A Novel Mechanism for Cyclic Adenosine 3',5'-Monophosphate Regulation of Gene Expression by CREB-Binding Protein

Kerstin Zanger, Laurie E. Cohen, Koshi Hashimoto, Sally Radovick and Fredric E. Wondisford

Divisions of Endocrinology Children’s Hospital (K.Z., L.E.C., S.R.) and Beth Israel Deaconess Medical Center (K.H., F.E.W.) and Harvard Medical School Boston, Massachusetts 02215


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The pituitary-specific transcription factor, Pit-1, is necessary to mediate protein kinase A (PKA) regulation of the GH, PRL, and TSH-ß subunit genes in the pituitary. Since these target genes lack classical cAMP DNA response elements (CREs), the mechanism of this regulation was previously unknown. We show that CREB binding protein (CBP), through two cysteine-histidine rich domains (C/H1 and C/H3), specifically and constitutively interacts with Pit-1 in pituitary cells. Pit-1 and CBP synergistically activate the PRL gene after PKA stimulation in a mechanism requiring both an intact Pit-1 amino-terminal and DNA-binding domain. A CBP construct containing the C/H3 domain [amino acids (aa) 1678–2441], but not one lacking the C/H3 domain (aa 1891–2441), is sufficient to mediate this response. Neither construct augments PKA regulation of CRE-containing promoters. Fusion of either CBP fragment to the GAL4 DNA-binding domain transferred complete PKA regulation to a heterologous promoter. These findings provide a mechanism for CREB-independent regulation of gene expression by cAMP.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pit-1 is a member of a family (POU) of transcription factors, and when bound to DNA, activates GH, Pit-1, PRL, and TSH-ß subunit gene expression (1, 2, 3, 4, 5, 6, 7, 8, 9). In addition to activating basal expression, Pit-1 is also necessary for cellular regulation of these target genes by hypothalamic hormones such as GHRH, TRH, and dopamine in a process requiring Pit-1 (8, 10, 11, 12, 13, 14). In the anterior pituitary, the GHRH receptor is known to activate (15), while the dopamine receptor is known to inhibit the cAMP/protein kinase A pathway (16); the TRH receptor is known to activate phospholipase C, leading to calcium mobilization and protein kinase C (PKC) activation (17). Interestingly, GH, PRL, and TSH-ß subunit genes do not contain consensus cAMP response elements (CREs) or AP-1 sites; instead, Pit-1 DNA response elements mediate induction by these pathways through a previously unknown mechanism.

On genes containing CREs, CREB binds as a homodimer and, after phosphorylation by protein kinase A (PKA), binds to CREB-binding protein (CBP) and a closely related coactivator (P300) (18, 19, 20, 21, 22, 23). CBP, via an N-terminal domain, binds to CREB and functions to increase transcription through a more C-terminal domain, which is proposed to both activate histone acetyltransferase (24) and displace nucleosomes as well as recruit RNA polymerase II to the transcription complex (25, 26, 27). The possibility that CBP could function independently of its recruitment to the transcription complex was recently suggested (28).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Since CBP is known to integrate a number of diverse cell-signaling pathways (29), we wanted to determine whether CBP acts as a cofactor for Pit-1-dependent gene regulation within the anterior pituitary. A cotransfection assay of the proximal PRL promoter reporter in a Pit-1-deficient cell line (CV-1) was performed to test this hypothesis. To activate the PKA pathway, a protein kinase A expression vector (30) was used; and to activate the PKC pathway, phorbol ester (TPA) treatment or a reconstituted TRH-signaling pathway was employed (31). The proximal PRL promoter contains four well defined Pit-1 DNA-binding sites (10). In this cell line, basal expression of the reporter construct was low but measurable. CBP or Pit-1 transfection activated this construct to a small extent after stimulation by these mediators (Fig. 1AGo). However, the combination of both CBP and Pit-1 expression vectors synergistically activated the proximal PRL reporter in the presence of PKA, TPA, or TRH receptor and TRH. Treatment of transfected cells with a cAMP analog (8-Br-cAMP) elicted similar responses (data not shown). These responses clearly required Pit-1 DNA-binding, as a Pit-1 DNA-binding mutant (W261C, Ref. 32) was completely defective in this assay (Fig. 1AGo). An intact Pit-1 N terminus was also necessary as the isoform Pit-1a (also referred to as Pit-1ß or GHF-2), which contains a 26-amino acid (aa) insertion in the N terminus and is defective in activating pituitary gene expression in vitro (33, 34, 35), was without stimulatory effect (Fig. 1AGo). A mutant PKA expression vector or TRH treatment in the absence of cotransfected TRH receptor did not activate this promoter after CBP and Pit-1 cotransfection (data not shown). These PKA, TPA, and TRH responses with cotransfected Pit-1 and CBP were not observed on a control promoter (TK, Fig. 1BGo). These data indicate that CBP functions as a Pit-1 cofactor to mediate PKA, TPA, and TRH induction of the PRL gene.



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Figure 1. Cotransfection Assay of the Proximal PRL and TK Promoters in CV-1 Cells

CV-1 cells were transfected with a SV-40 expression vector (pSG5) containing either CBP or Pit-1 (wt, Pit-1a, or W261C) and a RSV expression vector containing either a wt PKA catalytic subunit cDNA or a mouse TRH receptor cDNA in the presence of either (A) the bovine PRL promoter (Prl) or (B) the thymidine kinase promoter (TK) fused upstream of a luciferase reporter gene. These are representative experiments (one of four) performed in triplicate, and data are shown as mean ± SE. Fold basal activity is determined relative to cotransfection of the reporter construct with "empty" pSG5 and RSV expression vectors.

 
Since CBP significantly and specifically activated the proximal PRL reporter in a Pit-1-dependent manner, the ability of Pit-1 to bind to CBP was next evaluated. Figure 2AGo is a glutathione S-transferase (GST) pull-down assay using CBP fragments fused in-frame and C-terminal to GST. In data not shown, the quality and quantity of each GST-protein was determined on SDS-PAGE to ensure equivalence of the preparations. As can be seen, 35S-labeled Pit-1 bound specifically to two regions of CBP (aa 118–737 and aa 1677–2441). As shown in Fig. 2BGo, 35S-labeled Pit-1, regardless of PKA treatment, bound to the C/H1 (aa 362–429) and C/H3 (aa 1676–1844) domains of CBP but not to either GST alone or GST-CREB-binding domain (aa 463–661). In contrast, 35S-labeled CREB bound only to the CREB domain and only after in vitro phosphorylation by the catalytic subunit of PKA. These data indicate that Pit-1 can bind specifically to CBP in vitro.



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Figure 2. Protein Interactions between CBP and Pit-1

A, GST pull-down assay of radiolabeled Pit-1 and fragments of the CBP protein. GST-CBP fusions proteins were synthesized, purified, and exposed to either 35S-labeled Pit-1 or unprogrammed 35S-labeled lysate (C). After extensive washing, proteins trapped by the resin were resolved on SDS-PAGE and detected by autoradiography. B, GST pull-down assay of radiolabeled Pit-1 or CREB, before or after in vitro phosphorylation with the catalytic subunit of PKA (PKA cat.) and fragments of the CBP protein. GST-CBP proteins were synthesized as in panel A. C/H1 contains aa 1–450 of CBP, CREB contains aa 450–720, and C/H3 contains aa 1676–1891. C, GST pull-down assay of Pit-1 from GH3 cells using GST-CBP protein. GST alone or GST-CBP, containing aa 118–737 of CBP, was exposed to WCE before (C) or after treatment with 8-Br-cAMP or TRH. After extensive washing, proteins were eluted from the resin and resolved by SDS-PAGE, and Western blot for Pit-1 was performed. WCE without exposure to GST resin demonstrates the 33-kDa Pit-1 protein (arrow). D, GST pull-down assay of CBP from GH3 cells using a GST-Pit-1 protein. GST alone or GST-Pit-1, containing full-length rat Pit-1, was exposed to whole cell extract before or after treatment with 8-Br-cAMP or TRH. After extensive washing, proteins were eluted from the resin and resolved by SDS-PAGE, and Western blot analysis for CBP was performed. WCE without exposure to GST resin demonstrates the ~270-kDa CBP protein (arrow). E, Coimmunoprecipitation of CBP and Pit-1 from intact GH3 cells. Whole cell extract was immunoprecipitated with an anti-CBP or a control (anti-GST) polyclonal rabbit antibody followed by a Western blot analysis for Pit-1. The arrow marks the location of Pit-1 coimmunoprecipitated with CBP.

 
To prove that Pit-1 and CBP interact in pituitary cells, a GST pull-down assay using CBP aa 118–737 was performed, followed by a Western blot analysis for Pit-1 immunoreactivity. Figure 2CGo demonstrates that GST-CBP, but not GST alone, interacted equally well with Pit-1 from a rat pituitary cell line (GH3), expressing both GH and PRL, before or after treatment with either 8-Br-cAMP, or TRH. The converse experiment followed by a Western blot analysis for CBP immunoreactivity showed that GST-Pit-1, but not GST alone, interacted equally well with CBP from the same pituitary cell line, before or after treatment with either 8-Br-cAMP, or TRH (Fig. 2DGo). Finally, whole-cell extract (WCE) from untreated, 8-Br-cAMP-, or TRH-treated GH3 cells was immunoprecipitated with a CBP antibody followed by a Western blot analysis of the immunoprecipitate for Pit-1 immunoreactivity (Fig. 2EGo). CBP antiserum coimmunoprecipitated Pit-1, indicating that Pit-1 and CBP interact in vivo in pituitary cells.

Given that CBP and Pit-1 markedly activated the proximal PRL reporter in response to PKA, TPA, or TRH treatment and that CBP binds to Pit-1, we next determined what domains of CBP were responsible for this effect. In Fig. 3AGo, CBP deletion mutants were compared with wild-type (wt) CBP in a cotransfection assay in CV-1 cells. The {Delta} 8–1457 construct, which lacks the entire N-terminal half of CBP, was completely sufficient in mediating PKA stimulation of the proximal PRL reporter but was unable to mediate a normal TRH response. In contrast, the 1–1334 construct was defective in mediating a PKA effect but sufficient in mediating a TRH effect. The former result was expected since the known transactivation domain of CBP, contained within the carboxy terminus of CBP, was deleted in this construct. The latter result proves that the 1–1334 construct is functional and indicates that the TRH signaling pathway utilizes a different CBP domain, located in the N-terminal half of the molecule, to mediate transactivation. Finally the 1–450 construct, which lacks the CREB-binding site but contains the C/H1 domain, was fully sufficient to mediate the TRH response, indicating that the transactivation domain for this pathway is located in this amino-terminal region of CBP. In data not shown, treatment with a cAMP analog (8-Br-cAMP) or TPA produced a response pattern similar to PKA or TRH stimulation, respectively.



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Figure 3. Determination of the CBP Domains Required for PKA and TRH Induction of the Proximal PRL Reporter

A, This is a representative transfection experiment (one of three) each performed in triplicate showing CBP domains required for PKA and TRH induction. Data are shown as mean ± SD of activity relative to the wt CBP induction included in each experiment. To the left of the graph is a schematic representation of the CBP constructs used. C/H domains are indicated by black boxes. B, Western blots of CV-1 cells transfected with the indicated CBP expression vectors: V, pSG5 empty vector; 1, wt CBP; 2, {Delta} 8–1457; 3, {Delta} 142–705; 4, 1–1334; and 5, 1–450. The antibody used in the Western blot is indicated at the bottom of the figure; and a molecular mass marker in kilodaltons is indicated to the left.

 
In Fig. 3BGo, a Western blot of cellular lysate from CV-1 cells transfected with these same constructs and probed with an antibody directed at either the amino or carboxy terminus of CBP. In data not shown, nuclear localization of each of these proteins was confirmed using enhanced green fluorescent protein-tagged CBP deletion constructs. Note that CBP deletion constructs are expressed at similar levels in transfected CV-1 cells so that differences in protein expression of these constructs can not explain the results displayed in Fig. 3AGo.

Since the CREB binding domain of CBP is not required for the PKA response of the PRL gene, we next wanted to determine whether CREB was required for this response. Transfections were repeated using a human common glycoprotein {alpha}-subunit reporter gene construct, which contains two well defined CREs (36) (Fig. 4AGo). CBP, but not Pit-1, markedly enhanced the PKA induction of this reporter vs. transfection of empty vector alone. Since CV-1 cells are relatively deficient in CBP vs. GH3 cells (compare Fig. 3BGo with Fig. 2DGo) and CBP cotransfection markedly increased CBP levels in CV-1 cells (compare lanes 1 and 2, Fig. 3BGo), this result was expected. Cotransfection of an expression vector containing a mutation of the PKA phosphorylation site of CREB (CREBm, S133A, Ref. 37) completely blocked both the PKA response of the common glycoprotein {alpha}-subunit reporter in the absence (data not shown) and in the presence of CBP cotransfection (Fig. 4AGo). Cotransfection of CREBm had no significant effect on PKA induction of the PRL reporter (Fig. 4BGo). Cotransfection of wt CREB had no effect on induction of either reporter (Fig. 4Go, A and B). Since CV-1 cells are not deficient in CREB, it is not surprising that wt CREB cotransfection did not augment the PKA response. We next tested whether known PKA sites on Pit-1 mediated the PKA effect seen on the PRL gene. However, mutation of all three PKA phosphorylation sites on Pit-1 (aa 115, 219, 220, Ref. 38) had no significant effect on PKA induction of the PRL gene (data not shown).



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Figure 4. PKA Activation of the Proximal PRL or Common Glycoprotein {alpha}-Subunit Promoter in CV-1 Cells Using wt and Mutant CREB (S133A) Expression Vectors

CV-1 cells were transfected with SV-40 expression vectors (pSG5) containing either wt PKA catalytic subunit, CBP, or Pit-1 cDNAs in the presence of either a human common glycoprotein {alpha}-subunit (A) or proximal PRL (B) promoter fused upstream of a luciferase reporter gene. In some transfections, a pSG5 expression vector containing either a wt CREB or CREB mutant cDNA (S133A, CREBm) was also included. Data are shown as mean ± SE of two independent experiments each performed in triplicate. Fold basal activity is determined relative to cotransfection of the reporter construct with empty pSG5 expression vector.

 
To determine what C-terminal region of CBP was sufficient for the PKA response, CBP fragments were fused downstream and in-frame of the GAL4 DNA-binding domain. Figure 5AGo demonstrates that GAL4-CBP constructs containing either aa 1677–2441 or aa 1891–2441 were sufficient to mediate a robust PKA response when tested on a UAS-thymidine kinase (TK) reporter, and these responses were similar to those observed on the PRL gene (Fig. 5BGo). The isolated C/H3 domain (aa 1677–1891) mediated only a small PKA response. Mutation of the one consensus PKA site in CBP (S1772A) in the context of either the 1677–2441 or 1677–1891 construct had no effect on PKA induction, indicating that phosphorylation at this site is not important for this effect. As shown in Fig. 5BGo, only the 1677–2441 construct was active on the PRL reporter, whereas the 1891–2441 and 1677–1891 constructs were not active. Note also that the 1677–2441 construct alone was inactive on the PRL reporter and required cotransfection of Pit-1 to mediate a PKA effect. These data indicate that the C/H3 domain, which binds Pit-1, is required for the synergistic activation of the PRL reporter (compare aa 1677–2441 to aa 1891 to 2441) but was not active in isolation (aa 1677–1891). On a CRE reporter (four copies of a CRE upstream of the TK promoter), these constructs were all defective as compared with wt CBP in activating this reporter after PKA transfection (Fig. 5CGo).



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Figure 5. CBP Fusion Protein Mediates PKA Induction of a GAL or PRL Reporter

A, GAL4-CBP fusion proteins as indicated without and with Pit-1 were transfected in CV-1 cells, and PKA induction compared with GAL4 DNA-binding domain alone (vector) on the UAS-TK reporter (A), proximal PRL reporter (B), or a minimal thymidine kinase promoter containing four consensus CREs (C) is shown. The GAL4-CBP fusion construct used in shown to the left of the figure. Fold basal activity is determined relative to cotransfection of the reporter construct with empty GAL4 DNA-binding domain expression vector.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This study suggests a general mechanism for cAMP regulation of genes without CREs whereby CBP, bound constitutively to a cell-specific transcription factor directly or via additional cofactor interactions, stimulates gene expression. A CBP construct containing aa 1677–2441 is sufficient to mediate the PKA induction. Pit-1 presumably recruits this CBP fragment to the transcription complex by binding to aa 1677–1891, and subsequently aa 1891–2441 mediate the PKA induction. Interestingly, although this fragment does not contain intrinsic histone acetyltransferase (HAT) activity, it could recruit other cofactors such as p/CAF or p/CIP with known HAT domains.

Our data do not support a role for CBP phospho-rylation in this process as mutation of the only consensus PKA site (S1772A) in this C-terminal fragment did not affect PKA induction; these data are in contrast to a recent paper by Xu et al. (39). However, it is possible that another unknown PKA phosphorylation site in the C terminus of CBP could mediate this effect. Alternatively, PKA may phosphorylate other cofactors facilitating their recruitment to the C terminus of CBP. Experiments are in progress to test this hypothesis.

In contrast to the PKA effect, TRH stimulation of the PRL gene requires an N-terminal region of CBP (aa 1–450). Again, like the PKA effect, this CBP fragment is without intrinsic HAT activity, but other cofactors such as p/CAF could also be recruited to this fragment. Xu et al. also suggested that this same region of CBP could mediate induction by another growth factor (insulin) on a Pit-1 response element. Thus, CBP acts as a transcriptional cofactor for Pit-1 to permit regulation of the PRL gene by TRH-, TPA-, and PKA-signaling pathways. As other genes in the pituitary are regulated by Pit-1, including GH, TSH-ß subunit, and Pit-1 genes, CBP may subserve a similar function on these genes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transfection Constructs
Luciferase reporter constructs contain either 1000 bp of 5'-flanking DNA of the bovine PRL promoter, 109 bp of 5'-flanking DNA of the herpes simplex TK promoter (minimal TK construct), 846 bp of 5'-flanking DNA of the human common glycoprotein {alpha}-subunit promoter (glycoprotein {alpha}-subunit), five copies of the GAL4 DNA-binding site upstream of TK (UAS-TK), or four copies of a consensus CRE upstream of TK. All Pit-1, CBP, and CREB constructs were in the SV40 expression construct, pSG5. Pit-1a and W261C were modifications of the original wt Pit-1 cDNA and therefore contained the same translation initiation site. CBP deletion mutant were made using restriction enzyme digestion and removal of wt mouse CBP domains as indicated: {Delta} 8–1457, RsrII; 1–1334, BstXI; {Delta}142–705, ApaI; and 1–450, EcoRI. The reading frame and orientation of each mutant were confirmed in pSG5 by DNA sequencing.

Transfection Assays
In a 24-well format, 0.8 µg of reporter with 1 µg of pSG5 and/or 1 µg of rous sarcoma virus (RSV) expression vectors were transfected per plate; 16 h after transfection, cultures were treated with serum-free medium for 8 h. For TRH stimulation, 50 nM TRH was added to the medium. All transfections were balanced for the same amount of expression vector using empty vector as needed. For analysis of the expression of transfected CBP constructs, CBP expression vectors were transfected as above, and a Western blot of total cellular lysate was performed using a CBP antibody that recognizes the extreme N terminus (aa 2–22, Santa Cruz Biotechnology, Santa Cruz, CA) or C terminus (aa 1736–2179, Upstate Biotechnology, Lake Placid, NY) of CBP.

GST and Immunoprecipitation Assays
Regions of the CBP protein were fused in-frame with GST in the pGEX4T2 vector (Pharmacia Biotech, Piscataway, NJ). Recombinant proteins were synthesized in JM109 bacteria and purified on glutathione-Sepharose resin under nondenaturing conditions. GST proteins were analyzed on SDS-PAGE before use in the assay to ensure equivalence of preparations. 35S-labeled Pit-1 or CREB was generated in an in vitro transcription/translation system (TNT, Promega Biotech, Madison, WI) and exposed to the indicated GST protein. As a control, an unprogrammed translation with [35S]methionine was employed. In some experiments the translated protein was phosphorylated in vitro using the catalytic subunit of PKA (Boehringer Mannheim, Indianapolis, IN) and 1 mM ATP for 30 min at 37 C before exposure to the GST protein. After extensive washing with NET (150 mM NaCl, 1 mM EDTA, 0.5% NP40) at 4 C, the proteins trapped by the resin were resolved on SDS-PAGE and detected by autoradiography.

GST alone or GST-CBP, containing aa 118–737 of CBP, was exposed to whole cell GH3 extract (WCE) made from individual 100-mm dishes before or after 10 min of treatment with 1 mM 8-Br-cAMP or 50 nM TRH. After extensive washing with NET at 4 C, proteins were eluted from the resin and resolved by SDS-PAGE, and Western blot analysis was performed with a mouse monoclonal anti-Pit-1 antibody made against the full-length rat Pit-1 molecule (Transduction Laboratories, Lexington, KY). GST alone or GST-Pit-1, containing full-length rat Pit-1, was exposed to whole-cell GH3 extract made from individual 100-mm dishes before or after 10 min of treatment with 1 mM 8-Br-cAMP or 50 nM TRH. After extensive washing with NET at 4 C, proteins were eluted from the resin and resolved by SDS-PAGE, and Western blot analysis was performed with a rabbit polyclonal anti-CBP antibody made against aa 1736–2179 of mouse CBP (Upstate Biotechnology).

Whole-cell GH3 extracts from 100-mm plates untreated or treated with either 8-Br-cAMP or TRH were immunoprecipitated with an anti-CBP or a control GST polyclonal rabbit antibody (anti-GST) and protein A/G resin. After extensive washing in NET and PBS, a Western blot analysis for Pit-1 using the monoclonal Pit-1 antibody described above was performed.


    ACKNOWLEDGMENTS
 
We would like to thank M. C. Gershengorn, R. H. Goodman, J. L. Jameson, R. A. Maurer, and M. Montminy for plasmids used in this study.


    FOOTNOTES
 
Address requests for reprints to: Fredric E. Wondisford, Research North 330C, 99 Brookline Avenue, Boston, Massachusetts 02215. e-mail: fwondisf@bidmc.harvard.edu.

This work was supported by grants from the NIH, March of Dimes, and Deutsche Forschungsgemeinschaft.

Received for publication October 14, 1998. Revision received November 9, 1998. Accepted for publication November 19, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Rosenfeld MG 1991 POU-domain transcription factors: pou-er-ful developmental regulators. Genes Dev 5:897–907[CrossRef][Medline]
  2. Bodner M, Castrillo JL, Theill LE, Deerinck T, Ellisman M, Karin M 1988 The pituitary-specific transcription factor GHF-1 is a homeobox-containing protein. Cell 55:505–518[Medline]
  3. Ingraham HA, Chen R, Mangalam HJ, Elsholtz HP, Flynn SE, Lin CR, Simmons DM, Swanson L, Rosenfeld MG 1988 A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell 55:519–529[Medline]
  4. Theill LE, Castrillo J-L, Wu D, Karin M 1989 Dissection of functional domains of the pituitary-specific transcription factor GHF-1. Nature 342:945–948[CrossRef][Medline]
  5. Mangalam HJ, Albert VR, Ingraham HA, Kapiloff M, Wilson L, Nelson C, Elsholtz H, Rosenfeld MG 1989 A pituitary POU domain protein, Pit-1, activates both growth hormone, prolactin promoters transcriptionally. Genes Dev 3:946–958[Abstract]
  6. Chen R, Ingraham HA, Treacy MN, Albert VR, Wilson L, Rosenfeld MG 1990 Autoregulation of pit-1 gene expression mediated by two cis-active promoter elements. Nature 346:583–586[CrossRef][Medline]
  7. McCormick A, Brady H, Theill LE, Karin M 1990 Regulation of the pituitary-specific homeobox gene GHF1 by cell-autonomous, environmental cues. Nature 345:829–832[CrossRef][Medline]
  8. Steinfelder HJ, Hauser P, Nakayama Y, Radovick S, McClaskey JH, Taylor T, Weintraub BD, Wondisford FE 1991 Thyrotropin-releasing hormone regulation of human TSH-ß expression: role of a pituitary-specific transcription factor (Pit-1/ GHF-1), potential interaction with a thyroid hormone-inhibitory element. Proc Natl Acad Sci USA 88:3130–3134[Abstract]
  9. Supowit SC, Ramsey T, Thompson EB 1992 Extinction of prolactin gene expression in somatic cell hybrids is correlated with the repression of the pituitary-specific trans-activator GHF-1/Pit-1. Mol Endocrinol 6:786–792[Abstract]
  10. d’Emden MC, Okimura Y, Maurer RA 1992 Analysis of functional cooperativity between individual transcription-stimulating elements in the proximal region of the rat prolactin gene. Mol Endocrinol 6:581–588[Abstract]
  11. Keech CA, Jackson SM, Siddiqui SK, Ocran KW, Gutierrez-Hartmann A 1992 Cyclic adenosine 3',5'-monophosphate activation of the rat prolactin promoter is restricted to the pituitary-specific cell type. Mol Endocrinol 6:2059–2070[Abstract]
  12. Steinfelder HJ, Radovick S, Mroczynski MA, Hauser P, McClaskey JH, Weintraub BD, Wondisford FE 1992 Role of a pituitary-specific transcription factor (pit-1/GHF-1) or a closely related protein in c AMP regulation of human thyrotropin-ß subunit gene expression. J Clin Invest 89:409–419[Medline]
  13. Fischberg DJ, Chen X, Bancroft CA 1994 pit-1 phosphorylation mutant can mediate both basal, induced prolactin, growth hormone promoter activity. Mol Endocrinol 8:1566–1573[Abstract]
  14. Okimura Y, Howard PW, Maurer RA 1994 Pit-1 binding sites mediate transcriptional responses to cyclic adenosine 3',5'-monophosphate through a mechanism that does not require inducible phosphorylation of pit-1. Mol Endocrinol 8:1559–1565[Abstract]
  15. Baringa M, Yamamoto G, Rivier C, Vale W, Evans R, Rosenfeld MG 1983 Transcriptional regulation of growth hormone gene expression by growth hormone-releasing factor. Nature 306:84–85[Medline]
  16. Lew AM, Yao H, Elsholtz HP 1994 Gi{alpha}2-, G0{alpha}-mediated signaling in the Pit-1-dependent inhibition of the prolactin gene promoter. J Biol Chem 269:12007–12013[Abstract/Free Full Text]
  17. Gershengorn MC 1986 Mechanism of thyrotropin-releasing hormone stimulation of pituitary hormone secretion. Annu Rev Physiol 48:515–526[CrossRef][Medline]
  18. Chrivia JC, Kwok RP, Lamb N, Hadiwara M, Montminy MR, Goodman RH 1993 Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365:855–859[CrossRef][Medline]
  19. Arany Z, Sellers WR, Livingston DM, Ecker R 1994 E1A-associated p 300, CREB-associate CBP belong to a conserved family of coactivators. Cell 77:799–800[Medline]
  20. Arias J, Alberts AS, Brindle P, Claret FX, Smeal T, Karin M, Feramisco J, Montminy M 1994 Activation of cAMP, mitogen responsive genes relies on a common nuclear factor. Nature 370:226–228[CrossRef][Medline]
  21. Eckner R, Ewen ME, Newsome D, Gerdes M, DeCaprio JA, Lawrence JE, Livingston DM 1994 Molecular cloning, functional analysis of the adenovirus E1A-associated 300-kD protein (p300) reveals a protein with properties of a transcriptional adapter. Genes Dev 8:869–884[Abstract]
  22. Kwok RP, Lundblad JR, Chrivia JC, Richards JP, Bachinger HP, Brennan RG, Roverts SGE, Green MR, Goodman RH 1994 Nuclear protein CBP is a coactivator for the transcription factor CREB. Nature 370:223–226[CrossRef][Medline]
  23. Lundblad JR, Kwok RP, Laurance ME, Harter ML, Goodman RH 1995 Adenoviral E1A-associated protein p300 as a functional homologue of the transcriptional co-activator CBP. Nature 374:85–88[CrossRef][Medline]
  24. Ogryzko VV, Schilz L, Russanova V, Howard BH, Nakatni Y 1996 The transcriptional coactivators p300, CBP are histone acetyltransferases. Cell 87:953–959[Medline]
  25. Sharp ZD 1995 Rat Pit-1 stimulates transcription in vitro by influencing pre-initiation complex assembly. Biochem Biophys Res Commun 206:40–45[CrossRef][Medline]
  26. Kee BL, Arias J, Montminy MR 1996 Adaptor-mediated recruitment of RNA polymerase II to a signal-dependent activator. J Biol Chem 271:2373–2375[Abstract/Free Full Text]
  27. Nakajima T, Uchida C, Anderson SF, Lee C-G, Hurwitz J, Parvin JD, Montminy M 1997 RNA helicase A mediates association of CBP with RNA polymerase II Cell 90:1107–1112[Medline]
  28. Chawla S, Hardingham GE, Quinn DR, Bading H 1998 CBP: a signal-regulated transcriptional coactivator controlled by nuclear calcium, CaM kinase IV Nature 281:1505–1509
  29. Kamei Y, Xu L, Heinzel T, Torchia J, Kukokawa R, Gloss B, Lin S-C, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator complex mediates transcriptional activation, AP-1 inhibition by nuclear receptors. Cell 85:403–414[Medline]
  30. Maurer RA 1989 Both isoforms of the cAMP-dependent protein kinase catalytic subunit can activate transcription of the prolactin gene. J Biol Chem 264:6870–6873[Abstract/Free Full Text]
  31. Straub RE, Frech GC, Joho RH, Gershengorn MC 1990 Expression cloning of a cDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor. Proc Natl Acad Sci USA 87:9514–9518[Abstract]
  32. Li S, Crenshaw EB, Rawson EJ, Simmons DM, Swanson LW, Rosenfeld MG 1990 Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 347:528–533[CrossRef][Medline]
  33. Konzak KE, Moore DD 1992 Functional isoforms of Pit-1 generated by alternative mRNA splicing. Mol Endocrinol 6:241–247[Abstract]
  34. Morris AE, Kloss B, McChesney RE, Bancroft C, Chasin LA 1992 An alternatively spliced Pit-1 isoform altered in its ability to trans-activate. Nucleic Acids Res 20:1355–1361[Abstract]
  35. Theill LE, Hattori K, Lazzaro D, Castrillo J-L, Karin M 1992 Differential splicing of the GHF 1 primary transcript gives rise to two functionally distinct homeodomain proteins. EMBO J 11:2261–2269[Abstract]
  36. Deutsch PJ, Jameson JL, Habener JF 1987 Cyclic AMP responsiveness of the human gonadotropin-{alpha} gene transcription is directed by a repeated 18-base pair enhancer. J Biol Chem 262:12169–12174[Abstract/Free Full Text]
  37. Gonzalez GA, Montminy MR 1989 Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59:675–680[Medline]
  38. Kapiloff MS, Farkash Y, Wegner M, Rosenfeld MG 1991 Variable effects of phosphorylation of Pit-1 dictated by the DNA response elements. Science 253:786–789[Medline]
  39. Xu L, Lavinsky RM, Dasen JS, Flynn SE, McInerney EM, Mullen T-M, Heinzel T, Szeto D, Korzus E, Kurokawal R, Aggarwal AK, Rose DW, Glass CK, Rosenfeld MG 1998 Signal-specific co-activator domain requirements for Pit-1 activation. Nature 395:301–306[CrossRef][Medline]