Atbf1 is required for the Pit1 gene early activationYingchuan Qi*, Jeffrey A. Ranish†, Xiaoyan Zhu*, Anna Krones*, Jie Zhang*, Ruedi Aebersold†‡, David W. Rose§,Michael G. Rosenfeld*¶, and Catherine Carriere¶�

*Howard Hughes Medical Institute and Department of Medicine, School of Medicine, University of California at San Diego, La Jolla, CA 92093; †Institute forSystems Biology, 1441 North 34th Street, Seattle, WA 98103; ‡Institute of Molecular Systems Biology, Swiss Federal Institute of Technology (ETH)Honggerberg and Faculty of Sciences, University of Zurich, CH-8093 Zurich, Switzerland; §Department of Medicine, Division of Endocrinology andMetabolism, School of Medicine, University of California at San Diego, La Jolla, CA 92093; and �Department of Medicine, Dartmouth Medical School,Norris Cotton Cancer Center, 1 Medical Center Drive, Lebanon, NH 03756

Contributed by Michael G. Rosenfeld, December 24, 2007 (sent for review November 28, 2007)

Enhancers have been functionally described for >35 years, but themolecular principles underlying the integration of regulatory in-puts to alternate gene enhancers used during mammalian orga-nogenesis remain incompletely understood. Using a combinationof in vivo enhancer mapping and proteomics approaches, we haveestablished that two distant and distinct early enhancers, eachrequiring different transcription complexes, are required for fullactivation of the gene encoding the pituitary lineage determiningfactor, Pit1. A transcription factor belonging to the ‘‘giant, multi-ple-homeodomain and zinc finger family,’’ Atbf1, serves as a novelpituitary regulator for one of the two required enhancers as shownby genetic and in vitro analysis.

T he essence of embryonic development is to generate aregulated increase in cell number and diversity, with the

identity of each cell type dictated by specific patterns of signalsand expression of sets of genes Thus, a hierarchy of regulatoryfactors controls activation or repression of such genes withspatial/temporal precision, and such control is central toaccurately unfolding genetic information to create precisepatterns of developmental complexity. It involves evolution-arily conserved cis-regulatory sequences that are highly struc-tured and organized to recruit sets of transcription activators/repressors. These transcription complexes will determine therate and frequency of transcription initiation, generating finecontrol of gene expression in development, homeostasis, anddisease.

The anterior pituitary gland provides an excellent model systemto analyze molecular programs governing cell-specific gene regu-lation during embryonic development. The mature pituitary glandcontains five distinct hormone-producing cell types: corticotropes,somatotropes, lactotropes, thyrotropes, and gonadotropes. All ofthese cell types arise from a common primordium, the Rathke’spouch, which initially develops by mouse embryonic day 9 (E9) asa single layer of epithelium; it undergoes a fast expansion attrib-utable to intense cell proliferation and gives rise to a series of celllineages, which eventually develop into the five cell types inresponse to precise spatial/temporal patterns of overlapping signal-ing gradients and involves several transcription factors (reviewed inrefs. 1–6). Pit1, a POU-homeodomain transcription factor, is thelineage regulator responsible for the generation of somatotropes,lactotropes, and thyrotropes (7). Pit1 expression is initiated byE13.5 and is maintained in adulthood, being directly involved in thetranscriptional control of the genes encoding growth hormone(GH), prolactin (Prl), and thyroid-stimulating hormone (TSH�)(8). Our previous studies revealed that 14.8 kb of 5�-flankingsequence of the Pit1 gene was sufficient to direct the robustexpression of a reporter in an identical spatial and temporal patternas endogenous Pit1, whereas its minimal promoter (�327 bp to �13bp) was insufficient to drive detectable reporter expression intransgenic mice (9). A distal enhancer (DE) located at �10.2 kbfrom Pit1 transcription start site and containing multiple functionalPit1-binding sites was shown to be involved in autoregulation (9).The 14.8 kb of 5�-flanking sequence minus the distal enhancercould still drive the activation of a reporter gene in both wt and

Pit1-defective Snell (dw/dw) mutant mice, demonstrating that theelements required for Pit1 early activation were present in the samegenomic region (10). Further mapping showed that the regionsrequired for the Pit1 early activation were located between �10 kband �3.5 kb (10). Thus, Pit1 expression appears to be under thecontrol of multiple enhancers, at least one for its activation and onefor its maintained expression. As such, Pit1 gene regulation appearsas an ideal system to study fine regulation of transcription via distalcis-acting regulatory regions.

Here, we present the in vivo characterization of one of Pit1early regulatory region/enhancers, referred to as EE�, exempli-fying the complex interenhancer functional interactions requiredfor initial developmental gene activation. A quantitative pro-teomics approach allowed us to identify factors capable ofinteracting, directly or indirectly, with this element and one ofthese factors, Atbf1, a transcription factor containing multiplezinc finger and homeodomain motifs, proved able to bind andactivate Pit1 EE� in vitro and in vivo. Analysis of Atbf1 gene-trapmutant mice demonstrated that there was a direct geneticinteraction between Pit1 and Atbf1, and that Atbf1 was requiredto achieve full initial activation of Pit1 gene, providing a linkagebetween activation of early Pit1 enhancer and subsequentPit1-dependent autoregulatory events.

Results and DiscussionMapping of EE�, a cis-Regulatory Element Required for Pit1 EarlyActivation. Previous studies suggested that the �10.2-kb/�5.1-kbsequence upstream Pit1 coding region could be sufficient to drivethe expression of a reporter in an identical spatial and temporalpattern as Pit1 (10). A computational approach revealed a highnumber of highly conserved regions with lengths varying from 50bp to 500 bp, providing few clues for the identification of specificdevelopmental regulatory regions and making it critical to use invivo enhancer mapping as the most reliable approach in search-ing for key cis-regulatory information. Analysis of a transgenecontaining the �10.2-kb/�5.1-kb region using human growthhormone (hGH) as reporter and the Pit1 promoter as minimalpromoter showed that this 5-kb region directed pituitary-specificexpression of a reporter gene (Fig. 1A). We then determinedwhich specific subregions might be responsible for early activa-tion. For each transgene studied, at least three integration eventswere generated, and the analysis was performed on the founderanimals taken at E14.5–E15.5; the reporter activation was mea-sured by in situ hybridization with a specific hGH probe. The

Author contributions: M.G.R. and C.C. designed research; Y.Q., J.A.R., X.Z., A.K., J.Z., R.A.,D.W.R., and C.C. performed research; Y.Q., J.A.R., R.A., D.W.R., and C.C. analyzed data; andY.Q. and C.C. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

¶To whom correspondence may be addressed. E-mail: mrosenfeld@ucsd.edu orcatherine.carriere@newvixen.dartmouth.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0712196105/DC1.

© 2008 by The National Academy of Sciences of the USA

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analysis of the embryos was performed 1 or 2 days after initialPit1 gene activation (E13.5) to preclude any technical problems,because transgene injection occasionally delays embryonic de-velopment by 1–2 days. The analysis of the first two overlappingconstructs, �8.5 kb/�5.1 kb and �10 kb/�6.7 kb, showed thatonly the �8.5-kb/�5-kb region drove strong reporter activationin the pituitary gland of the transgenic embryos (Fig. 1 A).Enhancing cis-elements appeared to be located in the �6.7-kb/�5.1-kb regions, suggesting that these 1.6 kb were sufficient forreporter activation. The 1.6-kb region was subcloned in twononoverlapping fragments of 900 bp and 700 bp that were usedfor transgenic analysis. Only the transgenic embryos that hadintegrated the 900-bp-containing (�6.7 kb/�5.7 kb) transgenedisplayed reporter expression (Fig. 1 A), demonstrating thatenhancing activity was located between �6.7 kb and �5.7 kb.

Further mapping was performed by generating systematic100-bp deletions in the context of the 900-bp fragment. Onlydeletion 8 failed to direct reporter expression in the pituitarygland of transgenic embryos (Fig. 1B). Interestingly, the trans-genes carrying the flanking deletions 7 and 9 displayed adecrease of activation, suggesting that the sequences surround-ing region 8 could be required for a high level of activation.Because deletion 9 overlapped with deletion 8 by 9 bp, wededuced that the 9 bp were not required. These results permittedus to define a 91-bp core region that would be required for Pit1early activation. Consistent with the 100-bp deletion analysis, the

91-bp element alone was not sufficient to drive detectablereporter expression in the pituitary gland (Fig. 1C). The additionof 50 bp on each side of the 91-bp core, however, was sufficientfor obtaining strong reporter activation. The defined 191-bpsequence contains sufficient information to permit its earlyactivation during pituitary development and as such will bereferred to as the EE� region (Fig. 1C).

To independently assess the role of EE� in early Pit1 activa-tion, we constructed a transgene containing the original 14.8 kbwith deletions of both the DE and EE�. No expression of thereporter could be detected in six out of seven transgenicembryos, and only a modest expression was observed in the finalfounder mouse (Fig. 1D). The fact that only a single embryodisplayed a modest reporter activation suggested that, in specificinsertional contexts, other regulatory regions might exert addi-tional effects. Although EE� is required for efficient early Pit1activation, other regulatory elements would be predicted tocontribute to the initial activation of Pit1 promoter. This is inagreement with our findings that another well conserved region,EE�, located at �8.3 kb, is also initially involved in Pit1 earlyactivation (11). Prop1, an early pituitary-specific transcriptionfactor binds to the EE� region, and its interaction with the�-catenin coactivator is required to initiate Pit1 gene transcrip-tion. Genetic analysis demonstrated that Prop1 and �-cateninwere required for Pit1 gene activation and the subsequentdifferentiation of Pit1-dependent cell lineages (11). Interest-ingly, a �10.2-kb/�6.7-kb transgene, which encompasses EE�alone, failed to display significant reporter activity when ana-lyzed in three integration events, which correlates the dataobtained with the 14.8-kb/�D�/�EE� transgene. These dataargue that, although it is required, EE� alone is not sufficient forrobust initial activation of the Pit1 gene, and both EE� and EE�are combinatorially required to achieve effective, full earlyactivation of the Pit1 transcription unit.

Multiple Sites Within EE� Contribute to Its Function. Sequencealignment of 5�-flanking genomic regions of the Pit1 gene betweenmultiple species showed that EE� was located in a highly conservedregion [supporting information (SI) Fig. 6]. To define requiredbinding sites, we inserted segmented mutations across the entirelength of the 91-bp core region in the context of the 191-bp minimalpromoter hGH reporter, replacing 5 to 10 bp at a time with anonrelevant sequence devoid of any biological activity (8) (Fig. 2A).Transgene analysis showed that only the mutations of sites 5a, 6, and7 caused dramatic decrease of reporter activity (Fig. 2A). Interest-ingly, none of the mutations completely abolished promoter acti-vation, and the three sites appeared to be critical in facilitating ahigh level of pituitary-gland-specific reporter expression. Thesedata were confirmed by the fact that a double mutation of sites 6and 7 was sufficient to completely abolish reporter expression (Fig.2A). That the three sites were adjacent suggested that they could actcooperatively in instructing EE� function.

Identification of ATBF1 as the Main EE� Binding Factor by QuantitativeProteomics. To identify factors that bind EE� and potentiallycontribute to regulating early Pit1 activation, we developed abiochemical approach using GHFT1 cells. These cells are de-rived from pituitary tumors that were induced by a transgeneexpressing the SV40 viral transforming protein large T undercontrol of the 14.8-kb Pit1 regulatory region (12). They expresslow levels of Pit1 but none of the hormone genes (e.g., GH andPRL), suggesting that GHFT1 cells may reflect an early stage inPit1 lineage.

We tested by transient transfection whether EE� wouldrespond in GHFT1 cells as it did in vivo. Wt EE� could drive aconsistent 3- to 5-fold activation of a Luciferase reporter whencompared with Pit1 minimal promoter alone, whereas mutationswithin EE� of sites 5a, 6, and 7 markedly reduced reporter

Fig. 1. In vivo enhancer mapping identifies a 200-bp region sufficient forinitial Pit1 gene activation. (A) Scheme of successive deletions used to map a900-bp enhancer region, located at �6.6/�5.7 kb sufficient for hGH expres-sion. (B) Systematic deletions done in the 900-bp region: Deletion 8 abolishedreporter expression, and deletions 7 and 9 mildly affect reporter expression.(C) The 91-bp core region alone was not sufficient to activate the Pit1 pro-moter, whereas the addition of 50 bp of 5� and 3� information was sufficientto drive high (hGH) expression. (D) The 14.8 kb of the Pit1 upstream sequences,carrying deletions of both DE and 191-bp ���, abolished hGH expression. Theexpression level of each transgene was determined by in situ hybridization byusing a hGH probe at E14.5.

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activity (SI Fig. 7). Mutations in other sites did not decreasereporter activity (SI Fig. 7). Interestingly, mutation of site 4increased the activation by 2-fold, which would not be detectedin the transgene studies because in situ hybridization is onlysemiquantitative (SI Fig. 7). Only mutation of site 5 behaveddifferently than it did in vivo by showing a decreased reporteractivity (data not shown). Despite these few quantitative dis-tinctions, EE� behaved similarly enough in GHFT1 cells incomparison with the in vivo data to permit us to use these cellsfor the biochemical purification of factors binding to EE� thatmight be potentially involved in early Pit1 gene activation.

To purify the EE�-binding proteins, we performed a single-step procedure of DNA affinity purification of interactingprotein complexes from GHFT1 nuclear extracts followed by aquantitative proteomic approach. Protein complexes were firstbound to biotinylated DNA probes containing specific or non-specific sequence. The specific probe was a trimer of thesequence encompassing the 5a, 6, and 7 sites plus 5 bp on eachend (Fig. 2B). The nonspecific probe was of the same length andcontained tandem arrays of the nonrelevant sequence used tomutate sites in EE�. The protein–DNA complexes were isolatedon streptavidin beads, and the bound factors were eluted andlabeled with cleavable cysteine-reactive ICAT (isotope-codedaffinity tag) reagents. Mass spectrometry allowed us to compare

the relative abundance of tryptic peptides derived from the twoprotein samples, permitting the identification of specific factorsagainst a high background of copurifying molecules (13). TheICAT analysis identified a large number of peptides with asignificant ratio between specific and nonspecific probes. A highnumber of peptides purified appeared to be transcription-relatedas part of basic transcriptional machinery, chromatin remodel-ing, or transcription factors (Fig. 2C). A smaller number wereinvolved in signal transduction and cell structure, and a numberof unknown molecules were present (data not shown). ByRT-PCR, we showed that several of the molecules characterizedin this biochemical screen displayed pituitary expression, con-firming the potential relevance of proteins identified by thisapproach (data not shown).

Of the numerous candidate proteins identified, only three werepresent in �2-fold higher abundance in the specific-versus-nonspecific sample, similar to the data obtained in another similarscreen (14). Atbf1 exhibited a 2.5-fold increase (Fig. 2C) and wasselected for further investigation, because it is a transcription factorthat might exert regulatory roles in development.

Atbf1 Is Expressed in the Developing Pituitary Gland. Atbf1 wasoriginally identified as a factor that binds the AT-rich element inthe human �-fetoprotein gene (AFP) enhancer (15). The Atbf1gene encodes two proteins of 306 and 404 kDa, respectively. Thepeptide characterized in the proteomic analysis corresponded tothe 404-kDa isoform that contains four homeodomains and 23zinc fingers (16). Atbf1 mRNA is highly expressed in thedeveloping brain (17). In the mouse, its expression peaks aroundE13–E15 and progressively decreases to an undetectable level bypostnatal day 28 (18). In the developing pituitary gland, Atbf1transcripts appeared around E11.5, peaked by E12.5–E13.5, anddiminished after E14.5 (Fig. 3A), which coincided with thetiming of Pit1 initial activation. Immunostaining experimentsconfirmed that Atbf1 protein was specifically expressed inpituitary cells, although at a lower level than in the brain (Fig.3B). Costaining of Pit1 and Atbf1 at E13.5 revealed that virtuallyall Pit1-expressing cells were Atbf1-positive. However, manyAtbf1-positive cells in dorsal-anterior and intermediate pituitarywere Pit1-negative, suggesting that Atbf1 was also present innon-Pit1 lineages.

Atbf1 Binds and Activates EE�. To confirm the in vivo binding ofAtbf1 to EE�, we carried out chromatin immunoprecipitation(ChIP) analysis first in GHFT1 cells. ChIP assays showed thatAtbf1 was present specifically on EE�, but not on Pit1 DE orminimal promoter, demonstrating that ATBF1 binds EE� in vivo(Fig. 3C). We then examined Atbf1 presence on Pit1 EE� duringpituitary development by performing ChIP analysis on micro-dissected embryonic E14.5 pituitary glands. The ChIP assaysrevealed that Atbf1 was present exclusively on EE�, as observedin GHFT1 cells (Fig. 3C). These results confirmed that Atbf1,despite being purified by an in vitro biochemical approach, wasactually present in vivo on the Pit1 EE� during pituitary devel-opment. A direct functional interaction between the Pit1 EE�and Atbf1 was demonstrated by single-cell nuclear microinjec-tion assays in GHFT1 cells. An EE�–LacZ reporter constructwas consistently activated when microinjected into GHFT1 cellsas compared with Pit1 minimal promoter construct (Fig. 3D).This activation was dramatically reduced by coinjection ofanti-Atbf1 IgG. As a control for functional specificity, anti-Atbf1IgG did not affect the basal activation of Pit1 minimal promoter(Fig. 3D). Therefore, Atbf1 appeared to be a main componentof Pit1 EE� activation. Moreover Atbf1 could directly activatean EE�–Luciferase reporter construct by 6-fold as shown incotransfection experiments in 293 cells, a heterologous cell line(Fig. 3E). In parallel transfection experiments, Atbf1 failed toactivate EE� reporter constructs carrying the mutations 5a, 6, or

Fig. 2. Characterization of functional sites within EE� and purification ofEE� binding factors by ICAT analysis. (A) Mutations replacing 5- to 10-bpsegments of the 91-bp core region in the context of 191-bp EE�: Mutations ofsites 5a, 6, or 7 severely decreased reporter expression. Double mutation ofsites 6 and 7 completely abolished hGH expression. (B) DNA sequence used forDNA affinity protein purification encompasses sites 5a, 6, and 7. (C) List of thetranscription-related factors isolated by quantitative proteomics approachwith the 5a/6/7 regions.

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7, whereas mutations on other sites of EE� did not appear tosignificantly alter the activation (Fig. 3E and data not shown).We concluded that Atbf1 was directly involved in EE� activationand that all three sites, 5a, 6, and 7, were required for Atbf1 tobe fully functional in regulating EE�. Because Atbf1 was orig-inally identified as a factor that binds to the AT motif of humanAFP (15), we assumed that Atbf1 would bind the AT-rich site 5a.However, given the complexity the protein, with 4 homeodo-mains and 23 zinc fingers, Atbf1 might well interact with multiplefactors on the EE� site, which would explain the requirement forsuch large binding site. Moreover, Atbf1 is expressed in asignificant number of tissues during development (19–21), it is

likely that it synergizes with other transcription factors toregulate a variety of developmental processes. Atbf1 also dis-plays DNA/RNA-dependent ATPase activity (15). We aretempted to speculate that the simultaneous binding of thesethree sites by Atbf1 might lead to its enzymatic activation,perhaps with a switch of protein complexes associated with EE�,leading to Pit1 activation.

Generation of Atbf1 Mutant Mice by Using Gene-Trap ES Cells. Toassess the genetic interaction between Atbf1 and Pit1, we gen-erated an Atbf1 mutant mouse line by using the Atbf1 gene-trapES cells (RRJ548, BayGenomics). Analysis of the 5� RACEsequence (BayGenomics) showed that the insertion occurred in60 kb of intronic region between the 3rd and 4th exons of theAtbf1 gene (Fig. 4A). This insertion should result in the expres-sion of a protein truncated at amino acid 963 that would containonly 5 zinc fingers instead of 23 and no homeodomains and thatmight be expected to be functionally impaired. To genotype theAtbf1 trap allele, dot blots of genomic DNA isolated fromembryos were hybridized with �-geo (present in the trap vector)and wt Atbf1 probes. The signal intensity of �-geo was thenmeasured and normalized by that of Atbf1 (Fig. 4B). Atbf1

Trap/Trap

embryos bearing two �-geo copies were easily differentiatedfrom Atbf1Trap/� embryos bearing only one �-geo copy. Thelevels of mutant and wt transcripts in trap mutants were deter-mined by quantitative PCR (q-PCR). Surprisingly, �10% oftotal Atbf1 transcripts were wt in Atbf1Trap/Trap embryos atE13.5, 13% at E15.5, and 20% at E17.5 (Fig. 4C and data notshown). These ratios of wt/mutant transcripts were consistentlyobserved among many litters of embryos at the same embryonicstages. Also, the truncated Atbf1 protein did not appear tofunction as a dominant-negative as heterozygotes exhibited noabnormalities when compared with wt. Furthermore, cotrans-fections of various fragments of Atbf1 with full-length Atbf1 andEE� reporter in 293 cells showed that none of the fragmentsincluding region of 1–963 could affect Atbf1 activation of EE�(data not shown). We thus believe that the Atbf1 gene-trapmutation generated in this study generated a hypomorphic alleleof Atbf1 where posttranscriptional splicing occasionally skippedthe gene trap exon, thus producing a small amount of the wtAtbf1 transcript.

Differentiation Defects in Pit1 Lineage in Atbf1 Gene-Trap MutantsDemonstrate That Atbf1 Is Epistatic to Pit1. Knowing that low levelsof wt Atbf1 transcripts were still present in Atbf1 mutant mice,we assessed Atbf1 protein expression in the pituitary gland byimmunostaining with an antibody raised against the C terminusof Atbf1 protein. At E13.5, Atbf1 was barely detectable inmutant pituitary glands (Fig. 4D). A few Atbf1-positive cellscould still be detected in the brain of mutant embryos, but theyhad a much weaker signal compared with wt embryos, inaccordance to the q-PCR data. These data confirmed a residualAtbf1 expression in the mutant pituitary gland.

Despite a significant decrease of Atbf1 transcripts, the pitu-itary gland of the mutant animals displayed no major morpho-logical defects during embryonic development (data not shown).Labeling with the proliferation marker Ki67 also showed asimilar number of proliferating cells in Atbf1 mutants and wtembryos at E13.5 (Fig. 4E). The analysis of factors involved inthe early stages of pituitary development such as Lhx3, Prop1,and Tbx19 (23) showed no alterations in expression in Atbf1mutant mice (Fig. 4E and data not shown). Altogether itsuggested that in Atbf1 mutant mice, initial pituitary organcommitment, cell proliferation, and early cells lineages specifi-cation were not altered. However, at E13.5, Pit1 mRNA levelswere markedly decreased in the mutant pituitary gland (Fig. 4D),

Fig. 3. Atbf1 is expressed in the developing pituitary gland and binds EE� invivo. (A) Atbf1 expression peaks at E12–E13 as shown by in situ hybridization.(B) Double immunostaining indicates that Pit1 (red) colocalizes with Atbf1(green). Note the high level of Atbf1 expression in the adjacent brain tissue.(C) ChIP assays showed that Atbf1 specifically binds to EE� in both GHFT1 cellsand E14.5 microdissected pituitaries. (D) Microinjection experiments in GHFT1cells show that the EE�//Pit1 minimal promoter drives the activation of a LacZreporter. Coinjection with anti-Atbf1 suppresses this activation. In contrast,coinjection of anti-Atbf1 did not affect the basal level of activation driven byonly the Pit1 minimal promoter. (E) Cotransfection of Luciferase reporterscontaining EE� (wt or mutants) in front of the Pit1 minimal promoter with anATBF1 expression vector in 293 cells showed that Atbf1 can activate EE�

reporters, except for those harboring mutations 5a, 6, and 7. Error barsindicate the SEM. *, P � 0.001, the statistically significant difference betweenthe control and anti-Atbf1 or Atbf1 expression plasmid (Student’s t test).

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and only a few Pit1-expressing cells could be detected in the mostventral pituitary (Fig. 4D), demonstrating that Pit1 expressionwas affected at the transcriptional level. We next examined thedifferentiation of Pit1-dependent cell types. At E17.5, GH, thesomatotrope marker was modestly decreased, whereas TSH-�,the thyrotrope marker, was almost absent in Atbf1 mutant mice(Fig. 4G). In contrast, the expression of POMC, marker ofcorticotropes and melanotropes, non-Pit1-dependent cell types,was not modified in Atbf1 mutant mice at E13.5 or E15.5 (Fig.4F). Together, these data demonstrate that Atbf1 is directlyrequired for early Pit1 early transcriptional activation.

Atbf1 has been reported to exert roles in control of cellproliferation (24). Although Atbf1 gene-trap mutants failed todisplay overt defect in cell proliferation in early pituitary devel-

opment, it remains possible that a trace amount of wt Atbf1protein may adequately function to regulate subsets of its targets,whereas Pit1 ��� would be particularly sensitive to Atbf1 levels.

Thus, to date, three major regulatory elements—EE�, EE�,and an autoregulatory enhancer (DE)—have now been identi-fied in the Pit1 5�-f lanking region. The high interspecies se-quence conservation in the �10-kb/�5-kb region strongly sug-gests that additional regulatory sites may exist. However, of thesethree regions, EE� alone is able to provide efficient earlyactivation of Pit1 transcription in vivo with only low levels ofindependent activation by other putative regulatory elements.Early Pit1 gene activation, therefore, appears to be subject tocombinatorial regulation involving no less than three differentgenomic regions, including EE�, EE�, and DE. These differentenhancers each appear to recruit specific combinations of DNA-binding transcription factors and cofactors that drive the dy-namic, highly regulated expression of Pit1 (Fig. 5), and they linkthe ‘‘giant zinc finger family’’ to developmental regulatoryevents.

Materials and MethodsGeneration and Analysis of Transgenic Animals. All Pit1 reporter transgenescontained Pit1 minimal promoter and hGH as reporter gene (10). A hGH probewas used to genotype the embryos by Southern blotting. Cloning informationis provided in SI Materials and Methods.

DNA Affinity Purification. DNA affinity purification is described in SI Materialsand Methods.

Quantitative Proteomics Analysis by ICAT. Proteins were labeled with cleavableICAT reagents and analyzed as described (13).

In Situ Hybridization and Immunolabeling. In situ hybridization and immu-nolabeling were done as described (10, 11). Probes are described in

Fig. 4. Atbf1 is required for the Pit1 gene early activation. (A) Genomicstructure of Atbf1 mouse gene and location of the gene-trap vector insertionat amino acid 963. It should generate a protein fusion of truncated Atbf1(1–962) and �-geo. (B) Quantification of the dot-blot analysis of the genomicDNA isolated from gene-trap embryos, hybridized with �-geo probe. (C) q-PCRanalysis of relative amounts of wt and trapped Atbf1 mRNA on total RNAextracted from the tissue of gene-trap mouse embryos showed residualpresence of wt Atbf1 mRNA. (D) Immunostaining of Atbf1 and Pit1 on adja-cent sections of E13.5 pituitary showed that both factors were greatly de-creased in Trap mutant, whereas DAPI staining indicated that the overallmorphology of mutant pituitary was intact. Pit1 RNA levels were also de-creased as shown by in situ hybridization. (E) Immunostaining of cell prolif-eration marker Ki67 and Lhx3 showed no difference between wt and mutantpituitary glands, suggesting that early pituitary development is intact. (F)Immunostaining of POMC showed similar expression level between wt andmutant. (G) Immunostaining of GH and TSH-� showed a dramatic decrease ofexpression in gene-trap mutants. Error bars indicate the SEM. *, P � 0.001, thestatistically significant difference between the genotype Trap/� and Trap/Trap or �/� (Student’s t test).

Fig. 5. A model of early activation of the Pit1 gene. By E13–E13.5, Prop1/�-catenin complexes are recruited to EE�, where, although they are absolutelyrequired, they activate transcription at a barely detectable level; at the samestage, Atbf1 is recruited to EE�, where it could potentially synergize withProp1 (red arrow), and the Pit1 gene is then highly activated. Once Pit1 isexpressed at sustained levels, Prop1/�-catenin and ATBF1 are no longer re-quired on their respective enhancers; in addition, by E17.5, Atbf1 and Prop1are down-regulated in the pituitary gland, and Pit1 expression dependsessentially on the DE.

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SI Materials and Methods. ATBF1 antibodies were a generous gift fromMakoto Kawaguchi (Niigata Rosai Hospital, Johetsu, Japan).

ChIP. ChIP assays were performed as described (25) except for the fixation ofthe tissue in 2% formaldehyde for 30 min. PCR primers are described in SIMaterials and Methods.

Transfections and Nuclear Microinjection. Transfection experiments were per-formed by using Superfect (Invitrogen) in 293 and GHFT1 cells. ATBF1 expres-sion vector was a gift from Yutaka Miura (Nagoya Citu University GraduateSchool of Medical Sciences, Nagoya, Japan).

Microinjection assays were carried out as described in ref. 26 and SI Mate-rials and Methods.

Isolation of RNA and q-PCR. Details are described in SI Materials andMethods.

ACKNOWLEDGMENTS. We thank M. Kawaguchi for the Atbf1A antibodiesand Y. Miura for the Atbf1a cDNA. We thank C. Nelson, F. Hooshman, and H.Taylor for their technical assistance. We thank J. Hightower and M. Fisher forart and manuscript preparation. Y.Q. was supported by the U.S. Army MedicalResearch & Materiel Command (Grant PC40247-W81XWH-05-1-0100). C.C. issupported by the National Institutes of Health (NIH)/National Cancer Institute(Grant CA 127095) and by a Junior Faculty Development Award from Dart-mouth Medical School. M.G.R. is an investigator with the Howard HughesMedical Institute. This work was supported by National Heart, Lung and BloodInstitute Grant NO1-HV-28179 (to J.A.R. and R.A.) and by NIH GrantsDK018477, DK39949, and NS34934 (to M.G.R.).

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