced levels,of CBFA2T3 expression is associated with breast oncogenesis(22). CBFA2T3 has also been shown to interact with the solubleintracellular domain, termed s80, of ERBB4 and to have a role inERBB4-dependent differentiation (24). Reporter assays haveshown ERBB4 also regulates CBFA2T3-mediated transcrip-tional repression. ERBB4 and CBFA2T3 are both expressed innormal breast tissue, and changes in their levels are associatedwith breast cancer. Interaction between CBFA2T3 and ERBB4may therefore have an important role in the normal develop-ment of themammary gland, and their deregulationmay lead tobreast cancer (24). On the whole, although the original descrip-tion of the ETO family was based on their involvement in leu-

kemia, these proteins have now been shown to have a range ofother functions (14).The ETO proteins act as transcriptional repressors by form-

ing multiprotein complexes with the corepressor proteinsN-CoR, SMRT, mSin3A, and ATN1, which act as scaffolds forrecruiting various combinations of HDACs (14). The recruitedHDACs are critical for the potent repressor function of theETOcomplexes (14). The three ETOproteins exhibit variationsin their direct interactions with HDACs, with CBFA2T1 inter-acting directly with HDAC1, -2, and -3 (25), CBFA2T2 onlywithHDAC3 (17), andCBFA2T3withHDAC1, -2, -3, -6, and -8(25).ETO proteins do not bind DNA. Gene-specific repression by

ETO-containing multiprotein complexes is mediated throughtheir interaction with transcription factors that can binddirectly to the promoters of target genes. These DNA-bindingtranscription factors are predominantly classical C2H2 zinc fin-ger proteins. It is remarkable that all of the identified ETO-interacting DNA-binding zinc finger proteins are involved incancer. For example, BCL6 (B-cell lymphoma 6 protein) is fre-quently translocated and mutated in diffuse large cell lym-phoma (26), and PLZF (promyelocytic leukemia zinc finger) isdisrupted by a t(11;17) rearrangement in acute promyelocyticleukemia (27). The zinc finger protein Gfi-1 is required for thedevelopment of neuroendocrine cells, sensory neurons, andboth T- and B-lymphocytes (28). Germ-line mutations in theGfi-1 coding sequence are associated with severe congenitalneutropenia or non-immune chronic idiopathic neutropenia inadults (29). Recent findings also suggest that Gfi-1 can act aseither an oncogene or tumor suppressor gene (28, 30). There-fore, the ETO family of proteins generates a diverse constella-tion of transcriptional repressor complexes through interactionwith various transcription factors and corepressors.We have recently shown that the classical C2H2 zinc finger

DNA-binding protein ZNF652 specifically and functionallyinteracts with the ETO protein CBFA2T3 to repress transcrip-tion (31). ZNF652 has seven classical C2H2 zinc finger motifsconforming to the consensus CX2CX12HX3H sequence, withthree of these joined by part or all of a consensus TGEKP linkersequence, suggesting a role as a DNA-binding protein (32, 33).TheCBFA2T3-ZNF652 complexwas proposed to repress tran-scription of genes that have roles in the oncogenesis of breast(31). As an approach to discovering ZNF652 target genes, wehave identified the ZNF652 consensus DNA binding sequenceand subsequently have determined that the promoter of theHEB gene contains a functional ZNF652 DNA binding motif.We present data that HEB expression is regulated by theCBFA2T3-ZNF652 repressor complex. In addition, we alsohave determined the minimal interaction domains for ZNF652and CBFA2T3.

EXPERIMENTAL PROCEDURES

Cell Lines and Antibodies—HEK293T, CHO, HuT78, Jurkat(human T-cell leukemia), MCF7 and ZR75-1 (breast cancer),and HeLa (cervical carcinoma) cells were purchased from theAmerican Type Culture Collection and grown in the recom-mendedmedia at 37 °C in 5%CO2.Antibodies usedwere: rabbitaffinity-purified anti-ZNF652 (31); rat anti-HA (12CA5, Roche

CBFA2T3-ZNF652 Mediates Transcriptional Repression of HEB

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Diagnostics); rabbit anti-HEB (sc-357, Santa Cruz Biotechnol-ogy); mouse anti-Myc (9E10; sc-40, Santa Cruz Biotechnology);and rabbit anti-rat-IgG-HRP (DakoCytomation), sheep anti-mouse-IgG-HRP, and donkey anti-rabbit-IgG-HRP (Amer-sham Biosciences). A rabbit polyclonal anti-CBFA2T3 anti-body was generated using regions of the protein that divergedamong the ETO family (15). CBFA2T3 DNA fragmentsexpressing amino acids 354–392 and 505–567 were combinedby an overlap-PCR method and cloned in-frame into the bac-terial expression vector pET14b (Novagen). The His6-CBFA2T3-(354–392 � 505–567) protein induced fromBL21pLysS bacteria harboring the expression construct waspurified on nickel-nitrilotriacetic acid SuperFlow beads (Qia-gen) and injected into New Zealand White rabbits. The rabbitpolyclonal antibody was affinity-purified using bacteriallyexpressed GST-CBFA2T3-(354–392 � 505–567) fusion pro-tein conjugated to a CNBr-activated Sepharose 4B matrix (GEHealthcare).Plasmids—The FLAG-ZNF652 expression construct was gen-

eratedby subcloningaZNF652openreading frame fromtheexist-ing construct (31) into the self-inactivating bicistronic retroviralexpressionvectorpQCXIN(BDBiosciences). Forco-immunopre-cipitation assays, constructs expressing HA-tagged ZNF652 (full-length, amino acids 1–606), ZNF652-(243–606) (ZNF652-2), andZNF652-(498–606) (ZNF652-4) have been reported (31).Constructs expressing HA-tagged ZNF652-(565–568)(PPVP3AAAA) and ZNF652-(571–574) (PPPP3AAAA)mutant proteins were generated by overlap-PCR using thefollowing pairs of primers: ZNF652-(565–568)-mut1-F (5�-ACA-CCACCTTCCCATCGCAGCGGCCGCTCACCTCCCGCCA-CCTCCAGCTCTCT-3�), ZNF652-(565–568)-mut1-R (5�-TGG-AGGTGGCGGGAGGTGAGCGGCCGCTGCGATGGGAA-GGTGGTGTGGGTGGTGA-3�), ZNF652-(571–574)-mut2-F(5�-TCCCTCCAGTCCCTCACCTCGCGGCCGCAGCTGC-TCTCTTTAAGAGTGAGCCTTTAAATC-3�), and ZNF652-(571–574)-mut2-R (5�-AGGCTCACTCTTAAAGAGAGCAGC-TGCGGCCGCGAGGTGAGGGACTGGAGGGATGGGA-3�).To generate constructs expressing various regions of theCBFA2T3 protein, the relevant fragments of cDNA were PCR-amplified from an existing expression construct, using primerswith sequence extensions that created BamHI restriction sites,and cloned into the BglII restriction site of pCMV-myc (BDBiosciences). CBFA2T3 expression constructs generated wereCBFA2T3 (full-length, amino acids 1–567), CBFA2T3-(201–393) (CBFA2T3-1), CBFA2T3-(341–447) (CBFA2T3-2),CBFA2T3-(341–567) (CBFA2T3-3), and CBFA2T3-(447–567)(CBFA2T3-4). All CBFA2T3 constructs express theCBFA2T3bisoform. For the CASTing protocol, a DNA fragment encodingfull-length ZNF652 was cloned in-frame into the bacterialexpression vector pMAL-c2X (New England Biolabs). TheMBP-ZNF652 fusion protein induced in DH5� bacteria carry-ing the expression construct was purified using amylose resin(New England Biolabs). Myc-CBFA2T1 and Myc-CBFA2T2expression constructs used in testing the specificity of anti-CBFA2T3 antibody were as reported earlier (31).For luciferase reporter assays, a pGL2-4�ZNF652-TK-Luc

construct carrying four tandem copies of the ZNF652 consensusbinding sequence was generated by annealing two oligonucleo-

tides (5�-CGAAAGGGTTAATCGATCCGAAAGGGTTAATC-GATCCGAAAGGGTTAATCGATCCGAAAGGGTTAATCC-TCGAG-3� and 5�-TCGACTCGAGGATTAACCCTTTCGGA-TCGATTAACCCTTTCGGATCGATTAACCCTTTCGGAT-CGATTAACCCTTTCGGTAC-3�), and cloning at KpnI andXhoI sites located upstream of the herpes simplex virus-thymi-dine kinase (TK) promoter in a pGL2-TK-Luc reporter vector(Promega). TheHEB promoter region (1043 bp) containing theZNF652 consensus DNA-binding site was amplified from thehuman genomic DNA with forward (5�-CACACACACACA-CAACGCGTCGGTCTTTACTGCACTCACTTACCTA-3�)and reverse (5�-CACACACACACACAACGCGTAATAGAA-CTTGTGGGTCTCTTCAG-3�) primers using Platinum TaqDNA polymerase High Fidelity and cloned at the MluI site ofpGL3-Enhancer-Luc (hereafter called pGL3-E-Luc) reportervector (Promega) to generate pGL3-HEB-E-Luc construct. Thesingle ZNF652 DNA-binding site within the HEB promotersequence was mutated by overlap-PCR to generatepGL3-HEB-mut-E-Luc. HEBmut-F (5�-AGATGGGCCAT-GGTCCGCGGCTCTTCCCGGCGCGGAGGGAT-3�) andHEBmut-R (5�-GACCATGGCCCATCTCCCCGACCCGC-TCCGCCGCCTAGGAAGT-3�), and vector-based primerswere used. The Renilla luciferase vector (pRL-TK) was fromPromega. N-terminal Myc-tagged CBFA2T3 cloned intopcDNA3.1 (Invitrogen) has been described (31).CASTing (Cyclic Amplification and Selection of Targets)

protocol—To identify ZNF652 DNA binding sequence byCASTing (34), the bacterially expressed MBP-ZNF652 fusionprotein was affinity-purified using amylose-coated resin undernondenaturing conditions. A library of double-stranded DNA(dsDNA) molecules containing 18 degenerate nucleotidesflanked by known sequences was generated according to thepublished method (35). Briefly, random sequence oligonucleo-tide 5�-CAGGTCAGTTCAGCGGATCCTGTCG(N)18GAG-GCGAATTCAGTGCAACTGCAGC-3� was annealed to5�-GCTGCAGTTGCACTGAATTCGCCTC-3� and filled inby DNA polymerase I, large fragment (Klenow) to generate apool of dsDNA for the binding assay. This pool of dsDNA(250 ng) was incubated with 1 �g of affinity-purified MBP-ZNF652 in 1� DNA-binding buffer (20 mMHepes-KOH, pH7.9, 100 mM KCl, 2 mM MgCl2, 10 �M ZnSO4, 0.5 mM DTT,0.2 mM EDTA, 0.1% Nonidet P-40, 10% glycerol, 250 �g/mlBSA) in a 50-�l final volume at 4 °C for 1 h. MBP-ZNF652-DNA complexes were captured on amylose resin, eluted, andthen PCR-amplified using the primers 5�-GCTGCAGTTG-CACTGAATTCGCCTC-3� and 5�-CAGGTCAGTTCAGC-GGATCCTGTCG-3� with Platinum Taq DNA polymeraseHigh Fidelity (Invitrogen), initially for 2 min at 94 °C and then15 cycles of 94 °C for 10 s, 58 °C for 10 s, and 68 °C for 10 s. Theamplified DNA from each round was used for the subsequentround of binding and ZNF652-sequence specific enrichment.Five rounds of selection were performed. DNA amplified fromthe fifth round of selectionwas digested with EcoRI and BamHIand cloned at the same restriction enzyme sites in BlueScriptvector pKS(�) (Stratagene), and a total of 27 clones weresequenced.Nuclear Extracts—For nuclear extracts, nuclei from ZR75-1

breast cancer cells (4 � 107) known to express endogenous




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ZNF652 (31) were isolated according to the published method(36). Nuclei were resuspended in cold buffer containing 20 mMHepes-KOH, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mMEDTA, 0.5 mM DTT, and 25% glycerol (37) and sonicated on aVibra-Cell VCX 130 (Sonics) with three 10-s pulses at 30%amplitude. Nuclear lysates were then centrifuged, and thesupernatants were assayed for protein content using a BCAprotein assay kit (Pierce). Aliquots were stored at �80 °C untilrequired. Nuclear extracts were also prepared from Jurkat andHEK293T cells transiently expressing HA-ZNF652.ElectrophoreticMobility Shift Assay (EMSA)—All EMSA gels

were 4% polyacrylamide made in TGE buffer (12.5 mM Tris-HCl, pH 8.5, 85 mM glycine, 0.5 mM EDTA). DNA fragmentsfrom 3–5 rounds of CASTing were end-labeled with[�-32P]ATP in the presence of T4 DNA polynucleotide kinase(New England Biolabs), and purified on QIAquick nucleotideremoval columns (Qiagen). Equal amounts of DNA from thethree roundswere incubatedwith eitherMBP orMBP-ZNF652in 1� DNA-binding buffer (20 mM Hepes-KOH, pH 7.9, 100mM KCl, 2 mM MgCl2, 10 �M ZnSO4, 0.5 mM DTT, 0.2 mMEDTA, 0.1% Nonidet P-40, 10% glycerol, 250 �g/ml BSA, 5�g/ml sheared salmon sperm DNA) in a final volume of 20 �land loaded onto gel. For remaining EMSAs, annealedoligonucleotides with wild type (5�-GATCCTGTCGCGAA-AGGGTTAATCGAGGCGAATTC-3� and 5�-GATCGAATT-CGCCTCGATTAACCCTTTCGCGACAG-3�) and mutatedZNF652 (5�-GATCCTGTCGTAGCGTATCCGGCGGAGG-CGAATTC-3�) and 5�-GATCGAATTCGCCTCCGCCGGA-TACGCTACGACAG-3�) binding sequenceswere radiolabeledwith Klenow polymerase in the presence of [�-32P]dCTP andpurified usingQIAquick nucleotide removal columns (Qiagen).Radiolabeled DNA probes were incubated with either MBP orMBP-ZNF652 proteins in 1� DNA-binding buffer andresolved on EMSA gels. For EMSAs performed on nuclearextracts, short, annealed DNAs carrying either consensus ormutated sequences were incubated with different amounts ofprotein in 20 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 85 mM NaCl,10 �M ZnSO4, 10% glycerol, 100 �g/ml BSA, and 50 �g/mlsheared salmon sperm DNA in a final volume of 10 �l. Forsuper-shift EMSA, appropriate antibodies were added to bind-ing reactions 5 min before loading onto gels.Co-immunoprecipitations—Approximately 2� 106 HEK293T

cells were transiently transfected with 4 �g of each relevantplasmid using Lipofectamine 2000 (Invitrogen) according tothe manufacturer’s instructions. After 24 h, cells were har-vested and lysed in 50 mM Tris-HCl, pH 8.0, 150 mMNaCl, and1% Triton X-100 supplemented with complete protease inhib-itor mixture (Roche Applied Science). Lysates were sonicatedand cell debris removed by centrifugation at 16,000 � g. Super-natants were incubated with 500 ng ofmousemonoclonal 9E10anti-Myc antibody (Roche Applied Science) overnight at 4 °Cfollowed by the addition of 15 �l of sheep anti-mouse IgGDynabeads (Invitrogen). Beads were washed three times withlysis buffer, twice with wash buffer (50 mM Tris-HCl, pH 8.0, 150mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%SDS), and then twice with low salt buffer (20 mM Tris-HCl, pH7.5). Proteinswere elutedwith 50�l of SDSprotein-loading buffer(62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% �-mercap-

toethanol) (38) for 5min at 95 °C. Inputs and co-immunoprecipi-tateswere subjected to SDS-PAGEand transferred to a nitrocellu-lose membrane (Hybond C-Extra, Amersham Biosciences).Western Blotting—Western blots carrying input and immu-

noprecipitated samples were first incubated with primary anti-body (rat anti-HA or mouse anti-Myc) followed by the appro-priate HRP-conjugated antibodies. Proteins were visualizedusing the enhanced chemiluminescence kit (ECL, AmershamBiosciences).Dual-Luciferase Reporter Assays—Approximately 1.75� 105

CHO cells were transiently transfected using Lipofectamine2000 with 200 ng of pGL2-4�ZNF652-TK-Luc, constructsexpressing varying amounts of ZNF652 and/orMyc-CBFA2T3,and 25 ng of pRL-TK plasmid (Promega) as an internal trans-fection control. The cells were harvested after 16 h, lysed, andassayed using the Dual-Luciferase reporter assay system (Pro-mega). Luciferase values were normalized to Renilla luciferaseactivity and expressed as relative light units. For reporter assayson the HEB promoter, HeLa cells (1.5 � 105) were transfectedwith 200 ng of luciferase reporter together with varyingamounts of constructs expressing Myc-ZNF652 and/or Myc-CBFA2T3 and 50 ng of pRL-TK plasmid (Promega). Cells wereharvested and analyzed as described above. All luciferase reporterassays were performed in triplicate and repeated at least threetimes with the data presented as mean � S.E. ZNF652 andCBFA2T3 expression was confirmed by Western blot analysisusing the appropriate primary and secondary antibodies.Reverse Transcription Real-time PCR—Total RNA was

extracted using an RNeasy mini kit (Qiagen) along with On-Column RNase-free DNase digestion. cDNAs were generatedby reverse transcribing the total RNA using oligo(dT)24 andMoloney murine leukemia virus reverse transcriptase (H�)(Promega). Real-time PCRwas performed on these cDNA sam-ples to measure the levels of HEB transcript using forward (5�-GTCACTACTTCAAGCACAGACCTGA-3�) and reverse (5�-GCGTTCTCTGGCATTGTTAGCCAT-3�) primers and ofthe housekeeping gene cyclophilin A using forward (5�-GGC-AAATGCTGGACCCAACACAAA-3�) and reverse (5�-CTA-GGCATGGGAGGGAACAAGGAA-3�) primers. Real-timePCRs were carried out using iQ SYBR Green Supermix (Bio-Rad) on an iQ real-time PCR detection system under the fol-lowing conditions: 95 °C for 3 min; 40 cycles of 94 °C for 15 s,60 °C for 15 s, and 72 °C for 30 s. HEB expression was normal-ized against the levels of cyclophilin A.Promoter Precipitation—Wild type and mutant HEB pro-

moter fragments were PCR-amplified from the pGL3-HEB-E-Luc and pGL3-HEB-mut-E-Luc constructs, respectively,using HEB-F (5�-TGCTTCTCGAGGTGCCTGCGAGCGC-CTCAT-3�) and the biotinylated GLprimer2-R (5�-CTTTA-TGTTTTTGGCGTCTTCCA-3�) primer, which annealswithin the vector sequences. PCR products were gel-purifiedand immobilized on streptavidin-coated M-280 Dynabeads.HuT78 total cell extracts were used for the endogenousZNF652 and CBFA2T3 binding assay. For the second assay,total cell extracts from HEK293T cells ectopically expressingthe FLAG-ZNF652 and Myc-CBFA2T3 were used. Unchargedbeads or beads conjugated with the HEB promoter fragmentcarrying the wild type or mutant ZNF652 DNA binding




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sequence were incubated with the appropriate cell extracts inbinding buffer (20 mM Tris-HCl, pH 8.0, 10% glycerol, 6 mMMgCl2, 5mMDTT, 0.1mMEDTA, 0.01%Nonidet P-40) (20) for2 h at room temperature and then washed three times with thesame buffer. The proteins bound to the immobilizedDNAwererecovered by incubating the beads with 10 mM Tris-HCl, pH7.5, 1 mM EDTA, and 1 M NaCl buffer, resolved on SDS-PAGE,and analyzed by Western blotting using the appropriate pri-mary and secondary antibodies.Chromatin Immunoprecipitation (ChIP) Assay—A modified

ChIP assay protocol (Upstate Biotechnology) was used.Approximately 2 � 107 HEK293T cells were cross-linked for 9min at room temperature using 1% formaldehyde in suspensionon a rotor, terminated with cold 0.125 M glycine, and washedtwice with cold phosphate-buffered saline. Cells were lysed onice with lysis buffer (50mMTris-HCl, pH 8.0, 10mM EDTA, 1%SDS) for 10 min and then sonicated on a Vibra-Cell VCX 130(Sonics) using a 3-mm tip with seven 15-s pulses at 30% ampli-tude. Cells were centrifuged and the supernatant diluted with20 volumes of ChIP dilution buffer (16.7 mM Tris-HCl, pH 8.0,167mMNaCl, 1.2mMEDTA, 1.1%Triton-X-100). Thismixwasthen incubated with either rabbit IgG or affinity-purified anti-ZNF652 antibody overnight at 4 °C. Protein A beads, preincu-bated for 30min with 500 �g/ml sonicated salmon spermDNAand 1.25 mg/ml BSA in ChIP wash buffer (20 mM Tris-HCl, pH8.0, 150 mM NaCl, 1.7 mM EDTA, 1% Triton-X-100, 0.05%SDS), were used to collect the protein-DNA complexes. Beadswere washed three times with the ChIP wash buffer and twicewith TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Protein-DNAcross-linkswere reversed by treating themwith 200mMNaCl at65 °C for 5 h, and DNA was isolated according to a publishedmethod (39). PCRswere performedusingAmpliTaqGoldDNApolymerase (Applied Biosystems) under the following condi-tions: 94 °C for 12min; 56 cycles of 94 °C for 20 s, 56 °C for 20 s,and 72 °C for 30 s for the HEB promoter (forward, 5�-TCACC-CGACCGCGGGCTTTGTCCA-3�, and reverse, 5�-AGAACT-TGTGGGTCTCTTCAGGCGGACA-3� primers (HEB Gen-BankTM accession number NC_000015)) and 31 cycles for thenegative control �-globin sequence (forward, 5�-GAAGAGC-CAAGGACAGGTAC-3� (nucleotides 671–690), and reverse,5�-CAACTTCATCCACGTTCACC-3� (nucleotides 938–919)primers; �-globin GenBankTM accession number L26462).siRNA-mediated ZNF652 Knockdown—MCF7 breast cancer

cells were transfected with either ZNF652-specific (targetsequences, 5�-GUAGAGAAAGUCAGCGUUA-3� or 5�-GAG-AAGCACAUGAACGUUA-3� (Dharmacon RNA Technolo-gies)) or scrambled control (Qiagen) siRNA at 100 nM usinglipitoid transfection reagent according to the publishedmethod(40). Cells were collected 48 h after transfection and analyzedfor levels of ZNF652 and HEB protein using Western blotting.Protein loading was confirmed by probing with anti-�-actinantibody.

RESULTS

Identification and Validation of ZNF652 Consensus DNABinding Sequence—The binding sequence for ZNF652 wasidentified using a CASTing protocol (34). Bacterially expressedMBP-ZNF652 fusion proteinwas used in five sequential rounds

of binding and elution cycles to enrich for ZNF652 bindingsequences from a random pool of dsDNA. This random poolwas generated by annealing two complementary oligonucleo-tides with 18 degenerate bases (see “Experimental Proce-dures”). The enrichment of theZNF652DNAbinding sequencewas monitored by performing EMSA on equal amounts of thepools of DNA amplified from rounds 3 to 5 of selection (Fig.1A). Binding of MBP-ZNF652 protein to the enriched DNA

A.

MBP-ZNF652 (ng) - - 10- 40 100 - - -10 40 100 10 40 100MBP (ng)

Enrichment cycles- - - - - -- - - - - -3 34 45 5 5

MBP-ZNF652-DNA Complex

Free Probe

1 2 3 144 5 6 7 8 12 13 159 10 11

10 40 100

G C G A C G G A A G G G T T A A A C C G A A A G G G T T A A T A A G A T A C C G A A A G G G T T A A A T T C G C G A C G A A G G G G T T A A T C G T G A C G A A A G G G T T A A C C T G A A A G G G T T A A T A C A A T C T G G C A C A A A G G G T T A A T C G A A A G G G T T A A C A C G A G G C C G A A A G G G T T A A T C C C G C C G C G A A A G G G T T A A T G G G C A A C G A A A G G G T T A A T G C A C C G A A G G G G T T A A A G G G C G G T A A A G G G T T A A T G G G T A G T G A A C G G G T T A A T C C G A A G G G G T T A A T A A T C G G C G C A C A A A G G G T T A A T C G A A G G G G T T A A G T C C G G C C G C T G A A A G G G T T A A A C G T G C T G A A G G G G T T A A T G G T A C C G A A G G G G T T A A A T A A T T A A A G G G T T A A T C A T G C A C A A A G G G T T A A A C C T G A C A A G A A G G G G T T A A T C G C G T A A G G G T T A A T G G T C C G C G C A G A A G G G G T T A A T G T C A A T C A C G G G T T A A T G C C G A A A G G G T T A A T A T C G

C G A A A G G G T T A A T Consensus1 7 862 125 1011 133 94

B.

FIGURE 1. ZNF652 recognizes a specific DNA response element. A, sequen-tial enrichment of ZNF652 DNA binding sequences was performed using theCASTing protocol, where a pool of dsDNA containing 18 bp of randomizedsequence was incubated with purified recombinant MBP-ZNF652 fusion pro-tein. DNA bound to the MBP-ZNF652 fusion protein was PCR-amplified andused in subsequent cycles of selection and amplification. An equivalentamount of PCR-amplified DNA derived from the third, fourth, and fifth cyclesof selection was 32P-labeled, incubated alone (lanes 1–3) or with increasingamounts of MBP-ZNF652 fusion protein (lanes 4 –12) or MBP (lanes 13–15),and subjected to EMSA. B, alignment of ZNF652 DNA binding sequences.Fifth-round PCR products were cloned, and 27 individual clones weresequenced and aligned. The consensus ZNF652 DNA binding sequence and aschematic diagram showing relative frequency of nucleotides at each posi-tion within this consensus sequence are presented.




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pools sequentially increased with the three successive cycles ofselection (Fig. 1A, compare lanes 6, 9, and 12). No such bindingwas detectedwith theMBPprotein alone (Fig. 1A, lanes 13–15).PCR products from the fifth round of selectionwere cloned andsequenced. Sequence alignment of 27 individual clonesrevealed the consensus binding sequence CGAAAGGGT-TAAT (Fig. 1B).

To confirm that MBP-ZNF652specifically binds to this consensussequence, complementary oligonu-cleotides with wild type or mutantZNF652 DNA binding sequenceswere designed. These oligonucleo-tides were annealed to generateshort DNA fragments and used inEMSA. Whereas MBP-ZNF652bound to the short dsDNA carryingthe wild type sequence, no suchbinding was detected with themutated sequence (Fig. 2A). In addi-tion, binding to the wild type probedecreased in correspondence to thepresence of increasing amounts ofunlabeled wild type probe DNA(Fig. 2A, lanes 4–6). However, nochange in the binding to the wildtype probewas observed in the pres-ence of increasing amounts of unla-beled mutant probe DNA (Fig. 2A,lanes 9–11). MBP protein alone didnot bind to the wild type probe (Fig.2A, lane 2). In a separate EMSA,both the recombinant MBP-ZNF652 and endogenous-ZNF652proteins (from ZR75-1 breast can-cer cells) were observed to bind tothe wild type probe. The specificityof this binding was evident as boththeMBP-ZNF652-DNAand endog-enous ZNF652-DNA complexessupershifted in the presence of ananti-ZNF652 antibody (Fig. 2B).However, such a supershift was notobserved in the presence of a non-specific antibody. These resultsshow that the ZNF652 consensusDNA binding sequence identifiedfrom the CASTing protocol bindsthe ZNF652 protein.To further confirm that endoge-

nous mammalian nuclear ZNF652binds to the identified consensussequence, competitive EMSAexperiments were performed usingZR75-1 nuclear extracts (Fig. 2C).These results were similar to thoseobtained using the bacteriallyexpressed MBP-ZNF652 fusion

protein (Fig. 2A). Finally, the specificity of nuclear ZNF652 pro-tein binding to the consensus ZNF652DNA sequence was con-firmed by supershift EMSA using nuclear extracts of ZR75-1and HEK293T cells transiently expressing HA-ZNF652 (Fig.2D). ZNF652-DNA complexes underwent a supershift in thepresence of an anti-ZNF652 antibody but not in the presence ofa nonspecific antibody (Fig. 2D, compare lane 3 with 5 and 9

A.

MBP-ZNF652 (40 ng)

- - - 20 80 400

MBP (800 ng)

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Probe (WT)Probe (MT)

Competitor (WT; ×)Competitor (MT; ×)

- - - 20 80400 - -- - - - -- - - - - - + + - - -+ + + + + + - - + + +- - + + + + -

-+ + +

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1 2 3 4 5 6 7 8 9 10 11

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Probe (WT) + + + + + + + + + +

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ZNF652-DNAComplex

Non

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Anti-ZNF652 antibodyNon-specific Antibody - - +

- + -- - +- + -

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Nuclear Extract (mg)

- - - 40 80 400- - - - -

Probe (WT)Probe (MT)

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- - - - + + + - - -+ + + + + +- - + + +- - +

1- 3 6

- - - 20 80 400- - - -- - -

- --

-

- 31 6 6 6 6 6 6 6

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ZNF652-DNAComplex

1 2 3 4 5 6 7 8 9 10 11 12 13 14

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--

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Ant

i-ZN

F652

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ody

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293T/HA-ZNF652A

nti-Z

NF6

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ZNF652-DNAComplex

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FIGURE 2. ZNF652 specifically binds to the consensus DNA response element. A, recombinant MBP-ZNF652 fusion protein specifically binds to the consensus DNA binding sequence. Competitive EMSA wasperformed using a 32P-labeled WT probe consisting of the consensus ZNF652 binding sequence shown in Fig.1B. The MT probe contained a randomized sequence of same length. WT (lanes 1– 6 and 9 –11) or MT (lanes 7and 8) probes were incubated with MBP-ZNF652 fusion protein (lanes 3– 6 and 8 –11), MBP alone (lane 2), or noadded protein (lanes 1 and 7). Competition treatments included MBP-ZNF652 fusion protein incubated in thepresence of a 20 – 400 molar excess (�) of either unlabeled WT (lanes 4 – 6) or MT (lanes 9 –11) probe. B, recom-binant MBP-ZNF652 and endogenous ZNF652 bind to the consensus ZNF652 DNA binding sequence. EMSAwas performed using 32P-labeled WT probe. Probe was incubated with MBP (lanes 1–3), MBP-ZNF652 (lanes4 – 6), ZR75-1 (lanes 7–9), or no added protein (lane 10) either in the absence (lanes 1, 4, and 7) or presence ofanti-ZNF652 (lanes 2, 5, and 8) or nonspecific (lanes 3, 6, and 9) antibody. Both the recombinant MBP-ZNF652-DNA and endogenous ZNF652-DNA complexes supershifted (SS) only in the presence of anti-ZNF652 antibody(lanes 5 and 8). The supershifted MBP-ZNF652-DNA complexes did not migrate into the gel. C, endogenousZNF652 specifically binds to the consensus DNA binding sequence. Competitive EMSA was performed using32P-labeled WT (lanes 1– 4 and 9 –14) or MT (lanes 5– 8) probes. Probes were incubated with either no addedprotein (lanes 1 and 5) or different amounts of ZR75-1 nuclear extract (lanes 2– 4 and 6 –14). Nuclear extractswere also incubated with WT probe in the presence of 20 – 400 molar excess (�) of unlabeled WT (lanes 9 –11)or MT (lanes 12–14) probe. D, ZNF652-DNA complex supershifts in the presence of anti-ZNF652 antibody.Nuclear extracts from either ZR75-1 (lanes 1– 6) or HEK293T cells ectopically expressing HA-ZNF652 (lanes 7–12)were incubated with either 32P-labeled WT (lanes 1, 3, 5, 7, 9, and 11) or MT (lanes 2, 4, 6, 8, 10, 12) probe. TheDNA-protein complexes were incubated either without an antibody (lanes 1 and 2 and 7 and 8) or in thepresence of an anti-ZNF652 (lanes 3 and 4 and 9 and 10) or a nonspecific antibody (lanes 5 and 6 and 11 and 12).ZNF652-DNA complexes were observed only with WT probe (lanes 1, 5, 7, and 11) and underwent a supershift(lanes 3 and 9) only in the presence of an anti-ZNF652 antibody. WT (lane 13) and MT (lane 14) probes withoutany added protein were included as controls.




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with 11). Nuclear extracts of the breast cancer cell line MCF7and leukemic cell lines KU-812 and K-562 were also seen tobind specifically to the wild type ZNF652 probe (data notshown). Taken together, these results show that the identifiedZNF652 DNA binding sequence (hereafter called ZNF652response element (RE)) is biologically authentic. Therefore, wehypothesized that genes containing the ZNF652 RE in theirpromoters would be transcriptionally repressed by theCBFA2T3-ZNF652 complex.The CBFA2T3-ZNF652 Complex Mediates Transcriptional

Repression through a ZNF652 RE—Dual-Luciferase assays wereused to determine whether the CBFA2T3-ZNF652 interactioncomplex was able to mediate transcriptional repressionthrough the identified ZNF652 RE. For these assays, the pGL2-4�ZNF652-TK-Luc construct carrying four tandem copies ofthe ZNF652 RE was used. Luciferase activity of the pGL2-4�ZNF652-TK-Luc reporter was considered as the basal tran-scriptional activity. Reporter assays showed a dose-dependentdecrease in transcription of the luciferase gene in response toincreasing levels of ZNF652; this repression was enhanced inthe presence of increasing levels of CBFA2T3 (Fig. 3). However,expression of CBFA2T3 alone had no significant effect on lucif-erase activity from the pGL2-4�ZNF652-TK-Luc reporter.These results show that ZNF652 and CBFA2T3 act as func-tional corepressors of transcription.HEB (TCF12) Is a Target of CBFA2T3-ZNF652-mediated

Transcriptional Regulation—The Eukaryotic Promoter Data-base (EPD release 84) was used to search for gene promotersthat contain the consensus ZNF652 RE and thus are potentiallyregulated by ZNF652. The human HEB promoter contains a

putative ZNF652 RE (AAGGGTTAA) located at position�306upstream of the translational start site that is conserved in themouse HEB promoter (located at position �332).Reporter assays using a construct in which this HEB pro-

moter sequence drives the luciferase gene (pGL3-HEB-E-Luc)showed a dose-dependent transcriptional repression in thepresence of ectopically expressed ZNF652. Ectopic expressionof CBFA2T3 further increased this repression (Fig. 4). ZNF652-mediated repression was abrogated in the construct pGL3-HEB-mut-E-Luc carrying the mutated ZNF652 RE (Fig. 4).These results suggest that ZNF652 protein binds the ZNF652RE within the HEB promoter sequence to induce transcrip-tional repression, which is enhanced in the presence of theCBFA2T3 corepressor.The CBFA2T3-ZNF652 Complex Specifically Binds to the

ZNF652 RE within the HEB Promoter Region—A polyclonalrabbit anti-CBFA2T3 antibody was raised and affinity-purified.This antibodywas seen to specifically detect the CBFA2T3 pro-tein from the HEK293T cells ectopically expressing Myc-CBFA2T1, Myc-CBFA2T2, or Myc-CBFA2T3 protein (Fig.

0.0

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U)

anti-HA (ZNF652)anti-myc (CBFA2T3)

Reporter: pGL2-4×ZNF652-TK-Luc Luciferase TK Promoter 4×ZNF652 REs

anti-β-actin2 3 4 5 6 7 8

2 3 4 5 6 7 81

5050 50

500 200 200200 20020

---

- -- 10CBFA2T3 (ng)

p < 0.05

p < 0.05

FIGURE 3. ZNF652 transcriptional repression is enhanced by CBFA2T3.Increasing levels of ZNF652 result in a dose-dependent decrease in reportergene expression; this is further reduced in the presence of CBFA2T3. Dual-Luciferase reporter assays were performed to assess the effect of ZNF652 ontranscriptional activity of the pGL2-4�ZNF652-TK-Luc construct (schematicshown below the graph). CHO cells were transfected with pGL2-4�ZNF652-TK-Luc together with other expression constructs as indicated below eachcolumn. The total DNA in each treatment was equalized by supplementing itwith varying amounts of empty vector. pRL-TK-Renilla luciferase vector wasused as a transfection control. The -fold repression of transcription was calcu-lated in relative light units (RLU) from the ratio of firefly to Renilla luciferaseactivities and relativized to the base-line activity of the pGL2-4�ZNF652-TK-Luc. Reporter activity was significantly enhanced in the presence of ZNF652alone (bars 2– 4) or in combination with CBFA2T3 (bars 6 and 7) but was notrepressed in the presence of CBFA2T3 alone (bar 8). The levels of ZNF652 andCBFA2T3 expression were verified by Western blotting with antibodiesagainst the appropriate tags (inset). Data shown are representative of threeindependent experiments and are presented as mean � S.E. (n � 3).

0.0

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)

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ZNF652 (ng)-- - --

250 25 10010 5025 25 25 100

CBFA2T3 (ng) - - - -- -

pGL3-E-LucpGL3-HEB-E-LucpGL3-HEB-mut-E-Luc

-2 3 4 5 6 7 81 9 10

2 3 4 5 6 7 81 9 10

anti-ZNF652anti-myc (CBFA2T3)anti-β-actin

pGL3-HEB-E-Luc WT ZNF652 RE

Reporters:pGL3-E-Luc

-306-1247

p < 0.05

pGL3-HEB-mut-E-LucLuciferase MT ZNF652 RE

HEB Promoter SV40 Enhancer

HEB Promoter

-205

FIGURE 4. ZNF652-mediated transcriptional repression of HEB isenhanced in the presence of CBFA2T3. Increasing levels of ZNF652 areinversely related to the luciferase reporter gene activity driven by the HEBpromoter; this activity is further reduced in the presence of CBFA2T3. Dual-Luciferase reporter assays were performed to assess the effect of ZNF652 onthe transcription of the luciferase gene driven by the 1043-bp HEB promotercontaining either a single WT (pGL3-HEB-E-Luc) or MT ZNF652 DNA-bindingsite (pGL3-HEB-mut-E-Luc). Reporter constructs are diagrammed below thegraph. HeLa cells were transfected with pGL3-E-Luc (bars 1 and 2), pGL3-HEB-E-Luc (bars 3–7), or pGL3-HEB-mut-E-Luc (bars 8 –10) reporters with varyingamounts of ZNF652 and CBFA2T3 expression constructs as indicated. Thetotal DNA in each treatment was equalized by supplementing with varyingamounts of empty vector. The pRL-TK-Renilla luciferase vector was used as atransfection control. The -fold repression of transcription was calculated inrelative light units (RLU) from the ratio of firefly to Renilla luciferase activities.The transcriptional activity of each reporter plasmid was used as base line.The activity of the pGL3-HEB-E-Luc was significantly repressed in the pres-ence of increasing amounts of ZNF652 (lanes 4 and 5) and was furtherrepressed in the presence of CBFA2T3 (lane 6). Reporter activity of pGL3-HEB-mut-E-Luc did not show significant repression in the presence of ZNF652(lanes 9 and 10). The levels of ZNF652 and CBFA2T3 expression were verifiedby Western blotting with antibodies against the appropriate tags (inset). Datashown are representative of three independent experiments and are pre-sented as mean � S.E. (n � 3).




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5A). Anti-CBFA2T3 antibody could also specifically detect theendogenous CBFA2T3 protein from MCF7 and Jurkat nuclearextracts (Fig. 5B).In vitro promoter binding assays were used to show the bind-

ing of CBFA2T3-ZNF652 to the single ZNF652 RE within theHEB promoter. Firstly, promoter binding assays were per-formed using protein lysates from HEK293T cells exogenouslyexpressing FLAG-ZNF652 and Myc-CBFA2T3. Both ZNF652and CBFA2T3 bound to the HEB promoter carrying the wildtype ZNF652 RE but did not bind to the HEB promoter when

anti-CBFA2T3

anti-myc

myc-C

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Promoter precipitate

anti-ZNF652

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β-globin1 2 43

IP: I

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IP: a

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FIGURE 5. Affinity-purified rabbit anti-CBFA2T3 antibody specificallydetects the CBFA2T3 protein, and ZNF652 and CBFA2T3 bind to the sin-gle ZNF652 RE within the HEB promoter. A, affinity-purified rabbit anti-CBFA2T3 polyclonal antibody specifically detects the CBFA2T3 protein.Lysates from HEK293T cells ectopically expressing Myc-CBFA2T1, Myc-CBFA2T2, or Myc-CBFA2T3 were Western blotted with either anti-CBFA2T3(upper panel) or anti-Myc (lower panel) antibody. Anti-CBFA2T3 antibodydetected only the Myc-CBFA2T3 protein (upper panel). B, affinity-purified rab-bit anti-CBFA2T3 polyclonal antibody specifically detects the endogenousCBFA2T3 protein. Nuclear extracts of MCF7 (9 �g) and Jurkat (6 �g) cells wereWestern blotted with anti-CBFA2T3 antibody. Lysate from HEK293T cellsectopically expressing Myc-CBFA2T3 was used as a positive control. C, exog-enously expressed ZNF652 and CBFA2T3 proteins bind to the ZNF652 RElocated within the HEB promoter. WT and MT HEB promoter sequences werePCR-amplified from the appropriate reporter constructs using a pair of prim-ers (one of which was biotinylated at the 5�-end) and immobilized to strepta-vidin-coated magnetic beads. Magnetic beads charged with promotersequences (lanes 2 and 3) or uncharged beads (lane 4) were incubated withlysates from HEK293T cells ectopically expressing FLAG-ZNF652 and

Myc-CBFA2T3 and washed, and bound proteins were eluted with 1 M NaCl. Input(lane 1) and eluted (lanes 2– 4) proteins were analyzed by Western blotting withanti-ZNF652 (upper panel) and anti-Myc (lower panel) antibodies. D, endogenousZNF652 and CBFA2T3 bind to the ZNF652 RE located within the HEB promoter.Promoter precipitation assay was performed as described in C except that totalprotein lysates from HuT78 cells were used. Input (lane 1) and protein eluted fromthe beads charged with WT (lane 2) or MT (lane 3) promoter sequence oruncharged beads (lane 4) were analyzed by Western blotting with anti-ZNF652(upper panel) and anti-CBFA2T3 (lower panel) antibodies. Minor backgroundbands (lanes 3– 4) were due to a low level of nonspecific binding of the ZNF652and CBFA2T3 to the magnetic beads. E, ZNF652 binds in vivo to the ZNF652 RElocated within the HEB promoter. A ChIP assay was performed on HEK293T cellsusing either rabbit IgG control (lane 1) or an anti-ZNF652 (lane 2) antibody. DNAisolated from the chromatin immunoprecipitates (IP; lanes 1 and 2) or input (lane3; positive control) were PCR-amplified using primers flanking the ZNF652 REwithin the HEB promoter (upper panel). Amplification of a �-globin sequence wasused as a negative control (lower panel). Non-template negative controls are alsoshown (lane 4).

anti-ZNF652

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FIGURE 6. CBFA2T3-ZNF652 complex represses endogenous HEB tran-scription. A, HEK293T cells ectopically expressing FLAG-ZNF652 (lane 2),FLAG-ZNF652 and Myc-CBFA2T3 (lane 3), or Myc-CBFA2T3 (lane 4) were used.After 24 h, cells were harvested and total RNA extracted. Levels of HEB mRNAwere determined by quantitative real-time reverse transcription-PCR andnormalized to cyclophilin A. Values are expressed as -fold repression relativeto HEK292T, set at 1. The expression levels of ZNF652, HEB, and CBFA2T3 wereconfirmed by Western blotting the total cell lysates with the indicated anti-bodies (inset). HEB bands were quantified by densitometry and expressedrelative to lane 1, set at 1; lane 2, 0.42; lane 3, 0.26; and lane 4, 0.74. B, MCF7 cellswere transfected with either ZNF652-specific (lanes 1 and 2) or scrambledcontrol (lane 3) siRNA oligos. After 48 h, cells were harvested, and lysates wereWestern blotted with the indicated antibodies.




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this RE was mutated (Fig. 5C). Secondly, promoter bindingassays were performed using protein lysates from the humanT-cell line that expresses both endogenous ZNF652 andCBFA2T3. Endogenous CBFA2T3 and ZNF652 also specifi-cally bound to the HEB promoter carrying the wild typeZNF652 RE. However, no such binding was observed with theHEB promoter containing amutatedZNF652RE (Fig. 5D). Fur-thermore, the stoichiometric level of ZNF652 binding washigher than that of CBFA2T3. Finally, ChIP assay was used toconfirm in vivo binding of ZNF652 to its cognate RE locatedwithin the HEB promoter region (Fig. 5E). PCR data showedthat the HEB promoter sequence was enriched with an anti-ZNF652 antibody but not with rabbit IgG. Negative control�-globin sequences were also not enriched by either anti-ZNF652 antibody or control IgG (Fig. 5E). These results sug-gested that ZNF652 binds to the HEB promoter in vivo. AsCBFA2T3 does not directly bind DNA, these assays confirmedthat ZNF652 binds to the single wild type ZNF652 RE withintheHEB promoter region and is associatedwithCBFA2T3 (31).HEB Is a Direct Transcriptional Target of ZNF652—To fur-

ther confirm that ZNF652 mediates transcriptional repressionof HEB in vivo, HEK293T cells ectopically expressing FLAG-ZNF652 alone or with Myc-CBFA2T3 were used. The relativelevels of HEB transcription were assayed using real-timereverse transcription-PCR. The HEK293T cells expressing

exogenous ZNF652 had a reducedlevel of HEB expression comparedwith the empty vector-transfectedHEK293T cells (Fig. 6A). ZNF652-mediated repression of HEB wasenhanced in the presence ofCBFA2T3. Exogenous expression ofCBFA2T3 alone in the HEK293Tcells also resulted in a minimalrepression of HEB, an observationthat can be attributed to the pres-ence of endogenous ZNF652 (Fig.6A, lane 4). Similar to the transcriptlevels, HEB protein levels were alsorepressed in HEK293T cells ectopi-cally expressing either ZNF652 orCBFA2T3 or both (Fig. 6A). Theseresults show that ZNF652 mediatestranscriptional repression of HEBin vivo and that this repression isenhanced in the presence ofCBFA2T3.To further demonstrate that

ZNF652 represses HEB transcrip-tion, the effect of knockdown ofZNF652 expression on levels ofHEB protein was investigated.MCF7 cells were transfected witheither scrambled or two differentZNF652-specific siRNAs, and HEBand ZNF652 protein levels weredetermined subsequently by West-ern blot analysis. As predicted,

higher levels of HEB protein were detected in cells with ablatedZNF652 expression, but no such increase was detected in thepresence of scrambled siRNA (Fig. 6B).NHR3 and NHR4 Motifs of CBFA2T3 Are Both Required for

Interaction of CBFA2T3 with ZNF652—We have shown previ-ously that CBFA2T3 interacts with ZNF652 to repress tran-scription (31). To more precisely define the regions ofCBFA2T3 that interacted with ZNF652, various fragments ofCBFA2T3 were used in co-immunoprecipitation assays (Fig.7A). Full-length CBFA2T3 and the fragment CBFA2T3-3, con-taining both the NHR3 and NHR4 domains, interacted withZNF652 (Fig. 7B, lanes 9 and 11), whereas interaction was notobserved with the fragments CBFA2T3-1 (containing NHR2alone), CBFA2T3-2 (containing NHR3 alone) and CBFA2T3-4(containing NHR4 alone) (Fig. 7B, lanes 7, 8, and 10). Theseresults suggest that both the NHR3 and NHR4 domains ofCBFA2T3 are required for interaction with ZNF652.ZNF652 Interacts with the CBFA2T3 via Its C-terminal

Region—Immunoprecipitation experiments were undertakento define the region of ZNF652 that interacts with theCBFA2T3-3. Immunoprecipitations on HEK293T cellsexpressing CBFA2T3-3 and either full-length or two differentC-terminal fragments of ZNF652 were performed (Fig. 8A).ZNF652-2 weakly interacted with CBFA2T3-3, but no suchinteraction was detected with ZNF652-4 (Fig. 8B), suggesting

myc-CBFA2T3-1myc-CBFA2T3-2myc-CBFA2T3-3myc-CBFA2T3-4

myc-CBFA2T3

HA-ZNF652

+

WB: anti-HA

WB: anti-myc

Input IP: anti-myc

85kDa

78kDa

36kDa

22kDa

+ ++ + + + + ++ + +

- - +- - - - - +- - -

- - -+ - - - - -+ - -

- + -- - -

- - -- + - - - -- + -

- + -- - -

+ - -- - - + - -- - -

1 2 43 5 6 7 8 109 11 12

CBFA2T3

PST NHR1 NHR2 NHR3 NHR4

201aa 393aaCBFA2T3-1 -

CBFA2T3-2447aa341aa

-

567aa

448aa

CBFA2T3-4 -

567aa341aaCBFA2T3-3 +

Interaction with ZNF652

+

447aa

567aa1aa 85 181 308 334 399 447 470 506A.

B.

FIGURE 7. Both the NHR3 and the NHR4 domains of CBFA2T3 are required for interaction with ZNF652.A, diagram showing various CBFA2T3 regions used to define the minimal region of interaction with ZNF652 byco-immunoprecipitation assays. The PST (proline/serine/threonine-rich) domain and four evolutionarily con-served NHR1, -2, -3, and -4 (also called MYND) domains are shown. B, HEK293T cells were transfected withcombinations of constructs expressing full-length Myc-tagged CBFA2T3 (lane 5) or various fragments(CBFA2T3-1 to -4) (lanes 1– 4) together with HA-tagged ZNF652 protein (lanes 1–12) as indicated. Protein lysateswere co-immunoprecipitated (IP) with anti-Myc antibody. Inputs (lanes 1– 6) and immunoprecipitates (lanes7–12) were Western blotted (WB) with anti-HA (upper panel) or anti-Myc (lower panel) antibodies.




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that CBFA2T3-ZNF652 interaction is stabilized in the presenceof the zinc finger region (31). These results further confirm thatZNF652 interacts with CBFA2T3 via the NHR3 and NHR4domains.ZNF652 Interacts with CBFA2T3 via a Proline-rich Stretch

within its C-terminal Region—Recently it was demonstratedthat a proline-rich motif of N-CoR/SMRT has high affinity forthe MYND domain located within the NHR4 motif ofCBFA2T1. This finding was consistent with the data on thesolution structure of the NHR4-SMRT/N-CoR peptide com-plex (12) and an earlier report showing that NHR3-4 domainsof CBFA2T1 interact with N-CoR repression domain III, aregion of N-CoR that contains a conserved PPLXP bindingmotif for corepressor proteins (7). We aligned the amino acidsequences of ZNF652, N-CoR, and SMRT (Fig. 9A). We rea-soned that CBFA2T3 interacts with ZNF652 through a similarproline-rich region (amino acids 565–574) within the C termi-nus of ZNF652. To test this possibility, we determined theinteraction of ZNF652 with either PPVP mutated to AAAA(amino acids 565–568; HA-ZNF652-mut1) or PPPPmutated toAAAA (amino acids 571–574; HA-ZNF652-mut2) andCBFA2T3 using co-immunoprecipitation assays. Consistentwith this premise, neither of the ZNF652 mutants interactedwith the CBFA2T3 protein, suggesting that a proline-richregion located within the C terminus of ZNF652 (amino acids565–574) is the minimal region of interaction with CBFA2T3(Fig. 9B). To confirm that the two ZNF652 mutant proteins

retained their three-dimensionalstructure, we determined their abil-ity to bind the ZNF652 RE withinthe HEB promoter using promoterprecipitation assays. Both the wildtype and mutant ZNF652 proteinswere able to bind the ZNF652 RE,indicating that alanine substitutionsdo not impair the structure or DNAbinding ability of the ZNF652mutants (Fig. 9C). Taken together,these results suggest that a C-termi-nally located proline-rich region ofZNF652 interacts with the NHR3-NHR4 domain of CBFA2T3.

DISCUSSION

DNA-binding activator or repres-sor proteins play a critical role inregulation of gene expression. Dys-regulation of their functional activ-ity can lead to a number of malig-nancies, including breast cancer (1).The identification of transcriptionfactor binding sequences is neces-sary to identify target genes anddetermine their precise biologicalfunction.ETOproteins donot directly bind

DNA but perform their repressiverole through interaction with tran-

scription factors that are capable of binding their cognateresponse elements located within promoter sequences of thetarget genes. The ETO family of proteins has been shown tointeract with the zinc finger proteins Gfi-1, BCL6, PLZF, andGATA-1, as well as with transcription factors such as HEB andSCL/TAL-1 (stem cell leukemia/T-cell acute lymphocytic leu-kemia-1), to repress gene expression. In addition, we haveshown that ZNF652 is a novel ETO-interacting protein thatfunctions as a DNA-binding transcription factor and providesfurther complexity to the ETO family of repressor complexes(31). The major established function of these ETO-based com-plexes is in hematopoiesis, with dysregulation leading to leuke-mia (41). However, the expression of the ETO proteins andtheir interacting DNA-binding transcription factors are notlimited to the hematopoietic lineages, as more diverse roles arecurrently emerging. For example, the CBFA2T3-ZNF652 com-plex has a role in suppressing breast oncogenesis (31), and theBCL6 oncoprotein is implicated in the pathogenesis of B-celllymphomas (42). In addition, BCL6 is expressed in a higherproportion of clinically aggressive breast tumors and has beenshown to prevent mammary epithelial differentiation (43).The novel classical zinc finger protein ZNF652 specifically

interacts with the breast tumor suppressor CBFA2T3 (31). Thisis a transcriptional repressor, because a GAL4-ZNF652 fusionprotein, when tethered to DNA via a GAL4 DNA bindingsequence, represses transcription in reporter assays, and thisrepression is enhanced in the presence of the CBFA2T3 protein

ZNF6521aa

N-terminal Zinc Fingers C-terminal

Interaction withCBFA2T3

Interaction withCBFA2T3-3

ZNF652-2

ZNF652-4

497 606aa246+

++

+

+

?

Input IP: anti-myc

85kDa

50kDa

36kDa

18kDa

myc-CBFA2T3-3 - - +- + + - - +- + +

HA-ZNF652-4

HA-ZNF652HA-ZNF652-2

- - -+ - + - - -+ - +- + -- + - - + -- + -+ - +- - - + - +- - -

1 2 43 5 6 7 8 109 11 12

WB:anti-HA

WB:anti-myc

A.

B.

Kumar et al (31)

FIGURE 8. Zinc finger and C-terminal regions of ZNF652 are required for interaction with CBFA2T3-3.A, diagram showing various ZNF652 regions used to define minimal region of interaction with CBFA2T3-3 byco-immunoprecipitation assays. B, HEK293T cells were transfected with constructs expressing HA-taggedZNF652 (lanes 1, 4, 7, and 10), ZNF652-2 (lanes 2, 5, 8, and 11) or ZNF652-4 (lanes 3, 6, 9, and 12) proteins aloneor with Myc-CBFA2T3-3 (lanes 4 – 6 and 10 –12) as shown. The lysates were co-immunoprecipitated (IP) withanti-Myc antibody. Inputs (lanes 1– 6) and immunoprecipitates (lanes 7–12) were Western blotted (WB) withanti-HA (upper panel) or anti-Myc (lower panel) antibodies.




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(31). To assist the identification of gene targets of theCBFA2T3-ZNF652 complex, CGAAAGGGTTAAT was iden-tified as the ZNF652 consensus DNA binding sequence (Fig.1B). The authenticity of the identified ZNF652 DNA bindingsequence is supported by multiple assays including normal,competitive and supershift EMSAs using purified recombinantMBP-ZNF652 and endogenous and ectopically expressedmammalian ZNF652 proteins (Fig. 2). Furthermore, CBFA2T3and ZNF652 form a transcriptional repressor complex on thisZNF652 RE (Fig. 3).Our in vivo assays show that ZNF652 represses transcription

of HEB by binding to a single ZNF652 RE within the HEB pro-moter sequence, and this repression is enhanced in the pres-ence of CBFA2T3 (Fig. 4). This result was further supported byour in vivo and in vitro promoter binding assays showing thatthe CBFA2T3-ZNF652 complex is recruited to the HEB pro-moter (Fig. 5). Furthermore, ZNF652 ablation resulted in anincreased level ofHEB expression (Fig. 6). Based on these find-ings, it is proposed that CBFA2T3 and ZNF652 form a complexon the HEB promoter, subsequently recruiting corepressorsand HDACs and resulting in repression of transcription.Our results show that HEB is a direct target of CBFA2T3-

ZNF652-mediated transcriptional repression. It is of interestthat all of the ETO family proteins can interact directly with

HEB to generate transcription com-plexes and modulate the transcrip-tional activation function of HEB(8). The three-dimensional struc-ture of theCBFA2T1NHR1 domaininteracting with a 17-amino acidpeptide derived from the HEB acti-vation domain-1 (AD1) wasreported recently (44). As theNHR1domains of the ETO proteins arehighly conserved, it is predicted thatboth CBFA2T2 and CBFA2T3would also interact directly withHEB through their NHR1 domain.NHR1-mediated CBFA2T1 interac-tion with HEB activation domain-1inhibits its transcriptional activa-tion function by blocking the p300/CBP (CREB-binding protein) occu-pancy of HEB and promoting therecruitment of HDAC containingcomplexes (8).HEB is involved in both hemato-

poiesis and myogenesis (45, 46) andis associated with variousmalignan-cies including gliomas (47). HEB,along with E2-2, E12, and E47,belongs to a family of evolutionarilyconserved basic helix-loop-helixproteins called E-box transcriptionfactors. HEB and E2-2 proteins areencoded by unique genes, whereasE12 and E47 result from differentialsplicing of the E2A gene transcript.

HEB (along with E2A) plays an essential role by positively reg-ulating a number of target genes critical in B- and T-cell lineagedifferentiation and development (48). Systematic gene replace-ment experiments show that signals leading to transcriptionalactivation of HEB versus E2A are crucial for B- and T-cell line-age commitment and differentiation (49).Our results show thatthe CBFA2T3-ZNF652 complex represses HEB expression.Therefore, we predict that CBFA2T3-ZNF652-mediated HEBrepression may determine the relative levels of HEB and thusthe ratio of E2A-E2A homodimers to E2A-HEB heterodimers,critical in B- and T-cell development.CBFA2T3 performs its transcriptional repressor function in

erythropoiesis as a component of a multiprotein complex.Within this complex, CBFA2T3 associates with the hematopoi-etic-specific E-box SCL protein and a number of other tran-scription factors (for example, HEB, GATA-1, E2A, E12/47,E2.2, Lbd-1, LMO2, and SSDP2) (20, 21). The stoichiometry ofCBFA2T3 relative to the level of SCL, E2A, or HEB activatorswithin this multiprotein complex varies during erythroid dif-ferentiation, and levels of CBFA2T3 determine both the abilityto activate or repress transcription of essential SCL target genesand the timing of their expression (20). Therefore, it is evidentthat HEB has a complex relationship with CBFA2T3, as theCBFA2T3-ZNF652 transcriptional complex represses HEB

A.

N-CoR 606-615 EPPPPLPPPPN-CoR 1031-1040 RPPPPLIPSSSMRT 1103-1112 SNPPPLISSASMRT 1664-1673 LYPPYLIRGY

PPVPHLPPPPZNF6521 574565 606aa247 463

C.

N-terminal Zinc Fingers C-terminal

anti-HA

HA-ZNF652HA-ZNF652-mut1HA-ZNF652-mut2

Beads with HEB promoter

InputPromoter

precipitate

- - -+ -

1 2 43 5 6

- - -+ -- -+ - -

- -- ++

++ -

-+

Uncharged beads++ + -

7

PPPP

PPPPPL

LLL

PP

myc-CBFA2T3HA-ZNF652

Input IP: anti-myc

- - -+ -

1 2 43 5 6 7 8 109 11 12

HA-ZNF652-mut1HA-ZNF652-mut2

WB: anti-HA

WB: anti-myc

- - -+ -- - -+ - - - -+ -

- -+ - - - -+ - -- -- + + +

++

+

- -- + + ++

++

85kDa

78kDa

B.

85kDa

FIGURE 9. ZNF652 interacts with the CBFA2T3 through a C-terminal proline-rich region. A, diagram show-ing structure of the ZNF652 protein and alignment of the proline-rich regions of ZNF652, N-CoR, and SMRTproteins. Amino acids (aa) identical to those of ZNF652 are shaded. B, HEK293T cells were transfected withconstructs expressing HA-tagged wild type ZNF652 (lanes 1, 4, 7, and 10), ZNF652-mut1 (lanes 2, 5, 8, and 11;PPVP converted to AAAA, amino acids 565–568), or ZNF652-mut2 (lanes 3, 6, 9, and 12; PPPP converted toAAAA, amino acids 571–574) proteins alone (lanes 1–3 and 7–9) or with Myc-CBFA2T3 (lanes 4 – 6 and 10 –12) asshown. The lysates were co-immunoprecipitated (IP) with anti-Myc antibody. Inputs (lanes 1– 6), and immuno-precipitates (lanes 7–12) were Western blotted (WB) with anti-HA (upper panel) or anti-Myc (lower panel) anti-bodies. C, wild type and mutated ZNF652 proteins retain the ability to bind the ZNF652 RE within the HEBpromoter. Streptavidin beads conjugated to the biotinylated PCR product carrying the wild type HEB promotersequence were prepared as described for Fig. 5C. Magnetic beads charged with the HEB promoter sequence(lanes 4 – 6) or uncharged beads (lane 7) were incubated with the total cell lysates from HEK393T cells trans-fected with constructs expressing HA-tagged wild type ZNF652 (lanes 4 and 7), ZNF652-mut1 (lane 5), orZNF652-mut2 (lane 6) and washed, and bound proteins were eluted with 1 M NaCl. Inputs (lanes 1–3) andpromoter precipitates (lanes 4 – 6) were analyzed by Western blotting with anti-HA antibody.




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expression, and the CBFA2T3 protein directly interacts withthe HEB protein to inhibit its activator function.Although CBFA2T3 does not interact with the ZNF652 zinc

finger region (amino acids 243–491), this region is required inorder to stabilize the interaction of the C-terminal region ofZNF652 (amino acids 498–606) with CBFA2T3 (31). Findingspresented in this study define the NHR3 andNHR4 domains ofCBFA2T3 as the regions that interact with ZNF652 (Fig. 7). Ithas been shown for CBFA2T1 that both the NHR3 and NHR4domains were also required for its interaction with the core-pressor N-CoR (7, 50). Additional studies have shown thatCBFA2T1 interacts through a conserved proline-rich PPLXPmotif within N-CoR, and reporter assays confirmed that thisinteraction is functionally important (7). A number of proteinshave been reported to interact with MYND domains throughtheir PXLXP peptide motif. MYND domains are defined by aC4-C2HCconsensus and are frequently implicated in transcrip-tional repression (12, 51). Therefore,we predict that theMYNDdomain of CBFA2T3 interacts with ZNF652 through a proline-rich PXLXP motif (amino acids 565–574; PPVPHLPPPP)located within the C-terminal region of ZNF652 (Fig. 9). Insummary, both the CBFA2T3-ZNF652 and CBFA2T1-N-CoRinteractions are mediated through the NHR3 and NHR4regions. This interaction occurs through the MYND-typedomain located within the NHR4 and respective proline-richPXLXP motifs of either ZNF652 or N-CoR. It is of importanceto note that SMRT lacks a PXLXP motif but interacts withMYND through a PPPLI sequence (12). This suggests that vari-able proline-rich sequences can interact with MYND domains;further work will be required to define precisely the functionalresidues within these proline-rich motifs.ETOs have a modular structure due to the presence of con-

served NHR domains that facilitate multiple complex proteininteractions (41). ETOs perform their transcriptional repressorrole by interacting with various DNA-binding transcriptionfactors. Each ETO-interacting DNA-binding transcription fac-tor interacts through a specific region within the ETO proteins(Fig. 10). The interaction of CBFA2T3 with ZNF652 occursthrough its NHR3-NHR4 domains. This may reflect a uniquefunctional relationship between the CBFA2T3 and ZNF652proteins, as other known ETO-interacting zinc finger tran-

scription factors interact through the NHR1-NHR2 domains(8, 26, 27, 30, 31) (Fig. 10).We are currently working toward genome-wide identifica-

tion of ZNF652 gene targets, particularly ZNF652-responsivegenes directly associatedwith breast oncogenesis, using aChIP-ChIP approach. Such investigations are critical in determininghow ZNF652 regulates normal gene expression in cell fate,development, and differentiation and how ZNF652-mediatedtranscriptional alterations of target genes can lead to cell pro-liferation and/or apoptosis.

Acknowledgment—We thank Dr. Ronald N. Zuckermann for the lipi-toid transfection reagent.

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CBFA2T1

PLZF

BCL6

HEB

ZNF652

Gfi-1

1aa 604aa

399 506444 552

73 213 348108 248

217 385

217 345

120 226

291335 392

120 216 352 378 444 492 515 552

CBFA2T3567aa1aa 85 181 308 334 399 447 470 506

PST NHR1 NHR2 NHR3 NHR4

FIGURE 10. ZNF652 is unique in its interaction with the CBFA2T3 NHR3-NHR4 motifs. The published regions of CBFA2T1 or CBFA2T3 that interactwith the DNA-binding transcription factors HEB (8), PLZF (27), BCL6 (26), Gfi-1(30), and ZNF652 (31) are shown. Except for HEB, all of these transcriptionfactors are zinc finger proteins. The locations of the respective interactingregions of CBFA2T1 and CBFA2T3 with these proteins are depicted by theamino acid (aa) numbers above or below the horizontal lines, respectively.




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Jaclyn Lee, Juliane Cohen, Grant W. Booker and David F. CallenRaman Kumar, Kelly M. Cheney, Ross McKirdy, Paul M. Neilsen, Renèe B. Schulz,

HEBGene CBFA2T3-ZNF652 Corepressor Complex Regulates Transcription of the E-box

doi: 10.1074/jbc.M709136200 originally published online May 2, 20082008, 283:19026-19038.J. Biol. Chem.

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