Jun 25, 1986 - (cell cycle/electrophoretic mobility shift assay/DNase I "footprinting"/chromatography). LISA DAILEY, SARAH M. HANLY, ROBERT G. ROEDER, ...
Proc.. Natl. Acad. Sci. USA Vol. 83, pp. 7241-7245, October 1986 Biochemistry
Distinct transcription factors bind specifically to two regions of the human histone H4 promoter (cell cycle/electrophoretic mobility shift assay/DNase I "footprinting"/chromatography)
LISA DAILEY, SARAH M. HANLY, ROBERT G. ROEDER, AND NATHANIEL HEINTZ Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10021
Communicated by David Luck, June 25, 1986
ABSTRACT Two proteins specifically binding to separate regions of the human histone H4 promoter were identified in nuclear extracts prepared from synchronized S-phase HeLa cells. Competition experiments with H4 promoter mutants and DNase protection assays ("footprinting") demonstrate that these factors bind to regions of the H4 promoter that are essential for maxmal expression in vitro. One of these factors (H4TF-1) binds to sequences between -80 and -110 base pairs upstream of the H4 cap site, whereas the other (H4TF-2) binds to the H4 subtype-specific sequence element immediately upstream from the "TATA" homology. Neither of these activities can efficiently bind to any of the other histone gene subtypes or simian virus 40 DNA. Binding of H4TF-1 to the distal region of the pHu4A histone H4 promoter is inhibited competitively with varying efficiency by four of six human histone H4 genes cloned in this laboratory, whereas efficient competition for binding of H4TF-2 is exhibited by five of the six H4 genes. Since both of these factors bind to signficant regions of the pHu4A histone H4 promoter and can be bound by several different human H4 genes, we believe that they are important for maximal transcription of the gene and that they may be involved in its regulated expression during the cell cycle. A central question of cellular proliferation concerns the mechanisms by which the various temporal events occurring during the cell cycle are controlled. One opportunity for studying such temporal control is provided by the histone genes. This multigene family consists of five classes, each of which is repeated several times (for review, see ref. 1). Some of the genes within a particular class are expressed at a low but constant basal level throughout the cell cycle (2, 3). However, the expression of most of the histone genes is tightly coupled to DNA synthesis (4, 5). During the S phase in HeLa cells, histone mRNA levels increase 15-fold over their non-S-phase values as a combined result of increases in the rate of synthesis and the stability of histone mRNA (6, 7). In the interest of understanding this in vivo cell cycle control, the transcriptional regulation of a human histone H4 gene was reproduced in vitro (8). These experiments showed that regulation in vitro is manifest by a 3- to 10-fold stimulation of transcription of this gene in nuclear extracts derived from synchronized HeLa cells in S phase relative to transcription in non-S-phase extracts. H4 gene transcription is also stimulated upon mixture of the two extracts, indicating that the S-phase extract contains a positive regulatory activity(ies) (8). Further studies using mutants of the H4 gene with deletions of either 5' or 3' portions of the promoter localized a region between -70 and -100 base pairs (bp) from the cap site as necessary for optimal transcription of this gene in S-phase extracts (9). These combined results indicate the existence of an S-phase-specific factor(s), whose activity is The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
cell cycle regulated, and a region of the H4 template required for maximal functioning of this activity in vitro. Since removal of sequences upstream of -70 in the 5' deletion mutants reduced transcription so drastically, it was not possible to independently assess the involvement of any promoter elements downstream from this position. For example, the role of an H4 subtype-specific element between -40 and -57, which contains sequences that are highly conserved among H4 genes of several species, was not assayed (10-13). Thus, there may be additional components of the H4 promoter that were not identified by the deletion mutant analysis. To understand fully the way in which H4 transcription is controlled in the cell cycle, the factor(s) interacting with this promoter must be identified and analyzed. In the present report, we describe the identification of two such proteins (H4TF-1, H4TF-2) that bind to significant regions of the H4 promoter. Both of these factors can specifically interact with several cloned human histone H4 genes. Neither of these activities can efficiently bind to the other histone gene subtypes or simian virus 40 (SV40) DNA, although H4TF-2 can weakly bind to a previously characterized histone H3 gene. Thus, these proteins are important, and possibly specific, for the expression of human histone H4 genes and may be directly involved in the transcriptional regulation of these genes in vivo.
MATERIALS AND METHODS Plasmids. The 5' and 3' deletion mutants of the histone H4 gene pHu4A (11) used in this study have been described elsewhere (9). Recent resequencing of the promoter has revealed three additional cytidine residues between -89 and -90. The numerical positions representing the deletion end point of the mutants used in the present manuscript have been revised to take into account this addition. Mutant CF-3 was constructed by cutting wild-type H4 DNA at -40 with Ava II, filling in the 3' ends with Klenow fragment and dNTPs, and religating. The resulting CF-3 DNA contains a 3-bp insertion between positions -41 and -42. The other histone genes used in this work have also been described elsewhere (refs. 11 and 14; unpublished data for the H1 genes) as has the isolation of several other human histone H4 genomic clones (15). The SV40 DNA used was the SV40 early promoter region into which the chloramphenicol acetyltransferase coding sequences were inserted and cloned in a pBR322 vector [pSV2-cat (16)]. Preparation of Labeled DNA Fragments. H4 promoter DNA fragments from the 5' deletion mutant A2806 or A2603 were obtained by double digestion with EcoRI and Sac II (Bethesda Research Laboratories). The EcoRI site is within the M13 polylinkerjust upstream of the H4 DNA sequences. Because A2806 and A2603 are 5' deletion mutants of H4, the Abbreviations: bp, base pair(s); SV40, simian virus 40.
7241
7242
Biochemistry: Dailey et al.
Proc. Natl. Acad. Sci. USA 83 (1986)
H4 DNA sequences joined to the M13 polylinker differ in different mutants. The A2806 fragment (designated probe A) thus extends from the polylinker at the H4 position - 192 to the Sac II site at +46. The A2603 fragment (designated probe B) extends from -71 to +46. The gel-purified DNA fragments were 3' end labeled at the EcoRI sites with Klenow fragment and [a-32P]dATP (New England Nuclear). Extract Preparation and Chromatography. Cell culture and preparation of S-phase extracts from HeLa cells were as described by Heintz et al. (6). S-phase nuclear extract was loaded onto a heparin/agarose column (Heparin Ultrogel, LKB) that had been equilibrated with TM100 (50 mM Tris HCl, pH 7.9/1 mM EDTA/12.5 mM MgCl2/20% glycerol/0.5 mM dithiothreitol/100 mM KCl) at 10 mg of protein/ml of resin. After washing with TM100, bound proteins were eluted with TM400 (TM400 is the same as TM100 except it contains 400 mM KCl). After dialysis against buffer B (20 mM TrisHCl, pH 7.9/0.2 mM EDTA/20% glycerol/0.5 mM phenylmethylsulfonyl fluoride/0.5 mM dithiothreitol) in 100 mM KCl, the 0.4 M KCl/heparin/agarose fraction was loaded onto a phosphocellulose (Whatman) column at 10 mg of protein/ml of resin. After washing with 0.1 M KCl in buffer B, the column was sequentially eluted with 2-3 column vol of 0.35 M, 0.5 M, and 1.0 M KCl in buffer B. Peak protein-containing fractions [as determined by Bradford reactions (17)] were pooled and dialyzed against buffer B with 100 mM KCl. Binding Assays. Binding assays were essentially as originally described (18, 19) with minor modification (19). Twenty-microliter binding reaction mixtures contained 1 ng of labeled DNA fragment (20-50 x 103 cpm) and 5-15 jig of crude extract protein or 2-3 pug of chromatographic fraction protein. Two to 3 gg of sonicated salmon sperm DNA was included to minimize nonspecific binding to the labeled DNA. The reaction mixtures were incubated in a binding buffer of 20 mM Hepes, pH 7.9/4% (wt/vol) Ficoll/40 mM KCl/1 or 6 mM MgCl2 for 20-30 min at room temperature. Ten microliters of each reaction mixture was then loaded onto a 4% polyacrylamide (acrylamide:bisacrylamide, 30:1.5) gel in 6.25 mM Trizma base/6.25 mM boric acid/0.25 mM EDTA and run at 150-180 V for 1-1Y2 hr at room temperature. Gels were then dried and exposed to x-ray film. Binding of H4TF-2 was optimal in the presence of low (i.e., 0-1 mM) MgCl2, whereas H4TF-1 binding was greatly enhanced by raising the concentration to 6 mM MgCl2. Binding of the H4TF-2 was nearly undetectable in 6 mM MgCl2.
5'
3
A _L
cll
_-
1 2
__
C4
0w - co c
_
4-
,a
Conditions for binding competition experiments were as above except an -25-fold molar excess of unlabeled uncut competitor DNA was included in the reaction mixture prior to addition of the protein. DNase I Protection Assay ("Footprinting"). Footprinting reactions were performed in a fashion similar to published procedures (20). After performing binding reactions as described above, DNase I (Bethesda Research Laboratories) was added to a final concentration of 5 ,g/ml and the digestion was allowed to proceed for 1 min at room temperature. Samples were then immediately loaded onto a running acrylamide gel and electrophoresed as usual. The wet gel was exposed to XAR-5 film for 2 hr and the regions on the gel corresponding to bound and unbound DNA fragments were identified and separately isolated by electroelution in 6.25 mM Trizma base/6.25 mM boric acid/0.25 mM EDTA. Buffer containing the DNA fragments was passed through a glass wool plug, the volume was reduced to 400 ,ul by repeated extractions with 1-butanol, and the DNA was precipitated with ethanol with 1 ,g of carrier DNA. After centrifugation, the dried pellets were assayed for radioactivity and resuspended in formamide loading buffer. Samples containing equal amounts of radioactivity (4000-8000 cpm) were loaded onto an 8% acrylamide/50% urea sequencing gel, along with a G reaction mixture (21) of the probe DNA fragment.
RESULTS Identification of Binding Proteins and Mapping of Bound Regions on the H4 Promoter. To identify proteins that interact specifically with the human histone H4 promoter, we employed the gel electrophoresis retardation assay (17, 18). The first probe (probe A) used in this assay was an end-labeled H4 promoter fragment extending from the EcoRI (-192) to Sac II (+46) sites. This fragment contains all of the DNA promoter sequences necessary to give maximal levels of transcription in S-phase extracts in vitro (9). When this DNA fragment was mixed with a HeLa S-phase extract, two bands that migrated slower than the free DNA were seen (Fig. LA, lane 1). To determine if the bound protein was specific for the H4 promoter, an excess of unlabeled competitor H4 DNA [p2806 (9)] was added. As seen in Fig. 1A, lane 2, the intensity of the upper band (bold arrow), was reduced when excess unlabeled H4 promoter-containing DNA was included in the reaction mixture, thus indicating that this band represented a 5t
B O
-
cd '=
C" L
IC.D
1 2
C2.-
4.*'S
__
,
__-_
--S -.> _w
w __
_
-
FIG. 1. Electrophoretic mobility shift analysis of H4 promoter-specific protein-DNA complexes by competition with H4 deletion mutant DNAs. The end-labeled EcoRI (-192) to Sac II (+46) DNA fragment from H4 mutant A2806 (probe A, A) or the EcoRI (-71) to Sac II (+46) fragment from H4 mutant A2603 (probe B, B) was incubated with HeLa S-phase nuclear extract and electrophoresed on a 4% acrylamide gel. Bands representing specific H4 promoter DNA-protein complexes (in lanes 1) were identified, and the region of the DNA bound mapped by competition for binding of the factor by inclusion of a 25-fold molar excess of unlabeled H4 A2806 plasmid DNAs (lanes 2). To map the regions on the H4 promoter bound by factor, unlabeled competitor H4 promoter mutant DNAs with deletions originating from either the downstream (indicated as 3') or upstream (indicated as 5') side of the promoter were added to the reaction mixture. The deletion end points of competitor DNAs are indicated at the top of each lane. Thus, a 3' mutant competitor DNA whose deletion extends to -38 contains all H4 DNA sequences upstream of this position. CF-3 contains a 3-bp insertion between positions -41 and -42 on the H4 promoter. Lane P contains nonspecific pUC DNA competitor in addition to the salmon sperm DNA. The bold arrows indicate a specific DNA-protein complex, whereas the smaller arrows indicate a nonspecific complex. Free, unbound probe DNA is at the bottom of the gel.
Biochemistry: Dailey et al. specific protein-DNA complex. The lower band, on the other hand, apparently resulted from nonspecific binding of a protein to the H4 DNA since it remained essentially unchanged even after the addition of excess unlabeled H4 competitor DNA in most experiments. The presence of this nonspecific protein-DNA complex was variable and dependent on the protein preparation used in the assay. To map the region on the H4 promoter to which the specific protein bound, competition experiments for this binding were performed by using a series of H4 promoter deletion mutants (9). These mutants have deletions of varying length from the 5' (upstream) or 3' (downstream) side of the promoter. Only mutant DNAs in which the specific binding site has not been deleted should compete for binding. In addition, a mutant (CF-3) containing a 3-bp insertion (GTC) between positions -41 and -42 of the wild-type H4 promoter was employed to assay for specific binding to the H4 subtype-specific consensus element. As shown in Fig. LA, DNA sequences down-
stream of -102 but upstream of -79 must be present on the competitor DNA to effectively compete for binding of this protein to the probe. (The apparent inefficient competition of the 3' deletion ending at -1 in Fig. LA is an aberration. In several repetitions of this assay, the -1 mutant competes as effectively as wild-type DNA.) The 3' deletion mutant with sequences upstream of -88 can compete but not as well as the mutant with sequences extending to -79. This weak competition by the -88 3' deletion mutant was reproducible
in several experiments. The experiments map the binding site of this protein to a position that lies between -79 and -102 bp from the H4 cap site. In addition, since the 3' deletion mutant -88 competes weakly, the binding site for this factor may have a component upstream of -88. To further confirm this result, the binding of this protein was visualized by DNase I footprinting. As described elsewhere (for example, ref. 20), protein-DNA complexes were treated with DNase I prior to electrophoresis. The retarded and free DNase I-treated DNA bands were separately eluted from the gel, denatured, and run on an 8% polyacrylamide/50% urea sequencing gel alongside a G reaction mixture (21) of probe A to map the protected region. As seen in Fig. 2A, an area of protection from DNase I cleavage was observed in the lane containing DNA derived from the retarded band. This area maps between -82 and -105 bp upstream from the H4 cap site. A band of enhanced cleavage is also seen around position -108. These results confirm those obtained in the deletion mutant competition analysis and demonstrate that this protein, designated H4 transcription factor 1 (H4TF-1), specifically binds to sequences shown to be essential for maximal H4 transcription (9). In addition to probe A, a second labeled H4 promoter fragment, probe B, was used. Probe B was derived from H4 mutant A2603 and extends from its EcoRI site at -71 (where it is 3' end labeled) to the unlabeled Sac II site at +46. Thus, probe B does not contain the upstream binding site for H4TF-1 identified in the previous section. Use of probe B in the electrophoretic mobility shift assay (Fig. 1B) also gave two bands (Fig. 1B, lane 1), one of which (Fig. 1B, bold arrow) proved to be H4 specific, as judged by a competition assay with the A2806 H4 promoter (Fig. 1B, lane 2). As before, the binding site was mapped by competition experiments with the H4 promoter deletion mutants. The H4 promoter mutants were effective competitors for this protein only if DNA sequences between -38 and -57 were present (Fig. 11). Furthermore, mutant CF-3, which has the 3-bp insertion at -41, is unable to compete for binding of this protein. No H4 DNA segments containing only the H4TF-1 binding site identified in the previous section competed for binding of this factor. DNase I footprinting of this second protein-DNA complex revealed a region of protection between -35 and -65 bp from the cap site (Fig. 2B). This region
Proc. Natl. Acad. Sci. USA 83 (1986)
7243
B
A
F B G
FEB
if li .I
-
70
2f
.3 7..63 -
0-. 'I -
-
-90
4ww
t-i00 1.00-
ddb:
.LI
f
- -110
FIG. 2. DNase I footprinting of the H4TF-1 and H4TF-2 binding proteins on the histone H4 promoter. Binding reaction mixtures using the labeled H4 promoter DNA fragments described in the legend to Fig. 1 were treated with DNase I and electrophoresed. The bound (lanes B) and free (lanes F) DNAs were isolated separately from the gel, denatured, and run on an 8% acrylamide/50% urea sequencing gel with a G reaction mixture (20) of the labeled DNA fragment (lane G). (A) Reaction using the labeled probe A promoter fragment. (B) Reaction using the labeled probe B promoter fragment. The G reaction for the probe A fragment is not shown. The H4 promoter is depicted in the center and distance from the cap site (in bp) from -20 to -110 is shown on the right. Open box, TATA box; hatched box, conserved H4 consensus sequence. Arrows indicate the two common histone-specific GACTTC sequences and the related GATTTC sequence at -100 (11). The numbers expressed as decimals to the left of the promoter show the level of in vitro transcription efficiency (where 1.00 = 100o or wild-type level) in S-phase extract of 5' deletion mutants whose deletions extend to the indicated position (9).
of the H4 promoter exhibits extraordinary sequence conservation and positional conservation among H4 genes from several species (10-13). Binding Competition by Other Genes. It is well documented that the expression of several genes, histone and nonhistone, increases during S phase, and it is possible that this is coordinated by common transcription factors. Thus, we sought to determine whether either ofthe H4 binding proteins could bind other histone genes. As shown in Fig. 3 A and B, competition for binding of the H4TF-1 and H4TF-2 proteins was tested for several other human histone genes (refs. 11 and 14; F. LaBella and N.H., unpublished work) as well as SV40 (16). None of the genes tested could compete for binding of H4TF-1 (Fig. 3A), whereas an H3 gene was able to compete weakly for binding of H4TF-2 (Fig. 3B). Thus, neither of these factors can efficiently bind to any of the several different human H3, H2a, H2b, and H1 genes assayed. To determine whether binding of these factors was specific for the pHu4A human H4 gene or whether they could interact with other human H4 genes, competition assays employing five other human histone H4 genes characterized in this laboratory (15) were performed. As shown in Fig. 4, the majority were able to compete for H4TF-1 (Fig. 4A) and H4TF-2 (Fig. 4B) factors. pHh5C bound only H4TF-2 (lane 6), whereas clone 7A (lane 7) was unable to bind either factor. Since the level of expression of each of these genes in vivo is unknown, it is possible that some of the clones that are unable
7244
Biochemistry: Dailey et al. A
Proc. Natl. Acad. Sci. USA 83 (1986) A
B 0
S Extract HA
0
er I~ ~r-mr cCNa~n mNlM n C)>
co >
et_ _
zCM mm Ia:I m=
n
0.1 0.4
Pi1
w
S_
___m
-)I-
s
0.1
U;
B
0.35 0.5
1.0
C _
FIG. 3. Competition analysis of other histone genes and SV40. Binding reaction mixtures were assembled with the inclusion of a 25-fold molar excess of the unlabeled competitor DNA indicated on the top of each lane. Samples were then electrophoresed. The bold arrows indicate the specific protein-DNA complex of H4TF-1 (A) or H4TF-2 (B) with the H4 promoter. The competitor DNAs have been described elsewhere (11, 14-16). H4, pHu4A; H1, either pHh8 (A and B) or pHh9 (B) (F. LaBella and N.H., unpublished work); H2a, pHh5G; H2b, MP11PE; H3, pHh5B; SV40, pSV2-cat.
to bind one or both factors may not be cell cycle regulated or even expressed in vivo. However, it is evident from these data that both of the proteins bound by the pHu4A H4 promoter can also bind to at least several other human histone H4 genes. Separation by Chromatography. To determine whether the factors can bind to their respective target sites independently, they were isolated from one another and assayed for DNA binding. Thus, the S-phase HeLa nuclear extract was chromatographed on a heparin/Ultrogel column and proteins in the flow-through (0.1 M KCl) and 0.4 M KCl step fractions were assayed by the gel retardation assay. Both binding activities were present in the 0.4 M KC1 fraction (not shown). After dialysis to 0.1 M KCl, this 0.4 M KCl fraction was then loaded onto a phosphocellulose column and 0.1 M, 0.35 M, 0.5 M, and 1.0 M KCI step fractions were assayed for the H4 binding proteins. As shown in Fig. 5B, a shifted band corresponding to the position of the H4TF-1-DNA complex appeared when the assay was performed with the P11 0.5 M KCl fraction and probe A. Specific H4 DNA competition experiments confirmed that this was the H4TF-1-DNA complex (not shown). A band also appeared in the P11 flow-through fraction using probe A. This band predominantly represents the nonspecific DNA-protein complex seen in the nuclear extracts (also see below). When probe B was used A
B 1 2 3 4 5 6 7
1 2 3 4 5 6 7
A..... * ..."M.
a
.. 4~ mw
FIG. 4. Competition analysis with several independent genomic human histone H4 gene isolates. Binding reaction mixtures were assembled with the inclusion of a 25-molar excess of a plasmid bearing a different human histone H4 genomic clone (15). The reaction mixtures were then electrophoresed. The bold arrow indicates the specific protein-DNA complex of H4TF-1 with probe A in A and H4TF-2 with probe B in B. Each reaction mixture had the following unlabeled competitor DNA: pUC19, lanes 1; H4 clone pHu4A, lanes 2; pHhlB, lanes 3; pHh2A, lanes 4; pHh4B, lanes 5; pHh5C, lanes 6; pHh7A, lane 7.
I.
-_ FIG. 5. Separation of the two human histone H4 factors by chromatography. (A) Fractionation scheme. S-phase extract (S Extract) was put over a heparin/agarose (HA) column at 0.1 M KCl in buffer B and bound proteins were eluted at 0.4 M KCl in buffer B. This bound material was then dialyzed and passed over a phosphocellulose (P11) column, and 0.1 M KCI flow-through, 0.35 M KC1, 0.5 M KC1, and 1.0 M KCl step fractions were collected. Each of the phosphocellulose fractions was analyzed by electrophoretic mobility shift assay using probe A (B) or probe B (C). Two to 3 Itg of protein from each fraction was added to the binding reaction mixture.
to analyze the P11 fractions, a band corresponding to the H4TF-2-DNA complex (Fig. SC, arrow) was detected in the 0.1 M KCl flow-through (Fig. 5C) fraction and its identity was confirmed by competition with appropriate H4 mutants (not shown). In addition to this complex, nonspecific proteinDNA complexes were seen in the flow-through and 0.35 M step fractions (Fig. SC). The nonspecific complex seen in the 0.35 M step in Fig. 5C occurred when the binding was done at 1 mM MgCl2 (the condition for probe B) but not when the binding reactions are done in 6 mM MgCl2 (as for probe A). Analysis with similar fractions revealed binding of H4TF-2 in the P11 flow through to probe A at 1 mM MgCl2 when the amount of nonspecific protein binding was minimal and that nonspecific and H4TF-2 protein-DNA complexes comigrate when probe A is used. In summary, therefore, these results clearly demonstrate that the factors H4TF-1 and H4TF-? are distinct species and that they can bind the H4 promoter
independently.
DISCUSSION In this paper we describe the resolution of two proteins that bind to distinct portions of the human histone H4 transcriptional regulatory region. H4TF-1 binds to sequences between -80 and -110 bp from the cap site and H4TF-2 interacts with the DNA immediately upstream from the TATA box. The precise contacts of these factors with the H4 DNA have not been determined; however, several interesting sequence elements are present within the binding domains of these proteins. The demonstrated H4TF-1 binding region includes the sequence GATTTC, which is closely related to a hexameric sequence, GACTTC, present in several human histone genes (11). It is possible that this element may be important for binding of H4TF-1, since a mutant H4 promoter containing only this half of the H4TF-1 binding domain can compete with the wild-type promoter in the band shift assay. Since two additional copies (at -79 to -74 and -68 to -63)
Biochemistry: Dailey et al. of the histone hexamer are present proximal to the H4TF-1 binding site mapped in this study, it seems possible that this factor may interact with lower affinity to those sites. In this context it is relevant that one copy of this element is located at precisely the same site relative to the TATA box between
-70 and -60 in the H4 and H2b promoters and that it has been demonstrated to function in transcription of the H2b gene (11, 14). The demonstrated binding site of H4TF-1 also covers the sequence 5' CGGCGGG 3', which is found upstream from several mammalian histone H4 genes (10-13). Although it is closely related to the consensus sequence identified for binding of transcription factor Spl (22), the inability of the SV40 early promoter to compete for binding of H4TF-1 indicates that H4TF-1 is not Spl. Resequencing of the pHu4A promoter has revealed the presence of three additional cytidine residues between positions -89 and -90. This addition creates a 13-bp stretch of pyrimidines within the H4TF-1 binding region, which may also be relevant to the binding of this factor. The binding of H4TF-2 to the subtype-specific consensus element in the H4 promoter most probably involves the GGTCC core sequence since this sequence is found in many histone H4 genes (10-13), and a 3-bp insertion at this position in the wild-type promoter destroys the ability of the mutant CF-3 to compete for binding of the protein. However, this is probably not the only sequence required for binding H4TF-2 since the 5' deletion mutant containing DNA downstream of -49 (which has the GGTCC sequence) is unable to compete for binding. Failure to detect H4TF-2 bound to the full-length H4 promoter probe in crude extracts was mainly due to the comigration of the complex with a nonspecific DNA-protein
complex.
The first issue to be addressed is whether these proteins have a function relevant to transcription of the H4 gene. In the case of the H4TF-1, this seems certain since it is bound by most H4 genomic DNAs and progressive deletion of sequences within its binding site gradually reduces transcription in S-phase nuclear extracts from 100%o (when the site is intact) to 16% (when the site is completely removed) (ref. 9; as summarized in Fig. 2). The gradual reduction in transcription activity of the H4 promoter as the sequences are deleted may indicate that H4TF-1 can interact weakly with a partial binding site lacking the distal GAT"TC element or that this factor can bind with reduced affinity to the more proximal GACTTC repeats. The fact that the H2B promoter, which contains a perfect hexamer at -70, cannot compete for binding of H4TF-1 between - 80 and -100 in the H4 promoter is consistent with either of these possibilities. Preliminary data using a DNA fragment containing H4 promoter sequences downstream of position -88 (63% of wild-type transcription activity in vitro) suggest that H4TF-1 may interact weakly with this altered promoter. The importance of H4TF-2 for transcription of the H4 gene is implied by the conservation of nucleotide sequence and position of the subtype-specific consensus element in many different H4 genes. Although previous studies of this gene using 5' deletion mutants failed to demonstrate an independent role for this sequence in the absence of more distal elements (9), preliminary recent data indicate that it is indeed required for transcription in vitro (unpublished data). It seems apparent, therefore, that the role of H4TF-2 in transcription is influenced by and dependent upon the presence of upstream factors or sequences. Furthermore, Clerc et al. (23) have shown that this element is essential for transcription of a Xenopus H4 gene upon injection into oocytes. For the reasons stated above, we believe that H4TF-1 and H4TF-2 are required for expression of the H4 gene and that
Proc. Natl. Acad. Sci. USA 83 (1986)
7245
they are necessary, if not sufficient, for cell cycle regulation. However, based on a comparison of the DNA sequences bound by these factors with the phenotypes of the H4 deletion mutants we have previously characterized (9), there remains a distinct possibility that there may be elements not included in the binding sites mapped for H4TF-1 and H4TF-2 that are important for transcription from this promoter. Specifically, sequences between -71 and -84 nucleotides upstream from the cap site can result in a >2-fold increase in transcription in vitro (from 16% to 37% of wild type) but include less than one-third of the region protected by H4TF-1 from DNase attack. As an alternative to the suggestion that H4TF-1 may interact with less affinity to this region of the promoter, it is possible that there is a third factor whose action is effected through this medial region of the promoter and that it is required in addition to H4TF-1 and H4TF-2 for maximal transcription. Since in vitro regulation of this gene is dependent only on sequences from the cap site to the distal H4TF-1 binding site, we suggest that one of these activities (H4TF-1, H4TF-2, or yet another factor) is critical for cell cycle regulation in vivo. Obviously, changes in the activity of this factor could result either from alterations in its absolute levels or activity during S phase. Future experiments involving transcription of the H4 gene upon addition of the binding proteins to a reconstituted system of purified transcription factors and RNA polymerase II should help to determine the role of each of the H4 promoter binding proteins in H4 gene transcription and to define the critical elements that distinguish the activity of the S-phase and non-S-phase extracts in vitro. We thank Imre Kovesdi for helpful suggestions in the initial setting up of the binding assays and Michele Sawadogo for many interesting discussions. We also thank Olga Capasso for critical discussion and reading of the manuscript. This work was supported by National Institutes of Health Grants GM 32544 (to N.H.) and CA 34891 (to R.G.R.). N.H. was also supported by Pew Scholars Award. L.D. was supported by American Chemical Society Postdoctoral Fellowship PF-2739. 1. Maxson, R., Cohn, R., Kedes, L. & Mohun, T. (1983) Annu. Rev. Genet. 17, 239-277. 2. Groppi, V. E., Jr., & Coffino, P. (1980) Cell 21, 195-204. 3. Wu, R. S. & Bonner, W. M. (1981) Cell 27, 321-330. 4. Robbins, E. & Borun, T. W. (1967) Proc. Natl. Acad. Sci. USA 58, 1977-1983. 5. Butler, W. B. & Mueller, G. C. (1973) Biochim. Biophys. Acta 294, 481-496. 6. Heintz, N., Sive, H. L. & Roeder, R. G. (1983) Mol. Cell. Biol. 3, 539-550. 7. Sittman, D. B., Graves, R. A. & Marzluff, W. F. (1983) Proc. Natl. Acad. Sci. USA 80, 1849-1853. 8. Heintz, N. & Roeder, R. G. (1984) Proc. Natl. Acad. Sci. USA 81, 2713-2717. 9. Hanly, S. M., Bleecker, G. C. & Heintz, N. (1985) Mol. Cell. Biol. 5, 380-389. 10. Seiler-Tuyns, A. & Birnsteil, M. L. (1981) J. Mol. Biol. 151, 607-625. 11. Zhong, R., Roeder, R. G. & Heintz, N. (1983) Nucleic Acids Res. 11, 7409-7425. 12. Sugarman, B. J., Dodgson, J. B. & Engel, J. D. (1983) J. Biol. Chem. 258, 9005-9016. 13. Sierra, F., Stein, G. & Stein, J. (1983) Nucleic Acids Res. 11, 7069-7086. 14. Sive, H. L., Heintz, N. & Roeder, R. G. (1986) Mol. Cell. Biol., in press. 15. Heintz, N., Zernik, M. & Roeder, R. G. (1981) Cell 24, 661-668. 16. Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982) Mol. Cell. Biol. 2, 1044-1051. 17. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 18. Fried, M. & Crothers, D. M. (1981) Nucleic Acids Res. 9, 6505-6525. 19. Garner, M. M. & Revzin, A. (1981) Nucleic Acids Res. 9, 3047-3060. 20. Topol, J., Ruden, D. M. & Parker, C. S. (1985) Cell 42, 527-537. 21. Maxam, A. & Gilbert, W. (1980) Methods Enzymol. 65, 499-580. 22. Dynan, W. S. & Tjian, R. (1985) Nature (London) 316, 774-778. 23. Clerc, R. G., Bucher, P., Strub, K. & Birnstiel, M. L. (1983) Nucleic Acids Res. 24, 8641-8657.
