Proc. Natl. Acad. Sci. USA Vol. 84, pp. 3982-3986, June 1987 Biochemistry Proximal and distal regulatory elements that influence in vivo expression of a cell cycle-dependent human H4 gene (gene regulation//S1 nuclease analysis/) P. KROEGER, C. STEWART, T. SCHAAP, A. VAN WUNEN, J. HIRSHMAN, S. HELMS, G. STEIN, AND J. STEIN University of Florida College of Medicine, Gainesville, FL 32610 Communicated by Sidney Weinhouse, February 6, 1987

ABSTRACT We have examined the sequences required in To establish in vivo the functional properties of 5' flanking vivo to promote transcription of a cell cycle-regulated human sequences associated with the F0108 human H4 histone gene, H4 histone gene. Deletion mutants of the 5' flanking region we constructed a series of mouse cell lines carrying H4 genes were assayed in mouse cells or fused with the chloramphenicol with deletions in the region. These mouse cells acetyltransferase (CAT) gene for assay in HeLa cells. The have been shown to correctly express transfected human functional limits of the regulatory sequences were shown to histone genes (16, 17), suggesting that there are trans-acting extend at least 6.5 kilobases (kb) upstream. Sequences suffi- factors that regulate the expression of these heterologous cient for correctly initiated transcription were found in the 70 genes. Analysis of these deletion mutants has allowed us to base pairs (bp) immediately 5' to the cap site. A proximal identify both proximal and distal elements that are respon- element located 200-400 bp upstream increased the level of sible for initiation and maintenance of transcription in vivo. transcription several times above the basal level, although not Our results also indicate that 5' sequences located 6.5 to maximal levels. Maximal levels of expression were achieved kilobases (kb) upstream from this H4 gene may positively with 6.5 kb of 5' flanking sequence adjacent to the proximal influence the level of transcription. promoter sequences or when a distal enhancer element with both position- and orientation-independent function was MATERIALS AND METHODS moved proximal to the promoter. Our results indicate that a series of 5' cis-acting sequences are functionally related to the Construction of Deletion Mutants. BAL-31 deletions of fidelity and level of expression of this human H4 histone gene. pF0108A were prepared as reported (15). pFOO05, pFOO04, and pF0003 were constructed by standard cloning procedures The human histone genes encode a set of proteins essential using fragments from XHHG41 or other subclones (Fig. 1). for maintaining the integrity of eukaryotic chromosomal The 3' deletion mutants of the promoter region (see Fig. 2B) structure (1-4). The combined studies of several investiga- were made by BAL-31 deletion from a unique Sst II site in the tors have now established two basic facts: (i) most histone coding region followed by addition of Pst I linkers. Samples genes are expressed coordinately during the cell cycle in were digested with an excess of Pst I and then with BamHI. conjunction with DNA synthesis, and (ii) there are many Fragments were isolated and ligated to the 5' end of the different copies of the core and H1 histone genes being chloramphenicol acetyltransferase (CAT) gene (18) cloned in expressed concomitantly (5-8). This leads one to conclude pUC19. The distance from the TATA box to the cap site of that histone gene expression on a larger scale is coordinately the CAT gene in the A3 construct was determined by controlled, but that at the level of the single gene there may sequencing to be 26 base pairs (bp), a distance that mimics its be differences in the extent and timing of expression. Our natural location. previous results with H4 histone genes support this conten- Construction of Stable Cell Lines. C127 mouse cells were tion that not all human histone genes are expressed to the cotransfected with 10 ,g of histone plasmid and 10 ug of same degree during S phase (5). We and others have shown pSV2neo (19) using the CaPO4 precipitation/glycerol shock that the regulation of histone gene expression occurs at both method (18, 20). The cells were grown in Dulbecco's modi- the transcriptional and post-transcriptional levels. The two fied minimal essential medium containing 500 ,ug of G418 per levels of regulation together serve to increase significantly ml (GIBCO) for selection of resistant cells. G418-resistant the amount ofhistone messenger RNA during S phase (6-11). single clones (monoclones) were selected or entire plates Taking these results into account, we have initiated in vivo were pooled and passaged as mixed cell populations ("poly- studies to identify transcriptional regulatory sequences as- clones"). All experiments were performed with exponential- sociated with a human H4 histone gene. The gene we have ly growing cells. The presence and expression of integrated chosen to study has been previously shown to be one of the H4 histone gene constructs was confirmed by Southern most highly expressed human H4 histone genes (5). blotting of isolated (21) and S1 nuclease analysis of Many studies have established that there are consensus whole-cell RNA preparations (22). sequences present in RNA polymerase II promoters that are Short-Term Transient Expression. Transfection of mouse necessary for expression (12-14). Sequence analysis of the LTK- or HeLa S3 cells with histone plasmids was performed F0108A H4 histone gene has demonstrated the presence of by the CaPO4 precipitation/glycerol shock method (18). The several such sequences in the 5' flanking region, including cells were harvested 36-48 hr after glycerol shock and total two "CAAT" boxes and a "TATA" box (15). In vitro cellular RNA was prepared (6, 23, 24). transcription studies of this gene using whole-cell lysates S1 Nuclease Analysis of RNA Isolated from Stable Cell Lines have indicated that only the TATA box is necessary for or Short-Term Transient Assays. RNA (25-50 pzg) isolated correct transcript initiation (15). from short-term transient assays or stable cell lines was ethanol-precipitated with an excess of mouse and/or human The publication costs of this article were defrayed in part by page charge probes (see figure legends). S1 nuclease protection analysis payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: CAT, chloramphenicol acetyltransferase.

3982 Downloaded by guest on September 29, 2021 Biochemistry: Kroeger et al. Proc. Natl. Acad. Sci. USA 84 (1987) 3983 A HHG41 ,.Hi 9v Q. .9§9** + 9 If p

J67 Q P Q _ -45 C _ J56 w Q P -71 Q * Q _ -91 J50 B _ f Qw_-9146 Bal31 deletions of FO1O8A K8 Q iQ -146 aw Q L14 LP 9-166 A _ J40 Q Q -191bepro FO108A 2 Q _ F0005 F0004 Q s 9 F0003 gm_++ + v v s "O V ()probe go 4po scale

FIG. 1. Schematic diagram of human H4 histone gene 5' deletion 2 3 4 5 6 7 8 9 constructs. The original genomic clone, XHHG41, is shown for reference. Six BAL-31 deletions of F0108A (-210 bp) are shown, start to the B Histone Promoter with the 5' distance from the transcription site displayed '1'ct right. Construct F0005 has 410 bp of 5' flanking sequence. F0004 has the 210 bp of F0108A fused to the upstream 1.5-kb EcoRI fragment P CAT of XHHG41. F0003 contains 6.5 kb of 5' sequence, which covers almost the entire right end of XHHG41. The scale represents 2 kb on

the XHHG41 diagram and 1 kb on all others. o, HindIII; *, Pst I; m, Xba I; e, EcoRI; *, BamHI; o, Nco I. A.4 lTfC,1e, was carried out by a modification of the method of Berk and Sharp (5, 22). D3 RESULTS P PE B To address regulation oftranscription ofthe F0108 human H4 E2 -lO0bp histone gene, we determined the requirement of 5' flanking sequences for correctly initiated transcription and for the FIG. 2. Deletions ofthe promoter region from the 3' end. (A) CAT extent to which the gene is transcribed. Our experimental assay ofHeLa cells transfected with promoter 3' deletion constructs. strategy was to construct a series of deletion mutants with Extracts were prepared from HeLa cells transfected with 10 ,ug of each construct and 10 utg of salmon sperm and assayed as various amounts ofupstream sequence and then to determine DNA described (15). The three acetylated forms of chloramphenicol are genes in vivo the cellular levels oftranscription of these after designated A, B, and C. Lanes: 1, control with no CAT enzyme; 2, transfection into mouse cells. 0.9 unit of purified CAT enzyme; 3, 0.09 unit of purified CAT Deletions of the Promoter Region from the 3' End. To enzyme; 4, extract from cells transfected with salmon sperm DNA; understand the in vivo role of proximal sequences in the 5, extract from pSV2cat-transfected (18) cells; 6, A3 extract; 7, C1 transcriptional regulation of H4 histone gene expression, we extract; 8, D3 extract; 9, E2 extract. (B) Diagram of 3' deletion first conducted short-term transient expression experiments constructs. Histone promoter sequences with 3' deletions were fused with a series of 3' deletions of the promoter region. These to the CAT structural gene. Relevant 5' promoter consensus se- constructs (Fig. 2B) are fusions ofhistone promoter deletions quences are noted. P, Pst I; E, EcoRI; B, BamHI. with the CAT structural gene and contain -1 kb ofH4 histone 5' flanking sequence. The fusion constructs were transfected tion has occurred. Cellular levels of correctly initiated into HeLa S3 cells, and 36-48 hr later whole cell freeze-thaw from the transfected H4 histone gene constructs were deter- supernatants were assayed for the level of CAT activity (18). mined by an S1 nuclease protection assay. Because all The results shown in Fig. 2A along with appropriate controls experiments were carried out using exponentially growing indicate that progressive deletion in the 5' direction from the cells and the mRNA coding regions of all constructs were TATA box through the promoter region in constructs A3, C1, identical, cellular levels of mRNA should be an accurate D3, and E2 (lanes 6-9) resulted in decreasing amounts of reflection of transcription. Equimolar concentrations of pro- CAT activity until, in E2, where all proximal consensus moters were maintained in each transfection to ensure that sequences have been deleted, activity was no longer detect- the levels of mRNA accurately reflected the degree to which able. There was a considerable drop in the level of CAT the human H4 histone promoter could function in vivo. activity when the TATA box was deleted in Cl (compare Fig. 3 shows a representative example of the results we lanes 6 and 7). These results implicate both the TATA box have obtained for the 5' deletion constructs using transient and the region of the second CAAT box as important in the expression assays. Our results indicate that sequences cor- transcription offunctional mRNA from this H4 histone gene. responding to the 90 bp 5' to the transcription start site 5' Deletions-Transient Assays. To complement the set of including both CAAT boxes and the TATA box were re- deletions extending through the promoter region from the 3' quired for initiation of transcription in vivo (lanes 9 and 10). end, a series of5' promoter deletions of the F0108 H4 histone The presence ofadditional 5' sequences extending to -210 bp gene was prepared. These deletions were assayed in a (pF0108A) (lanes 5-8) produced a 4-fold increase over the short-term transient expression system in mouse LTK- cells level of transcription from J50 (lane 9), which extends only to determine the level of human H4 histone transcripts. The through the second CAAT box (-90 bp). For the purpose of short-term transient assay reflects the level of transcription comparison, we have defined the basal level of transcription from transfected DNA before stable chromosomal integra- as that observed for construct J50. However, when we Downloaded by guest on September 29, 2021 3984 Biochemistry: Kroeger et al. Proc. Natl. Acad. Sci. USA 84 (1987) 6 7 8 9 10 protection assays. In Fig. 4 are shown examples ofthe results 4c we obtained with this assay from both polyclonal (Fig. 4A) a an 0 X 0 0 I o q 0 r- *1 00 00 0 v in and monoclonal (Fig. 4B) cell lines. A mouse H4 histone I 0 -l X1 -o UL Ye Ji n probe was included in each S1 nuclease assay to serve as an internal control. This allowed us to express the level of *X .. transcript from the deletion mutants as a function of endog-

s ;. enous cellular levels of mouse H4 histone gene transcript. In k.;f, Table 1 we have presented the results of multiple determi- .7 nations with each polyclonal or monoclonal cell line. In most cases, analysis was carried out on more than one indepen- 1I dently isolated cell line for each construct. The data are A.^ a1 expressed relative to the level of transcripts from F0003, which is the highest-expressing construct. Overall, the results we obtained from the polyclonal and '.4 monoclonal cell lines were consistent with the results of the .il.'! _. 29 short-term transient assays. In the stable cell lines, the presence of at least 410 nucleotides of 5' flanking sequence ,low (F0005, F0003) yielded significantly increased levels of Lz mRNA transcribed from this H4 histone gene compared with 221 F0108A and 'other constructs that have <210 nucleotides of J~~~~~~~~~~~~~~~~~ 5' flanking sequence (compare Fig. 4A, lanes 1 and 2 with lanes 4-9 and Fig. 4B, lanes 1 and 2 with lanes 4-6). As was seen in the transient assays, when only 50 bp of sequence 5' to the transcription start site were present, no correctly FIG. 3. S1 nuclease analysis of short-term transient assays of H4 initiated transcription was observed (Fig. 4A, lane 10). There histone 5' deletion constructs. The probe was single end-labeled at were, however, significant quantitative differences between the Nco I site in the H4 histone coding region and extends to the the short-term transient assays and the stable cell lines in the BamHI site. Markers (lane M) were pBR322 digested with Hinfl. level of message from constructs F0003, F0004, and F0005 Fifty micrograms of RNA from each transient transfection was (compare Fig. 4 A and B, lanes 1-3 with Fig. 3, lanes 2-4). assayed. Lanes: 1, probe fragments protected by 25 tug of total HeLa The most notable difference between the short-term transient cell RNA (SS, salmon sperm-transfected cells); 2-10, 5' deletion and stable line was to the constructs transiently expressed in mouse LTK- cells. Upper bands assays the cell results with respect in each lane correspond to transcripts initiated in the vector and map level of H4 mRNA transcribed from F0003. This construct, to the end of the homology between probe and construct. Each which has 6.5 kb of 5' flanking sequence, expressed high construct was transfected and assayed for expression at least three levels of H4 histone mRNA (50-fold above the basal level) times. when integrated in a stable cell line compared with a 4-fold increase above the basal level in transient assays (compare assayed the construct F0005 (lane 3), which contains 410 Fig. 4A with Fig. 3). This difference between transient assays nucleotides upstream from the cap site, we observed a and stable cell lines may be a reflection of the chromatin significant (10-fold) increase in the expression of this H4 structure of nonintegrated versus integrated genes (26). histone'gene above the basal level. Extension of the 5' region When the F0003 construct, with 6.5 kb of upstream to 6.5 kb in construct F0003 (lane 2) resulted in lower levels sequence, was compared to F0005 (-410 bp) (Fig. 4A, lanes ofhistone gene transcripts when compared with F0005. When 1 and 2; Table 1), we observed a 3-fold increase in the level the distal 5' sequence located 6.5 kb upstream was moved to of H4 histone mRNA. These results suggested that an within 210 nucleotides of the cap site in F0004 (lane 4), the upstream sequence positively influences expression. In level of transcription was enhanced 3-fold over F0108A. For F0004, a distal sequence element (-6.0 kb to -7.5 kb) was comparison, logarithmic phase HeLa cell RNA was subject- placed into a proximal position (at -210 bp) with respect to ed to S1 nuclease protection analysis (lane 1) using the human the human H4 coding region, and this construct expresses at H4 histone probe. These experiments suggested that there nearly the level of F0003 (Fig. 4A, lane 3; Fig. 4B, lane 3), were proximal and distal elements functioning in the short- indicating that this far upstream region may contain se- term transient assay to regulate the level ofhuman H4 histone quences that influence transcription of the F0108 H4 gene. mRNA. 5' Deletions-Stable Cell Lines. To determine whether DISCUSSION integration of the transfected human H4 histone gene into the host-cell genome influenced expression, we prepared stable Our studies of the cell cycle-regulated F0108 human H4 mouse cell lines containing the 5' deletion constructs. The histone gene'have elucidated proximal and distal elements regulated expression of human histone genes in mouse cells that influence transcription. The proximal elements consist of has been documented (16, 17) and suggests that murine and conserved sequences that have been shown to be required for human H4 histone gene regulatory sequences and trans- transcription in many systems (12-15). The TATA box acting factors are compatible. Both polyclonal and monoclo- homology, which is -30 bp from the cap site, has been shown nal cell lines were prepared to preclude the possible influence to function in vitro and in vivo to specify the start of of integration sites on expression. Southern blot analysis of transcription. In our in vivo system, we have shown with the the stable cell lines indicated that most of the integrated deletion mutant J67 (-45 bp) that the TATA box is not constructs had their coding regions and flanking sequences sufficient for the production of correctly initiated histone intact (data not shown). We also determined the copy number mRNA. Previous in vitro work with the J67 construct of each cell line and did not observe a direct correlation demonstrated that it was accurately transcribed in a cell-free between copy number and the level of expression of the system (ref. 15; K. Wright, personal communication). Most transfected genes (data not shown). likely, the J67 construct does not contain sufficient histone The quantitation of human H4 histone gene transcripts in promoter sequence to allow correct in vivo recognition by the various polyclonal and monoclonal cell lines was deter- proteins required for transcription. Notably, this deletion mined by densitometry of autoradiograms from S1 nuclease mutant lacks the sequences shown in our laboratory (27, 28) Downloaded by guest on September 29, 2021 Biochemistry: Kroeger et al. Proc. Natl. Acad. Sci. USA 84 (1987) 3985 A Table 1. Quantitation of H4 histone mRNA in 1 2 3 4 5 6 7 8 9 10 11 12 human levels CO an le co transfected cell lines o 0 a o I* #I-. oa n J- co in*n*so ?C-P61 O 0 00 * Construct % % .7 ..1- i.. .3 .3 0 ±c a Polyclones, Monoclones, U. U. Ui. F0003 100 (4) 100 (5) *& 309 F0004 84 (3) 52 (3) .1. F0005 35 (3) 25 (4) I_, HHuman F0108A 2 (1) ND J40 4(2) 8 (2) I 242 0 L14 2 (2) 3 (2) I * 217 K8 3 (2) 1(2) J50 5 (1) ND * 201 J56 2 (1) ND - 190 J67 0 (1) ND '3* " " 180 The relative level of expression of histone mRNA from each H4 histone gene construct was compared for polyclonal and monoclonal 0 4p 160 cell lines. Percentage expression represents the average of several

147 cell lines and is based on levels of H4 mRNA detected in cells transfected with F0003, which was the highest-expressing construct in both polyclonal and monoclonal cell lines. The number ofcell lines 0 .. used for each determination is listed in parentheses; each cell line

d was assayed at least twice. Quantitation of the steady-state message * f levels was performed using autoradiographic exposures in the linear * J 110 ranges of the x-ray film and the densitometer. The total amount of 1.4mtst *ftt - Mouse RNA in each sample was determined by scanning the endogenous mouse message signal. ND, not done. and others (29) to be required for binding histone-specific B 1 2 3 4 transcription factors. 5 6 7 8 Our studies have implicated the two CAAT boxes located at -70 bp and -90 bp 5' to the transcription initiation site in Cl un 0 0To 0 I* N the in vivo expression of this human H4 histone gene. In all o a a aU. . u -r -i x 0 assays, the CAAT boxes were required for accurate initiation 309 of transcription and maintenance of the basal level of tran- scription. When the CAAT box has been altered in other eukaryotic promoters (30, 31), a substantial decrease in to _Human 242 was seen. The recent purification offactors 238 9 that interact with the CAAT box in vitro (31, 32) points especially to its role in the transcriptional regulation of RNA 217 polymerase II promoters. Analysis of in vivo sites ofprotein- 201 DNA interaction carried out in our laboratory has shown that 190 a region spanning the CAAT box is protected from DNase I 180 digestion, and this may be functionally related to the expres- sion of this human H4 histone gene (28). 0 160 Many genes contain the proximal regulatory sequences we have described here (TATA and CAAT). These regulatory 147 sequences have in most cases been shown to be necessary for maximal transcription of their accompanying gene (30). We have delineated two areas of the human H4 histone gene 123 promoter that function to stimulate transcription in vivo many 110 times above a basal level. The first area is found in the EcoRI/HindIII fragment between -210 and -410 bp, and in ^ Mou a a 0 _ both the stable polyclonal and monoclonal cell lines this fragment increased the level of transcription 10-fold above basal level. The second area shown to stimulate the tran- scription of this H4 histone gene in both polyclonal and monoclonal cell lines is located -6.5 kb upstream of the cap FIG. 4. S1 nuclease analysis of RNA from cell lines containing stably integrated H4 histone 5' deletion constructs. Two DNA probes site. Several lines of evidence have led us to postulate that were used in these studies. The human H4 histone gene probe was this far upstream element has enhancer-like activity. The labeled at the Nco I site with polynucleotide kinase and [y32P]ATP distal area we have identified increased the expression of the and extends to the HindIl site in the 5' flanking region. The total H4 histone gene not only in its natural location 6.5 kb length is 695 nucleotides and the protected fragment is 280 nucleo- upstream, but also when the distal segment was fused 210 tides. The mouse H4 histone probe, used as a control for quantita- nucleotides upstream of the cap site in either orientation. tion, was derived from pBR-mus-hi-I-H4-HinfI (25). The probe Therefore, we have shown that the distal element can exert fragment is 1000 nucleotides and was labeled at the BstNI site in the is inde- coding region. The protected fragment is 110 nucleotides. Total influence over a long distance and that its activity cellular RNA (25 ,ug) was hybridized to an excess of human and pendent ofits exact location and orientation. Other studies in mouse probe at 550C for 3 hr, digested with S1 nuclease and analyzed on a 6% denaturing polyacrylamide gel. (A) Polyclonal cell lines. fragments protected by RNA isolated from cell lines containing the Lanes: 1-10, probe fragments protected by RNA isolated from cell 5' deletion constructs; 7, HeLa cell RNA; 8, C127 RNA. Markers are lines containing integrated H4 histone deletion mutants; 11, C127 cell pBR322-digested with Hpa II (lane M). The respective human and RNA; 12, HeLa RNA. (B) Monoclonal cell lines. Lanes: 1-6, probe mouse signals are indicated. Downloaded by guest on September 29, 2021 3986 Biochemistry: Kroeger et al. Proc. Natl. Acad. Sci. USA 84 (1987)

our laboratory have linked this distal element to the 3' end of man, S., Stein, J. & Stein, G. (1984) Proc. Nati. Acad. Sci. the CAT structural gene and demonstrated a 4-fold increase USA 81, 434-438. in CAT activity (37). 8. Heintz, N., Sive, H. C. & Roeder, R. G. (1983) Mol. Cell. are the ofthis Biol. 3, 539-550. There two plausible explanations for activity 9. Baumbach, L., Marashi, F,, Plumb, M., Stein, G. & Stein, upstream element. First, it may function as a general acti- J. L. (1984) Biochemistry 23, 1618-1625. vator of the region. Histone genes are clustered but not 10. Morris, T., Marashi, F., Weber, L., Hickey, E., Greenspan, tandemly repeated (33, 36). Some H4 histone genes that have D., Bonner, J., Stein, J. & Stein, G. (1986) Proc. Natl. Acad. been characterized do not have 7.5 kb of uninterrupted Sci. USA 83, 981-985. flanking sequence, suggesting perhaps that not all H4 genes 11. Sittman, D. B., Graves, R. A. & Marzluff, W. F. (1983) Proc. would be associated directly with this element. Second, this NatI. Acad. Sci. USA 80, 1849-1853. element may serve to enhance this particular H4 histone 12. McKnight, S. L. & Kingsbury, R, (1982) Science 217, 316-324. gene, and we note that from our previous work this H4 gene 13. Benoist, C., O'Hare, K., Breathrach, R. & Chambon, P. (1980) Nucleic Acids Res. 8, 127-142. is one ofthe most highly expressed (5). In another human H4 14. Breathrach, R. & Chambon, P. (1981) Annu. Rev. Biochem. histone gene examined by Hanly et al. (34), sequences that 50, 349-383. influence transcription were shown to reside within 110 15. Sierra, F., Stein, G. & Stein, J. (1983) Nucleic Acids Res. 11, nucleotides upstream of the transcription start site. Howev- 7069-7086. er, because these studies were carried out in vitro, a direct 16. Green, L., Schlaffer, I., Wright, K., Moreno, M. L., Berand, comparison with our in vivo results is difficult. The possibility D., Hager, G., Stein, J. & Stein, G. (1986) Proc. NatI. Acad. must be considered that different copies of the human H4 Sci. USA 83, 2315-2319. histone genes are regulated by different types of transcrip- 17. Capasso, D. & Heintz, N. (1985) Proc. Natl. Acad. Sci. USA tional control elements. 82, 5622-5626. 18. Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982) Mol. In summary, we have examined in vivo the proximal and Cell. Biol. 2, 1044-1051. distal elements required for transcription of a human H4 19. Southern, P. J. & Berg, P. (1982) J. Mol. Appl. Genet. 1, histone gene. Three classes of 5' regulatory sequences were 327-341. identified. Minimal proximal sequences that were necessary 20. Graham, F. & van der Eb, A. (1973) J. Virol. 52, 456-457. for initiation of transcription were determined to include the 21. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. CAAT box and the TATA box. A "distal-proximal" se- 22. Berk, A. J. & Sharp, P. A. (1978) Proc. Nati. Acad. Sci. USA quence located between -210 and -410 bp upstream stim- 75, 1274-1278. ulated transcription 10-fold above basal level. Inclusion of6.5 23. Tullis, R. H. & Rubin, H. (1980) Anal. Biochem. 107, 260-264. kb of 5' sequence resulted in additional enhancement of 24. Bird, C. R., Jacobs, F. A., Stein, G. S., Stein, J. L. & Sells, B. H. (1985) Proc. Natl. Acad. Sci. USA 82, 6760-6764. transcription by severalfold. These results are consistent 25. Seiler-Tuyns, A. & Birnstiel, M. L. (1981) J. Mol. Biol. 151, with the hypothesis that the control of histone gene tran- 607-625. scription is a regulated mechanism that allows periodic 26. Reeves, R., Gorman, C. M. & Howard, B. (1985) Nucleic stimulation of transcription above a basal level. Previous Acids Res. 13, 3599-3615. results from our laboratory have shown that the transcription 27. van Wijnen, A. J., Stein, J. L. & Stein, G. S. (1987) Nucleic of this H4 histone gene at the onset of S phase is stimulated Acids Res. 15, 1679-1698. -3-fold above the basal level of transcription that occurs 28. Pauli, U., Chrysogelos, S., Stein, G. S., Stein, J. L. & Nick, throughout the cell cycle (35). H. (1987) Science, in press. 29. Dailey, L., Hanly, S. M., Roeder, R. G. & Heintz, N. (1986) Proc. NatI. Acad. Sci. USA 83, 7241-7245. We wish to thank our colleagues Urs Pauli and Susan Chrysogelos 30. Myers, R. M., Tilly, K. & Maniatis, T. (1986) Science 232, for helpful discussions during the course of these studies. These 613-618. studies were supported by grants from the National Institutes of 31. Graves, B. J., Johnson, P. F. & McKnight, S. L. (1986) Cell Health (GM 32010), National Science Foundation (PCM83-18177), 44, 565-576. and the March of Dimes Birth Defects Foundation (1-813). 32. Jones, K. A., Yamamoto, K. R. & Tjian, R. (1985) Cell 42, 559-572. 1. Isenberg, I. (1979) Annu. Rev. Biochem. 48, 159-171. 33. Heintz, N., Zernik, M. & Roeder, R. G. (1981) Cell 24, 2. McGhee, J. D. & Felsenfeld, G. (1980) Annu. Rev. Biochem. 661-668. 49, 1115-1156. 34. Hanly, S. M., Bleecker, G. C. & Heintz, N. (1985) Mol. Cell. 3. Kornberg, R. D. (1977) Annu. Rev. Biochem. 46, 931-954. Biol. 5, 380-389. 35. Baumbach, L., Stein, J. & Stein, G. (1987) Biochemistry, in 4. Cartwright, I. L., Abmayr, S. M., Fleischmann, G., Lowen- haupt, K., Elgin, S. C. Z., Keene, M. A. & Howard, G. C. press. (1982) CRC Crit. Rev. Biochem. 13, 1-86. 36. Sierra, F., Lichtler, A., Marashi, F., Rickles, R., Van Dyke, 5. Lichtler, A. L., Sierra, F., Clark, S., Wells, J. R. E., Stein, T., Clark, S., Wells, J., Stein, G. & Stein, J. (1982) Proc. Natl. J. L. & Stein, G. S. (1982) Nature (London) 298, 195-198. Acad. Sci. USA 79, 1795-1799. 6. Plumb, M., Stein, J. & Stein, G. (1983) Nucleic Acids Res. 11, 37. Helms, S. R., van Wijnen, A. J., Kroeger, P. E., Shiels, A., 7927-7945. Stewart, C., Hirshman, J., Stein, J. L. & Stein, G. S. (1987) J. 7. Plumb, M., Marashi, F., Green, L., Zimmerman, A., Zimmer- Cell. Physiol., in press. Downloaded by guest on September 29, 2021