Cell, Vol. 61, 1021-1033,

June

15, 1990, Copyright

0 1990 by Cell Press

The PO&Specific Domain of Pit-l Is Essential for Sequence-Specific, High Affinity DNA Binding and DNA-Dependent Pit-l-Pit-l Interactions Holly A. Ingraham:t Sarah E. Flynn,‘t Jeffrey W. Voss,? Vivian R. Albert,*t Michael S. Kapiloff,t Laura Wilson,7 and Michael G. Rosenfeld*t * Howard Hughes Medical Institute t Eukaryotic Regulatory Program and Center for Molecular Genetics School of Medicine M-013 University of California, San Diego La Jolla, California 92093

Summary Pit-l is a member of a family of transcription factors sharing two regions of homology: a highly conserved POU-specific (PO&) domain and a more divergent homeodomain (POU”c). Analysis of mutant Pit-l proteins suggests that, while the POllHo is required and sufficient for low affinity DNA binding, the PO& domain is necessary for high affinity binding and accurate recognition of natural Pit-l response elements. Pit-l is monomeric in solution but associates as a dimer on its DNA response element, exhibiting DNAdependent protein-protein interactions requiring the PO& domain. Analysis of a-helical domains and conserved structures in Pit-l suggests that POU domain proteins interact with their DNA recognition sites differently than classic homeodomain proteins, with both the POUHD and the POUs domain contacting DNA. Transcriptional activity of Pit-l on enhancer elements is conferred primarily by a Ser- and Thr-rich N-terminal region unrelated to other known transcription-activating motifs. Introduction Recently, a structural motif referred to as the POU domain has been identified in a large family of tissue-specific transcription factors. This motif contains two regions, 67-70 and 60 amino acids in length and separated by a nonconserved sequence, termed the PO&specific (POUs) domain and the POU homeodomain (POUH~), respectively (Herr et al., 1988). Initial recognition of this family resulted from the molecular cloning of cDNAs encoding several tissue-specific frans-activators, including the pituitaryspecific transcription factor Pit-l (Ingraham et al., 1988; Bodner et al., 1988) which has been shown to be capable of regulating transcription of both prolactin and growth hormone genes (Ingraham et al., 1988; Mangalam et al., 1989; Larkin et al., 1990; Fox et al., 1990; 2. D. Sharp, personal communication); the B cell-specific factor Ott-2 (Ko et al., 1988; Clerc et al., 1988; Scheidereit et al., 1988; Mijller et al., 1988a); the related, widely expressed transcription factor Ott-1 (Sturm et al., 1988); and the Caenorhabditis elegans gene product uric-86, established genetically to be critical for accurate neuronal lineage formation (Finney et al., 1988). While the POUs domain is

unique to members of this protein family, the POUHD, although quite divergent, shares both sequence homology (33%) and the predicted a-helical structures with classic homeodomain-containing proteins. With the identification of multiple members of the POU domain protein family in mammals (He et al., 1989), Drosophila (Johnson and Hirsch, 1990; M. N. Treaty, H. Xi, V. Hartenstein, and M. G. Rosenfeld, submitted), and C. elegans (Burglin et al., 1989) it is clear that the POU domain is remarkably conserved among all known members. The importance of homeodomain proteins in establishing the precise temporal and spatial pattern of development has been clearly documented by genetic studies (reviewed in McGinnis et al., 1984a, 1984b; Scott and Carroll, 1987; Gehring, 1987; Ingham, 1988; Scott et al., 1989). Recently, it has been demonstrated that Drosophila homeodomain proteins can specifically bind to promoter regions in genes encoding their own and other homeodomain proteins (e.g., Hoey and Levine, 1988; Desplan et al., 1988; Beachy et al., 1988; Winslow et al., 1989). This ability to bind DNA has been shown to reside in the homeodomain region of these proteins (Hall and Johnson, 1987; Mihara and Kaiser, 1988; Hoey et al., 1988; Miiller et al., 1988b). The classic homeodomain is predicted to form a helix-turn-helix motif (Laughon and Scott, 1984) similar to the DNA binding domain established for prokaryotic repressors (for review see Ptashne, 1986). In addition, high resolution NMR analyses of purified Antennapedia homeodomain protein has suggested the importance of three helical structures and two clusters of basic residues flanking these helices (Miller et al., 1988b; Otting et al., 1988). Similarities between classic homeodomain-containing proteins and POU domain proteins predicted that DNA binding would be confined to the POUHo. However, in vitro translation products of mutant Ott-1 proteins lacking only the POUs domain were reported incapable of binding DNA (Sturm and Herr, 1988). It therefore became important to determine the potential functions of the POUs domain and its contributions in distinguishing these related gene families. Here, the role of the POUs domain has been investigated using Pit-l as a representative member of this gene family. We report that although the Pit-l POUHo is sufficient for low affinity binding (Ko ~10~~ M) with relaxed specificity, the POUs domain confers sitespecific, high affinity binding (Ko ~0.5 x 1O-g M) and contributes to DNA-dependent Pit-l-Pit-l interactions. Based on analyses of predicted helical motifs, it is suggested that the requirements for high affinity binding of POU domain proteins differ from those of the classic homeodomain proteins, Results The POUs Domain Is Required for High Affinity Binding Pit-l binds to a series of sequence-related c&active elements in the rat growth hormone and prolactin genes that partly confers pituitary-specific expression of these two

Cell 1022

transcription units (Nelson et al., 1988; lngraham et al., 1988). Initial experiments were designed to establish the precise domains in Pit-l required for specific, high affinity binding. Bacterially expressed wild-type and mutant Pit-l proteins were examined for binding activity using a response element from the rat prolactin promoter region (Prl-lP, bp -85 to -42). Deletions of the N-terminal residues (AN, A8-128) or C-terminal residues (AC, A273-291) had no effect on DNA binding activity (Figures 1A and 1B). Native wild-type Pit-l gave rise to dominant and minor species termed Cp and C,, respectively. These species are also observed with both AN and AC proteins and in all cases are selectively competed with a lOO-fold excess of unlabeled Prl-1P but not with an unrelated oligonucleotide, the engrailed binding site (see Figure 1B). Deletion of the POUHo (APOUHD, A213-272) entirely abolished DNA binding, indicating that the POUs domain alone possessed no DNA binding properties (Figure 1B). In contrast, selective deletion of the entire POUs domain (APOlJs, A128-198) did not abolish DNA binding (Figure lB), but did result in a marked decrease in affinity, as demonstrated by the requirement for 40-fold more protein and a 8-fold longer exposure time for observation of even a weak signal. Marked relaxation of site specificity was demonstrated by the requirement of a lO,OOO-fold excess of either Prl-1P or engfailed DNA sites in order to observe significant competition (data not shown). Enhancement of this binding was achieved by the addition of a second homeodomain region in substitution of the POUs domain (APOUs-HD2), but did not restore wild-type affinity and exhibited some competition with a nonspecific site. Previous data showing that the POUs region of Ott-1 was absolutely required for DNA binding (Sturm and Herr, 1988) may reflect the low concentrations of in vitro translation product used in the assays. Consistent with this explanation, APOUs protein failed to yield detectable DNA binding when extremely low concentrations were employed (data not shown). Protein levels were quantitated prior to DNA binding experiments by Western blot analysis using independent antibodies recognizing both the N- and C-terminal regions of Pit-l (data not shown). Binding constants were determined at equilibrium for wild-type Pit-l, APOUs, and APOUs-HD2 using limiting protein concentrations, radiolabeled DNA at concentrations well below the expected Ko, and increasing amounts of unlabeled DNA. Binding saturation curves are shown in Figure 2A with the corresponding Scatchard analyses. Wild-type Pit-l protein, assayed with a Prl-1P site, revealed a K. of 0.5 + 0.06 x 10eg M. This same relative Ko was also obtained using another independent assay, the avidin-biotin complex DNA assay (Glass et al., 1989), in which labeled Pit-l protein was incubated with unlabeled DNA (described below and in Experimental Procedures). Both assays revealed a similar binding constant, as is shown in Figure 2A. Nearly equivalent binding constants were obtained with AN protein, KD = l-2.5 x 10mg M. A similar analysis of the c&active element of the rat growth hormone promoter GH-1 (-96 to -66), established to bind Pit-l (Mangalam et al., 1989), indicated a comparable binding constant with an estimated value of KD 0.7-l x 10Vg M.

Blndmg +++ +++ +++ +++ +

+++ ++

Figure 1. DNA Binding Proteins

Characteristics

(A) Diagrams of deletion constructions. matically; hatched and shaded regions

of Wild-Type

and Mutant

Pit-l

Wild-type Pit-l is shown schedepict the POUs domain and

the POUHD,respectively. Specific deletions, constructed as described in Experimental Procedures, are indicated. Relative levels of binding on a Prl-1P oligonucleotide, CCTGATTATATATATATTCATGAA, corresponding to coordinates -65 to -42 in the rat prolactin gene are shown with a plus sign for each construct ‘(B) Examples of binding are shown using the indicated amount of relative bacterial Pit-l proteins for wild-type Pit-l (WT), AN, AC, APO&-HD2, APOUHD,and APOlls. Binding to labeled Prl-IP probe was with a lOO-fold excess of unlabeled Prl-1P (P) or unlabeled engrailed site (E) (see Figure 3A for sequence), except in the case of APO& and APO&HD2, where a lO,OOO-fold excess was used. Cp and C, are also shown (analyzed in Figure 6). Exposure time of the autoradiogram was &fold longer for APO& and APO&D as indicated by the asterisks. Also note the increased amounts of protein used to obtain protein-DNA complexes in the cases of APOUs and APOUs-HD2. Aliquots of extracts used in binding studies were run on a 12% SDS-polyacrylamide gel, transferred to niVocellulose, and bound with an antibody reactive to the N- or C-terminal peptide to quantitate Pit-l protein (data not shown).

However, the removal of the POUs domain greatly reduced the affinity to a value of Ko 1 f 0.15 x 10m6, and full saturation was not observed even at levels approaching 10m5 M, consistent with the marked decrease in measured affinity and the compromised gel mobility shift pattern, as documented in Figure 1B. As was observed previously in a gel retardation assay, inserting a second homeodomain in this mutant (APOUs-HDZ) stabilized binding, as seen by the lo-fold lower Ko (1 f 0.1 x lo-’ M). DNAase I footprint analysis of the rat GH-1 promoter region (-180 to +l) (Figure 28) or the rat prolactin promoter region (data not shown) using an excess of proteins in which the first portion (APOUs-A, A128-160) or the entire POUs domain was removed revealed a complete absence of binding; in con-

POU-Specific 1023

Domain

2

of Pit-l

4

i

6

DNA CONCENTRATION (nkl)

APOU,

t

i

i

APOU~ -HD, _______-I

i

DNA CONCENTRATION (nM)

.

.06 .06 .04 .02 hW ,““l 1

DNA CONCENTRATION (UN)

Figure

2. DNA Binding

Affinity

Is Greatly

2

3

4

DNA CONCENTRITION (uM)

Reduced

after

Removal

of the POUs Domain

(A) Measurement of binding constants for wild-type and mutant Pit-l. Binding affinities were determined under conditions of limiting protein concentration with increasing probe concentrations of oligonucleotides representing Pit-l binding sites (Prl-1P or GH-1). Retarded complexes and unbound probe were measured by a computer-controlled imaging system that directly quantitates radiation (AMBIS). Binding constants for wild-type Pit-l (WT) on Prl-1P were determined by both gel retardation (closed circles) and the avidin-biotin DNA assay (open circles) (described in legend to Figure 5). Each saturation binding curve is representative of at least two independent sets of experiments, and the calculated binding constant is presented in the lower right corner of each graph. (B) DNAase I footprinting was performed as previously described (Nelson et al., 1988) using the GH-1 promoter region (-180 to +I). generated by polymerase chain reaction using oligonucleotides corresponding to positions -180 to -188 and -21 to +I, with the former end labeled at position -180. Relative amounts of protein used in each assay are indicated.

trast, footprints were clearly observed with 200-fold less wild-type Pit-l protein (Figure 28). The lack of apparent footprints provided a third, independent demonstration of reduced binding affinities for Pit-l mutants with full or partial deletion of the POUs domain. The POUs Domain Contributes to Site Recognition The observation that proteins containing only the POUnc could bind to DNA with very low affinity raised the possibility that the POUs domain contributed in a critical fashion to binding site specificity. Binding experiments were therefore performed on a series of sites in which Pit-l is known to bind with varying affinities, to determine whether removal of the POUs domain would alter the relative hierarchy of binding. Binding sites and their relative hierarchy of binding by wild-type Pit-l are shown in Figure 3A. As previously described, Pit-l binds with about equal affinity to a GH-1 or Prl-IP site; their patterns of binding are identical with formation of two different migrating complexes, referred to as C2 and C,. However, when the core consensus site T/AT/ATATNCAT was altered to include an octamer sequence, PRL-OCT (Elsholtz et al., 1990), or the 5’ AT-rich region preceding the TT/ATATTCAT sequence was altered to include GC residues (PRLATT MUT) (Figure 3), binding was greatly reduced, and the nature of

binding was altered such that a single species (C,) is observed rather than two. Removing the POUs domain revised this binding hierarchy. While binding was still observed with a Prl-1P site using APOUs and APOUs -HDs proteins, little or no binding was observed with either the GH-1 or the PRL-A/T MUT sites (see Figure 3A). These data suggested that the POlJnc, by itself, preferentially bound to the AT-rich region and not the TTIATATTCAT region. Consistent with this interpretation was the apparent binding by APO& to the engrailed site, which also contains a long uninterrupted stretch of AT-rich sequence. These data strongly suggest that the POUs domain has an important role in establishing correct DNA site recognition. To analyze directly the specific sequences recognized by wild-type and mutant Pit-l proteins, an in situ copperphenanthroline footprinting technique was used. Complexes identical to those shown in Figure 16 were isolated and analyzed as previously described (Kuwabara and Sigman, 1987). Cleavage patterns of unbound probe, the Pit1, Cpr and C, complexes, and APO& complexes were analyzed and compared (Figure 36). As expected, the protected sequence observed with wild-type Pit-l protein centered over the TT/ATATTCAT region (shown in the antisense direction). In contrast, Pit-l protein lacking the

Cell 1024

PRkOCT PRL-A,T

MUT

++ ++

++

EN-1

WT GH

P

APO&HD,

it

APOU,'

OPJTEGHPOA’TEGHPO~E

B.

C

Figure

3. Deletion

of the POUs

Domain

Affects

DNA Site Specificity

(A) Relative binding affinities of wild-type and mutant Pit-l proteins minus the POUs domain are shown for each site: GH-6x1 (GH), Prl-1P (P), the two bp mutant octamer binding site, P-Ott (0), a mutant Prl-1P site in which AT residues are replaced by GC residues (A/T), and the engrailed binding site (E). The relative amounts of protein used in binding sites are equivalent to those used in Figure 16, and the exposure time for APOUs is B-fold longer, as noted by the asterisk. (B) Footprint analysis of wild-type and mutant Pit-l DNA complexes. In situ copper-phenanthroline footprinting was carried out as described in Experimental Procedures using protein-DNA complexes generated by gel retardation, examples of which are shown in (A). The cleavage pattern of unbound probe (Free), wild-type species (C, and C,), and the major species of APO& and APOUs-HDs are shown for the Prl1P probe labeled in the sense or antisense direction. The corresponding sequence as determined by a G+A ladder is shown on either side of the cleavage patterns. Areas of footprints are depicted schematically for wild-type (filled bars) and APOUs mutants (hatched bars) above and beside the sequences in (A) and (B), respectively.

POUs domain showed little or no protection of this same region (antisense), but exhibited significant footprinting in the AT-rich region. The sense and antisense cleavage patterns (Figure 36) of wild-type and mutant Pit-i proteins (APO& domain) partially overlap, and while precise boundaries are difficult to assign, the region protected by wild-type Pit-l, in comparison to APOUs protein, is distinctly different, supporting the DNA binding studies described above. Chimeras of Pit-l and Ott-1 that bind to distinct, welldefined sequences were generated totest further the hypothesis that the POUs domain contributed to binding specificity. Sequences encoding the POUs domain, the POUH~, or the entire POU domain of Ott-1 were inserted into the corresponding positions of Pit-l cDNA, as schematically diagrammed in Figure 4A. Binding studies used Prl-1P or an altered site that contained the two bp alterations required to generate the canonical octamer binding sequences (P-Ott). These analyses revealed a similar pattern of preference for the Prl-1P and P-Ott sites by both wild-type Pit-l and the chimera containing the Ott-1 P0tJ~o region (Figure 48). Similar data showed that replacement of only the POUs region from Ott-I with that of Pit-l reduced affinity to an octamer DNA sequence (Stern et al., 1989). The analysis of chimeras in which the Ott-1 POUs domain replaces the Pit-l POUs domain is complicated by the fact that preference of Ott-1 for a P-Ott site (Figure 4) is only slightly higher than for a Prl-1P site (Elsholtz et al., 1990; data not shown), and, indeed, substituting the entire POU domain of Pit-l with that of Ott-1 (Octpous/Octnc) reversed the pattern of site preference, with the P-Ott site slightly preferred to the Prl-1P site (Figure 48). When the POUs domain of Ott-1 replaced comparable Pit-l sequences (OctPOus/PitHD), binding to both recognition sequences was observed (Figure 48). To examine the relative affinities in a more precise fashion, competition experiments were performed using radiolabeled Prl-1P mixed with either unlabeled Prl-1P or P-Ott oligonucleotides. The competition curves observed with wild-type Pit-l and a chimera containing an Ott-1 POUH~ were similar, such that Prl-1P but not P-Ott competed readily, implying that both proteins preferred a Prl-1P site (Figure 4C). When the POUs domain was contributed by Ott-1, both the Prl-1P and P-Ott sites competed almost equivalently, displaying a slightly higher affinity for the Prl1P site (Figures 48 and 4C). Although these data imply that the POUs region is crucial in conferring specificity of binding, the lack of a complete reversal by the OctpouJ PitHo chimera for a P-Ott site implies that the POUs domain functions in concert with the POUHD to achieve correct DNA contact. Pit-l Is Monomeric in Solution, but Associates As a Dimer on DNA To test whether Pit-l is bound as a dimer on DNA, Prl-1P and wild-type Pit-l were mixed with a truncated Pit-l (AN, A8-128) that was still competent to bind DNA as shown in Figure 1B. A unique intermediate sized band resulted from gel mobility shift analysis, as illustrated in Figure 5A, suggesting that Pit-l monomers associate with one another

PO&Specific 1025

Domain

of Pit-l

Figure 4. DNA Binding of Pit-1-Ott-1 Fusion Proteins to Prl-1P and Cktamer Sequences

Siles Prl-1 P (P) P-0~1

(0)

(A) Pit-l-Got-1 fusion constructs are shown with the POUs domain and the POUno from Pit-l (hatched pattern) and the corresponding regions from Ott-1 (shaded areas). Coordinates for domains within Ott-I were originally described by Sturm and Herr (1988), and all fragments were engineered to permit fusion inframe within the Pit-l cDNA with compatible BstEll DNA fragments obtained by polymerase chain reaction corresponding to amino acid positions 280354 (Octpoup). 376-439 (Dctno), and 280-439 (Oc1pouJOti~o). (B) Gel mobility shift patterns, obtained using equivalent amounts of protein as judged by Western analysis (data not shown), were performed using 0.1 nM of the Prl-1P site(P) or the two bp mutant octamer binding site, P-Ott (0). (C) Competition studies were done for wildtype Pit-l (Pitpou$Pituo) or chimeric proteins Octpou$Pitno and Pitpou&ctno by incubation with radiolabeled Prl-1P site in the presence of increasing amounts of unlabeled Prl-1P site or the unlabeled variant site (P-O@.

TATATATATATTCATG TATATATATTTGCATG

on DNA. This interaction was examined further by use of an avidin-biotin complex DNA assay (Glass et al., 1989) in which radioactive Pit-l was prepared by translation in vitro and incubated with a biotinylated Prl-1P oligonucleotide. The protein-DNA complexes were precipitated with strepavidin-agarose, washed, chemically reacted with a homobifunctional cross-linking reagent, bismaleimidohexane (BMH), and analyzed by SDS-PAGE (Figure 58). Addition of BMH to binding reactions produced a novel species migrating at ~66 kd, which was not observed in the

A.

beled HD and is distinct

DNA

-

-

+

BMH

-

+

-+++-

+

+

+

+

C.

2

4

6

6

10

12

14

16

18

20

22

Fraction No. Figure

5. Pit-l

Is a Dimer on DNA but Monomeric

in Solution

(A) Equivalent amounts of wild-type (WT) and AN Pit-l proteins were bound separately or together to radiolabeled Prl-1P Binding of equivalent amounts of each protein alone are shown, and the new, intermediate sized species, observed following addition of both proteins. is la-

from the faster

migrating

species

of wild type

(Cl). (B) Equimolar amounts of 35S-labeled wild-type Pit-l (WT) and AN were incubated with 3 nM of the Prl-1P site containing biotin-ll-dUTP for 20 min at room temperature as described in Experimental Procedures. Protein-DNA complexes were precipitated with strepavidinagarose, washed in 100 mM NaCI, 20 mM HEPES, and 20% glycerol three times, resuspended in 40 j~l of 20 mM HEPES, 20% glycerol, and 1 mM BMH (Pharmacia), reacted for 10 min at 22OC, and quenched by addition of 580 mM j3-mercaptoethanol before analysis by SDS-PAGE. Wild-type Pit-1 without cross-linking reagent is shown as indicated at the bottom. The percentage of homodimeric species was equivalent for both wild type and AN; however, for this experiment the AN homodimerit species is not well visualized since the specific activity was reduced by almost half in order to achieve equal masses of protein. (C) Rate-zonal sedimentation analysis of Pit-l. Bacterially expressed, partially purified Pit-i (100 nM) (described in Experimental Procedures) was loaded onto a linear V/0-20% (v/v) glycerol gradient containing 150 mM KCI, 20 mM HEPES, 5 mM MgCIa, and 0.1% NP40 and subjected to centrifugation for 38 hr at 6OC and 45,000 rpm. Fractions (200 ~1) were collected and 3 PI was assayed for DNA binding activity by gel mobility shift assays as shown. Fractions containing external sedimentation markers, bovine serum albumin (4.49S), hemoglobin (4.45), lysozyme (1.9s) and RNAase Tl (1.62s) were determined by measurements of Aas and are indicated above the corresponding lanes.

Cell 1026

absence of BMH or DNA (Figure 58). Mixing wild-type Pit1 and a truncated Pit-l containing only the POU domain (ANAC, A8-128 and 11273-291) gave rise to a prominent species of 48-49 kd, approximating the theoretical value of a heterodimeric form. The estimated homodimer (34 kd) of ANAC was also faintly observed, and for this experiment the monomer of the ANAC protein migrated with the dye front. Similar data were obtained using a GH-1 element. Cross-linked species could be obtained with several reagents that react with sulfhydryl groues (i.e., cysteine) at distances ranging from 6 to 16 A (data not shown). Because both gel mobility shift and cross-linking data demonstrated protein-protein association on DNA, it was important to determine whether Pit-l dimers occurred in solution. This possibility was investigated by cross-linking experiments using high concentrations of Pit-l in the absence of DNA (5 x lo-’ M). No detectable cross-linking was observed (data not shown). Another method of analysis was therefore applied, using rate-zonal sedimentation analysis. Pit-l exhibited a sedimentation coefficient equal to ~2.2S, characteristic of a monomeric protein, as assayed by Pit-l binding activity (Figure 5C) and Western blot analysis (data not shown). In addition, gel filtration using a high performance gel filtration medium, Superose 6, precalibrated with protein markers also indicated that Pit-l is a monomer in solution, migrating at a position corresponding to a calculated molecular mass of 34 kd (data not shown). We conclude that Pit-l dimers form on DNA and not in solution. The POUs Domain Is Required for Protein-Protein Interactions As previously noted, two distinct retarded species, C2 and C,, were observed by gel mobility shift analysis when using limiting amounts of Pit-l protein. Because the formation of the more rapidly migrating species, Cl, was an apparent prerequisite for formation of the second, more slowly migrating species, Cz, it was concluded that these represented one and two molecules of Pit-l, respectively. To confirm this hypothesis, a5S-labeled Pit-l was incubated with bacterial Pit-l and bound to a Prl-1P site at protein concentrations that allowed formation of Cz and Cr. Complexes were isolated and cross-linked while still in the gel and analyzed by SDS-PAGE. Addition of BMH to C2 resulted in an ~66 kd species, but not in the case of C, (Figure 6A). A similar result was obtained if crosslinking was performed in solution before the complexes were separated (data not shown). The rapid rate at which Cp formed suggested that the binding of the second Pit-l molecule is cooperative. Dissociation measurements of C2 and C, as a function of time following addition of a lOOO-fold excess of cold DNA showed that, while CZ remained largely intact for up to 15 min, C, readily disappeared within 2 min (Figure 6B). Therefore, the apparent stability of C2 as compared to Cl is consistent with the notion that two Pit-l molecules are binding to Prl-1P in a cooperative manner. Furthermore, binding studies in which Pit-l concentrations were varied also suggested cooperative interactions. Formation of the

-68

BMH

Figure

+

-

6. Cooperative

-

+

Binding

of Pit-l to the Prl-1P Sequence

(A) Analysis of C2 and C,. Isolated wild-type C2 and C, complexes were generated using a mixture of 35S-labeled and bacterially expressed Pit-l, cross-linked as previously described with (+) or without (-) BMH, and analyzed by SDS-PAGE. (B) Dissociation of Pit-l complexes. Nuclear extracts isolated from HeLa cells permanently transfected with Pit-l (Mangalam et al., 1989) were bound to labeled Prl-1P with a lOOO-fold excess of unlabeled PrlIP and loaded immediately onto a 0.5x TBE, 5% polyacrylamide nondenaturing gel during time intervals from O-15 min. Note that the curvature of the Cs pattern results from the fact that electrophoresis was continuous during the time course of the experiment. (C) Increasing amounts of diluted Pit-l were assayed for DNA binding activity by gel retardation assays. Unbound probe and Pit-l complexes were quantitated as described in Figure 2, and these data are plotted below as percentage of total counts for each complex versus increasing amounts of Pit-l added to the reactions.

second retarded complex exhibited a sigmoidal equilibrium binding curve (Figure 6C), and when the fraction of occupied sites are plotted as a function of relative protein concentration, a Hill coefficient with a slope of 2.7 f 0.67 is obtained, suggesting that cooperativity exists (Figure 6C). A more detailed analysis of cooperativity using avariety of sites is forthcoming (V. R. A. et al., unpublished data). Pit-l mutants lacking the POUs domain consistently yielded a gel mobility shift pattern that was similar to the mobility of C, (see Figures 1B and 2A) and thus were in-

POU-Specific 1027

Domain

of Pit-l

A. APOU~-HD,

BMH-+--f-t

Figure 7. Deletion of the Pit-l-Pit-l Interactions

POUs

Domain

Prevents

DNA-Dependent

(A) Titration of APOUs-HDs protein. Increasing amounts of APOUs-HDs protein were assayed for binding to Prl-1P as shown at left. Equivalent amounts of AN and APOUs -HDs protein were bound separately or together to radiolabeled Prl-1P site. Binding of each protein, alone or mixed, is shown. Note that a new, intermediate sized species is not observed, unlike in Figure 5A. (B) %-labeled wild-type Pit-i (WT) and the C263-Q and APOlJsHDs mutants were reacted with biotin-11-dUTP-labeled PrllP crosslinked as previously described with (+) or without (-) BMH (see Experimental Procedures and Figure 4) and analyzed using SDS-PAGE.

terpreted to be associated as monomers on DNA. Protein titration with the variant Pit-i protein (APOUs-HD,) failed to produce a slower migrating species as was observed with Pit-l, nor were any intermediate species observed A.

PO” SPEClFCDOMAIN

when mixed with N-terminally truncated Pit-l protein (AN), as shown in Figure 7A. Consistent with these data, crosslinking experiments with APOUs-HDs failed to result in a dimeric species as was observed with wild-type protein or the C263+Q point mutation (Figure 78). Comparable titration curves or cross-linking experiments with APOUs also failed to produce a slower migrating species (data not shown). We conclude that the POUs domain of Pit-l is required in mediating DNA-dependent protein-protein interaction. Further experimentation is needed to map the precise residues in the POUs domain that interact and to discover what role, if any, the PO&c exerts in this interaction. A Predicted a-Helical Structure within the POUs Domain Is Required for Binding Chou-Fasman algorithms suggested the presence of three a helices in the POUuc and two in the POUs domain (Figure 8A). The importance of these predicted a helices was assessed by selective mutagenesis followed by gel mobility shift analysis (Figure 8). Interruption of the first predicted helix of the POUs domain by introduction of a proline residue (helix A) abolished DNA binding, while a similar mutation of the second helix did not (Figure 8B). Replacing the cysteine with a proline at residue 174 (adjacent to SQTTI) had no effect. Mutants with the order of conserved amino acid clusters in the POUs domain rePO” HOME0 DOMAlN

;t;g

.L--

Figure

8. POU Domain

Mutants

(A) u helices predicted by the Chou-Fasman algorithms (Chou and Fasman, 1974) are shown for the POU domain of Pit-l. Analyses before (solid line) and after (broken line) prolines are introduced at different residues in the Pit-l protein are shown and positions of the mutations indicated underneath the plotted data (University of Wisconsin program). Other cluster and point mutations in both the POUs domain and the PO&o are indicated below the normal amino acid sequence. (B) DNA binding data using the Prl-1P site and the mutant proteins are shown. Gel mobility shift assays were performed using comparable amounts of proteins, as quantitated by Western analysis (data not shown). Larger deletions of helical regions correspond to residues 128-159 (APOUA); 159-199 (APO&B); 213-229 (AHelixl); 229-253 (AHelixP); and 253-275 (AHelix3). (C) Competition binding analysis of Pit-l mutants. Wild-type (WT) and mutant Pit-l proteins (C2m-Q and Q s6/-M) were bound to radiolabeled Prl-IP probe in the absence or presence of lOO-fold molar excess of unlabeled Prl-1P or an oligonucleotide containing the engrailed consensus sequence (En) (see Figure 3A).

Cdl 1028

A.

Figure

versed (GLKIRR and CITTQS) or basic residues removed (GHLMIS) exhibited no binding (Figure 86). As expected, interfering with the third helix (helix 3-P) or the so-called “recognition helix” in the POLlno resulted in complete loss of binding; however, replacement of nonconserved amino acid residues with prolines in the predicted first and second helices had little (helix 2-P) or no (helix 1-P) effect on binding, a8 shown in Figure 8B. Several point mutations were also examined for effects. The cysteine at residue 263 in the WFC region of the recognition helix is conserved among all POU domain proteins and corresponds to the ninth residue in the third helix of classic homeodomain-containing proteins, which has been shown to be critical for DNA binding specificity (Hanes and Brent, 1989; Treisman et al., 1989). In contrast with these observations, the C26s-Q Pit-l mutant bound DNA with normal affinities and specificity (Figure 8C). A glutamine (0) was chosen to be representative of the Antennapedia or engrailed class of homeodomain proteins, known to bind to engrailed binding sites (Hoey and Levine, 1988; Desplan et al., 1988). However, the C263-Q mutant did not effectively bind the engrailed element (Figure 8C). Similar binding characteristics were also observed when the conserved glutamine (Q) residue at position 267 was altered to a methionine (Q267+M). However, substituting the basic arginine (R) at position 265 (R265-+G) entirely abolished DNA binding. These data suggest that regions rich in basic amino acids and a-helical structures in both the POUs domain and POUno are critical for binding.

9. Mapping

the Transcriptional

Domain

of Pit-l

(A)The reporter plasmid contained the rat prolactin distal enhancer (bp -1531 to -1830) fused to the proximal promoter (bp -189 to +33) and the luciferase reporter cDNA, as diagrammed at the top. Wild-type and mutant Pit-l constructs placed in the pCMV1 expression vector as described in Experimental Procedures were assayed for their transcriptional effects by cotransfection of 5 ug of both expression and reporter plasmids into CV1 cells. Fold induction is expressed as the ratio of activity with Pit-l plasmid to a control plasmid containing antisense Pit-l. Fold induction obtained for wild-type Pit-l = 180 f 20. Levels of activity observed with the antisense Pit-l construct averaged between 3- and 7-fold higher than with reporter plasmid alone. In different experiments maximal and minimal light units observed were 340,000 and 95,000, respectively, for wild-type Pit-i using ~100 ug of whole-cell protein. Data represent an average obtained from six separate experiments. Single amino acid substitutions were made in the region of the third predicted a-helical structure (cylinders) of the P0lJ~o (Qzo’+M, C263-Q, and R-G), and their respective transcriptional efficacies were also determined. (B) The ability of Pit-l regions to transfer trans-activation function to the transcriptionally inactive LexA DNA binding region was assessed for a promoter with or without LexA binding sites. The LexA reporter plasmid was constructed as described in Experimental Procedures. Fragments of Pit-l cDNA were fused in-frame C-terminal to residues l-87 of LexA and cloned into the same expression vector as above. These regions correspond exactly to those deleted in the mutants depicted in (A). The fold inductions represent the ratio of activity of a cotransfection with either the LexA-Pit-l fusion protein or the LexA protein alone and are adjusted for the nonspecific activation of the control reporter plasmid lacking LexA sites. Results represent the average of four inde-

The Major Trans-Activation Domain Resides in the N-Terminal Region To identify the location of the frans-activation function in Pit-l, the deletion mutants described in Figure 1A were placed in a mammalian expression vector under the control of the cytomegalovirus intermediate-early enhancer/ promoter and the SV40 enhancer. These constructs were transfected into a heterologous cell line (CV-1) with a reporter plasmid containing the distal enhancer and proximal promoter regions of the rat prolactin gene fused to firefly luciferase cDNA, as previously described (Nelson et al., 1988). Fold induction was calculated by comparison of the various Pit-l constructs with a control vector containing antisense Pit-l cDNA and expressing these values as a fraction of the maximal induction produced by wild-type Pit-l; levels of activity with wild-type Pit-l averaged 180fold greater than the activity observed using antisense Pit1 (Figure 9A). Mutant Pit-l molecules lacking the C-terminal portion of Pit-l resulted in transcriptional stimulation equivalent to wild-type protein, whereas any substantial deletion in the N-terminal portion of Pit-l reduced transcriptional levels by 85% or more (Figure 9A). Deletion of amino acids 8-80 resulted in this reduction, despite the observed ability of this truncated protein to bind DNA with high affinity (data not shown) and the presence of an acidic cluster with a net negative charge of -6 located bependent experiments. It should be noted, however, that there was a 4-fold stimulation with the POLlHo-Lex fusion not dependent on the LexA recognition elements, as indicated by the asterisk.

POWSpecific 1029

Domain

of Pit-l

*

Figure 10. Schematic Representation tional Domains of Pit-l c

L

Transactivation Domain SerfThr Rich (21%) n

V

POLLSpeafic 1

is Required

for:

High affinity DNA binding Site soecificitv Protein-protein interactions

L

POWHome0 -J

G DNA Binding Low affinity Relaxed specificity

of Func-

The functional domains of the pituitary transcriptional factor Pit-l are diagrammed, showing the basic amino acid clusters and helices. The amino acid sequence lo-40 is shown for the major trans-activation domain. The POUs domain plays important roles in high affinity DNA binding and site specificity and is involved in protein-protein interactions on DNA.

aa IO-40

DTFIPLNSDASAALPL RMHHSAAEGLPASNH -

tween residues 117 and 128. While the N-terminus was quantitatively critical for transcriptional activity on the distal and proximal promoter, the POU domain by itself was competent to activate transcription when bound to elements immediately adjacent to the TATA box (J. Holloway, unpublished data). Little, if any, transcriptional activity was demonstrated when the POUno was deleted. However, some residual activity was still observed with removal of the POUs region (Figure 9A). Surprisingly, the C263-+Q mutation in the WFC region of the PO&, considerably compromised transcriptional function (~25% of wild-type Pit-l) (Figure 9A), while exhibiting wild-type binding, as shown in Figure 8C. This further supports the idea that the POU domain by itself permits some transcriptional activation and therefore represents a secondary transcription domain analogous to that observed in steroid receptors (Hollenberg and Evans, 1988). Point mutations of other residues in this region, @‘jr-+M and R265+G, stimulated transcription to levels consistent with their observed ability or inability to bind DNA, respectively, as shown in Figure 8C. All proteins were expressed at comparable levels as assayed by gel mobility shift analyses using nuclear extracts (data not shown). To confirm the function of putative tfans-activating sequences, regions were transferred to a heterologous DNA binding domain, provided by the N-terminal 87 residues of bacterial LexA (Brent and Ptashne, 1985) and analyzed by cotransfection assays in CV-1 cells with reporter plasmids bearing the LexA operator (Brent and Ptashne, 1985). As shown in Figure 96, LexA-Pit-l fusion constructs containing all or some of the 72 N-terminal amino acids of Pit-l (residues 10-40, 40-80, 8-80, or 8-128) stimulated the reporter plasmid carrying two LexA recognition elements 4-to 12-fold, with residues IO-40 almost twice as effective as residues 40-80. In contrast, the POUs, PO&o, and C-terminal regions were ineffective in stimulating transcription. Therefore, the N-terminal residues 8-80, by both methods of analysis, compose the dominant region responsible for frans-activation functions of Pit-l. The region encompassing residues 8-80 is particularly rich in serine and threonine (>21%). It is noted that the N-terminal region of Ott-2 has the same percentage of serine and threonine residues as that found in Pit-l, as does a com-

parable region in the Drosophila POU domain protein, dPOU-1 (M. N. Treaty, H. Xi, V Hartenstein, C. Zuker, and M. G. Rosenfeld, submitted). When this region of Ott-2 (amino acids 3-78) is placed in front of LexA, a notable stimulation is observed, as shown in Figure 98. Discussion The data presented in this study provide evidence that the POUs domain and the PO&o work in concert to confer high affinity, sequence-specific DNA binding properties to the POU class of transcriptional regulators (summarized in Figure IO). Based on our analysis of Pit-l, it appears that the POUHo alone is sufficient to permit DNA binding, but with an affinity that is lOOO-fold weaker than that of wildtype Pit-l. The POUs domain not only serves to increase the affinity of DNA binding but also imparts specificity of DNA recognition, as documented by an altered hierarchy of binding on different sites, specific DNA base pair contacts, and the effects of substituting POU domains from Ott-1. Thus, the POUs domain is suggested to contact directly DNA. Initial studies using in vitro translation products of Ott-1 mutants were interpreted to suggest that the POUs domain was absolutely required for DNA binding (Sturm and Herr, 1988). However, the reported ability of Oct.2 fusion proteins to bind DNA (Ko et al., 1988) and predicted homologies to bacterial DNA binding proteins (GarciaBlanc0 et al., 1989) were used to argue that the critical DNA binding functions would reside primarily in the POUHo. Theill et al. (1989) independently concluded that the Pit-l/GHF-1 homeodomain is itself sufficient for sequence-specific DNA binding despite the fact that their analysis used a TrpE-Pit-l fusion protein with the second portion of the POUs domain still present. Furthermore, it was suggested that the ability of this same fusion protein to bind DNA was only diminished 2- to 3-fold based on a footprinting analysis. The failure to remove all of the POUs domain and the uncharacterized contribution of the TrpE protein in these studies (Theill et al., 1989) most likely account for their conclusions, which inadvertently disregarded the role of the POUs domain in sequence specificity and binding affinity. We have shown in this study that full or partial deletion of the POUs domain in

Cell 1030

the context of native Pit-l results in a protein unable to bind the growth hormone promoter. The ability of the POUHD to permit very low affinity DNA binding is coincident with the ability of several Drosophila homeobox proteins to bind DNA sites (Hoey et al., 1988; Miiller et al., 1988b). However, one curious observation is that point mutations or small deletions in the POUs region fully eliminated DNA binding, whereas removal of the entire POUs domain enabled the POUHD to bind with minimal but reproducible binding activity, exhibiting a preference for AT-rich sequences. This would suggest that the presence of the full POUs domain configures the POUH~, such that both structures are now required for correct DNA contact. Supporting this notion is the finding that the Anrennapedia homeobox inserted into Pit-l with a POUs domain does not bind to its recognition element, but does bind if the POUs is deleted (H. A. I., unpublished data). Although Pit-l appears to be monomeric in solution as determined by rate-zonal sedimentation, gel filtration, and cross-linking studies, it associates as a dimer on DNA and can be crest-linked by reagents that react at distances of less than 8 A. Highly purified Ott-1 and Ott-2 have been shown to behave as apparent monomers using sedimentation analysis (F’oellinger and Roeder, 1989). Interestingly, Antennapedia protein is also reported to exist as a monomer in solution (Miller et al., 1988b). The POUs domain appears to be required for the observed protein-protein interaction on DNA, and although replacement of the POUs domain with a second copy of the POUH~ increased binding affinity, DNA-dependent protein-protein interaction was not restored. This protein-protein interaction appears cooperative on certain natural response elements. Indeed, by analysis of specific synthetic sites, it has been elegantly demonstrated that Ott-2 can bind cooperatively to two adjacent octamer elements (LeBowitz et al., 1989). Cooperative interactions that functionally increase transcriptional activation (Poellinger et al., 1989) would presumably allow large responses in promoter activity to small fluctuations in functional Pit-l protein levels. The data presented in this study strongly suggest that cooperative interactions are likely to be mediated by the POUs domain and may be a general feature of most POU domain proteins on DNA response elements. Identification of the precise residues involved in mediating this function requires further dissection of the POUs domain. In this regard, it seems likely from studies using POU domain fusion proteins (Ko et al., 1988; Theill et al., 1989) that the heterologous portion (i.e., kgalactosidase, TrpE) can substitute for the function of the POUA domain (Herr et al., 1988), therefore enabling specific DNA binding to occur. The simplest interpretation of these data is that protein-protein interactions depend on the A portion of the POUs domain, while the site specificity functions reside in the B portion. This would be consistent with the fact that the first a helix of the POUs domain appears necessary for binding and suggests that this may be the region responsible for mediating protein-protein contact. The possibility that two heterologous POU proteins can inter-

act seems feasible since a chimeric Pit-l protein containing the Oct.1 POUs domain can associate with Pit-l (AN) on DNA (H. A. I., unpublished data). This would suggest that heterodimers of POU domain proteins are able to bind DNA in vitro, and, in fact, heterodimers of Pit-l and Ott-1 have been observed in vivo (J. W. V., unpublished data). The physiological and functional significance of this event remains to be determined. The major transcriptional domain of Pit-l is located in the N-terminus and is distinct from the DNA binding domain, similar to separable DNA binding and transcriptional activation domains found in other transcription factors (for reviews see Mitchell and Tjian, 1989; Johnson and McKnight, 1989). The N-terminal serine- and threonine-rich trans-activation domain depicted in Figure 10 is apparently unrelated to previously described motifs, including highly acidic (Struhl, 1987), proline-rich (Mermod et al., 1989), or glutamine-rich (Courney and Tjian, 1988) domains. Further dissection of this domain reveals that the major activity resides in residues 10-40, as shown in Figures QB and 10. Mutations of the serine residues in this region greatly reduced transcriptional activity, despite the presence of three negatively charged residues, suggesting that this transcriptional activity is not conferred by these acidic residues. It is also possible that an underlying specific motif exists, since the region from 40-80 is considerably richer in serine and threonine residues but far less efficient at trans-activation. While the N-terminal residues are established to be the major trans-activation domain in Pit-l (this study; Theill et al., 1989), the POU domain by itself exhibits low levels (~10%) of trans-activation. It is likely, as shown for other transcription factors (Rosenfeld et al., 1988; Tora et al., 1989), that there is more than one transcriptional domain for Pit-l that may contribute differently in various promoter contexts. Homeodomains, including those of POU domain proteins, have been noted to exhibit structural similarities with the DNA binding regions of classic bacterial repressors (Laughon and Scott, 1984; Otting et al., 1988; GarciaBlanc0 et al., 1989). Although these studies indicate that residue 9 of the second helix makes nonspecific contact with the sugar-phosphate backbone in phage repressor protein, two independent observations demonstrate the importance of amino acid 9 in the recognition helix (helix 3) of the classic homeodomain proteins paired, Antennapedia, and bicoid in conferring DNA specificity (Hanes and Brent, 1989; Treisman et al., 1989). In contrast, when the comparable residue, a cysteine in all known POU domain proteins, is changed to glutamine, specificity and affinity of binding are unchanged, but transcriptional stimulation is markedly (>70%) impaired, implying that the putative recognition helix in Pit-l may, directly or indirectly, modulate function of the N-terminal trans-activation domain. Although remarkable sequence conservation in this third helix (KENVVIIRVWFCN) is observed among all known POU domain proteins, their DNA binding specificities are dissimilar, consistent with the role of the POUs domain in conferring binding specificity, perhaps by directly contacting DNA. We suggest that the roles of amino acids corresponding to homeodomain helices 1 and 2

POU-Specific 1031

Domain

of Pit-l

may differ from those of classic homeodomain proteins, since the introduction of proline residues in these regions of Pit-l had little effect on binding. We conclude that the POUs domain confers high affinity binding and site specificity of DNA recognition and permits protein-protein interactions that may be important for trans-activation events, thereby providing unique properties to the large family of POU domain proteins that appear to play a critical role in development. Experimental

Procedures

Plasmld Constructions The original pZL7.4 Pit-l plasmid in PBSK- (Stratagene) has been previously described (Ingraham et al., 1988) and was used to generate all mutant constructs (see below). All subsequent mutants were transferred into a bacterial expression vector (described below) by excising a 685 bp PpuMI-Pstl fragment, except for constructs involving deletions of the N-terminal region in which in vitro mutagenesis was employed to achieve the appropriate Ndel restriction site. Pit-l and derivatives were inserted in the T7 expression vector (generously provided by William Studier and described in Studier and Moffat, 1986), pET3a, which contains the promoter, the Shine-Dalgarno sequence, and the transcription terminator of phage T7 gene 70. An Ndel site was created in the original pZL7.4 Pit-l vector at the translation initiation site; the resultant 1.1 kb Ndel-Pstl fragment was excised and cloned into these unique sites in pET3a. Wild-type and mutant Pit-l constructs were placed in the mammalian expression vector pCMV1 (a generous gift from David W. Russell), derived from pTZ19R (Pharmacia), by inserting a 1.6 kb Kpnl-Xbal fragment from Pit-l into the polylinker preceded by the cytomegalovirus promoter region and followed by the human growth hormone termination and polyadenylation signal and the SV40 enhancer/promoter sequences. LexA-Pit-l fusion proteins were obtained by inserting an adapter linker containing a BstEll site and Xhol ends into the unique Sail site of YCp88-LexA-GCN4-A19 (a generous gift from Kevin Struhl), substituting a unique Kpnl for a Hindlll site. The subsequent Kpnl-BstEll fragment including residues l-87 of LexA was cloned into pCMV865, supplying the transcription terminator and 3’ untranslated sequences of Pit-l cDNA. Regions of Pit-l coding sequences (see above) with BstEll overhangs were inserted into a unique BstEll site of the pLex-Pit-l plasmid. The reporter plasmid pEAH has been previously described (Ingraham et al., 1968), and the Lex reporter plasmid, p2xLex, was constructed using two copies of the LexA operator site (5”GATCCAATTCTACTGTATGTACATACAGTATTCCAAA-3’) ligated and inserted into the unique BamHl site of the reporter plasmid pTaq-36. pTaq-36 includes the rat prolactin promoter region (-36 to +6) linked to the firefly luciferase gene as previously described (Ingraham et al., 1986; de Wet et al., 1967). Mutagenesis and Polymerase Chain Reaction Site-directed mutagenesis using the procedure of Kunkel (1985) was used for point and cluster mutations and for introducing BstEll sites at boundaries of all deletion mutants, thereby generating deletions and excisable fragments with BstEll ends. Replacing Val and Thr by the addition of a BstEll site avoided introduction of prolines or charged or bulky residues. An identical strategy was used to clone desired fragments from Ott-1 cDNA obtained from HeLa mRNA. Ott-I oligonucleotide sequences were used in the polymerase chain reaction to generate Ott-I fragments containing BstEll overhangs. cDNA (5 vg) synthesized from HeLa cell mRNA, oligonucleotides (1 a) corresponding to regions of Ott-1, and Cetus Taq polymerase were mixed and subjected to 30-40 cycles of polymerase chain reaction at 94OC for 1 min, 55% for 1 min, and 72OC for 3 min. Ott-1 fragments were cloned into the BstEll sites of Pit-i deletion constructs APOU, APO&, and APO&o, and all mutants were sequenced by the method of Sanger et al. (1977). Protein Preparations Proteins from all constructs were BL21(DE3) as described previously

expressed in the bacterial strain (Studier and Moffat, 1986). Crude

bacterial extracts were used for all experiments except where noted and were prepared essentially by the reported method of Hoey and Levine (1988), using small preparations (30 ml) induced at an ODm of 0.6-0.8, with 0.4 mM IPTG, and sonicated for 30 s in buffer Z, except that MgC12 was omitted and whole extracts were subjected to denaturation in 4 M guanidine-HCI. Pit-l was approximately lo%-30% of total protein. Experiments involving sedimentation analysis or gel filtration used Pit-l, partially purified by phosphocellulose chromatography as previously described (Mangalam et al., 1989); under these conditions peak fractions of Pit-l eluted between 250-350 mM KCI and were judged to be >50% pure by silver staining of an SDS-polyacrylamide gel (Laemmli, 1970). Protein preparations were stored at 4% for several months without noticeable loss in binding activity. mRNA transcripts were prepared using T7 RNA polymerase, and pET3a mutant derivatives were incubated in a rabbit reticulocyte lysate system in the presence of [35S]methionine. Transient Cotransfection Assays Green monkey kidney (CV-1) cells were plated at a density of 0.5 x IO6 per 100 mm plate in Dulbecco’s modified Eagle’s medium containing 10% newborn calf serum. One day later cells were transfected with 5 pg each of expression and reporter plasmid (pTaq36 or p2xLex) using the calcium phosphate coprecipitation method (Chen and Okayama, 1967). Cells were harvested 48 hr after transfection and assayed for luciferase activity as previously described (de Wet et al., 1987). In the cases of all constructs displaying reduced transcriptional activities, but known to bind DNA with high affinity (e.g., AN, C263-Q), a second independent plasmid preparation was evaluated. DNA Binding Studies DNA binding studies were carried out by standard gel retardation assays as described by Mangalam et al. (1989). All binding assays were performed at room temperature for 20 min, in a 20 +d volume of 20 mM HEPES (pH 7.6), 1 mM EDTA, 0.1% NP-40, 15% glycerol, 2 kg of poly(dl-dC), 0.5 vg of bovine serum albumin, and 0.25% dry nonfat milk (Carnation) using between 0.05 to 0.1 nM of double-stranded oligonucleotides (Figure l), radiolabeled using T4 kinase. One-fifth of each reaction was loaded on a 6% nondenaturing 05x TBE-polyacrylamide gel, electrophoresed at 250 V for 1 hr, and autoradiographed for 2-14 hr at -80%. DNA probes containing biotin-1%dUTP used in DNA binding and cross-linking studies were prepared as previously described (Glass et al., 1989). Two 39 bp oligonucleotides were synthesized, sense (5’-AAGGGGATCCACCTGATTATATATATATTCAlGAAGGTG-37 and antisense (S-GGACTAATATATATATAAGTACTlCCACT~AGAAGGA-3’); thus when S’overhangs are filled with Klenow fragment, four biotin-11-dUTP residues are incorporated in both sense and antisense directions. Low levels (

The POU-specific domain of Pit-1 is essential for sequence-specific, high affinity DNA binding and DNA-dependent Pit-1-Pit-1 interactions.

Pit-1 is a member of a family of transcription factors sharing two regions of homology: a highly conserved POU-specific (POUS) domain and a more diver...
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