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Originally published In Press as doi:10.1074/jbc.M006048200 on July 31, 2000

J. Biol. Chem., Vol. 275, Issue 40, 30977-30986, October 6, 2000
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The Pit-1beta Domain Dictates Active Repression and Alteration of Histone Acetylation of the Proximal Prolactin Promoter*

Scott E. DiamondDagger and Arthur Gutierrez-Hartmann§

From the § Department of Medicine and Department of Biochemistry and Molecular Genetics, Program in Molecular Biology and Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262

Received for publication, July 10, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A critical problem in current molecular biology is to gain a detailed understanding of the molecular mechanisms by which related transcription factor isoforms with identical DNA sequence specificity mediate distinct transcription responses. Pit-1 and Pit-1beta constitute such a pair of transcription factor isoforms. Pit-1 enhances the Ras signaling pathway to the prolactin promoter, and Pit-1beta represses basal prolactin promoter activity as well as Ras signaling to the prolactin promoter in pituitary cells. We have previously demonstrated that the beta -domain amino acid sequence dictates the transcriptional properties of Pit-1beta . Here, we show that five hydrophobic beta -domain residues are required for Pit-1 isoform-specific repression of Ras signaling, and we demonstrate that sodium butyrate and trichostatin A, pharmacological inhibitors of histone deacetylation, as well as viral Ski protein, a dominant-negative inhibitor of recruitment of N-CoR/mSin3 histone deacetylase complexes, specifically reverse beta  isoform-specific repression of Ras signaling. Moreover, we directly demonstrate, with a chromatin immunoprecipitation assay, that the Pit-1beta isoform alters the histone acetylation state of the proximal prolactin promoter. This differential analysis of Pit-1/Pit-1beta isoform function provides significant insights into the structural determinants that govern how different transcription factors with identical DNA sequence specificity can display opposite effects on target gene activity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Most transcription factors are members of extended families defined by conserved structural motifs, typically in the DNA-binding domain, yet differing in other domains, especially the transactivation domain (TAD).1 A number of transcription factors are expressed as a set of proteins derived from a single gene via alternative promoter usage or splicing events that result in virtually identical transcription factor isoforms that, nonetheless, can mediate distinct responses (e.g. PR-A versus PR-B; TRbeta 1 versus TRbeta 2; Oct 2.1 versus Oct 2.5; Ets-1 versus Ets-1Delta VII; and Pit-1 versus Pit-1beta ) (1-6). The molecular mechanisms, by which related transcription factor isoforms with identical DNA sequence specificity mediate distinct transcription responses, remain an area of active investigation.

Pit-1 is a pituitary-specific POU homeodomain transcription factor that governs both anterior pituitary cell identity and hormone gene expression (reviewed in Ref. 7). Pit-1 occurs in vertebrates, including humans, as two principal splice isoforms (Fig. 1). The beta  isoform arises from an alternative splice-acceptor sequence at the end of the first intron resulting in a 26-amino acid insertion, the beta -domain, at position 48 in the TAD, between the first and second exons, TAD1 and TAD2 (Fig. 1) (5, 6). The beta  isoform differs from Pit-1 only in the TAD and displays identical DNA sequence specificity with respect to the prolactin (PRL) promoter (5) but has dramatically different transcriptional properties than Pit-1, and these differences are dictated by the unique amino acid sequence of the beta -domain TAD insertion (8). The beta -domain insertion causes Pit-1beta to act as a pituitary-specific repressor of both of basal transcription of the rat (r) PRL and of Ras signaling to the rPRL promoter gene (reviewed in Ref. 8). Additionally, the beta -domain blocks functional interaction with Ets-1 (9) and functional interaction with the thyroid hormone and retinoic acid receptors (10) in nonpituitary cells. Nevertheless, this same 26-amino acid insertion endows Pit-1beta with even greater potency with regard to mediation of protein kinase A signaling to the rPRL promoter (8, 11).

In this article, we focus on identifying key beta -domain residues that are responsible for the beta  isoform-specific repression of Ras signaling to the rPRL promoter, as well as identifying a mechanism for this repression. We have utilized an epitope-scanning approach to replace sequentially 6 amino acid blocks of the beta -domain in order to identify a limited subset of functionally important residues. Replacement of each of these residues with alanine identified five hydrophobic residues that are required for the beta -domain to act as a transcriptional repressor of Ras signaling to the rPRL promoter. Moreover, we demonstrate that the beta -domain does not simply disrupt TAD structure but functions as an active repression domain, which modifies the acetylation state of the proximal PRL promoter in a manner dependent upon an N-CoR/mSin3-containing histone deacetylase complex (HDAC). Thus, analysis of the Pit-1/Pit-1beta isoform pair provides significant insight into the structural determinants of transcription activation versus repression mediated by two nearly identical transcription factor isoforms.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Monolayer cultures of HeLa human cervical carcinoma cells and GH4T2 rat pituitary tumor cells (12) were maintained in Dulbecco's modified Eagle's medium, 15% horse serum, 2.5% fetal bovine serum, and 50 µg/ml penicillin and streptomycin at 37 °C in 5% CO2. The medium was changed 16-18 h before each transfection. Cells used for transfections were harvested at approximately 60-80% confluence using 0.05% trypsin and 0.5 mM EDTA.

Plasmids-- The rat PRL promoter luciferase expression vector, pA3 PRL luc, contains the firefly luciferase coding region under the control of a 498-bp fragment (-425 to +73) of the rPRL promoter downstream of three polyadenylation termination sites in pA3 luc (12). Plasmid pSV Ras contains the T24 bladder carcinoma Harvey Ras valine 12 mutant oncogene (RasVal-12) under control of the SV40 early promoter. Plasmids pRSV HA Pit-1 and pRSV HA Pit-1beta express N-terminally hemagglutinin (HA)-tagged Pit-1 and Pit-1beta under the control of the RSV promoter (8), and plasmids pCGN2-Pit-1 and pCGN2 Pit-1Delta TAD express N-terminally HA-tagged Pit-1 and Pit-1 deleted for its TAD (amino acids 1-80) under the control of the CMV promoter, and they were the generous gift of Dr. David F. Gordon (University of Colorado Health Sciences Center, Denver, CO). Plasmid pAPR EtsZ encodes the DNA-binding domain (amino acids 334-466) of human c-ETS-2 fused to LacZ under the control of the actin promoter (13). Plasmid pRSVt3 v-Ski contains the avian Sloan-Kettering virus ski oncogene under the control of the RSV promoter (14) and was the generous gift of Dr. Edward Stavnezer (Case Western Reserve University, Cleveland, OH). Plasmid DNAs were prepared by Qiagen (Qiagen Inc., Chatsworth, CA) columns and quantified by fluorimetry.

Mutant Pit-1 Constructs-- The vectors pRSV HA Pit-1-ES1, pRSV HA Pit-1-ES2, pRSV HA Pit-1-ES3, pRSV HA Pit-1-ES4, pRSV HA Pit-1-ES5, and pRSV HA Pit-1-ES6, which encode HA-tagged Pit-1beta s with different epitope-scanning mutations of the 26-amino acid beta -domain, as well as the vectors pRSV HA Pit-1-AS1, pRSV HA Pit-1-AS2, pRSV HA Pit-1-AS3, pRSV HA Pit-1-AS4, pRSV HA Pit-1-AS5, pRSV HA Pit-1-AS6, pRSV HA Pit-1-AS7, pRSV HA Pit-1-AS8, and pRSV HA Pit-1-AS9, which encode HA-tagged Pit-1beta s with different alanine-scanning mutations of the 26 amino acid beta -domain, were constructed as follows.

All mutant beta -domain constructs were constructed by nested PCR mutagenesis of the Pit-1 transactivation domain as described previously (8, 11). The pRSV HA Pit-1 plasmid was used as a substrate for PCR mutagenesis in which the 26-amino acid beta -domain was substituted with six different epitope-scanning sequences (see Table I) or nine different alanine-scanning sequences (Table II), and an HA epitope tag was retained at the N terminus of all of the Pit-1 constructs. Common 5'- and 3'-deoxyoligonucleotides were utilized, as well as mutation-specific mutagenic deoxyoligonucleotides that encode the nucleotide substitutions in the beta -domain. Amplified DNA was initially subcloned into pCR 2.1 (Invitrogen). The commercially synthesized deoxyoligonucleotides (Life Technologies, Inc.) contained the following sequences: 5'-TAD, AAA AAG CAA GCT TCC ATG GGG TAC CCA TAC GAT GTT CCG GAT TAC GCT AGT TGC AAC CTT TC; 3'-TAD, GTT TGT CTG GGT GTA TC; 5'-ES-1, GTG TGC AAA CAT TTA GGA GTT TGG ATC AAT ATA TAG CGA TAG GTG TCT GTG GAC ATC ACG TTG; 3'-ES-1, CTA AAT GTT TGC ACA CAT ATT TCT CGA TGA CAA CGA TGG GAA ATA CAG CGA CAG GAC TTC ATT 5'-ES-2, GTG TGC AAA CAT TTA GGT ATA TAG CGA TAG GTG TCA ATA GAC GGG ACT GTG GAC ATC ACG TTG; 3'-ES-2, CTA AAT GTT TGC ACA CAT ATT TCT CGA TGA CAA CGA TGG GAA ATA CAG CGA CAG GAC TTC ATT; 5'-ES-3, GTG TGT ATA TAG CGA TAG GTG TCG ATC AAA GAC AAA ATA GAC GGG ACT GTG GAC ATC ACG TTG; 3'ES-3, ATC GCT ATA TAC ACA CAT ATT TCT CGA TGA CAA CGA TGG GAA ATA CAG CGA CAG GAC TTC ATT; 5'-ES-4, CGA TAG GTG TCT TTA GGA GTT TGG ATC AAA GAC AAA ATA GAC GGG ACT GTG GAC ATC ACG TTG; 3'-ES-4, CTA AAG ACA CCT ATC GCT ATA TAT CGA TGA CAA CGA TGG GAA ATA CAG CGA CAG GAC TTC ATT; 5'-ES-5, GTG TGC AAA CAT TTA GGA GTT TGG ATC AAA GAC AAA ATA GAC GGG ACT GTG GAC ATC ACG TTG; 3'-ES-5, CTA AAT GTT TGC ACA CAG ACA CCT ATC GCT ATA TAA TGG GAA ATA CAG CGA CAG GAC TTC ATT; 5'-ES-6, GTG TGC AAA CAT TTA GGA GTT TGG ATC AAA GAC AAA ATA GAC GGG ACT GTG GAC ATC ACG TTG; 3'-ES-6, CTA AAT GTT TGC ACA CAT ATT TCT CGA TGG ACA CCT ATC GCT ATA TAG CGA CAG GAC TTC ATT; 5'-AS-1, GTG TGC AAA CAT TTA GGA GTT TGG ATC GCG GAC AAA ATA GAC GGG ACT GTG GAC ATC ACG TTG; 3'-AS-1, CTA AAT GTT TGC ACA CAT ATT TCT CGA TGA CAA CGA TGG GAA ATA CAG CGA CAG GAC TTC ATT; 5'-AS-2, GTG TGC AAA CAT TTA GGA GTT TGC GCG AGA GAC AAA ATA GAC GGG ACT GTG GAC ATC ACG TTG; 3'-AS-2, CTA AAT GTT TGC ACA CAT ATT TCT CGA TGA CAA CGA TGG GAA ATA CAG CGA CAG GAC TTC ATT; 5'-AS-3, GTC GCG AGA CAT TTA GGA GTT TGG ATC AAA GAC AAA ATA GAC GGG ACT GTG GAC ATC ACG TTG; 3'-AS-3, CTA AAT GTC TCG CGA CAT ATT TCT CGA TGA CAA CGA TGG GAA ATA CAG CGA CAG GAC TTC ATT; 5'-AS-4, GCG TGC AAA CAT TTA GGA GTT TGG ATC AAA GAC AAA ATA GAC GGG ACT GTG GAC ATC ACG TTG; 3'-AS-4, CTA AAT GTT TGC ACG CGT ATT TCT CGA TGA CAA CGA TGG GAA ATA CAG CGA CAG GAC TTC ATT; 5'-AS-5, GTG TGC AAA CAT TTA GGA GTT TGG ATC AAA GAC AAA ATA GAC GGG ACT GTG GAC ATC ACG TTG; 3'-AS-5, CTA AAT GTT TGC ACA CCG CGT TCT CGA TGA CAA CGA TGG GAA ATA CAG CGA CAG GAC TTC ATT; 5'-AS-6, GTG TGC AAA CAT TTA GGA GTT TGG ATC AAA GAC AAA ATA GAC GGG ACT GTG GAC ATC ACG TTG; 3'-AS-6, CTA AAT GTT TGC ACA CAT ACG CCT CGA TGA CAA CGA TGG GAA ATA CAG CGA CAG GAC TTC ATT; 5'-AS-7, GTG TGC AAA CAT TTA GGA GTT TGG ATC AAA GAC AAA ATA GAC GGG ACT GTG GAC ATC ACG TTG; 3'-AS-7, CTA AAT GTT TGC ACA CAT ATT TGC CGA TGA CAA CGA TGG GAA ATA CAG CGA CAG GAC TTC ATT; 5'-AS-8, GTG TGC AAA CAT TTA GGA GTT TGG ATC AAA GAC AAA ATA GAC GGG ACT GTG GAC ATC ACG TTG; 3'-AS-8, CTA AAT GTT TGC ACA CAT ATT TGT CCG CGA CAA CGA TGG GAA ATA CAG CGA CAG GAC TTC ATT; 5'-AS-9, GTG TGC AAA CAT TTA GGA GTT TGG ATC AAA GAC AAA ATA GAC GGG ACT GTG GAC ATC ACG TTG; 3'-AS-9 CTA AAT GTT TGC ACA CAT ATT TCT CGA TGA CCG CGA TGG GAA ATA CAG CGA CAG GAC TTC ATT.

The presence of each introduced mutation and integrity of its TAD region were verified by dideoxy sequencing by the University of Colorado Health Sciences Cancer Center DNA Sequencing Core facility. HA-tagged Pit-1 TAD sequences were then excised from pCR2.1 by digestion with HindIII and PpuMI and ligated to the unique HindIII and PpuMI sites of pRSV-Pit-1 to produce pRSV HA Pit-1-ES1, pRSV HA Pit-1-ES2, pRSV HA Pit-1-ES3, pRSV HA Pit-1-ES4, pRSV HA Pit-1-ES5, and pRSV HA Pit-1-ES6, as well as pRSV HA Pit-1-AS1, pRSV HA Pit-1-AS2, pRSV HA Pit-1-AS3, pRSV HA Pit-1-AS4, pRSV HA Pit-1-AS5, pRSV HA Pit-1-AS6, pRSV HA Pit-1-AS7, pRSV HA Pit-1-AS8, and pRSV HA Pit-1-AS9,

Transfections-- DNA was introduced into HeLa or GH4 cells by electroporation as follows. Approximately 2-3 × 106 enzymatically dispersed cells were mixed with plasmid DNA in a sterile gene-pulse chamber and exposed to a controlled electrical field of 500 microfarads at 220 V, as described previously (15). Cells from individual transfections were then maintained in Dulbecco's modified Eagle's medium, 15% horse serum, 2.5% fetal bovine serum, and 50 µg/ml penicillin and streptomycin at 37 °C. The nonspecific effects of the RSV or CMV promoters upon transcription factor availability was controlled by including amounts of pRSV or CMV beta -globin plasmid DNA in all assays to render the total pRSV or CMV DNA concentration constant.

Luciferase Assays-- Transient transfections were performed in triplicate, in at least three separate experiments. After incubation for 24 h, cells were harvested with phosphate-buffered saline containing 3 mM EDTA, pelleted, and resuspended in 100 mM potassium phosphate buffer, pH 7.8, 1 mM dithiothreitol. Cells were lysed by three cycles of freeze-thawing and by vortexing between thaws. Cell debris was pelleted by centrifugation for 10 min at 10,000 × g at 4 °C, and the supernatant was used for subsequent assays. Luciferase activity in the supernatant was assayed as described previously (12). Samples were measured in duplicate using a Monolight 2010 Luminometer (Analytical Luminescence Laboratories, San Diego, CA). Relative light units for each transfection were calculated by normalizing for total protein. Protein assays were performed according to the method of Bradford (16) using commercially available reagents (Bio-Rad). Results are expressed as the fold activation of the rPRL promoter ± S.E. for at least three experiments, each in triplicate.

Western Blot Analysis of HA-tagged Pit-1 Proteins-- Transient transfections were performed as above. Cells were harvested with phosphate-buffered saline containing 3 mM EDTA, pelleted, and resuspended in a triethanolamine/SDS solubilization buffer (55 mM triethanolamine, 111 mM NaCl, 2.2 mM EDTA, and 0.44% SDS) (17). Lysed extracts were passed through a 25-gauge needle seven times. The protein content of each extract was assayed according to the method of Lowry et al. (18), using commercially available reagents (Bio-Rad).

Equal amounts (100 µg) of protein from each extract were separated on 15% SDS-polyacrylamide gels and transferred to Immobilon-P (polyvinylidene difluoride) membrane (Millipore, Bedford, MA). The HA-tagged Pit-1 proteins were detected with a mouse monoclonal anti-HA primary antibody (BAbCO, Richmond, CA), secondary sheep anti-mouse HRP-conjugated antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA), and ECL media (Amersham Pharmacia Biotech). Dilutions of 1:1,000 of the primary anti-HA monoclonal antibody and of 1:10,000 of the secondary sheep anti-mouse antibody preparation were used.

Chromatin Immunoprecipitation Studies-- Chromatin immunoprecipitation (ChIP) assays were performed according to the protocol for the acetyl-histone H4 ChIP Assay Kit (Upstate Biotechnology, Lake Placid, NY), as modified by Lambert and Nordeen (19). Transient transfections were performed as above. Twenty four hours after transfection, 2 × 107 GH4 cells were cross-linked by addition of formaldehyde into the medium at a final concentration of 1% and incubated for 15 min at room temperature. Cells were washed with ice-cold phosphate-buffered saline and resuspended in 500 µl of ChIP Lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0, with protease inhibitors). The lysates were sonicated utilizing a Branson Sonifier 450 at power setting 2 with three 10-s pulses at duty cycle 90 and diluted to 3 ml with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, 167 mM NaCl). 1 ml of each sample was precleared by incubating with 80 µl protein A-agarose beads for 30 min at 4 °C with rotation. 5 µl of anti-acetyl histone H4 antibody (Upstate Biotechnology, Lake Placid, NY) was added, and immunoprecipitation was done overnight at 4 °C with rotation. Immune complexes were collected with 60 µl of protein A-agarose and washed once with 1 ml each of the following buffers in sequence: Low Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl), High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 1500 mM NaCl), LiCl Wash Buffer (250 mM LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0); and twice with TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Immune complexes were eluted, and cross-links were reversed by heating at 65 °C and subjected to proteinase K treatment. DNA was recovered by phenol/chloroform extraction followed by ethanol precipitation and was used as a template for PCR (25 cycles) using pA3 -425 PRL Luc promoter-specific commercially synthesized deoxyoligonucleotides (Life Technologies, Inc.) that contain a PRL promoter-specific sequence, GCCTTTCTTTATGTTTTTGGC, and a luciferase-specific sequence, GACTCAAGATGTCAGTCAGC. In addition, internal control PCRs were performed with pSV Ras plasmid-specific commercially synthesized deoxyoligonucleotides (Life Technologies, Inc.) that contain an SV40 promoter-specific sequence, GCATCTCAATTAGTCAGC, and a Ha-Ras-exon-1-specific sequence, ACCAGCTTATATTCCGTC. Control reactions were performed to ensure that all PCR assays took place in the linear range of response to input DNA. PCR products were separated by agarose gel electrophoresis, and bands were imaged and quantified on an Alpha Imager 2000 Gel Documentation System (Alpha Innotech, San Leandro, CA).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The beta -Domain Does Not Simply Disrupt TAD Function to Repress Ras Signaling-- We have previously demonstrated that the beta -domain insertion converts Pit-1 from a co-activator to a repressor of Ras signaling to the rPRL promoter (9, 20). Moreover, we have shown that the amino acid sequence of the beta -domain dictates this repression (8, 11). One possible mechanism for beta -domain-specific repression of Ras signaling would be that beta -domain residues disrupt secondary or tertiary structures within the Pit-1 TAD that are important for Ras signaling. A prediction of this hypothesis would be that a particular Pit-1 TAD structure should be necessary for Ras signaling.

In order to test this prediction, HA-tagged wild-type Pit-1, Pit-1beta , and Pit-1 Delta TAD, which is deleted for amino acids 1-80 (Fig. 1), were introduced into GH4 pituitary cells by electroporation in the presence of a rPRL promoter-driven luciferase reporter and pSV Ras (Fig. 2). As documented previously, co-transfection with wild-type Pit-1 constructs enhanced the Ras response from 3-fold in its absence to 10-13-fold in its presence, and co-transfection of the Pit-1beta isoform not only failed to enhance the Ras response but actually reduced it by more than one-third. The deletion of the Pit-1 TAD, on the other hand, did not significantly interfere with enhancement of Ras signaling by Pit-1 but, in fact, enhanced the response to 9-fold. These data thus demonstrate that the TAD itself is not required for Pit-1 mediation of Ras signaling to the rPRL promoter.


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  		Fig. 1.   Structural organization of Pit-1 isoforms and constructs. Top, Pit-1 with its TAD, POU-specific, and POU homeodomains and their amino acid end points. PB and HDB represent POU-specific and POU-homeodomain basic domains; alpha 1-4 and alpha 1-3 represent their alpha -helices; Hinge represents the region between the TAD and the bipartite DNA-binding domain; FL represents the 15-amino acid flexible linker between the POU-specific and POU-homeodomains. Middle, Pit-1beta with the 26-amino acid beta -domain insertion in its TAD. Bottom, the location of the Delta TAD mutation is shown, a deletion of amino acids 2-80.


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  		Fig. 2.   The beta -domain does not simply disrupt TAD structure. Effects of wild-type and mutant Pit-1 and Pit-1beta constructs on activation of the rPRL promoter in GH4 cells. Plasmid pA3 PRL luc-425 (3 µg) and combinations of pSV Ras (2 µg), pRSV HA Pit-1 (10 µg), pRSV HA Pit-1beta (25 µg), pCGN2-Pit-1 (1 µg), and pCGN2-Pit-1Delta TAD were introduced into GH4 pituitary cells by electroporation. Total pRSV and pCMV plasmid amounts were maintained constant with pRSV beta -globin and pCMV beta -globin DNA. After 24 h, cells were harvested and total light units were measured (see "Experimental Procedures").

Epitope-scanning Mutagenesis of the Pit-1 beta -Domain-- In order to identify beta -domain residues that are functionally important for repression of Ras signaling, we took a two-step approach. In order to identify small regions required for repression, we sequentially altered overlapping 6 amino acid blocks of the beta -domain (Table I). Individual residues in functionally important regions identified were then subjected to alanine-scanning mutagenesis (see below).

                              
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Table I
Epitope-scanning beta -domains

We specifically chose the AU1 epitope (21) to replace 6 amino acid stretches of the beta -domain because it did not affect the ability of mutant Pit-1beta to function as a transcription factor when part of a beta -domain substitution mutant (8). Each mutant beta -domain is of the same size and in the same position as the wild-type beta -domain and differs from the wild type by at most 6 amino acids. The mutant and wild-type constructs were each N-terminally tagged with an HA epitope (8). Thus, all constructs expressed proteins that contained the same epitope in the same relative position to allow for their detection by Western blot analysis regardless of possible alterations of protein structure by the beta -domain substitutions.

Expression of Epitope-scanned Pit-1beta Proteins-- It has been previously shown that wild-type Pit-1 and Pit-1beta constructs express protein at different levels in transient transfection experiments (6, 8). In order to exclude the effect of differences in protein expression level on transcription potency, we carried out a series of transfection experiments to find levels of input DNA that would yield similar levels of protein expression from the wild-type and mutant Pit-1 vectors. In a preliminary experiment, 10 µg of each of the pRSV HA Pit-1 constructs were introduced into HeLa nonpituitary cells and GH4 pituitary cells by electroporation. Extracts from transfected cells were separated by SDS-PAGE, and Western blot analysis was used to determine the level of Pit-1beta protein expression (data not shown). Pit-1beta was expressed at lower levels than Pit-1 as observed previously (8); ES1, ES2, and ES5 were expressed at levels similar to but slightly higher than Pit-1beta ; ES6 was expressed at levels similar to but slightly lower than Pit-1beta ; whereas ES3 and ES4 were expressed at higher levels than Pit-1beta . Relative expression levels of Pit-1beta proteins did not differ between cell lines (data not shown). Guided by these results, varying amounts of each of the mutant constructs were then introduced into GH4 pituitary cells by electroporation, and DNA doses that result in similar levels of protein expression were identified as follows: 10 µg of Pit-1, 25 µg of Pit-1beta , 20 µg of ES1, 20 µg of ES2, 10 µg of ES3 and ES4, 20 µg of ES5, and 30 µg of ES6 (Fig. 3A). In the vector-only lane, protein migrating in the Pit-1 range of 30-33 kDa was not detected. Examination of the relative amounts of wild-type versus mutant Pit-1beta reveals that the levels of all mutant constructs (lanes 4-9) were roughly equivalent to that of wild-type Pit-1beta (lane 3). ES6 (lane 9), however, was expressed at a somewhat lower level than the other constructs. The DNA doses noted above were used for subsequent experiments.


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  		Fig. 3.   The epitope-scanning Pit-1beta proteins retain variable transcription function. A, the various pRSV HA Pit-1 constructs were introduced into GH4 cells by electroporation. In order to achieve equal levels of protein expression for the various HA Pit-1 constructs, varying amounts of each pRSV Pit-1 DNA were introduced, with pRSV levels held constant by the addition of pRSV beta -globin. After 24 h cells were harvested and analyzed by SDS-PAGE and Western blot (see "Experimental Procedures"). Lanes were loaded as follows: No pRSV HA Pit-1 (lane 1); 10 µg of pRSV HA Pit-1 (lane 2); 25 µg of pRSV HA Pit-1beta (lane 3); 20 µg of pRSV HA Pit-1-ES1 (lane 4); 20 µg of pRSV HA Pit-1-ES2 (lane 5); 10 µg of pRSV HA Pit-1-ES3 (lane 6); 10 µg of pRSV HA Pit-1-ES4 (lane 7); 20 µg of pRSV HA Pit-1-ES5 (lane 8); and 30 µg of pRSV HA Pit-1-ES6 (lane 9). B, mutant and wild-type pRSV Pit-1 constructs were introduced into HeLa nonpituitary cells by electroporation with 3 µg of pA3 PRL luc-425. pRSV HA Pit-1 plasmid DNA amounts were adjusted for equal protein expression. Total pRSV plasmid amount was maintained constant with pRSV beta -globin DNA. After 24 h, cells were harvested and total light units measured (see "Experimental Procedures").

Epitope-scanning Pit-1beta Proteins Retain Variable Transcription Function-- Epitope-scanning mutagenesis could have induced alterations in the three-dimensional structure of the mutant Pit-1beta s such that they could no longer activate transcription under any circumstances, and such a result would preclude the identification of functionally important beta -domain residues. To address this problem, we utilized an isoform-insensitive HeLa transcription reconstitution system, in which the Pit-1beta isoform retains basal transcription potency on the rPRL promoter (8).

The wild-type and mutant Pit-1/Pit-1beta constructs were introduced into HeLa nonpituitary cells with an rPRL promoter-driven luciferase reporter, and their ability to transactivate target promoter activity was measured. Pit-1beta displayed a stronger effect on transcription of the target promoter compared with Pit-1 (210- versus 102-fold, respectively) (Fig. 3B). All but one (ES2) of the epitope-scanning mutants resulted in diminished basal activity, compared with that of wild-type Pit-1beta (Fig. 3B), yet were expressed at levels comparable to the wild-type Pit-1beta (Fig. 3A). In particular, the almost 90-fold difference in transcription activation potency between ES2 (347-fold) and ES3 (5-fold) cannot be explained by any difference in protein expression and is likely due to a difference in intrinsic activity of the proteins. These data identify beta -domain amino acids altered by the ES3 mutation but not by the ES2 mutation, PKCL, as contributing to the basal transcription potency of Pit-1beta . Nevertheless, the key point is that the epitope-scanning beta -domain mutants are all capable of transactivating the rPRL promoter, albeit to a varying degree.

Two Regions of the beta -Domain Are Required for Repression of Ras-stimulated rPRL Promoter Activity in Pituitary Cells-- In HeLa nonpituitary cells, both Pit-1beta and Pit-1 activate transcription. However, in GH4 pituitary cells, the two isoforms have opposite transcriptional effects with regard to Ras signaling to the rPRL promoter; Pit-1 enhances and Pit-1beta represses the oncogenic RasVal-12 response (9, 22). Moreover, we have previously demonstrated that the amino acid sequence of the beta -domain dictates this repression (8) (Fig. 2). In order to identify small regions of the beta -domain that are functionally important in repressing the Ras response, the Pit-1beta mutant epitope-scanning constructs were introduced into GH4 pituitary cells by electroporation in the presence of a rPRL-driven luciferase reporter with and without pSV Ras (Fig. 4).


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  		Fig. 4.   Two small regions of the beta -domain mediate repression of Ras-stimulated rPRL promoter activity in pituitary cells. Mutant and wild-type pRSV HA Pit-1 constructs were introduced into GH4 pituitary cells by electroporation with 3 µg of pA3 PRL luc-425 and 5 µg of pSV Ras. Plasmid pRSV HA Pit-1 plasmid DNA amounts were adjusted for equal protein expression (see Fig. 3). Total pRSV plasmid amount was maintained constant with pRSV beta -globin DNA. After 24 h, cells were harvested and total light units measured (see "Experimental Procedures").

As documented previously, co-transfection of a Pit-1 construct enhanced the Ras response from 3-fold in its absence to 9-fold in its presence, whereas co-transfection of the Pit-1beta isoform not only failed to enhance the Ras response but reduced it to 50% below that achieved by Ras alone. This inhibitory effect of Pit-1beta on Ras signaling to the rPRL promoter in pituitary cells defines the stereotypical repressor phenotype of Pit-1beta in this assay (8). Therefore, we tested the Pit-1beta epitope-scanning mutants for their ability to reproduce the repressor phenotype. Three of the epitope-scanning mutations, ES1, ES3 and ES6, repressed the Ras response to the same extent as wild-type Pit-1beta (Fig. 4), despite displaying variable basal transcriptional potencies (Fig. 3B) and equivalent protein expression levels (Fig. 3A). The other three mutations, ES2, ES4, and ES5, switched the Pit-1beta repressor phenotype such that each no longer repressed but instead enhanced the Ras response, just as did Pit-1. These data identify two separate regions of the beta -domain required for repression of the Ras signaling (Table II).

                              
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Table II
Alanine-scanning beta -domains

Alanine-scanning Mutagenesis of the Pit-1 beta -Domain-- We next sequentially substituted alanines for nine implicated residues that reversed the Pit-1beta phenotype whenever changed (Table II). Alanine-scanning mutagenesis (23) was employed because alanine is an uncharged amino acid whose small CH3 side chain should have minimal effects on protein secondary structure. Moreover, since there are no alanines in the beta -domain, each alanine substitution is distinct from the endogenous beta -domain sequence (Table II). Alanine-scanning mutagenesis has been used to probe structure-function correlates in several systems (reviewed in Ref. 24). All constructs were N-terminally HA-tagged to allow identification of DNA doses that result in similar levels of protein expression.

Expression of Alanine-scanning Pit-1beta Proteins-- Ten µg of RSV HA Pit-1, 25 µg of RSV HA Pit-1beta , and 25 µg each of the mutant constructs were introduced by electroporation into HeLa nonpituitary cells (data not shown) and GH4 (Fig. 5A) pituitary cells. Extracts from transfected cells were separated by SDS-PAGE, and Western blot analysis was used to determine the level of Pit-1beta protein expression; the levels of all mutant constructs were roughly equivalent to that of wild-type Pit-1beta . AS3, AS5, and AS6 were expressed at a somewhat higher level than the other constructs, with AS3 displaying a second band of slightly slower mobility. Relative expression levels of Pit-1beta proteins did not differ between cell lines (data not shown). These DNA doses were used for all further experiments.


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  		Fig. 5.   The alanine-scanning Pit-1beta proteins retain transcription function. A, the various pRSV HA Pit-1 constructs were introduced into GH4 cells by electroporation. In order to achieve equal levels of protein expression for the various HA Pit-1 constructs, varying amounts of each pRSV Pit-1 DNA were introduced, with pRSV levels held constant by the addition of pRSV beta -globin. After 24 h cells were harvested and analyzed by SDS-PAGE and Western blot (see "Experimental Procedures"). Lanes were loaded as follows: no pRSV HA Pit-1 (lane 1); 10 µg of pRSV HA Pit-1 (lane 2); and 25 µg each of pRSV HA Pit-1beta (lane 3), pRSV HA Pit-1-AS1 (lane 4), pRSV HA Pit-1-AS2 (lane 5), pRSV HA Pit-1-AS3 (lane 6), pRSV HA Pit-1-AS4 (lane 7), pRSV HA Pit-1-AS5 (lane 8), pRSV HA Pit-1-AS6 (lane 9), pRSV HA Pit-1-AS7 (lane 10), pRSV HA Pit-1-AS8 (lane 11), and pRSV HA Pit-1-AS9 (lane 12). After 24 h cells were harvested and analyzed by SDS-PAGE and Western blot (see "Experimental Procedures"). B, mutant and wild-type pRSV Pit-1 constructs were introduced into HeLa nonpituitary cells by electroporation with 3 µg of pA3 PRL luc-425. Plasmid pRSV HA Pit-1 plasmid DNA amounts were adjusted for equal protein expression (see Fig. 5). Total pRSV plasmid amount was maintained at a constant level with pRSV beta -globin DNA. After 24 h, cells were harvested and total light units measured (see "Experimental Procedures").

Alanine-scanning Pit-1beta Proteins Retain Transcription Function-- All of the alanine-scanned Pit-1beta proteins retained basal transcriptional potency in the isoform-insensitive HeLa reconstitution assay (Fig. 5B). In contrast to the epitope-scanning (ES) proteins, for which transcriptional potency was variable (Fig. 3B), the alanine-scanning (AS) proteins displayed basal activity comparable to wild-type Pit-1beta (~140-fold) (Fig. 5B). The higher transcription potency of AS6 (478-fold) or the lower transcription activity of AS4 (~67-fold) cannot be explained by extreme levels of protein expression, since AS6 and AS4 are expressed at levels equivalent to the other AS mutants and wild-type Pit-1beta (Fig. 5A). In addition, AS3, despite the novel slower-migrating band, activated the rPRL promoter to the same extent as did wild-type Pit-1beta . Again, the key point is that the alanine-scanning beta -domain mutants are all capable of transactivating the rPRL promoter in an isoform-insensitive assay.

Five Hydrophobic beta -Domain Residues Mediate Repression of the rPRL Promoter Ras Response-- In order to identify specific beta -domain residues that are required for repression of Ras signaling, the alanine-scanning constructs were assessed for retention of the Pit-1beta repressor phenotype in GH4 pituitary cells. Again, Pit-1 enhanced the Ras response from 6-fold without Pit-1 to 13-fold with Pit-1, whereas the Pit-1beta isoform inhibited the Ras response by 50% (Fig. 6A). Four of the alanine-scanning proteins, AS3, AS4, AS7, and AS9, acted like wild-type Pit-1beta and repressed the Ras response (Fig. 6A). Despite the aberrantly sized band seen with AS3 (Fig. 5A), it had no effect on repression, and AS3 repressed the Ras response at least as well as Pit-1beta (Fig. 6A). Five mutations, AS1, AS2, AS5, AS6, and AS8, abrogated beta -domain mediated repression of the Ras response. Two of these mutations, AS5 and AS8, eliminated the beta -domain-mediated repression and restored the 6-fold Ras response but did not allow further enhancement of the Ras effect (Fig. 6A). In contrast, three mutations, AS1, AS2, and AS6, switched Pit-1beta into an enhancer of Ras signaling, such that they functioned essentially the same as Pit-1 (~13-fold) (Fig. 6A). These data identify five beta -domain amino acids (leucine 7, isoleucine 8, tyrosine 17, phenylalanine 18, and methionine 20) (Fig. 6B) that are required for repression of the Ras response of the rPRL promoter.


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  		Fig. 6.   Five hydrophobic beta -domain residues mediate repression of Ras-stimulated rPRL promoter activity in pituitary cells. A, mutant and wild-type pRSV Pit-1 constructs were introduced into GH4 pituitary cells by electroporation with 3 µg of pA3 PRL luc-425 and 5 µg of pSV Ras. Plasmid pRSV HA Pit-1 plasmid DNA amounts were adjusted for equal protein expression (see Fig. 5). Total pRSV plasmid amount was maintained constant with pRSV beta -globin DNA. After 24 h, cells were harvested and total light units measured (see "Experimental Procedures"). B, the five hydrophobic residues leucine 7, isoleucine 8, tyrosine 17, phenylalanine 18, and methionine 20, which are required for beta -domain-dependent repression of Ras signaling.

Pharmacological Inhibitors of Histone Deacetylation Reverse the beta -Domain-dependent Repressor Phenotype-- Two distinct models could explain beta -dependent repression of Ras signaling to the rPRL promoter. In one model, the Pit-1beta isoform acts as a classic dominant-negative inhibitor of Ras signaling, by binding to the Pit-1 site (FP IV) of the rPRL promoter composite Pit-1/Ets-1 Ras response element required for the Ras effect (9, 22). In the second model, the beta -domain imparts the repressor phenotype by modulating functional interaction with, or the function of, an HDAC, and thus altering the acetylation state of the PRL promoter.

To distinguish between these two models for beta -domain-dependent repression, we first tested the role of HDACs using sodium butyrate and trichostatin A. Both sodium butyrate and trichostatin A have been widely used as pharmacological inhibitors of histone deacetylase activity (25, 26) and are being considered for clinical use in managing certain cancers (reviewed in Ref. 27). We used EtsZ, a recombinant dominant-negative inhibitor, as a control. EtsZ consists of an Ets DNA-binding domain fused to LacZ and prevents transactivation of target promoters by competitively binding to Ets-binding sites (EBS) on DNA (13). Specifically, EtsZ blocks the Ras response of the rPRL promoter by interfering with endogenous Ets-1 binding to the rPRL promoter composite Pit-1/Ets-1 Ras response element (20, 28). EtsZ was therefore expected to be insensitive to treatment with histone deacetylase inhibitors.

In the absence of histone deacetylase inhibitors, Ras activated rPRL promoter activity by 4-fold, whereas Pit-1beta and EtsZ repressed the Ras response by 50 and 66%, respectively (Fig. 7). However, in the presence of 5 mM sodium butyrate, Pit-1beta enhanced the 5-fold activation by Ras to 16-fold, whereas EtsZ-mediated repression was unchanged. Addition of 100 ng/ml trichostatin A also switched Pit-1beta into an enhancer of Ras signaling. Trichostatin A and Pit-1beta increased the 7-fold activation by Ras alone to 28-fold but had no effect on EtsZ, which continued to strongly repress the Ras response. Thus, both sodium butyrate and trichostatin A reversed beta -domain-mediated repression of the Ras response yet had no effect on repression by EtsZ, the competitive inhibitor of EBS binding. Moreover, similar effects were seen with 50 and 200 ng/ml trichostatin A (data not shown). These results implicate histone deacetylation in the mechanism of beta -domain-dependent repression of Ras signaling and demonstrate that the mechanisms by which the beta -domain and the simple dominant-negative inhibitor, EtsZ, repress Ras signaling are distinct.


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  		Fig. 7.   Pharmacological inhibitors of deacetylation reverse beta -domain-dependent repression of Ras signaling. Plasmid pA3 PRL luc-425 (3 µg) and combinations of pSV Ras (2 µg), pRSV-Pit-1beta (25 µg), and pAPR EtsZ (5 µg) were introduced into GH4 pituitary cells by electroporation. Trichostatin A (TSA) (Sigma) or sodium butyrate (Butyrate) (Sigma) were added to the medium immediately post-transfection at concentrations of 50 ng/ml and 5 mM, respectively. After 24 h, cells were harvested and total light units measured. Results are expressed as the fold activation of the rPRL promoter ± S.E. for at least three experiments and nine transfections (see "Experimental Procedures").

v-Ski Protein Reverses beta -Domain-dependent Repression-- Pit-1 has recently been shown to interact with both a co-repressor complex that contains an N-CoR/mSin3 HDAC and a signal-dependent co-activator complex (29). The Sloan-Kettering virus Ski oncogene, v-ski (30), is a dominant-negative viral form of mammalian c-Ski protein and a specific inhibitor of transcriptional repression mediated by N-CoR/mSin3-containing HDACs. We examined the effect of v-Ski on Pit-1beta -mediated repression of the Ras response, in order to determine whether the Pit-1beta repressor phenotype is mediated by an N-CoR/mSin3-containing HDAC.

In the absence of v-Ski, Ras activated rPRL promoter activity by 3-fold, whereas Pit-1beta and EtsZ reduced it (Fig. 8). However, co-transfection of pRSV t3 v-Ski (5 µg) switched Pit-1beta from a repressor to an enhancer of the Ras response, increasing the 4-fold activation by Ras to 9-fold. EtsZ-mediated repression was unchanged. In addition, 2- and 10-µg doses of pRSV t3 v-Ski reversed the Pit-1beta repressor phenotype (data not shown) without affecting the EtsZ-mediated inhibition. Thus, v-Ski, like the pharmacological histone deacetylation inhibitors, sodium butyrate and trichostatin A, blocks the repressive effect of the beta -domain yet has no effect on repression by EtsZ.


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  		Fig. 8.   Dominant negative v-Ski reverses beta -domain-dependent repression of Ras signaling. Plasmid pA3 PRL luc-425 (3 µg) and combinations of pSV Ras (2 µg), pRSV-Pit-1beta (25 µg), pAPR EtsZ (5 µg), and pRSVt3 v-Ski (5 µg) were introduced into GH4 pituitary cells by electroporation. After 24 h, cells were harvested and total light units measured (see "Experimental Procedures").

Pit-1beta Alters the Acetylation State of the Proximal PRL Promoter-- In order to test directly the hypothesis that the Pit-1 beta -domain represses PRL promoter expression by altering the level of histone deacetylation of the proximal PRL promoter, we performed a ChIP assay. The ChIP assay has been used by a number of laboratories to probe the effects of hormones, DNA-binding factors, and transcription cofactors on the acetylation state of chromatin (reviewed in Ref. 19). In the assay, total cellular extracts are reversibly cross-linked, and DNA bound to acetylated histone proteins is immunoprecipitated with antibodies directed against acetylated histones. The cross-links are reversed and DNA-precipitated, and DNA sequences of interest are amplified by PCR. Changes in the amounts of specific PCR products reflect changes in the amount of acetylated histone bound to the DNA sequence.

We examined the effect of Pit-1 and Pit-1beta on the acetylation state of the proximal PRL promoter in two independent ChIP assays with internal control experiments to verify that the biological effect in question, repression of Ras activation of PRL promoter activity, had occurred (see "Experimental Procedures"). In addition we utilized the SV40 promoter of the pSV Ras plasmid as a control promoter to demonstrate that Pit-1beta -dependent changes in histone deacetylation were specific for the proximal PRL promoter and not global changes in histone deacetylation.

In two independent ChIP assays, Pit-1 increased the amount of proximal PRL promoter associated with acetylated histone H4 by 2-3-fold, whereas Pit-1beta decreased the amount of proximal PRL promoter associated with acetylated histone H4 by 60% (Fig. 9). Neither Pit-1 nor Pit-1beta had an appreciable effect on the amount of SV40 internal control promoter associated with acetylated histone H4. In parallel control experiments to verify Pit-1beta repression of Ras signaling, Pit-1 enhanced the Ras response from 2- to 4-fold, whereas the Pit-1beta isoform inhibited the Ras response by 80% (data not shown). Thus, the presence of the beta -domain endows Pit-1beta with the ability to increase histone deacetylation in a target promoter-dependent manner.


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  		Fig. 9.   Pit-1beta alters the acetylation state of the proximal PRL promoter. Plasmid pA3 PRL luc-425 (3 µg), pSV Ras (2 µg), and either pRSV-Pit-1 (10 µg) or pRSV-Pit-1beta (25 µg) were introduced into 10 100-mm plates of GH4 (2 × 107) pituitary cells by electroporation. After 24 h, eight plates were harvested for chromatin immunoprecipitation assays, and two plates were harvested for luciferase assays (see "Experimental Procedures"). A, a representative ChIP assay is shown; Blank lanes, negative control that had no template for the PCR; B, two independent ChIP assays are shown, expressed as relative amounts of target and control promoter DNA associated with acetylated histone H4 (in arbitrary densitometric units).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although a number of functionally distinct transcription factor isoforms with identical DNA specificities have been identified (1-6, 31), the molecular mechanisms by which structural alterations outside of DNA- or ligand-binding domains specify altered transcriptional function remains a relatively unstudied subject. The work presented here shows that the naturally occurring beta -domain insertion converts the Pit-1beta isoform into a transcriptional repressor by modulating functional interaction with histone deacetylases.

Only two other transcription factor splice isoforms, both nuclear receptors, are known to alter distinct interactions with co-activators or co-repressors. Alternate exon usage in the TRbeta 2 isoform of TRbeta introduces a novel N terminus that reverses a functional interaction with N-CoR, thus altering the mechanism of ligand-independent repression (32). The HNF4alpha 2 splice isoform of nuclear receptor hepatocyte nuclear factor 4alpha (HNF4alpha ) contains a 10-amino acid insertion in the inhibitory F-domain, which abrogates an F-domain block of interaction with the GRIP1 co-activator (33). Thus, the Pit-1/Pit-1beta isoform pair represents the first example of differential interaction with either co-repressors or co-activators by transcription factor splice isoforms outside of the nuclear receptor family.

Transcriptional repressors can act through either passive or active mechanisms (reviewed in Refs. 34 and 35). Passive transcriptional repressors act by blocking such functions as nuclear localization or DNA binding that are necessary for transcriptional activation but that are not directly involved in nuclear signal transduction. Such repressors as Drosophila knirps protein, (36), mammalian ATF-2 (37), and Drosophila I-POU protein (38) can function as passive repressors. EtsZ, the recombinant dominant-negative construct used in this article, also acts as a passive binding site competitor in the context of the rPRL FPIV composite Ras response element (28). Active repressors interfere with the molecular mechanism of signal transduction in an activator-specific manner by blocking activation functions of specific transcription factors, recruiting co-repressors, or by sequestering co-activators. Examples of active repressors include YY1 repression of CREB-mediated transcription (39), KRAB-KAP-1 repression (40), the t(8;21) AML-1/ETO fusion protein (41), Ume 6 (42), TR (43), PLZF protein (44), and Laz3/BCL6 oncoprotein (45).

The data presented here (Figs. 7-9) clearly demonstrate that the beta -domain endows the Pit-1beta isoform with the properties of an active repressor. Pit-1beta blocks Ras signaling through a mechanism quite distinct from that of EtsZ, the artificially constructed passive binding site competitor. Moreover, by four separate methodological approaches (site-specific mutagenesis, pharmacological inhibitors, co-expression of a dominant-negative effector, and chromatin immunoprecipitation assay), we show that five hydrophobic residues present as a bipartite element within the beta -domain and that the beta -domain mediates repression of the Ras response by modulating the acetylation state of the proximal PRL promoter in a manner dependent upon an N-CoR/mSin3-HDAC with Pit-1. The ability of two pharmacological inhibitors of histone deacetylase activity, trichostatin A and sodium butyrate, to reverse beta -domain repression of Ras signaling to the rPRL promoter is consistent with such a mechanism for beta -domain repression. In fact, sodium butyrate and trichostatin A appear to phenocopy some of the epitope- and alanine-scanning mutations of the beta -domain and convert Pit-1beta from a repressor to an enhancer of Ras signaling. By contrast, neither of these inhibitors affected the dominant-negative EtsZ inhibitory response (Fig. 7). This specificity of the pharmacological inhibitors for the beta -domain-dependent repressor phenotype supports the validity of this approach and our interpretation of the data.

The use of v-Ski as a molecular tool allowed us to address the following two concerns raised by the pharmacological inhibitor experiments used here: 1) the possibility that these inhibitors had nonspecific effects, and 2) that the pharmacological approach cannot distinguish between different HDAC subtypes. The c-Ski protein is a normal component of an HDAC subtype, and it serves to recruit N-CoR and mSin3 (14, 46). The oncogenic form, v-ski, lacks the mSin3-binding domain and inhibits transcriptional repression by Rb, Mad, and TRbeta (14, 46). Thus, v-Ski is a specific inhibitor of HDAC-mediated transcriptional repression and provides independent evidence for the specificity of the beta -domain repression mechanism.

The ChIP assays allowed us to demonstrate directly that the Pit-1beta isoform causes a decrease in acetylation of histones associated with the proximal PRL promoter in the presence of activated Ras. The lack of change in the acetylation state of the control promoter shows that the transcriptional effects of the Pit-1beta isoform are not global in nature but specific to Pit-1 target genes.

Recently it had been shown that an N-CoR/mSin3-containing-HDAC is known to associate with the POU domain of Pit-1 and that the balance of interaction between this HDAC and signal-specific co-activators with Pit-1 determines the transcriptional activity of Pit-1 (29). Taken together with these data, our findings that the Pit-1 beta  isoform modifies the acetylation state of the proximal PRL promoter and that dominant-negative v-Ski blocks the ability of the beta -domain to repress Ras activation of the proximal PRL promoter suggest a model in which the beta -domain modulates an already existing functional interaction between Pit-1/Pit-1beta and N-CoR/mSin3 HDAC. The exact mechanism by which the beta -domain may modulate this functional interaction remains to be determined.

The fact that the Pit-1beta isoform only acts as a repressor in pituitary cells, and actually is a superior mediator of protein kinase A signaling to the rPRL promoter in nonpituitary cells (8, 11), suggests that the beta -domain functions through a signal-specific (and possibly even cell-type-specific) rather than a global mechanism. It is important, then, to note that Pit-1 functions in Ras signaling as part of a multicomponent complex consisting of signal-specific CBP/p300 co-activators and an N-CoR/mSin3 co-repressor (29), as well as Ets-1, a prototypical member of the ETS transcription factor family. Ets-1 and Pit-1 functionally interact via the (Pit-1)/EBS (Ets-1) composite DNA element in the rPRL promoter (9, 22, 28), and Ets-1 is a direct nuclear target of the Ras/mitogen-activated protein kinase signaling pathway through phosphorylation of a conserved threonine 82 residue (9, 47, 48). Moreover, Ets-1 interacts with the same region of CBP as Pit-1 (29, 49). Therefore, the specific mechanism of beta -domain-dependent repression may take place in the context of a Pit-1·Ets-1 complex in balanced interaction with co-activator and co-repressor complexes.

It is of interest, then, that the beta -domain blocks synergy between Pit-1 and Ets-1 in HeLa nonpituitary cells (reviewed in Ref. 8). Our recent finding that the beta -domain can interact physically with Ets-1 in vitro (50) raises the possibility that the beta -domain might function by blocking the ability of Ets-1 to respond to Ras signaling, perhaps by blocking access to the crucial threonine 82 mitogen-activated protein kinase phosphorylation site. Thus, the beta -domain may function by modulating the ability of either a single component of the Pit-1·Ets-1 complex or a novel interaction surface generated by the complex to interact with co-repressor(s).

The role of the beta -domain in basal transcriptional activity of Pit-1beta is unclear at this time. For example, Pit-1 lacks the beta -domain yet retains basal transcription function in the HeLa reconstitution assay (Figs. 3A and 5A), and complete substitution mutations of the