Precise control of transcriptional programmes underlying metazoan development is modulated by enzymatically active co-regulatory complexes, coupled with epigenetic strategies. One thing that remains unclear is how specific members of histone modification enzyme families, such as histone methyltransferases and demethylases, are used in vivo to simultaneously orchestrate distinct developmental gene activation and repression programmes. Here, we report that the histone lysine demethylase, LSD1--a component of the CoREST-CtBP co-repressor complex--is required for late cell-lineage determination and differentiation during pituitary organogenesis. LSD1 seems to act primarily on target gene activation programmes, as well as in gene repression programmes, on the basis of recruitment of distinct LSD1-containing co-activator or co-repressor complexes. LSD1-dependent gene repression programmes can be extended late in development with the induced expression of ZEB1, a Krüppel-like repressor that can act as a molecular beacon for recruitment of the LSD1-containing CoREST-CtBP co-repressor complex, causing repression of an additional cohort of genes, such as Gh, which previously required LSD1 for activation. These findings suggest that temporal patterns of expression of specific components of LSD1 complexes modulate gene regulatory programmes in many mammalian organs.

During mammalian pituitary gland development, distinct cell types emerge from a common primordium. Appearance of specific cell types occurs in response to opposing signaling gradients that emanate from distinct organizing centers. These signals induce expression of interacting transcriptional regulators, including DNA binding-dependent activators and DNA binding-independent transrepressors, in temporally and spatially overlapping patterns. Together they synergistically regulate precursor proliferation and induction of distinct cell types. Terminal cell type differentiation requires selective gene activation strategies and long-term active repression, mediated by cell type-specific and promoter-specific recruitment of coregulatory complexes. These mechanisms imply the potential for flexibility in the ultimate identity of differentiated cell types.

A pituitary LIM homeodomain factor, PLim, is expressed as Rathke's pouch forms and as specific pituitary cell phenotypes are established, suggesting functional roles throughout pituitary development. While selectively expressed in Pituitary organ commitment appears to occur shortly after a region of the somatic ectoderm makes direct contact with neuroectodermal cells in an area of mesenchymal incompetence (1, 2). Subsequently, five distinct cell types, each characterized by the expression of a unique hormone, appear in a spatially and temporally specific fashion (reviewed in refs. 3 and 4). The pituitary-specific POU-domain transcription factor Pit-1 (3, 4) is selectively activated in the caudomedial part of the nascent gland at embryonic day 15.5 (e15.5) in the mouse and is required for activation of the prolactin (Prl), growth hormone (GH), and thyroid-stimulating hormone }3-subunit (TSHI3) genes in this region, as well as for somatotrope, lactotrope, and thyrotrope cell proliferation (5-7). Additional activating factors work with Pit-1 to achieve cellspecific gene activation (8)(9)(10)(11)(12)(13)(14)(15).A second family of homeodomain transcription factors, initially defined by RNA. These RT-PCR-generated DNA fragments were used to screen a mouse pituitary cDNA library and a mouse genomic library. RNase protection assays were performed with an intron 3-containing cDNA clone (no. 11) as template for T7RNA polymerase-directed synthesis of a radiolabeled antisense probe. In situ hybridization of 20-,um sagittal sections of mouse embryos and organs (e9, e9.5, elO.5, and e15.5) fixed in buffered 10% formalin was performed as described (1) by using T7 RNA polymerase to generate two separate 35S-labeled cRNA probes (486 and 550 nt) corresponding to fragments of N-terminal or C-terminal coding sequences of P-Lim.In Vitro Protein-Protein Interaction, DNA Binding, and Transfection Assays. Restriction fragments of P-Lim cDNA were ligated in frame into pGEX-KG (39) to yield glutathione S-transferase (GST) fusion proteins, and PCR was used to generate deletions of each LIM region separately (ALIM-1, A1-87; ALIM-2, A1-29 and A86-154) and of the entire LIM region (P-ALim = A1-151).[35S]Methionine-labeled in vitro translated protein was incubated for 20 min at 37°C with 2-3 jig of GST fusion protein bound to 25 p,l of glutathioneagarose beads in a total volume of 100 ,ul of 20 mM Hepes, pH 7.9/100 mM NaCl/1 mM EDTA/4 mM MgCl2/1 mM dithiothreitol, 0.02% (vol/vol) Nonidet P-40/10% (vol/vol) glycerol/0.5% (wt/vol) nonfat dry milk, with ethidium bromide at 50 ,ug/ml to eliminate potential protein-DNA interactions (32).Protein-mediated gel shift assays, in vitro culture, and transient transfection of cells were performed as described (9,14). For the mouse a-glycoprotein subunit (aGSU) reporter gene, a BamHI-Pst I fragment containing the promoter region of the aGSU starting from bp -440 was ligated to the luciferase gene in the pGL2 basic vector (Promega). Mouse TSH,B (from kb -1.2), rat Prl promoter/enhancer, and mous...

Reciprocal gene activation and restriction during cell type differentiation from a common lineage is a hallmark of mammalian organogenesis. A key question, then, is whether a critical transcriptional activator of cell type-specific gene targets can also restrict expression of the same genes in other cell types. Here, we show that whereas the pituitary-specific POU domain factor Pit-1 activates growth hormone gene expression in one cell type, the somatotrope, it restricts its expression from a second cell type, the lactotrope. This distinction depends on a two-base pair spacing in accommodation of the bipartite POU domains on a conserved growth hormone promoter site. The allosteric effect on Pit-1, in combination with other DNA binding factors, results in the recruitment of a corepressor complex, including nuclear receptor corepressor N-CoR, which, unexpectedly, is required for active long-term repression of the growth hormone gene in lactotropes.

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Pit-1 is a tissue-specific POU domain factor obligatory for the appearance of three cell phenotypes in the anterior pituitary gland. Expression of the pit-1 gene requires the actions of a cell-specific 390-bp enhancer, located 10 kb 5' of the pit-1 transcription initiation site, within sequence that proves essential for effective pituitary targeting of transgene expression during murine development. The enhancer requires the concerted actions of a cell-specific c/s-active element, Pit-1 autoregulatory sites, and atypical morphogen response elements. Pituitary ontogeny in the Pit-l-defective Snell dwarf mouse reveals that pit-1 autoregulation is not required for initial activation or continued expression during critical phases of Pit-1 target gene activation but, subsequently, is necessary for maintenance of pit-1 gene expression following birth. A potent 1,25-dihydroxyvitamin D3-responsive enhancer element defines a physiological site in which a single nucleotide alteration in the sequence of core binding motifs modulates the spacing rules for nuclear receptor response elements. Unexpectedly, the major retinoic acid response element is absolutely dependent on Pit-1 for retinoic acid receptor function. On this DNA element, Pit-1 appears to function as a coregulator of the retinoic acid receptor, suggesting an intriguing linkage between a cell-specific transcription factor and the actions of morphogen receptors that is likely to be prototypic of mechanisms by which other cell-specific transcription factors might confer morphogen receptor responsivity during mammalian organogenesis.

Targeted insertional disruption of the mouse estrogen receptor-alpha (ER alpha) gene has provided a genetic model in which to test hypotheses that estrogens exert important effects in development and homeostatic functions of the anterior pituitary gland, particularly in the lactotroph and gonadotroph cell types. Analysis of ER alpha gene-disrupted mice reveals a marked reduction in PRL mRNA and a decrease in lactotroph cell number, but normal specification of lactotroph cell phenotype. Gonadotropin mRNA levels in ER alpha gene-disrupted female mice are elevated, consistent with previously described transcriptional suppression of gonadotropin subunit gene expression in response to sustained administration of estrogen in wild type mice. These results provide genetic evidence that ER alpha plays a critical role in PRL and gonadotropin gene transcription and is involved in lactotroph cell growth, but is not required for specification of lactotroph cell phenotype.

Transcriptional activation of the prolactin and growth hormone genes, occurring in a cell-specific fashion, requires short-range synergistic interactions between the pituitary-specific POU domain factor Pit-1 and other transcription factors, particularly nuclear receptors. Unexpectedly, we find that these events involve the gene-specific use of alternative Pit-1 synergy domains. Synergistic activation of the prolactin gene by Pit-1 and the estrogen receptor requires a Pit-1 amino-terminal 25-amino-acid domain that is not required for analogous synergistic activation of the growth hormone promoter. The action of this Pit-1 synergy domain is dependent on the presence of two of three tyrosine residues spaced by 6 amino acids and can be replaced by a comparable tyrosine-dependent trans-activation domain of an unrelated transcription factor (hLEF). The gene-specific utilization of this tyrosine-dependent synergy domain is conferred by specific Pit-1 DNA-binding sites that determine whether Pit-1 binds as a monomer or a dimer. Thus, the critical DNA site in the prolactin enhancer, where this domain is required, binds Pit-1 as a monomer, whereas the Pit-1 sites in the growth hormone gene, which do not utilize this synergy domain, bind Pit-1 as a dimer. The finding that the sequence of specific DNA sites dictates alternative Pit-1 synergy domain utilization based on monomeric or dimeric binding suggests an additional regulatory strategy for differential target gene activation in distinct cell types.