Atbf1 Is Required for the Pit1 Gene Early Activation

Atbf1 is required for the Pit1 gene early activation

Yingchuan Qi*, Jeffrey A. Ranish†, Xiaoyan Zhu*, Anna Krones*, Jie Zhang*, Ruedi Aebersold†‡, David W. Rose§, Michael G. Rosenfeld*¶, and Catherine Carrie` re¶ʈ

*Howard Hughes Medical Institute and Department of Medicine, School of Medicine, University of California at San Diego, La Jolla, CA 92093; †Institute for Systems Biology, 1441 North 34th Street, Seattle, WA 98103; ‡Institute of Molecular Systems Biology, Swiss Federal Institute of Technology (ETH) Ho¨nggerberg and Faculty of Sciences, University of Zu¨rich, CH-8093 Zu¨rich, Switzerland; §Department of Medicine, Division of Endocrinology and Metabolism, School of Medicine, University of California at San Diego, La Jolla, CA 92093; and ʈDepartment 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 the Pit1-defective Snell (dw/dw) mutant mice, demonstrating that the molecular principles underlying the integration of regulatory in- elements required for Pit1 early activation were present in the same puts to alternate gene enhancers used during mammalian orga- genomic region (10). Further mapping showed that the regions nogenesis remain incompletely understood. Using a combination required for the Pit1 early activation were located between Ϫ10 kb of in vivo enhancer mapping and proteomics approaches, we have and Ϫ3.5 kb (10). Thus, Pit1 expression appears to be under the established that two distant and distinct early enhancers, each control of multiple enhancers, at least one for its activation and one requiring different transcription complexes, are required for full for its maintained expression. As such, Pit1 gene regulation appears activation of the gene encoding the pituitary lineage determining as an ideal system to study fine regulation of transcription via distal factor, Pit1. A transcription factor belonging to the ‘‘giant, multi- cis-acting regulatory regions. ple-homeodomain and zinc ﬁnger family,’’ Atbf1, serves as a novel Here, we present the in vivo characterization of one of Pit1 pituitary regulator for one of the two required enhancers as shown early regulatory region/enhancers, referred to as EE␣, exempli- by genetic and in vitro analysis. fying the complex interenhancer functional interactions required for initial developmental gene activation. A quantitative pro- he essence of embryonic development is to generate a teomics approach allowed us to identify factors capable of T regulated increase in cell number and diversity, with the interacting, directly or indirectly, with this element and one of identity of each cell type dictated by specific patterns of signals these factors, Atbf1, a transcription factor containing multiple and expression of sets of genes Thus, a hierarchy of regulatory zinc finger and homeodomain motifs, proved able to bind and ␣ factors controls activation or repression of such genes with activate Pit1 EE in vitro and in vivo. Analysis of Atbf1 gene-trap spatial/temporal precision, and such control is central to mutant mice demonstrated that there was a direct genetic accurately unfolding genetic information to create precise interaction between Pit1 and Atbf1, and that Atbf1 was required patterns of developmental complexity. It involves evolution- to achieve full initial activation of Pit1 gene, providing a linkage arily conserved cis-regulatory sequences that are highly struc- between activation of early Pit1 enhancer and subsequent tured and organized to recruit sets of transcription activators/ Pit1-dependent autoregulatory events. repressors. These transcription complexes will determine the Results and Discussion rate and frequency of transcription initiation, generating fine ␣ control of gene expression in development, homeostasis, and Mapping of EE ,acis-Regulatory Element Required for Pit1 Early Ϫ Ϫ disease. Activation. Previous studies suggested that the 10.2-kb/ 5.1-kb The anterior pituitary gland provides an excellent model system sequence upstream Pit1 coding region could be sufficient to drive to analyze molecular programs governing cell-specific gene regu- the expression of a reporter in an identical spatial and temporal lation during embryonic development. The mature pituitary gland pattern as Pit1 (10). A computational approach revealed a high contains five distinct hormone-producing cell types: corticotropes, number of highly conserved regions with lengths varying from 50 somatotropes, lactotropes, thyrotropes, and gonadotropes. All of bp to 500 bp, providing few clues for the identification of specific these cell types arise from a common primordium, the Rathke’s developmental regulatory regions and making it critical to use in pouch, which initially develops by mouse embryonic day 9 (E9) as vivo enhancer mapping as the most reliable approach in search- a single layer of epithelium; it undergoes a fast expansion attrib- ing for key cis-regulatory information. Analysis of a transgene Ϫ Ϫ utable to intense cell proliferation and gives rise to a series of cell containing the 10.2-kb/ 5.1-kb region using human growth lineages, which eventually develop into the five cell types in hormone (hGH) as reporter and the Pit1 promoter as minimal response to precise spatial/temporal patterns of overlapping signal- promoter showed that this 5-kb region directed pituitary-specific BIOLOGY

ing gradients and involves several transcription factors (reviewed in expression of a reporter gene (Fig. 1A). We then determined DEVELOPMENTAL refs. 1–6). Pit1, a POU-homeodomain transcription factor, is the which specific subregions might be responsible for early activa- lineage regulator responsible for the generation of somatotropes, tion. For each transgene studied, at least three integration events lactotropes, and thyrotropes (7). Pit1 expression is initiated by were generated, and the analysis was performed on the founder E13.5 and is maintained in adulthood, being directly involved in the animals taken at E14.5–E15.5; the reporter activation was mea- transcriptional control of the genes encoding growth hormone sured by in situ hybridization with a specific hGH probe. The (GH), prolactin (Prl), and thyroid-stimulating hormone (TSH␤) (8). Our previous studies revealed that 14.8 kb of 5Ј-flanking Author contributions: M.G.R. and C.C. designed research; Y.Q., J.A.R., X.Z., A.K., J.Z., R.A., sequence of the Pit1 gene was sufficient to direct the robust D.W.R., and C.C. performed research; Y.Q., J.A.R., R.A., D.W.R., and C.C. analyzed data; and expression of a reporter in an identical spatial and temporal pattern Y.Q. and C.C. wrote the paper. as endogenous Pit1, whereas its minimal promoter (Ϫ327 bp to ϩ13 The authors declare no conﬂict of interest. bp) was insufficient to drive detectable reporter expression in Freely available online through the PNAS open access option. Ϫ transgenic mice (9). A distal enhancer (DE) located at 10.2 kb ¶To whom correspondence may be addressed. E-mail: [email protected] or from Pit1 transcription start site and containing multiple functional [email protected]. Pit1-binding sites was shown to be involved in autoregulation (9). This article contains supporting information online at www.pnas.org/cgi/content/full/ The 14.8 kb of 5Ј-flanking sequence minus the distal enhancer 0712196105/DC1. could still drive the activation of a reporter gene in both wt and © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712196105 PNAS ͉ February 19, 2008 ͉ vol. 105 ͉ no. 7 ͉ 2481–2486 Downloaded by guest on September 26, 2021 91-bp element alone was not sufficient to drive detectable reporter expression in the pituitary gland (Fig. 1C). The addition of 50 bp on each side of the 91-bp core, however, was sufficient for obtaining strong reporter activation. The defined 191-bp sequence contains sufficient information to permit its early activation during pituitary development and as such will be referred to as the EE␣ region (Fig. 1C). To independently assess the role of EE␣ in early Pit1 activation, we constructed a transgene containing the original 14.8 kb with deletions of both the DE and EE␣. No expression of the reporter could be detected in six out of seven transgenic embryos, and only a modest expression was observed in the final founder mouse (Fig. 1D). The fact that only a single embryo displayed a modest reporter activation suggested that, in specific insertional contexts, other regulatory regions might exert additional effects. Although EE␣ is required for efficient early Pit1 activation, other regulatory elements would be predicted to contribute to the initial activation of Pit1 promoter. This is in agreement with our findings that another well conserved region, EE␤, located at Ϫ8.3 kb, is also initially involved in Pit1 early activation (11). Prop1, an early pituitary-specific transcription factor binds to the EE␤ region, and its interaction with the ␤-catenin coactivator is required to initiate Pit1 gene transcription. Genetic analysis demonstrated that Prop1 and ␤-catenin were required for Pit1 gene activation and the subsequent differentiation of Pit1-dependent cell lineages (11). Interest- ingly, a Ϫ10.2-kb/Ϫ6.7-kb transgene, which encompasses EE␤ alone, failed to display significant reporter activity when analyzed in three integration events, which correlates the data obtained with the 14.8-kb/⌬D⌭/⌬EE␣ transgene. These data Fig. 1. In vivo enhancer mapping identifies a 200-bp region sufficient for argue that, although it is required, EE␤ alone is not sufficient for initial Pit1 gene activation. (A) Scheme of successive deletions used to map a ␤ ␣ 900-bp enhancer region, located at Ϫ6.6/Ϫ5.7 kb sufficient for hGH expres- robust initial activation of the Pit1 gene, and both EE and EE sion. (B) Systematic deletions done in the 900-bp region: Deletion 8 abolished are combinatorially required to achieve effective, full early reporter expression, and deletions 7 and 9 mildly affect reporter expression. activation of the Pit1 transcription unit. (C) The 91-bp core region alone was not sufficient to activate the Pit1 promoter, whereas the addition of 50 bp of 5Ј and 3Ј information was sufficient Multiple Sites Within EE␣ Contribute to Its Function. Sequence to drive high (hGH) expression. (D) The 14.8 kb of the Pit1 upstream sequences, alignment of 5Ј-flanking genomic regions of the Pit1 gene between carrying deletions of both DE and 191-bp ⌭⌭␣, abolished hGH expression. The multiple species showed that EE␣ was located in a highly conserved expression level of each transgene was determined by in situ hybridization by region [supporting information (SI) Fig. 6]. To define required using a hGH probe at E14.5. binding sites, we inserted segmented mutations across the entire length of the 91-bp core region in the context of the 191-bp minimal analysis of the embryos was performed 1 or 2 days after initial promoter hGH reporter, replacing 5 to 10 bp at a time with a Pit1 gene activation (E13.5) to preclude any technical problems, nonrelevant sequence devoid of any biological activity (8) (Fig. 2A). because transgene injection occasionally delays embryonic de- Transgene analysis showed that only the mutations of sites 5a, 6, and velopment by 1–2 days. The analysis of the first two overlapping 7 caused dramatic decrease of reporter activity (Fig. 2A). Interest- constructs, Ϫ8.5 kb/Ϫ5.1 kb and Ϫ10 kb/Ϫ6.7 kb, showed that ingly, none of the mutations completely abolished promoter acti- only the Ϫ8.5-kb/Ϫ5-kb region drove strong reporter activation vation, and the three sites appeared to be critical in facilitating a in the pituitary gland of the transgenic embryos (Fig. 1A). high level of pituitary-gland-specific reporter expression. These data were confirmed by the fact that a double mutation of sites 6 Enhancing cis-elements appeared to be located in the Ϫ6.7-kb/ and 7 was sufficient to completely abolish reporter expression (Fig. Ϫ5.1-kb regions, suggesting that these 1.6 kb were sufficient for 2A). That the three sites were adjacent suggested that they could act reporter activation. The 1.6-kb region was subcloned in two cooperatively in instructing EE␣ function. nonoverlapping fragments of 900 bp and 700 bp that were used for transgenic analysis. Only the transgenic embryos that had ␣ Ϫ Ϫ Identification of ATBF1 as the Main EE Binding Factor by Quantitative integrated the 900-bp-containing ( 6.7 kb/ 5.7 kb) transgene Proteomics. To identify factors that bind EE␣ and potentially displayed reporter expression (Fig. 1A), demonstrating that contribute to regulating early Pit1 activation, we developed a Ϫ Ϫ enhancing activity was located between 6.7 kb and 5.7 kb. biochemical approach using GHFT1 cells. These cells are de- Further mapping was performed by generating systematic rived from pituitary tumors that were induced by a transgene 100-bp deletions in the context of the 900-bp fragment. Only expressing the SV40 viral transforming protein large T under deletion 8 failed to direct reporter expression in the pituitary control of the 14.8-kb Pit1 regulatory region (12). They express gland of transgenic embryos (Fig. 1B). Interestingly, the trans- low levels of Pit1 but none of the hormone genes (e.g., GH and genes carrying the flanking deletions 7 and 9 displayed a PRL), suggesting that GHFT1 cells may reflect an early stage in decrease of activation, suggesting that the sequences surround- Pit1 lineage. ing region 8 could be required for a high level of activation. We tested by transient transfection whether EE␣ would Because deletion 9 overlapped with deletion 8 by 9 bp, we respond in GHFT1 cells as it did in vivo.WtEE␣ could drive a deduced that the 9 bp were not required. These results permitted consistent 3- to 5-fold activation of a Luciferase reporter when us to define a 91-bp core region that would be required for Pit1 compared with Pit1 minimal promoter alone, whereas mutations early activation. Consistent with the 100-bp deletion analysis, the within EE␣ of sites 5a, 6, and 7 markedly reduced reporter

2482 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712196105 Qi et al. Downloaded by guest on September 26, 2021 the relative abundance of tryptic peptides derived from the two protein samples, permitting the identification of specific factors against a high background of copurifying molecules (13). The ICAT analysis identified a large number of peptides with a significant ratio between specific and nonspecific probes. A high number of peptides purified appeared to be transcription-related as part of basic transcriptional machinery, chromatin remodel- ing, or transcription factors (Fig. 2C). A smaller number were involved in signal transduction and cell structure, and a number of unknown molecules were present (data not shown). By RT-PCR, we showed that several of the molecules characterized in this biochemical screen displayed pituitary expression, con- firming the potential relevance of proteins identified by this approach (data not shown). Of the numerous candidate proteins identified, only three were present in Ͼ2-fold higher abundance in the specific-versus- nonspecific sample, similar to the data obtained in another similar screen (14). Atbf1 exhibited a 2.5-fold increase (Fig. 2C) and was selected for further investigation, because it is a transcription factor that might exert regulatory roles in development.

Atbf1 Is Expressed in the Developing Pituitary Gland. Atbf1 was originally identified as a factor that binds the AT-rich element in the human ␣-fetoprotein gene (AFP) enhancer (15). The Atbf1 gene encodes two proteins of 306 and 404 kDa, respectively. The peptide characterized in the proteomic analysis corresponded to the 404-kDa isoform that contains four homeodomains and 23 zinc fingers (16). Atbf1 mRNA is highly expressed in the developing brain (17). In the mouse, its expression peaks around E13–E15 and progressively decreases to an undetectable level by postnatal day 28 (18). In the developing pituitary gland, Atbf1 transcripts appeared around E11.5, peaked by E12.5–E13.5, and diminished after E14.5 (Fig. 3A), which coincided with the timing of Pit1 initial activation. Immunostaining experiments confirmed that Atbf1 protein was specifically expressed in Fig. 2. Characterization of functional sites within EE␣ and purification of pituitary cells, although at a lower level than in the brain (Fig. ␣ EE binding factors by ICAT analysis. (A) Mutations replacing 5- to 10-bp 3B). Costaining of Pit1 and Atbf1 at E13.5 revealed that virtually segments of the 91-bp core region in the context of 191-bp EE␣: Mutations of sites 5a, 6, or 7 severely decreased reporter expression. Double mutation of all Pit1-expressing cells were Atbf1-positive. However, many sites 6 and 7 completely abolished hGH expression. (B) DNA sequence used for Atbf1-positive cells in dorsal-anterior and intermediate pituitary DNA affinity protein purification encompasses sites 5a, 6, and 7. (C) List of the were Pit1-negative, suggesting that Atbf1 was also present in transcription-related factors isolated by quantitative proteomics approach non-Pit1 lineages. with the 5a/6/7 regions. Atbf1 Binds and Activates EE␣. To confirm the in vivo binding of Atbf1 to EE␣, we carried out chromatin immunoprecipitation activity (SI Fig. 7). Mutations in other sites did not decrease (ChIP) analysis first in GHFT1 cells. ChIP assays showed that reporter activity (SI Fig. 7). Interestingly, mutation of site 4 Atbf1 was present specifically on EE␣, but not on Pit1 DE or increased the activation by 2-fold, which would not be detected minimal promoter, demonstrating that ATBF1 binds EE␣ in vivo in the transgene studies because in situ hybridization is only (Fig. 3C). We then examined Atbf1 presence on Pit1 EE␣ during semiquantitative (SI Fig. 7). Only mutation of site 5 behaved pituitary development by performing ChIP analysis on micro- differently than it did in vivo by showing a decreased reporter dissected embryonic E14.5 pituitary glands. The ChIP assays activity (data not shown). Despite these few quantitative dis- revealed that Atbf1 was present exclusively on EE␣, as observed BIOLOGY tinctions, EE␣ behaved similarly enough in GHFT1 cells in in GHFT1 cells (Fig. 3C). These results confirmed that Atbf1, DEVELOPMENTAL comparison with the in vivo data to permit us to use these cells despite being purified by an in vitro biochemical approach, was for the biochemical purification of factors binding to EE␣ that actually present in vivo on the Pit1 EE␣ during pituitary devel- might be potentially involved in early Pit1 gene activation. opment. A direct functional interaction between the Pit1 EE␣ To purify the EE␣-binding proteins, we performed a single- and Atbf1 was demonstrated by single-cell nuclear microinjec- step procedure of DNA affinity purification of interacting tion assays in GHFT1 cells. An EE␣–LacZ reporter construct protein complexes from GHFT1 nuclear extracts followed by a was consistently activated when microinjected into GHFT1 cells quantitative proteomic approach. Protein complexes were first as compared with Pit1 minimal promoter construct (Fig. 3D). bound to biotinylated DNA probes containing specific or non- This activation was dramatically reduced by coinjection of specific sequence. The specific probe was a trimer of the anti-Atbf1 IgG. As a control for functional specificity, anti-Atbf1 sequence encompassing the 5a, 6, and 7 sites plus 5 bp on each IgG did not affect the basal activation of Pit1 minimal promoter end (Fig. 2B). The nonspecific probe was of the same length and (Fig. 3D). Therefore, Atbf1 appeared to be a main component contained tandem arrays of the nonrelevant sequence used to of Pit1 EE␣ activation. Moreover Atbf1 could directly activate mutate sites in EE␣. The protein–DNA complexes were isolated an EE␣–Luciferase reporter construct by 6-fold as shown in on streptavidin beads, and the bound factors were eluted and cotransfection experiments in 293 cells, a heterologous cell line labeled with cleavable cysteine-reactive ICAT (isotope-coded (Fig. 3E). In parallel transfection experiments, Atbf1 failed to affinity tag) reagents. Mass spectrometry allowed us to compare activate EE␣ reporter constructs carrying the mutations 5a, 6, or

Qi et al. PNAS ͉ February 19, 2008 ͉ vol. 105 ͉ no. 7 ͉ 2483 Downloaded by guest on September 26, 2021 likely that it synergizes with other transcription factors to regulate a variety of developmental processes. Atbf1 also dis- plays DNA/RNA-dependent ATPase activity (15). We are tempted to speculate that the simultaneous binding of these three 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. To assess the genetic interaction between Atbf1 and Pit1, we generated an Atbf1 mutant mouse line by using the Atbf1 gene-trap ES cells (RRJ548, BayGenomics). Analysis of the 5Ј RACE sequence (BayGenomics) showed that the insertion occurred in 60 kb of intronic region between the 3rd and 4th exons of the Atbf1 gene (Fig. 4A). This insertion should result in the expression of a protein truncated at amino acid 963 that would contain only 5 zinc fingers instead of 23 and no homeodomains and that might be expected to be functionally impaired. To genotype the Atbf1 trap allele, dot blots of genomic DNA isolated from embryos were hybridized with ␤-geo (present in the trap vector) and wt Atbf1 probes. The signal intensity of ␤-geo was then measured and normalized by that of Atbf1 (Fig. 4B). Atbf1Trap/Trap embryos bearing two ␤-geo copies were easily differentiated from Atbf1Trap/ϩ embryos bearing only one ␤-geo copy. The levels of mutant and wt transcripts in trap mutants were determined by quantitative PCR (q-PCR). Surprisingly, Ϸ10% of total Atbf1 transcripts were wt in Atbf1Trap/Trap embryos at E13.5, 13% at E15.5, and 20% at E17.5 (Fig. 4C and data not shown). These ratios of wt/mutant transcripts were consistently observed among many litters of embryos at the same embryonic stages. Also, the truncated Atbf1 protein did not appear to function as a dominant-negative as heterozygotes exhibited no abnormalities when compared with wt. Furthermore, cotrans- fections of various fragments of Atbf1 with full-length Atbf1 and EE␣ reporter in 293 cells showed that none of the fragments including region of 1–963 could affect Atbf1 activation of EE␣ (data not shown). We thus believe that the Atbf1 gene-trap mutation generated in this study generated a hypomorphic allele of Atbf1 where posttranscriptional splicing occasionally skipped Fig. 3. Atbf1 is expressed in the developing pituitary gland and binds EE␣ in the gene trap exon, thus producing a small amount of the wt vivo.(A) Atbf1 expression peaks at E12–E13 as shown by in situ hybridization. Atbf1 transcript. (B) Double immunostaining indicates that Pit1 (red) colocalizes with Atbf1 (green). Note the high level of Atbf1 expression in the adjacent brain tissue. Differentiation Defects in Pit1 Lineage in Atbf1 Gene-Trap Mutants (C) ChIP assays showed that Atbf1 speciﬁcally binds to EE␣ in both GHFT1 cells Demonstrate That Atbf1 Is Epistatic to Pit1. Knowing that low levels and E14.5 microdissected pituitaries. (D) Microinjection experiments in GHFT1 ␣ of wt Atbf1 transcripts were still present in Atbf1 mutant mice, cells show that the EE //Pit1 minimal promoter drives the activation of a LacZ we assessed Atbf1 protein expression in the pituitary gland by reporter. Coinjection with anti-Atbf1 suppresses this activation. In contrast, coinjection of anti-Atbf1 did not affect the basal level of activation driven by immunostaining with an antibody raised against the C terminus only the Pit1 minimal promoter. (E) Cotransfection of Luciferase reporters of Atbf1 protein. At E13.5, Atbf1 was barely detectable in containing EE␣ (wt or mutants) in front of the Pit1 minimal promoter with an mutant pituitary glands (Fig. 4D). A few Atbf1-positive cells ATBF1 expression vector in 293 cells showed that Atbf1 can activate EE␣ could still be detected in the brain of mutant embryos, but they reporters, except for those harboring mutations 5a, 6, and 7. Error bars had a much weaker signal compared with wt embryos, in Ͻ indicate the SEM. *, P 0.001, the statistically signiﬁcant difference between accordance to the q-PCR data. These data confirmed a residual the control and anti-Atbf1 or Atbf1 expression plasmid (Student’s t test). Atbf1 expression in the mutant pituitary gland. Despite a significant decrease of Atbf1 transcripts, the pitu- 7, whereas mutations on other sites of EE␣ did not appear to itary gland of the mutant animals displayed no major morpho- significantly alter the activation (Fig. 3E and data not shown). logical defects during embryonic development (data not shown). We concluded that Atbf1 was directly involved in EE␣ activation Labeling with the proliferation marker Ki67 also showed a and that all three sites, 5a, 6, and 7, were required for Atbf1 to similar number of proliferating cells in Atbf1 mutants and wt be fully functional in regulating EE␣. Because Atbf1 was orig- embryos at E13.5 (Fig. 4E). The analysis of factors involved in inally identified as a factor that binds to the AT motif of human the early stages of pituitary development such as Lhx3, Prop1, AFP (15), we assumed that Atbf1 would bind the AT-rich site 5a. and Tbx19 (23) showed no alterations in expression in Atbf1 However, given the complexity the protein, with 4 homeodo- mutant mice (Fig. 4E and data not shown). Altogether it mains and 23 zinc fingers, Atbf1 might well interact with multiple suggested that in Atbf1 mutant mice, initial pituitary organ factors on the EE␣ site, which would explain the requirement for commitment, cell proliferation, and early cells lineages specifi- such large binding site. Moreover, Atbf1 is expressed in a cation were not altered. However, at E13.5, Pit1 mRNA levels significant number of tissues during development (19–21), it is were markedly decreased in the mutant pituitary gland (Fig. 4D),

2484 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712196105 Qi et al. Downloaded by guest on September 26, 2021 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 absolutely required, they activate transcription at a barely detectable level; at the same stage, Atbf1 is recruited to EE␣, where it could potentially synergize with Prop1 (red arrow), and the Pit1 gene is then highly activated. Once Pit1 is expressed at sustained levels, Prop1/␤-catenin and ATBF1 are no longer required on their respective enhancers; in addition, by E17.5, Atbf1 and Prop1 are down-regulated in the pituitary gland, and Pit1 expression depends essentially on the DE.

opment, it remains possible that a trace amount of wt Atbf1 Fig. 4. Atbf1 is required for the Pit1 gene early activation. (A) Genomic protein may adequately function to regulate subsets of its targets, structure of Atbf1 mouse gene and location of the gene-trap vector insertion whereas Pit1 ⌭⌭␣ would be particularly sensitive to Atbf1 levels. at amino acid 963. It should generate a protein fusion of truncated Atbf1 Thus, to date, three major regulatory elements—EE␣,EE␤, ␤ (1–962) and -geo. (B) Quantiﬁcation of the dot-blot analysis of the genomic and an autoregulatory enhancer (DE)—have now been identi- DNA isolated from gene-trap embryos, hybridized with ␤-geo probe. (C) q-PCR fied in the Pit1 5Ј-flanking region. The high interspecies se- analysis of relative amounts of wt and trapped Atbf1 mRNA on total RNA Ϫ Ϫ extracted from the tissue of gene-trap mouse embryos showed residual quence conservation in the 10-kb/ 5-kb region strongly sug- presence of wt Atbf1 mRNA. (D) Immunostaining of Atbf1 and Pit1 on adja- gests that additional regulatory sites may exist. However, of these cent sections of E13.5 pituitary showed that both factors were greatly de- three regions, EE␣ alone is able to provide efficient early creased in Trap mutant, whereas DAPI staining indicated that the overall activation of Pit1 transcription in vivo with only low levels of morphology of mutant pituitary was intact. Pit1 RNA levels were also de- independent activation by other putative regulatory elements. creased as shown by in situ hybridization. (E) Immunostaining of cell proliferation marker Ki67 and Lhx3 showed no difference between wt and mutant Early Pit1 gene activation, therefore, appears to be subject to pituitary glands, suggesting that early pituitary development is intact. (F) combinatorial regulation involving no less than three different Immunostaining of POMC showed similar expression level between wt and genomic regions, including EE␣,EE␤, and DE. These different mutant. (G) Immunostaining of GH and TSH-␤ showed a dramatic decrease of enhancers each appear to recruit specific combinations of DNA- expression in gene-trap mutants. Error bars indicate the SEM. , P Ͻ 0.001, the * binding transcription factors and cofactors that drive the dy- BIOLOGY statistically signiﬁcant difference between the genotype Trap/ϩ and Trap/

namic, highly regulated expression of Pit1 (Fig. 5), and they link DEVELOPMENTAL Trap or ϩ/ϩ (Student’s t test). the ‘‘giant zinc finger family’’ to developmental regulatory events. and only a few Pit1-expressing cells could be detected in the most ventral pituitary (Fig. 4D), demonstrating that Pit1 expression Materials and Methods was affected at the transcriptional level. We next examined the Generation and Analysis of Transgenic Animals. All Pit1 reporter transgenes differentiation of Pit1-dependent cell types. At E17.5, GH, the contained Pit1 minimal promoter and hGH as reporter gene (10). A hGH probe somatotrope marker was modestly decreased, whereas TSH-␤, was used to genotype the embryos by Southern blotting. Cloning information is provided in SI Materials and Methods. the thyrotrope marker, was almost absent in Atbf1 mutant mice (Fig. 4G). In contrast, the expression of POMC, marker of DNA Affinity Purification. DNA afﬁnity puriﬁcation is described in SI Materials corticotropes and melanotropes, non-Pit1-dependent cell types, and Methods. was not modified in Atbf1 mutant mice at E13.5 or E15.5 (Fig. 4F). Together, these data demonstrate that Atbf1 is directly Quantitative Proteomics Analysis by ICAT. Proteins were labeled with cleavable required for early Pit1 early transcriptional activation. ICAT reagents and analyzed as described (13). Atbf1 has been reported to exert roles in control of cell proliferation (24). Although Atbf1 gene-trap mutants failed to In Situ Hybridization and Immunolabeling. In situ hybridization and immu- display overt defect in cell proliferation in early pituitary devel- nolabeling were done as described (10, 11). Probes are described in

Qi et al. PNAS ͉ February 19, 2008 ͉ vol. 105 ͉ no. 7 ͉ 2485 Downloaded by guest on September 26, 2021 SI Materials and Methods. ATBF1 antibodies were a generous gift from Isolation of RNA and q-PCR. Details are described in SI Materials and Makoto Kawaguchi (Niigata Rosai Hospital, Johetsu, Japan). Methods.

ChIP. ChIP assays were performed as described (25) except for the ﬁxation of ACKNOWLEDGMENTS. We thank M. Kawaguchi for the Atbf1A antibodies the tissue in 2% formaldehyde for 30 min. PCR primers are described in SI and Y. Miura for the Atbf1a cDNA. We thank C. Nelson, F. Hooshman, and H. Materials and Methods. Taylor for their technical assistance. We thank J. Hightower and M. Fisher for art and manuscript preparation. Y.Q. was supported by the U.S. Army Medical Research & Materiel Command (Grant PC40247-W81XWH-05-1-0100). C.C. is Transfections and Nuclear Microinjection. Transfection experiments were per- supported by the National Institutes of Health (NIH)/National Cancer Institute formed by using Superfect (Invitrogen) in 293 and GHFT1 cells. ATBF1 expres- (Grant CA 127095) and by a Junior Faculty Development Award from Dart- sion vector was a gift from Yutaka Miura (Nagoya Citu University Graduate mouth Medical School. M.G.R. is an investigator with the Howard Hughes School of Medical Sciences, Nagoya, Japan). Medical Institute. This work was supported by National Heart, Lung and Blood Microinjection assays were carried out as described in ref. 26 and SI Mate- Institute Grant NO1-HV-28179 (to J.A.R. and R.A.) and by NIH Grants rials and Methods. DK018477, DK39949, and NS34934 (to M.G.R.).

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