ANNUAL REVIEWS
Further
Quick links to online content Annu. Rev. Biochem. 1991. 60:827-61 Copyright © 1991 by Annual Reviews Inc. All rights reserved
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THE lREGULATION OF HISTONE SYNTHESIS IN THE CELL CYCLE M. A. Osley Program in Molecular Biology, Sloan Kettering Cancer Center, New York, New York
10021 KEY WORDS:
h i ston e RNAs: transcription , p rocessin g , turnov er .
CONTENTS SUMMARY AND PERSPECTIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
827
ORGANIZATION OF HISTONE GENES-STRUCTURE OF HISTONE mRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
828
OVERVIEW OF HIS TONE SYNTHESIS IN THE CELL CyCLE . . . . . . . . . . . . . . . . . . . . . . . .
829
TRANSCRIPTIONAL REGULATION . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Higher Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . ... . . . . . . . . Lower Eukaryotes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .
832 832
POSTTRANSCRIPTIONAL REGULATION . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . .... Higher Eukaryotes. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... . . . . . . . . . . . . . . . . . . . . . . . . Lower Eukaryotes . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . .
841 847 847 853
MULTIPLE FORMS OF REGULATION MODULATE HISTONE mRNA LEVELS IN THE CELL CyCLE . . . . . ........... . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . .
!l55
FUTURE PROSPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . .. . . . . . . . . . . . . . . . . . ... .. . . . . .. . . . . . . . . .
856
SUMMARY AND PERSPECTIVE The recent focus on the eukaryotic cell cycle has renewed interest in genes whose expression is regulated during the division cycle. The most prominent and best-studied examples of cell cycle-regulated genes are those encoding histones , proteins that are synthesized during S phase. Multiple levels of control are involved in restricting the synthesis of histones to this phase of the cell cycle , and their combined effect is to regulate the production of histone 827
0066-4154/9 1/0701 -0827$02.00
828
OSLEY
mRNAs. Three major pathways contribute to this regulation, and they act at the levels of transcription, pre-mRNA processing, and mRNA stability. During the past 10 years , each pathway has been subjected to detailed analysis; cis-acting regulatory elements and trans-acting regulatory factors have been identified, and in some instances, the mechanisms of regulation have begun to be defined. This review summarizes the major developments underlying the contribution of each pathway to the production of histone mRNAs in the eukaryotic cell cycle. Because significant differences are found
Annu. Rev. Biochem. 1991.60:827-861. Downloaded from www.annualreviews.org by Rice University on 04/22/13. For personal use only.
between higher and lower eukaryotes in the regulation of histone genes , these two groups of organisms are considered separately. Although this review emphasizes the control of histone synthesis during the cell cycle, histone synthesis is also regulated during development , and the interested reader is referred to ( 1-5) for discussions of this specialized aspect of regulation.
ORGANIZATION OF HISTONE GENES-STRUCTURE OF HISTONE mRNAs Two classes of histone genes are found in most eukaryotes. The most frequent class contains the replication-dependent histone genes whose expression is regulated in the cell cycle, while a second, less abundant class contains genes that encode the minor histone variants and whose expression occurs at a basal level throughout the cell cycle (6) . In all eukaryotes the replication-dependent core (H2A, H2B, H3, H4) histone genes are members of a multigene family, and in higher eukaryotes the linker (HI) histone genes are also repeated. The core histone gene family has as few as one to two members each, as exemplified by fungi (7- 14), to as many as several hundred per core histone gene in invertebrates (2, 3, 15, 16). The genomic organization of the histone genes differs widely among organisms. In invertebrates the genes are part of a reiterated tandem repeat encompassing the five histone genes (3, 16). This arrangement is not found in vertebrates or lower eukaryotes, where the genomic organization of histone genes is largely dispersive, albeit with some clustering (2 , 1 7-23). For example, in humans the H4 gene occurs in hetero geneous clusters , either unlinked to other histone genes or associated with as many as three other core histone genes ( 17 , 1 9 , 23). In budding yeast and other fungi, the genes encoding H2A and H2B are often linked and genetically separable from linked pairs of H3 and H4 genes (7, 8, 1 0, 1 3 , 14). A dispersive genomic organization implies that the histone genes are not regulated by far upstream enhancer elements. The regulatory sequences of these genes are in fact very compactly arranged. The 5 I sequences responsible for transcriptional control are usually found within several hundred bases upstream of the mRNA initiation site, and the 3 I processing signals in
REGULATION OF HISTONE SYNTHESIS
829
vertebrate histone genes are positioned close to the translation termination site
(24). Morl�over, vertebrate histone genes containing short 5' and 3' flanking sequences are correctly regulated following transfection into tissue culture cells (25-29), and yeast histone genes remain cell cycle regulated when placed in ectopic chromosomal locations (30). In
vertebrates,
two
remarkable features
distinguish
the replication
dependent histone genes from both the replication-independent histone genes and the majority of genes transcribed by RNA polymerase II. The first is the
Annu. Rev. Biochem. 1991.60:827-861. Downloaded from www.annualreviews.org by Rice University on 04/22/13. For personal use only.
absence of introns (24) . The second is the structure of the 3' terminus of mature histone RNAs. While most pol II transcripts are polyadenylated in vertebrate cells, the mRNAs produced by the replication-dependent histone genes do not contain poly(A) sequences and terminate with a highly con served stem-loop structure (24, 31). In most lower eukaryotes the histone genes are also free of introns (7, 11, 12), except for the histone genes of Neurospora (14), Aspergillus (13, 32), and Physarum (33). Another contrast between higher and lower eukaryotes is seen in th� structure of their mature histone RNAs. The histone mRNAs of some lower eukaryotes (fungi and ciliates) do not terminate with the stem loop structure that is characteristic of most eUkaryotic histone mRNAs, but instead are polyadenylated (7, 34-36). It is intriguing that the replication independent histone genes of vertebrates generally contain introns and are transcribed into polyadenylated mRNAs, since it is clear that neither of these features precludes the regulation of histone genes in the cell cycle of some lower eukaryotes.
OVERVIEW OF HISTONE SYNTHESIS IN THE CELL CYCLE The coupling of histone biosynthesis to the cell cycle was noted more than two decades ago by Robbins & Borun, who reported that histones were synthesized only in S phase cells (37). Subsequent studies showed that this pattern of histone protein synthesis is the direct consequence of the restriction of histone mRNA synthesis to the period of DNA replication (6, 38, 39). With the isolation of individual histone genes, the tools became available to study in detail the molecular basis of the coupling between histone biosynthesis and the DNA synthetic phase. What has emerged is a complicated picture of cell cycle regulation in which a combination of transcriptional and posttranscrip tional conltrois regulates the levels of histone mRNAs. Two general approaches have been taken to study histone mRNA metabo lism in the cell cycle. In the first, synchronized cell cultures have been used to evaluate at what point histone mRNAs accumulate during an uninterrupted cell cycle. In the second, inhibitors of DNA chain elongation have been
Annu. Rev. Biochem. 1991.60:827-861. Downloaded from www.annualreviews.org by Rice University on 04/22/13. For personal use only.
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OSLEY
employed to test whether ongoing DNA synthesis is a prerequisite for the continued production of histone mRNAs. The major conclusions of a large body of work are that histone mRNA production is temporally restricted to the S phase of the cell cycle and dependent on the presence of replicating DNA. During the cell cycle of most eukaryotes, histone mRNAs accumulate to maximal levels only during S phase. As cells pass from G1 into S phase, the levels of these mRNAs increase 15-30-fold, with peak accumulation occur ring in mid S phase (13, 18, 23, 40a-51). When DNA replication is in terrupted, histone mRNAs rapidly disappear from the cytoplasm (6, 40a, 41, 52-54). Three pathways regulate the production of histone mRNAs in the cell cycle of higher eukaryotes. The first regulates transcription at the G I-S phase boundary: as cells enter S phase, nascent histone mRNA synthesis increases three to five fold over basal levels of transcription characteristic of G1 phase cells (40a, 40b, 41-43, 52). The two remaining pathways regulate histone mRNA production at a posttranscriptional level. These latter pathways are responsible for the remaining five- to sixfold increase in histone mRNA levels that occurs during S phase and for the disappearance of histone mRNAs from the cytoplasm when chromosome replication is blocked. The two pathways of posttranscriptional regulation affect histone mRNA levels very differently in the cell cycle. The first acts in the cytoplasm to control the half-life of histone mRNAs. The major role of this pathway is to degrade histone mRNAs when DNA synthesis is inhibited (41, 52-55). The half-life of histone mRNAs in S phase has been estimated to be 30-60 minutes by several different techniques (40a, 40b). When chromosome replication is blocked with inhibitors of DNA chain elongation, the apparent half-life of these mRNAs drops to 10-15 minutes (40a, 41, 52-54, 56). Numerous studies have asked whether this pathway might also operate during the uninterrupted cell cycle. One point at which it might act is upon entry into S phase, where histone mRNAs show maximal levels of accumulation. Here, the pathway would be used to stabilize histone mRNAs in order to accumulate these transcripts. A compelling argument that changes in histone mRNA stability can take place during S phase arises from the observation that histone mRNA half-life can be modulated both rapidly and reversibly in this phase of the cell cycle. The destabilization of histone mRNAs that results from a replication block is abrogated when the block is removed; histone mRNAs quickly reaccumulate in the cytoplasm as the consequence of a lengthened half-life (40a, 52-54, 56). These results reinforce the notion that during an unperturbed cell cycle, histone mRNAs synthesized in late G I-early S phase are stabilized when cells enter S phase. Support for this view has come from the analysis of H3 mRNA stability during the HeLa cell cycle when transcrip tional and posttranscriptional controls were uncoupled (40c). H3 mRNA was
Annu. Rev. Biochem. 1991.60:827-861. Downloaded from www.annualreviews.org by Rice University on 04/22/13. For personal use only.
REGULATION OF HISTONE SYNTHESIS
831
almost three times more stable in early S phase cells than in G2-M-Gl phase cells. This proposition, however, has been challenged by the results of a study on the rellative half-life of histone mRNAs in the G l and S phases of synchronized CHO cells (40b). During both of these cell cycle stages, histone mRNAs had a half-life of 40-45 minutes, suggesting that the intrinsic stability of histone messengers may not be significantly different between u 1pe�urbed G l and S phase cells. In contrast, the degradation pathway apparrntly does act at the natural completion of chromosome replication. At this point in the cell cycle, the rate of transcription is very reduced (57), and the half-life of histone mlrnAs has been estimated to be only 10-20 minutes (40b, 40c). The second pathway of posttranscriptional regulation in higher eukaryotes acts in the nucleus to process histone pre-mRNAs. After their transcription, histone pre-mRNAs are processed by endonucleolytic cleavage at their 3' termini to produce the mature cytoplasmic mRNA species (58, 59). The processing pathway has been postulated to act primarily at the Gl-S phase
boundary, the same period of the cell cycle in which transcription is activated. This was fiirst demonstrated with a temperature-sensitive mouse mastocytoma mutant that could be blocked in G 1 phase at the restrictive temperature. In such blocked cells, transcription of histone genes continued at almost maxi mal levels, but the nascent histone transcripts were processed inefficiently and few mature histone mRNAs were found in the cytoplasm (40b , 60). Process ing was activated when the cells were released from the G 1 block, and the subsequent entry into S phase was correlated with an almost to-fold increase in the levels of mature histone RNAs (40b). The temporal relationship between efficient nuclear processing and the cytoplasmic accumulation of histone mRNAs suggests that the processing pathway may contribute sub stantially to the increase in histone mRNA levels as cells enter S phase. The observation that histone transcripts that could be correctly processed but not degraded continued to accumulate in S phase is consistent with this view (40b, 6 1) . It is therefore possible that the regulation of histone pre-mRNA processing is the major posttranscriptional pathway utilized in higher eu karyotes upon entry into S phase, while the regulation of histone mRNA stability may be a specialized response either to an interrupted S phase or at the natural completion of S. The discovery that three major pathways (one transcriptional and two posttranscr:iptional) regulate the levels of histone mRNAs in the vertebrate cell cycle does not necessarily extend to all eukaryotes . Since fungi and ciliates have polyadenylated histone mRNAs, these organisms lack the post transcriptional pathway that regulates pre-mRNA processing. The two remaining pathways are apparently common to all eukaryotes, although in some lower eukaryotes, transcription figures more prominantly than messen ger turnover in regulating the levels of histone mRNAs. In budding yeast, for
Annu. Rev. Biochem. 1991.60:827-861. Downloaded from www.annualreviews.org by Rice University on 04/22/13. For personal use only.
832
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example, transcription of the histone genes, which is hardly detectable in early Gl phase cells, is activated 10-20-fold in late G l phase cells (62). This degree of stimulation approximates the magnitude of accumulation of these mRNAs during S phase (46, 62). Moreover, when DNA synthesis is blocked in yeast, the major effect is not to destabilize histone mRNAs but to tum off transcription of the histone genes (63) . Thr; contribution of posttranscriptional regulation to the accumulation of histone mRNAs in the cell cycle of lower eukaryotes has been examined systematically only in budding yeast. When several different yeast histone genes were transcribed constitutively, the levels of their mRNAs showed significant fluctuations during the cell cycle, so that maximal accumulation of these rnRNAs continued to occur in S phase (63-65). Since yeast histone H2B mRNA can be degraded selectively under some circumstances (66), these fluctuations are believed to represent differen tial effects on histone mRNA stability during the GI and G2 phases of the cell cycle. Among the major forms of gene regulation found in eukaryotes, only translational control does not make a significant contribution to the synthesis of histones in the cell cycle . An exception to this rule, however, is found in the lower eukaryote, Physarum. Histone rnRNAs synthesized in G2 (the equivalent of GI phase in other eukaryotes) are transported to the cytoplasm and stored as a translationally inactive RNP particle (67). Translation com mences only when cells enter S phase (67). It is likely that proteins associated with histone mRNAs in the RNP particles mask translation, since these same mRNAs can be efficiently translated in vitro after the particles have been deproteinized (68). Similar translational regulation of histone biosynthesis hitherto has been noted only during embryogenesis (4, 69, 70) . TRANSCRIPTIONAL REGULATION
Higher Eukaryotes In vertebrates, where most of the studies to be described have been per formed, the histone genes are transcribed by RNA polymerase II, and the sequences that regulate transcription occur 5' to the site of transcription initiation. A number of approaches have been taken to identify discrete regulatory elements within these sequences and the proteins with which they interact. An initial approach has been to search the 5' flanking regions of histone genes to identify DNA sequences that are either common to the histone gene family or specific to a particular class of histone gene. A second has been to identify regulatory elements directly through functional analysis of upstream sequences . This analysis has been facilitated by the discovery that exogenous histone genes--either chimeric reporter genes containing histone specific 5' sequences (25, 27-29) or heterologous histone genes (26, 7 1 )-are
Annu. Rev. Biochem. 1991.60:827-861. Downloaded from www.annualreviews.org by Rice University on 04/22/13. For personal use only.
REGULATION OF HISTONE SYNTHESIS
833
correctly cell cycle regulated when they are transfected into cells in culture. With these: genes it has been possible to study the effects of deletion, linker substitution, and point mutations in upstream regulatory sequences in vivo. A third avenue has employed techniques that detect specific protein-DNA con tacts in vitro (72-79) and in vivo (80--82) to identify factors that interact with transcriptional regulatory elements . Finally, the development of a soluble transcription system that faithfully mimics S phase activation of histone genes in vitro (55) has permitted the assignment of critical regulatory roles to factors that interact with upstream regulatory elements. With the possible exception of a human H4 gene (83, 84), vertebrate histone genes do not contain enhancer-like regulatory sequences that act from a distance. Most of the regulatory elements in the promoters of these genes are located within several hundred base pairs upstream of a highly conserved mRNA capping site (24, 85). In many respects, the promoters of histone genes are not organized very differently from the promoters of other differen tially regulated genes. They are generally modular in nature, and contain a number of discrete, independently functioning sequence elements that con tribute in toto to transcriptional activity. Among the regulatory elements found in vertebrate histone genes are several general promoter elements common to genes transcribed by RNA polymerase II, e. g. the TATA box (15, 24, 86, 87) and CCAAT motif (15, 24, 88), elements found in a subset of genes [SPI sites (24, 89, 90)], elements that are specific to all classes of replication-dependent histone genes, and elements that occur only in particu lar classes of histone genes. The histone family-specific element is a hexa meric sequence (GACTTC) that is analogous to a pentameric sequence (GATCC) first noted in sea urchin histone genes (15, 24, 91). Histone gene or subtype··specific elements (24) occur in the promoters of HI (92, 93), H2A (91, 93-95), H2B (93, 96), and H4 (93, 97) genes. Because the transcription of the five vertebrate histone genes is coordinate ly activated upon entry into S phase, a unifying model to account for their simultaneous regulation would be that a unique transcription factor interacts with an element common to all histone genes. The likely candidate for such a element is the histone family-specific hexamer sequence. The surprising conclusion from a large body of work is that this is an overly reductionist view of cell cyde regUlation. Indeed, no known cell cycle-related function yet has been assigned to the hexamer element, which acts primarily to maintain maximal kvels of transcription. Rather, each kind of vertebrate histone gene harbors a different S phase regulatory element that binds a distinct regulatory factor with specific activation functions. Thus, although each of these dis parate transcription factors acts independently, they all must be regulated coordinatelly to elicit their S phase-specific functions . Extensive analysis has been performed on the transcriptional regulatory
834
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sequences and proteins of four of the five replication-dependent histone genes of human, rodent, and chicken origin. Data on the regulation of each of these genes is presented below and summarized in Figure 1. Five distinct sequence elements have been found in the promoters of HI genes examined to date (Figure 1). The most proximal is a TATA box motif located approximately -30 bp from the mRNA cap site (24, 98, 99) . A G-rich element (GGGCGGG) that is conserved across species lines and shows a good fit to the consensus SP l -binding site (G/TG/AGGCGGITG/AG/ACIT) is also found in most HI genes at -70 bp from the cap site (24, 98, 99). The two remaining sequence elements are ubiquitous in HI promoters. The first, the AC box, having a consensus sequence of AAACACA, is the most distal element in HI promoters (-100 bp from the cap site) (24, 92). The second is more proximal, interposed between the G-rich element and TATA box (24, 99, 100). Although the latter element contains a general CCAAT box motif, this motif is actually part of a more .extended, HI-specific homology box of GCACCAATCACAGCGCGC (78, 99, 100). Finally, the most distal promot er element is a copy of the histone family-specific hexamer sequence (100). The contribution of each of these elements to maximal or S phase-activated transcription has been determined by analysis of HI promoter mutations in vivo following transfection of mutant genes (79, 98, 100) and in an in vitro transcription assay (100). Progressive 5' deletions of a human HI promoter resulted in an incremental loss in transcriptional efficiency, suggesting a positive and additive role for each element in maximal gene expression (100). This theme is reiterated in the regulation of each of the different histone genes. The only element with a demonstrated role in cell cycle activation is the HI-specific AC box. Deletion of the AC element from a chicken HI promoter or 4 bp substitution mutations in the same motif greatly reduced the overall level of HI transcription and specifically abolished S phase activation (79). No equivalent cell cycle function has yet been assigned to the more proximal HI-specific element, although this element and the TATA box motif are juxtaposed, a positional context that has been noted for the S phase specific activation elements of other classes of histone genes (Figure 1). The potential significance of the AC box to transcriptional regulation is underscored by the observation that a site-specific DNA-binding protein [HI-SF in chickens (74); HlTFI in mammals (100)] interacts with this element. HI-SF DNA complexes were 12-fold more abundant in S phase extracts than in G 1 phase extracts (101), supporting a role for these complexes in cell cycle regulation. However, it has not yet been demonstrated by an in vitro transcription assay that the HI-SF factor can positively regulate the synthesis of HI mRNA, so its precise role in S phase activation remains unknown (J. Wells, personal communication).
Annu. Rev. Biochem. 1991.60:827-861. Downloaded from www.annualreviews.org by Rice University on 04/22/13. For personal use only.
Hl
Annu. Rev. Biochem. 1991.60:827-861. Downloaded from www.annualreviews.org by Rice University on 04/22/13. For personal use only.
-140 HEX
-100 Hl/AC
f-O-t/
-80
I
GACITC
-60
-40 HlICCAAT
G/C
c::::J
AACAAACACAAAT
CAT
GCACCAATCACAGCGCGC
-60 HEX
- :310 HEX
D GACIT
-
/�
190 H3/APl
-170
-150 G/C
CAT
-100 CAT
GACTTC
CCAAT
-100 HEX H4/GC
HI
H
CCAAT
-60 G/C
-80 -60 G/C HEX HEX
-20
+1
H2B
r------c:J TATATAAA
-40 -20 HEX TATA
H3
GGTCC TATAAA
-40 H4BOX
I� TCAGTTCGGTCC
-20 TATA
0
)
>
+1
CIJ
CCGCCCCG
GATTIC CCCTCCCCC GACTTC GGGGCG
�
o
'T.I
+1
H4
TATAAA
>
Regulatory elements in vertebrate histone gene promoters.
Regulatory elements that are characteristic of four different vertebrate histone gene promoters are indicated as boxes, with pert inent sequences indicated below each box. Underlined sequences point to specialized motifs within certain elements. Data for each gene have been adapted from the
Hi: human gene (100); H2B: human gene (29, 108); H3: hamster gene (76, 138); H4: human gene (144, 147, 149). The coordinates are relative to the rnRNA cap site.
following references:
§
::t V;
6 �
Vl -
Further
Quick links to online content Annu. Rev. Biochem. 1991. 60:827-61 Copyright © 1991 by Annual Reviews Inc. All rights reserved
Annu. Rev. Biochem. 1991.60:827-861. Downloaded from www.annualreviews.org by Rice University on 04/22/13. For personal use only.
THE lREGULATION OF HISTONE SYNTHESIS IN THE CELL CYCLE M. A. Osley Program in Molecular Biology, Sloan Kettering Cancer Center, New York, New York
10021 KEY WORDS:
h i ston e RNAs: transcription , p rocessin g , turnov er .
CONTENTS SUMMARY AND PERSPECTIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
827
ORGANIZATION OF HISTONE GENES-STRUCTURE OF HISTONE mRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
828
OVERVIEW OF HIS TONE SYNTHESIS IN THE CELL CyCLE . . . . . . . . . . . . . . . . . . . . . . . .
829
TRANSCRIPTIONAL REGULATION . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Higher Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . ... . . . . . . . . Lower Eukaryotes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .
832 832
POSTTRANSCRIPTIONAL REGULATION . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . .... Higher Eukaryotes. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... . . . . . . . . . . . . . . . . . . . . . . . . Lower Eukaryotes . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... . . . . . . . . . . . . . . . . . .
841 847 847 853
MULTIPLE FORMS OF REGULATION MODULATE HISTONE mRNA LEVELS IN THE CELL CyCLE . . . . . ........... . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . .
!l55
FUTURE PROSPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . .. . . . . . . . . . . . . . . . . . ... .. . . . . .. . . . . . . . . .
856
SUMMARY AND PERSPECTIVE The recent focus on the eukaryotic cell cycle has renewed interest in genes whose expression is regulated during the division cycle. The most prominent and best-studied examples of cell cycle-regulated genes are those encoding histones , proteins that are synthesized during S phase. Multiple levels of control are involved in restricting the synthesis of histones to this phase of the cell cycle , and their combined effect is to regulate the production of histone 827
0066-4154/9 1/0701 -0827$02.00
828
OSLEY
mRNAs. Three major pathways contribute to this regulation, and they act at the levels of transcription, pre-mRNA processing, and mRNA stability. During the past 10 years , each pathway has been subjected to detailed analysis; cis-acting regulatory elements and trans-acting regulatory factors have been identified, and in some instances, the mechanisms of regulation have begun to be defined. This review summarizes the major developments underlying the contribution of each pathway to the production of histone mRNAs in the eukaryotic cell cycle. Because significant differences are found
Annu. Rev. Biochem. 1991.60:827-861. Downloaded from www.annualreviews.org by Rice University on 04/22/13. For personal use only.
between higher and lower eukaryotes in the regulation of histone genes , these two groups of organisms are considered separately. Although this review emphasizes the control of histone synthesis during the cell cycle, histone synthesis is also regulated during development , and the interested reader is referred to ( 1-5) for discussions of this specialized aspect of regulation.
ORGANIZATION OF HISTONE GENES-STRUCTURE OF HISTONE mRNAs Two classes of histone genes are found in most eukaryotes. The most frequent class contains the replication-dependent histone genes whose expression is regulated in the cell cycle, while a second, less abundant class contains genes that encode the minor histone variants and whose expression occurs at a basal level throughout the cell cycle (6) . In all eukaryotes the replication-dependent core (H2A, H2B, H3, H4) histone genes are members of a multigene family, and in higher eukaryotes the linker (HI) histone genes are also repeated. The core histone gene family has as few as one to two members each, as exemplified by fungi (7- 14), to as many as several hundred per core histone gene in invertebrates (2, 3, 15, 16). The genomic organization of the histone genes differs widely among organisms. In invertebrates the genes are part of a reiterated tandem repeat encompassing the five histone genes (3, 16). This arrangement is not found in vertebrates or lower eukaryotes, where the genomic organization of histone genes is largely dispersive, albeit with some clustering (2 , 1 7-23). For example, in humans the H4 gene occurs in hetero geneous clusters , either unlinked to other histone genes or associated with as many as three other core histone genes ( 17 , 1 9 , 23). In budding yeast and other fungi, the genes encoding H2A and H2B are often linked and genetically separable from linked pairs of H3 and H4 genes (7, 8, 1 0, 1 3 , 14). A dispersive genomic organization implies that the histone genes are not regulated by far upstream enhancer elements. The regulatory sequences of these genes are in fact very compactly arranged. The 5 I sequences responsible for transcriptional control are usually found within several hundred bases upstream of the mRNA initiation site, and the 3 I processing signals in
REGULATION OF HISTONE SYNTHESIS
829
vertebrate histone genes are positioned close to the translation termination site
(24). Morl�over, vertebrate histone genes containing short 5' and 3' flanking sequences are correctly regulated following transfection into tissue culture cells (25-29), and yeast histone genes remain cell cycle regulated when placed in ectopic chromosomal locations (30). In
vertebrates,
two
remarkable features
distinguish
the replication
dependent histone genes from both the replication-independent histone genes and the majority of genes transcribed by RNA polymerase II. The first is the
Annu. Rev. Biochem. 1991.60:827-861. Downloaded from www.annualreviews.org by Rice University on 04/22/13. For personal use only.
absence of introns (24) . The second is the structure of the 3' terminus of mature histone RNAs. While most pol II transcripts are polyadenylated in vertebrate cells, the mRNAs produced by the replication-dependent histone genes do not contain poly(A) sequences and terminate with a highly con served stem-loop structure (24, 31). In most lower eukaryotes the histone genes are also free of introns (7, 11, 12), except for the histone genes of Neurospora (14), Aspergillus (13, 32), and Physarum (33). Another contrast between higher and lower eukaryotes is seen in th� structure of their mature histone RNAs. The histone mRNAs of some lower eukaryotes (fungi and ciliates) do not terminate with the stem loop structure that is characteristic of most eUkaryotic histone mRNAs, but instead are polyadenylated (7, 34-36). It is intriguing that the replication independent histone genes of vertebrates generally contain introns and are transcribed into polyadenylated mRNAs, since it is clear that neither of these features precludes the regulation of histone genes in the cell cycle of some lower eukaryotes.
OVERVIEW OF HISTONE SYNTHESIS IN THE CELL CYCLE The coupling of histone biosynthesis to the cell cycle was noted more than two decades ago by Robbins & Borun, who reported that histones were synthesized only in S phase cells (37). Subsequent studies showed that this pattern of histone protein synthesis is the direct consequence of the restriction of histone mRNA synthesis to the period of DNA replication (6, 38, 39). With the isolation of individual histone genes, the tools became available to study in detail the molecular basis of the coupling between histone biosynthesis and the DNA synthetic phase. What has emerged is a complicated picture of cell cycle regulation in which a combination of transcriptional and posttranscrip tional conltrois regulates the levels of histone mRNAs. Two general approaches have been taken to study histone mRNA metabo lism in the cell cycle. In the first, synchronized cell cultures have been used to evaluate at what point histone mRNAs accumulate during an uninterrupted cell cycle. In the second, inhibitors of DNA chain elongation have been
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employed to test whether ongoing DNA synthesis is a prerequisite for the continued production of histone mRNAs. The major conclusions of a large body of work are that histone mRNA production is temporally restricted to the S phase of the cell cycle and dependent on the presence of replicating DNA. During the cell cycle of most eukaryotes, histone mRNAs accumulate to maximal levels only during S phase. As cells pass from G1 into S phase, the levels of these mRNAs increase 15-30-fold, with peak accumulation occur ring in mid S phase (13, 18, 23, 40a-51). When DNA replication is in terrupted, histone mRNAs rapidly disappear from the cytoplasm (6, 40a, 41, 52-54). Three pathways regulate the production of histone mRNAs in the cell cycle of higher eukaryotes. The first regulates transcription at the G I-S phase boundary: as cells enter S phase, nascent histone mRNA synthesis increases three to five fold over basal levels of transcription characteristic of G1 phase cells (40a, 40b, 41-43, 52). The two remaining pathways regulate histone mRNA production at a posttranscriptional level. These latter pathways are responsible for the remaining five- to sixfold increase in histone mRNA levels that occurs during S phase and for the disappearance of histone mRNAs from the cytoplasm when chromosome replication is blocked. The two pathways of posttranscriptional regulation affect histone mRNA levels very differently in the cell cycle. The first acts in the cytoplasm to control the half-life of histone mRNAs. The major role of this pathway is to degrade histone mRNAs when DNA synthesis is inhibited (41, 52-55). The half-life of histone mRNAs in S phase has been estimated to be 30-60 minutes by several different techniques (40a, 40b). When chromosome replication is blocked with inhibitors of DNA chain elongation, the apparent half-life of these mRNAs drops to 10-15 minutes (40a, 41, 52-54, 56). Numerous studies have asked whether this pathway might also operate during the uninterrupted cell cycle. One point at which it might act is upon entry into S phase, where histone mRNAs show maximal levels of accumulation. Here, the pathway would be used to stabilize histone mRNAs in order to accumulate these transcripts. A compelling argument that changes in histone mRNA stability can take place during S phase arises from the observation that histone mRNA half-life can be modulated both rapidly and reversibly in this phase of the cell cycle. The destabilization of histone mRNAs that results from a replication block is abrogated when the block is removed; histone mRNAs quickly reaccumulate in the cytoplasm as the consequence of a lengthened half-life (40a, 52-54, 56). These results reinforce the notion that during an unperturbed cell cycle, histone mRNAs synthesized in late G I-early S phase are stabilized when cells enter S phase. Support for this view has come from the analysis of H3 mRNA stability during the HeLa cell cycle when transcrip tional and posttranscriptional controls were uncoupled (40c). H3 mRNA was
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REGULATION OF HISTONE SYNTHESIS
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almost three times more stable in early S phase cells than in G2-M-Gl phase cells. This proposition, however, has been challenged by the results of a study on the rellative half-life of histone mRNAs in the G l and S phases of synchronized CHO cells (40b). During both of these cell cycle stages, histone mRNAs had a half-life of 40-45 minutes, suggesting that the intrinsic stability of histone messengers may not be significantly different between u 1pe�urbed G l and S phase cells. In contrast, the degradation pathway apparrntly does act at the natural completion of chromosome replication. At this point in the cell cycle, the rate of transcription is very reduced (57), and the half-life of histone mlrnAs has been estimated to be only 10-20 minutes (40b, 40c). The second pathway of posttranscriptional regulation in higher eukaryotes acts in the nucleus to process histone pre-mRNAs. After their transcription, histone pre-mRNAs are processed by endonucleolytic cleavage at their 3' termini to produce the mature cytoplasmic mRNA species (58, 59). The processing pathway has been postulated to act primarily at the Gl-S phase
boundary, the same period of the cell cycle in which transcription is activated. This was fiirst demonstrated with a temperature-sensitive mouse mastocytoma mutant that could be blocked in G 1 phase at the restrictive temperature. In such blocked cells, transcription of histone genes continued at almost maxi mal levels, but the nascent histone transcripts were processed inefficiently and few mature histone mRNAs were found in the cytoplasm (40b , 60). Process ing was activated when the cells were released from the G 1 block, and the subsequent entry into S phase was correlated with an almost to-fold increase in the levels of mature histone RNAs (40b). The temporal relationship between efficient nuclear processing and the cytoplasmic accumulation of histone mRNAs suggests that the processing pathway may contribute sub stantially to the increase in histone mRNA levels as cells enter S phase. The observation that histone transcripts that could be correctly processed but not degraded continued to accumulate in S phase is consistent with this view (40b, 6 1) . It is therefore possible that the regulation of histone pre-mRNA processing is the major posttranscriptional pathway utilized in higher eu karyotes upon entry into S phase, while the regulation of histone mRNA stability may be a specialized response either to an interrupted S phase or at the natural completion of S. The discovery that three major pathways (one transcriptional and two posttranscr:iptional) regulate the levels of histone mRNAs in the vertebrate cell cycle does not necessarily extend to all eukaryotes . Since fungi and ciliates have polyadenylated histone mRNAs, these organisms lack the post transcriptional pathway that regulates pre-mRNA processing. The two remaining pathways are apparently common to all eukaryotes, although in some lower eukaryotes, transcription figures more prominantly than messen ger turnover in regulating the levels of histone mRNAs. In budding yeast, for
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example, transcription of the histone genes, which is hardly detectable in early Gl phase cells, is activated 10-20-fold in late G l phase cells (62). This degree of stimulation approximates the magnitude of accumulation of these mRNAs during S phase (46, 62). Moreover, when DNA synthesis is blocked in yeast, the major effect is not to destabilize histone mRNAs but to tum off transcription of the histone genes (63) . Thr; contribution of posttranscriptional regulation to the accumulation of histone mRNAs in the cell cycle of lower eukaryotes has been examined systematically only in budding yeast. When several different yeast histone genes were transcribed constitutively, the levels of their mRNAs showed significant fluctuations during the cell cycle, so that maximal accumulation of these rnRNAs continued to occur in S phase (63-65). Since yeast histone H2B mRNA can be degraded selectively under some circumstances (66), these fluctuations are believed to represent differen tial effects on histone mRNA stability during the GI and G2 phases of the cell cycle. Among the major forms of gene regulation found in eukaryotes, only translational control does not make a significant contribution to the synthesis of histones in the cell cycle . An exception to this rule, however, is found in the lower eukaryote, Physarum. Histone rnRNAs synthesized in G2 (the equivalent of GI phase in other eukaryotes) are transported to the cytoplasm and stored as a translationally inactive RNP particle (67). Translation com mences only when cells enter S phase (67). It is likely that proteins associated with histone mRNAs in the RNP particles mask translation, since these same mRNAs can be efficiently translated in vitro after the particles have been deproteinized (68). Similar translational regulation of histone biosynthesis hitherto has been noted only during embryogenesis (4, 69, 70) . TRANSCRIPTIONAL REGULATION
Higher Eukaryotes In vertebrates, where most of the studies to be described have been per formed, the histone genes are transcribed by RNA polymerase II, and the sequences that regulate transcription occur 5' to the site of transcription initiation. A number of approaches have been taken to identify discrete regulatory elements within these sequences and the proteins with which they interact. An initial approach has been to search the 5' flanking regions of histone genes to identify DNA sequences that are either common to the histone gene family or specific to a particular class of histone gene. A second has been to identify regulatory elements directly through functional analysis of upstream sequences . This analysis has been facilitated by the discovery that exogenous histone genes--either chimeric reporter genes containing histone specific 5' sequences (25, 27-29) or heterologous histone genes (26, 7 1 )-are
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correctly cell cycle regulated when they are transfected into cells in culture. With these: genes it has been possible to study the effects of deletion, linker substitution, and point mutations in upstream regulatory sequences in vivo. A third avenue has employed techniques that detect specific protein-DNA con tacts in vitro (72-79) and in vivo (80--82) to identify factors that interact with transcriptional regulatory elements . Finally, the development of a soluble transcription system that faithfully mimics S phase activation of histone genes in vitro (55) has permitted the assignment of critical regulatory roles to factors that interact with upstream regulatory elements. With the possible exception of a human H4 gene (83, 84), vertebrate histone genes do not contain enhancer-like regulatory sequences that act from a distance. Most of the regulatory elements in the promoters of these genes are located within several hundred base pairs upstream of a highly conserved mRNA capping site (24, 85). In many respects, the promoters of histone genes are not organized very differently from the promoters of other differen tially regulated genes. They are generally modular in nature, and contain a number of discrete, independently functioning sequence elements that con tribute in toto to transcriptional activity. Among the regulatory elements found in vertebrate histone genes are several general promoter elements common to genes transcribed by RNA polymerase II, e. g. the TATA box (15, 24, 86, 87) and CCAAT motif (15, 24, 88), elements found in a subset of genes [SPI sites (24, 89, 90)], elements that are specific to all classes of replication-dependent histone genes, and elements that occur only in particu lar classes of histone genes. The histone family-specific element is a hexa meric sequence (GACTTC) that is analogous to a pentameric sequence (GATCC) first noted in sea urchin histone genes (15, 24, 91). Histone gene or subtype··specific elements (24) occur in the promoters of HI (92, 93), H2A (91, 93-95), H2B (93, 96), and H4 (93, 97) genes. Because the transcription of the five vertebrate histone genes is coordinate ly activated upon entry into S phase, a unifying model to account for their simultaneous regulation would be that a unique transcription factor interacts with an element common to all histone genes. The likely candidate for such a element is the histone family-specific hexamer sequence. The surprising conclusion from a large body of work is that this is an overly reductionist view of cell cyde regUlation. Indeed, no known cell cycle-related function yet has been assigned to the hexamer element, which acts primarily to maintain maximal kvels of transcription. Rather, each kind of vertebrate histone gene harbors a different S phase regulatory element that binds a distinct regulatory factor with specific activation functions. Thus, although each of these dis parate transcription factors acts independently, they all must be regulated coordinatelly to elicit their S phase-specific functions . Extensive analysis has been performed on the transcriptional regulatory
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sequences and proteins of four of the five replication-dependent histone genes of human, rodent, and chicken origin. Data on the regulation of each of these genes is presented below and summarized in Figure 1. Five distinct sequence elements have been found in the promoters of HI genes examined to date (Figure 1). The most proximal is a TATA box motif located approximately -30 bp from the mRNA cap site (24, 98, 99) . A G-rich element (GGGCGGG) that is conserved across species lines and shows a good fit to the consensus SP l -binding site (G/TG/AGGCGGITG/AG/ACIT) is also found in most HI genes at -70 bp from the cap site (24, 98, 99). The two remaining sequence elements are ubiquitous in HI promoters. The first, the AC box, having a consensus sequence of AAACACA, is the most distal element in HI promoters (-100 bp from the cap site) (24, 92). The second is more proximal, interposed between the G-rich element and TATA box (24, 99, 100). Although the latter element contains a general CCAAT box motif, this motif is actually part of a more .extended, HI-specific homology box of GCACCAATCACAGCGCGC (78, 99, 100). Finally, the most distal promot er element is a copy of the histone family-specific hexamer sequence (100). The contribution of each of these elements to maximal or S phase-activated transcription has been determined by analysis of HI promoter mutations in vivo following transfection of mutant genes (79, 98, 100) and in an in vitro transcription assay (100). Progressive 5' deletions of a human HI promoter resulted in an incremental loss in transcriptional efficiency, suggesting a positive and additive role for each element in maximal gene expression (100). This theme is reiterated in the regulation of each of the different histone genes. The only element with a demonstrated role in cell cycle activation is the HI-specific AC box. Deletion of the AC element from a chicken HI promoter or 4 bp substitution mutations in the same motif greatly reduced the overall level of HI transcription and specifically abolished S phase activation (79). No equivalent cell cycle function has yet been assigned to the more proximal HI-specific element, although this element and the TATA box motif are juxtaposed, a positional context that has been noted for the S phase specific activation elements of other classes of histone genes (Figure 1). The potential significance of the AC box to transcriptional regulation is underscored by the observation that a site-specific DNA-binding protein [HI-SF in chickens (74); HlTFI in mammals (100)] interacts with this element. HI-SF DNA complexes were 12-fold more abundant in S phase extracts than in G 1 phase extracts (101), supporting a role for these complexes in cell cycle regulation. However, it has not yet been demonstrated by an in vitro transcription assay that the HI-SF factor can positively regulate the synthesis of HI mRNA, so its precise role in S phase activation remains unknown (J. Wells, personal communication).
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Hl
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-140 HEX
-100 Hl/AC
f-O-t/
-80
I
GACITC
-60
-40 HlICCAAT
G/C
c::::J
AACAAACACAAAT
CAT
GCACCAATCACAGCGCGC
-60 HEX
- :310 HEX
D GACIT
-
/�
190 H3/APl
-170
-150 G/C
CAT
-100 CAT
GACTTC
CCAAT
-100 HEX H4/GC
HI
H
CCAAT
-60 G/C
-80 -60 G/C HEX HEX
-20
+1
H2B
r------c:J TATATAAA
-40 -20 HEX TATA
H3
GGTCC TATAAA
-40 H4BOX
I� TCAGTTCGGTCC
-20 TATA
0
)
>
+1
CIJ
CCGCCCCG
GATTIC CCCTCCCCC GACTTC GGGGCG
�
o
'T.I
+1
H4
TATAAA
>
Regulatory elements in vertebrate histone gene promoters.
Regulatory elements that are characteristic of four different vertebrate histone gene promoters are indicated as boxes, with pert inent sequences indicated below each box. Underlined sequences point to specialized motifs within certain elements. Data for each gene have been adapted from the
Hi: human gene (100); H2B: human gene (29, 108); H3: hamster gene (76, 138); H4: human gene (144, 147, 149). The coordinates are relative to the rnRNA cap site.
following references:
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