Atbf1 Is Required for the Pit1 Gene Early Activation
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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 activa- tion, 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 addi- tional 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 transcrip- tion. 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 ana- lyzed 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.