TIBS 17 - MARCH 1992 2 Kinzler, K. W. and Vogelstein, B. (1990) Mol. Cell. Biol. 10, 634-642 3 Thiesen, H-J. and Bach, C. (1990) Nucleic Acids Res. 18, 3203-3209 4 Pollock, R. and Treisman, R. (1990) Nucleic Acids Res. 18, 6197-6204 5 Blackwell, T. K. and Weintraub, H. (1990) Science 250, 1104-1110 6 Blackwell, T. K. et al. (1990) Science 250, 1149-1151 7 Tuerk, C. and Gold, L. (1990) Science 249, 505-510 B Robertson, D. L. and Joyce, G. F. (1990) Nature 344, 467-468 9 Green, R., Ellington, A. D. and Szostak, J. W. (1990) Nature 347,406-408 10 Ellington, A. D. and Szostak, J. W. (1990) Nature 346, 818-822
11 Eigen, M., ed. (1991) Report on the Intemational Workshop 'Selection - Natural and Unnatural - in Biotechnology', Max-Planck-lnstitut
12 13 14 15 16 17 18
fur Biophysikalische Chemie, Gottingen, Germany Horwitz, M. S. Z. and Loeb, L. (1986) Proc. Natl Acad. Sci. USA 83, 7405-7409 Oliphant, A. R. and Struhl, K. (1987) Methods Enzymol. 155, 568-582 Gronostajski, R. M. (1987) Nucleic Acids Res. 15, 5545-5559 Kaiser, C. A., Preuss, D., Grisafi, P. and Botstein, D. (1987) Science 235, 312-317 Ma, J. and Ptashne, M. (1987) Cell 51, 113-119 Dube, D. K. and Loeb, L. (1989) Biochemistry 28, 5703-5707 Perkins, A. S., Fishel, R., Jenkins, N. A. and
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20 21 22 23 24 25
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Copeland, N.G. (1991) Mol. Cell. Biol. 11, 2665-2674 Chittendon, T., Livingston, D. M. and Keilin, Jr, W. G. (1991) Cell 65, 1073-1082 Andrake, M. et al. (1988) Proc. Natl Acad. Sci. USA 85, 7942-7946 Barrel, D., Zapp, M., Green, M, and Szostak, J. W. (1991) Cell 67, 529-536 Kruger, K. et al. (1982) Cell 31, 147-157 Kramer, F. R. et al. (1974) J. Mol. Biol. 89, 719-736 Miele, E. A., Mills, D. R. and Kramer, F. R. (1983) J. Mol. Biol. 171, 281-295 Guatelli, J. C. et al. (1990) Proc. Natl Acad. Sci. USA 87, 1874-1878 Abelson, J. (1990) Science 249, 488-489 Riordan, M. L. and Martin, J. C. (1991) Nature 350, 442-443
TALKINGPOINT EUKARYOTIC DNA is packaged with histone proteins into chromatin 1. Core histone proteins (H2A, H2B, H3 and H4) form an octameric complex around which is wound approximately two turns of DNA. This basic nucleosomal unit is highly conserved and is compacted into higher-order structures such as the 10 nm thin filament and the 30 nm thick fiber. How these structures are formed or how chromatin decondenses and recondenses to facilitate biological processes such as transcription, replication and recombination is unclear. A fifth histone, H1, binds to the nucleosome core particle, sealing the entry and exit points of the DNA. In vitro studies strongly suggest that electrostatic interactions between H1 and DNA play a paramount role in chromatin folding5. Most Hls have a tripartite structure consisting of a globular central domain flanked by lysine-rich, highly charged amino-terminal and carboxytermin~ tails 2. The central domain appears to interact with the nucleosome core particle while the positively charged tails interact with the linker DNA and are important in condensation 3,4. Post-translational phosphorylation of these tails may well influence their interaction with DNA. Changing levels of H1 phosphorylation could
s. Y. Roth is at the Laboratoryof Cellular and Developmental Biology, NIDDK,NIH, Bethesda, MD 20892, USA.C. D. Allis is at the Department of Biology, Syracuse University,Syracuse, NY 13244, USA. © 1992,ElsevierSciencePublishers, (UK)
In this article we describe three distinct biological systems where histone H1 phosphorylation is uncoupled from mitosis and highly condensed chromatin is enriched in dephosphorylated forms of HI: t,he amitotic macronucleus of Tetrahymena, terminally differentiated avian erythrocytes and sea urchin sperm. Each system offers informative contrasts to the idea that H1 hyperphosphorylation is causally related to mitotic chromosome condensation. Assuming that higher order chromatin folding is primarily driven by electrostatic interactions between H1 and DNA, an alternative model is presented for the role of H1 phosphorylation in chromatin condensation.
then, in turn, modulate chromatin condensation/decondensation.
a number of consistent observations have been made:
H1 phosphorylationand mitotic chromosome condensation:the original correlation
(1) Phosphorylation of H1 is increased in rapidly dividing cells and decreased in non-proliferating or quiescent cells. (2) Levels of H1 phosphorylation are usually lowest in the G1 phase of the cell cycle, and rise during both S phase (in which DNA replication also occurs) and mitosis. (3) During mitosis, phosphorylation of H1 and H3 becomes maximal just before or at metaphase and sharply decreases thereafter, reaching a minimum during anaphase or telophase. (4) Extracts from mitotic cells containing an 'H1 kinase' activity can stimu-
Phosphorylation of H1 during the cell cycle has been examined in a number of different organisms and cell types. Pioneering work was performed in two systems: the slime mold, Physarum polycephalum, which undergoes naturally synchronous mitotic divisions, and artificially synchronized Chinese hamster ovary cells in culture. Many other mammalian and non-mammalian systems have been studied subsequently ~ . Although the exact timing of events varies somewhat in different systems,
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late premature chromatin condensation when added to non-mitotic cells. (5) Inhibition of intracellular H1 kinase activity and subsequent dephosphorylation of H1 can cause partial chromosome decondensation. These correlations imply a role for H1 phosphorylation in mitotic chromosome
~
condensation and lead to a proposal that H1 hyperphosphorylation itself might serve as a mitotic 'trigger 'gJ°. Our understanding of the factors involved in regulation of the cell cycle has increased dramatically in the past few years with the characterization of various cyclin proteins and p34 cdc2
(a) Tetrahymena Micronuclei. ~ inactive ~
,.
Old macronuclei inactive condensed H1 dephosphorylated
active amitotic H1 phosphorylated
(b) Avian erythropoiesis
v
Erythroblast active H1 phosphorylated
Erythrocyte inactive condensed H5 dephosphorylated
Immature erythrocyte active H5 phosphorylated
(c) Sea urchin spermatogenesis and fertilization
Spermatocyte active H1 phosphorylated
Mature sperm inactive condensed H1 dephosphorylated
Male pronucleus reactivated decondensed H1 rephosphorylated
Figure 1 Exceptions to the long-held correlation between H1 phosphorylation and chromatin condensation are depicted schematically. In each case, the transcriptional activity (active or inactive), the condensation state of chromatin (condensed or decondensed), and the phosphorylation state of H1 (or H5) within particular nuclei are indicated. (a) Tetrahymenacontain germ-line micronuclei, which are transcriptionally inactive and divide mitotically, and macronuclei, which are transcriptionally active and divide amitotically. Macronuclei contain an Hl-like histone whose phosphorylation is modulated under different physiological conditions by a cdc2-1ike kinase activity. During conjugation (double cell figures), old macronuclei become highly pycnotic and transcriptionally inactive, as new macronuclei develop from micronuclei. Highly condensed chromatin in the old macronucleus contains only dephosphorylated H1. (b) During avian enjthropoiesis, nuclei are retained but become progressively more condensed. Another lysine-rich histone, H5, is synthesized and deposited in the immature erythrocyte in a highly p~sphonjlated form. In the mature erythrocyte, H5 is dephosphorylated as chromatin is compacted into a transcriptionally inactive state. (c) During sea urchin spermatogenesis, nuclei are compacted and chromatin becomes highly condensed, concomitant with the dephosphorylation of HI. These events are reversed upon fertilization, with decondensation of chromatin in the male pronucleus (white) and phosphorylation of HI.
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kinase homologues u. H1 serves as an important in vitro substrate for cdc2 kinase with phosphorylation occurring exclusively on the tail domains at conserved (K[S/T]PXK) or (K[S/T]PK) motifs ~2. However, it is not clear whether H1 is phosphorylated in vivo by cdc2 kinase ~3or what role H1 hyperphosphorylation plays in the condensation of chromosomes or the progression of mitosis. Direct evidence linking these processes is limited. In considering the 'effect' of H1 phosphorylation on mitotic chromosome condensation, we point out that addition of negatively charged phosphate groups to the tail domains of H1 should weaken interactions of H1 with DNA. Furthermore, in some systems H1 phosphorylation is uncoupled from mitosis and chromosome condensation; in these systems condensed chromatin is enriched in dephosphorylated forms of H1. In this article, we examine some of these 'exceptions' which are frequently overlooked. These include the amitotic macronucleus of Tetrahymena, the terminally differentiated nuclei of avian erythrocytes, and the highly condensed chromatin of sea urchin sperm. Each of these systems exhibits remarkable transitions between decondensed and condensed chromatin in the absence of mitosis (see Fig. 1). Each offers an informative contrast to the long-held notion that H1 phosphorylation is causally related to chromosome condensation during mitosis and suggests other functions that might be regulated by H1 phosphorylation/dephosphorylation.
Phosphorylation of H1 in the amitotic macronucleus of Tetrahymena . Ciliated protozoa such as Tetrahymena pr6vide a unique system in which to study H1 phosphorylation and transcriptional regulation outside mitosis. These organisms contain two distinct nuclei, differing both in function and mode of division (Fig. la) TM. The somatic macronucleus is transcriptionally active and divides amitotically without overt chromatin condensation. By contrast, the germ-line micronucleus is transcriptionally silent under most physiological conditions and undergoes normal mitotic divisions including the formation of mitotic chromosomes. The composition of histones differs significantly in these two nuclei. In particular, macronuclei contain an H1 histone which is highly phosphorylated during vegetative growth. In micronuclei, H1 is
TIBS 1 7 - MARCH 1992 missing and is replaced by specialized linker polypeptides which are also highly phosphorylated ~4. The level of macronuclear H1 phosphorylation changes dramatically in response to varying physiological conditions ~s.A high level of phosphorylation occurs in exponentially growing cells, and hyperphosphorylation is observed upon heat shock and induction of conjugation, the sexual cycle. Moderately phosphorylated H1 is observed upon starvation and in highdensity, stationary phase cultures. Most interesting, however, is the recent finding that H1 is rapidly and completely dephosphorylated during conjugation when the macronucleus ceases transcription and becomes highly condensed, prior to its eventual elimination from the cell (see Fig. la and Ref. 16). Since the highest levels of H1 phosphorylation are observed under conditions in which no division of the cell or macronucleus occurs (heat shock and conjugation), it appears that H1 phosphorylation is not, by itself, uniquely linked to either mitotic or amitotic division. Indeed, changes in the levels of H1 phosphorylation in Tetrahymena appear to occur whenever there are marked changes in gene expression due to altered physiological conditions, supporting a role for H1 phosphorylation in transcriptional regulationlS,16. Despite the lack of correlation between H1 phosphorylation and mitosis in macronuclei, macronuclear H1 is nonetheless phosphorylated by a cdc2-1ike kinase activity~7. This activity is highest in rapidly growing cells, and declines steadily as cells reach stationary phase. Importantly, the Tetrahymena kinase activity displays several hallmark features of cdc2 kinase in other systems, including binding to Suc1/p13 beads and recognition by polyclonal antibodies to fission yeast cdc2, indicating~hat it shares some conserved structural features with the yeast kinase. Macronuclear H1 is phosphorylated in vitro by mammalian cdc2 at sites remarkably similar to those phosphorylated by the Tetrahymena kinase in vivo and in vitro, suggesting it is a bona fide substrate for this activity in vivo. Phosphorylation of H1 by a cdc2-1ike kinase activity in amitotic macronuclei suggests that although H1 phosphorylation may be required for chromosome condensation, it is not sufficient to induce the condensation process. Macro-
nuclear H1 differs from other H1 molecules in that it lacks some features of the central globular domain, and this structural difference may underlie a different function for macronuclear H1 phosphorylation. However, phosphorylation of motifs believed to be involved in mitotic chromosome condensation is conserved in Tetrahymena H1, arguing against this possibility.
chromatin compaction during spermatogenesis does not represent a terminally differentiated state but instead is reversible upon fertilization (Fig. lc). Thus, changes in H1 phosphorylation can be studied both in regards to condensation of chromatin as spermatids develop into mature sperm and to decondensation of chromatin in the male pronucleus following fertilization 23.24. Unlike sperm development in many other species, that occurring in sea Phosphorylationof histone H5 in avian erythrocytes urchins does not involve replacement During the final stages of erythrocyte of core histones with protamines; development into mature red blood cells, somatic variants of histones H2A, H3 transcriptional inactivation occurs and H4 are maintained. Sperm-specific along with the formation of highly con- forms of H1 and H2B are deposited densed chromatin. In mammalian cells, early in the differentiation of the male nuclei are eventually discarded from germ line. These sperm-specific hismature erythrocytes. In avian species, tones contain extended amino-termini however, nuclei are retained and several consisting largely of repeats of the specialized proteins associate with the sequence SPKK24,25, the target phoshighly condensed chromatin (Fig. lb). phorylation site of cdc2 kinase24.2E H1 One of these proteins, H5, another type and H2B are highly phosphorylated in of lysine-rich histone, is present in spermatids, and are dephosphorylated low levels early in erythropoiesis and in the highly condensed chromatin of reaches maximal levels only in fully dif- mature sperm 24. Further, chromatin ferentiated red blood cells2E Newly syn- isolated from spermatids condenses thesized H5 is highly phosphorylated, less readily at increasing ionic strength but during the last stages of red cell dif- than does sperm chromatin, and ferentiation, when transcription ceases phosphorylation inhibits self-associand nuclei condense, H5 is quantitatively ation of oligonucleosomes27,28. One dephosphorylatedTM. Hence, dephos- explanation for these observations is phorylation of H5 rather than phos- that phosphorylation effectively neuphorylation is correlated with the com- tralizes the positive charge in the tail domain, perhaps lowering the affinity of paction of chromatin in these nuclei. Recent experiments further support H1 termini for DNA thereby uncovering a direct correlation between dephos- linker DNA phosphates and inhibiting phorylation of H5, cessation of tran- chromatin condensation. Following fertilization, sperm H1 and scription, and chromatin condensation. Introduction of H5 protein 19or the gene H2B are re-phosphorylated, prior to the encoding iF°-22into normal cells typically decondensation of chromatin and reaccauses nuclear condensation similar to tivation of gene expression in the male that observed in mature red blood pronucleus 2s and sperm H1 is replaced cells. However, Aubert et al.22 have with a phosphorylated, egg-specific shown that phosphorylation of H5 is (CS) form of H1. Thus, dephosphorylminimal in normal cells, but is more ation of sperm H1 appears necessary pronounced in transformed cells, which for the stabilization of highly condo not undergo nuclear compaction. densed chromatin within mature Thus, it appears that in cycling, trans- sperm, and rephosphorylation preformed cells, expression of a kinase cedes decondensation of chromatin in (perhaps cdc2) allows constitutive the male pronucleus and loss of sperm phosphorylation of H5, and that this H1 from chromatin. phosphorylation inhibits rather than Alternatives and explanations promotes chromatin condensation. In all three systems described above, phosphorylation of H1 correlates best Phosphorylationof H1 in sea urchin spermatogenesis and fertilization with decondensation of chromatin Compaction of chromatin during sea while H1 dephosphorylation is associurchin spermatogenesis in some ways ated with the formation or stabilization resembles that which occurs during of highly condensed structures. This is erythropoiesis. However, in contrast to exactly the opposite of the expectation erythropoiesis and the elimination of based on the correlation between parental macronuclei in Tetrahymena, hyperphosphorylation of H1 and mitotic
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(a) Decondensed
P-~1~(~H l ~ p H1 phosphorylated core histones acetylated
(b) Condensed
(c) Mitosis
P - ~ p -
_
H1 dephosphorylated core histories deacetylated
H1 phosphorylated core histones deacetylated
Figure2 Possible involvement of histone post-translational modifications in chromatin condensation. The positively charged tails of the core histones within the nucleosome (N) and histone H1 are shown in close association with linker DNA. (a) Upon acetylation (Ac) of the core histones and phosphorylation (P-) of H1, positive charges on the tails are reduced, favoring chromatin decondensation. (b) Conversely, shielding of charge in the DNA backbone through interactions with the positively charged (dephosphorylated, deacetylated) tails could facilitate chromatin condensation. (c) During mitosis, a unique configuration of modifications may exist wherein the core histones are deacetylated and histone H1 is phosphorylated. Partial shielding of linker DNA in this case may allow access to secondary factors involved in mitotic condensation (see Fig. 3). In addition, opposite charges on the core histone and H1 tails could facilitate interactions between these tails (within or between nucleosomes), allowing a high degree of compaction.
chromosome condensation. How might this apparent contradiction be reconciled? Firstly, it might be argued that all of the examples cited above involve variant forms of H1 which might have specialized roles in chromatin condensation/ decondensation. The altered globular domain in Tetrahymena H1, well known differences in structure and composition between H5 and H1, and the extended amino-terminal and carboxyterminal tails of sperm H1 could each alter the interaction of these molecules with DNA, with themselves, or with other components of chromatin. These changes, in turn, could affect consequences of phosphorylation on the formation or maintenance of higher order chromatin structures. Secondly, even if restricted domains within these Hls are functionally equivalent to more typical Hls, phosphorylation of different domains within them may affect their association with chromatin. Recently, Hill et al. 28 examined the effects of phosphorylation on the DNA binding affinity of intact H1 and of isolated amino-terminal and carboxyterminal H1 tails. Phosphorylation of tandemly repeated sites in the aminoterminal tail severely inhibited its interaction with DNA in vit;o, while phosphorylation of dispersed gites in the carboxy-terminal tail had little effect on its DNA binding capability. Surprisingly, phosphorylation of both amino-terminal and carboxy-terminal
96
sites did not affect the association of intact H1 with DNA but did weaken the binding of H1 to chromatin. These workers propose that different domains within H1 have separate functions which are differentially affected by phosphorylation. In particular, they suggest that the H1 tails are important in establishing intermolecular contacts between condensed chromatin filaments, and that such interactions are inhibited by phosphorylation. The idea that phosphorylation of different sites in H1 has different functional consequences can be taken a step further by supposing the involvement of multiple kinase activities which might phosphorylate specialized, individual sites. Many Hls, for example, contain sites recognized by cAMP-dependent protein kinase (PKA) 7. Interestingly, microinjection of a stable peptide inhibitor of PKA into mammalian cells rapidly induces chromatin condensation, regardless of the phase of the cell cycle29. Both the inactivation of PKA and the activation of cdc2 kinase, then, may be necessary for the full induction of mitotic chromosome condensation. A third possibility is that the dynamic nature of H1 phosphorylation/ dephosphorylation in chromatin might then influence the accessibility of other factors involved in condensation. Phosphorylation facilitates exchange of H1 in chromatin and so changes in the steady state level of H1 phosphoryl-
ation could result in changes in the level of association of other proteins with DNAo The differences observed between mitosis and the examples reviewed here might thus reflect a different composition of non-histone proteins present in different nuclei. For example, genetic and biochemical experiments indicate that topoisomerase II (topo II) is required for mitotic chromosome condensation ~. Interestingly, chicken erythrocyte nuclei contain low levels of endogenous topo 1I and are not condensed upon addition of topo lIdepleted mitotic extracts prepared from Xenopus eggs. HeLa cell nuclei, on the other hand, are well condensed in topo lI-depleted extracts, and contain high levels of endogenous topo I131,32. Thus, H1 may be able to regulate the topology of looped chromosomal domains indirectly by modulating the accessibility of topo I1 for its consensus sequences. The binding of other non-histone chromosomal proteins could also be modulated by H1 phosphorylation. Finally, we point out that other postsynthetic modifications, such as core histone acetylation, are known to modulate the structure of chromatin dramatically. As shown in Fig. 2, the overall level of specific sites of core histone acetylation/deacetylation may play an important, perhaps pivotal, role in the final outcome of the chromatin folding process (condensed or decondensed states). For example, when levels of core histone acetylation and H1 phosphorylation are both high (Fig. 2a), numerous histone-DNA contacts would be weakened, leading to an overall decondensed chromatin conformation. Under this set of conditions there may be exchange of free and chromatinbound H1 explaining numerous reports of reduced "(or missing) H1 in hyperacetylated transcriptionally competent chromatinL In contrast, stronger H1DNA interactions may occur when core histones are deacetylated and H1 is dephosphorylated (Fig. 2b). That core histones are largely deacetylated in highly condensed, mature avian erythrocytes 35 and in sea urchin sperm 24 is consistent with this idea. Interestingly, when macronuclei in Tetrahymena condense and undergo transcriptional inactivation, core histone amino termini are removed proteolytically and H1 is dephosphorylated 16. Removal of amino termini from core histones during this interval may mimic the reduction in charge which occurs upon acetylation and could promote H1-DNA interac-
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MARCH 1 9 9 2
tions by exposing regions of linker DNA previously engaged with core histone amino terminP6. Although only speculation at this point, a unique configuration of posttranslational modifications may exist during the transition into mitosis. In addition to phosphorylation of H1, deacetylation of the core histones appears to be required for mitotic progression, as suggested by genetic studies in yeast and biochemical studies in other sytems34. Under the set of conditions depicted in Fig. 2c, phosphorylated H1 tails might dissociate from the linker DNA while the positively charged tails of the core histones would continue to shield charges in the DNA phosphate backbone. In addition, the opposite charges on the tails of H1 and the core histones could conceivably facilitate interactions between the tails of neighboring nucleosomes (or within a nucleosome), further promoting condensation. Although a direct association in chromatin of H1 and core histone termini has not been reported, amino-terminal domains of the core histories are in contact with linker DNAs6 and the binding of H1 to nascent chromatin is influenced by core histone acetylation37. Whether direct linkage of H1 phosphorylation and core histone deacetylation occurs during mitotic chromosome condensation remains to be determined.
(a) Decondensed
Condensed H1 phosphorylated
H 1 dephosphorylated
Reversible f
S/T phosphorylation
(b) Condensed H1 dephosphorylated
• J~'~ ~
~ll
Decondensed H1 phosphorylated
II
Reversible
~,,
~..
S/T phosphorylation
Figure 3 Models for the involvement of H1 phosphorylation/dephosphorylation in chromatin condensation. (a) Adapted from Bradbury et al.9 Dephosphorylated H1 is depicted as having a stronger interaction with DNA than with other Hls favoring decondensation of chromatin. Upon phosphorylation, at serine or threonine (S/T) residues, H1 is less tightly bound to DNA and is in a conformation which favors H1-H1 interactions. Interactions between H1 molecules on the same, or different, chromatin fibers results in condensation of chromatin. (b) In an alternative model, the positive charge of the lysine-rich tails of H1 shields the negative charge of the phosphate backbone of DNA, allowing interactions between neighboring chromatin fibers and, ultimately, chromatin condensation. Target sequences for trans-acting factors (black boxes) would be inaccessible in this state. Upon phosphorylation, the increased negative charge of the H1 tails causes repulsion of the tails from DNA, unshielding the charged DNA backbone. Interactions between chromatin fibers would not be favored under these conditions, allowing local decondensation. Target sequences for trans-acting factors would be more accessible in this state, and upon binding, such factors could promote further condensation (as during mitosis) or decondensation (as during transcription) of chromatin. Particular phosphorylation sites (shown as larger Ps) might have specialized roles in chromatin condensation/decondensation. N represents a single nucleosome associated with a single molecule of H1.
A revised model for the role of H1 phosphorylation in chromatin condensation A model proposed by Bradbury and colleagues to explain how phosphorylation of H1 might result in chromosome condensation is summarized in Fig 3a9. During interphase, non-phosphorylated H1 present in decondensed chromatin was assumed to be loosely bound to DNA through interactions involving the amino-terminal and carboxy-terminal tails, as well as the central globular domaitr. Phosphorylation of H1 tails during mitosis was proposed to weaken H1-DNA interactions, thereby promoting H1-H1 interactions necessary for for- ated tails of H1. This situation is conmation of higher order chromatin sistent with the dephosphorylation of H1 (or H5) observed during chromatin structures. Taking into consideration features of condensation in old Tetrahymena macrothe systems described in this review, nuclei, avian erythrocytes and mature we propose an alternative model for the sperm. In keeping with the original role of H1 phosphorylation in chro- Bradbury model, we envisage that matin condensation outside of mitosis. In phosphorylation of the H1 tails should this model (Fig. 3b) much of the nega- weaken H1-DNA interactions, perhaps tive charge from linker DNA phosphates in a domain-specific manner. Unlike the is shielded in condensed chromatin by original model, however, we suppose the positively charged, non-phosphoryl- that the resulting increase in negative
charge and/or the exposure of negative charge in the DNA phosphate backbone would cause a repulsion of adjacent DNA fibers, leading to chromatin decondensation. Consistent with this idea is the finding that chromatin folding in vitro is largely electrostatic in nature and is governed by repulsions between DNA regions which are reduced upon binding of H1s. One attractive feature of this model is that it would provide a means where-
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by the cell could, through reversible H1 phosphorylation, 'reprogram' the DNA template with non-histone chromosomal proteins. For example, a transacting regulatory factor (black box, Fig. 2b) might be excluded from its target binding site in condensed chromatin. Transient phosphorylation of H1 could result in increased accessibility of the target sequence through chromatin decondensation. Once bound, such a factor might either promote further decondensation, as during transcription, or condensation, as during mitosis. Cycles of H1 phosphorylation/dephosphorylation could thus be viewed to function similarly to core histone acetylation/deacetylation (see Fig. 2). Both acetylation, and phosphorylation effectively decrease the amount of net positive charge able to interact with DNA, and acetylation has been proposed to 'open' the chromatin fiber by negating histone-DNA interactions. Is the model shown in Fig. 3b consistent with the correlation between H1 hyperphosphorylation and chromosome condensation observed during mitosis? H1 tail repulsion from DNA via phosphorylation may be an essential first step for mitotic chromosome condensation. In this regard, our model is similar to the original Bradbury model in which HI1 phosphorylation was proposed to initiate chromosome condensation. However, rather than play a direct, physical role in the condensation process, we propose that H1 phosphorylation actually allows a transient decondensation of chromatin which allows specific chromosome condensation factors (for example, topo II) to gain access to specific DNA sequences. Cell cycle control of the steady-state amount of such factors, coupled with H1 hyperhphosphorylation driven by an increase in cdc2 kinase activity, would insure that chromosome condensation occurs at the appropriate time. Thus, systems like macronuclei in Tetrahymena may not undergo mitotic chromosome condensation because they lack appropriate amounts of such factors necessary for the actual second step of the condensation process. The apparent lack of an Hl-like histone in yeast33'34may obviate the need for the first step, ~since the more open configuration of yeast chromatin may allow ready access to such condensing factors. Two features of this alternative model are worth emphasizing. First, there may be two distinct types of chro-
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matin condensation: (1) a type as in macronuclear degeneration, avian red blood cell maturation and sea urchin sperm formation which directly reflects the dephosphorylation of H1 (and possibly other chromosomal proteins) and (2) a factor-mediated process as in mitosis, requiring H1 phosphorylation for factor accessibility to DNA. Second, H1 phosphorylation may be a general first step mechanism for inducing chromatin decondensation throughout the cell cycle enabling access of specific DNA binding factors for processes such as gene activation or replication as well as chromosome condensation. In conclusion, the Bradbury model has provided a very useful framework for studies of H1 phosphorylation, but it does not account for the examples of non-mitotic chromatin condensation described here. The alternative ideas we present attempt to accommodate these unique systems. Perhaps a consideration of such 'exceptions' will provide a basis for the design of future experiments which will allow a more complete definition of the rules for chromatin condensation and of the role of H1 phosphorylation/dephosphorylation in this process.
Acknowledgements The authors acknowledge past and present associates whose work has made much of this review possible. In particular, we appreciate the critical review and valuable comments of Drs A. Annunziato, D. Clark, R. Green, M. Gorovsky, R. Morse, D. Poccia, R. Simpson and A. Wolffe. We also thank T. Hunt for his suggestion to write this article for TIBS and M. Fagliarone for her help in preparing the manuscript. Published work by the authors was supported by grants from NIH.
References Due to TIBS' policy of short reference lists we are only able to list a portion of the vast body of relevant literature. Other papers can be found through the reference lists of review articles cited. 1 Wolffe, A. P. (1991) Trends Cell Biol. 1, 61-66 2 van Holde, K. E. (1988) in Chromatin (Rich, A., ed.), pp. 69-180, Springer-Verlag 3 Allan, J., Hartman, P. G., Crane-Robinson, C. and Aviles, F. X. (1980) Nature 288, 675-679 4 Staynov, D. Z. and Crane-Robinson, C. (1988) EMBO J. 7, 3685-3691 5 Clark, D. J. and Kimuram T. (1990) J. Mol. Biol. 211, 88,3-896 6 Gurley, L. R. et al. (1978) in Cell Cycle Regulation (Jeter, J. R., Cameron, I. L., Padilla, G. M. and Zimmerman, A.M., eds), pp. 37-60, Academic Press 7 Hohmann, P. (1983) Mol. Cell. Biochem. 57,
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81-92 8 Roberge, M., Th'ng, J., Hamaguchi, J. and Bradbury, E. M. (1990) J. Cell Biol. 111, 1753-1762 9 Bradbury, E. M., Inglis, R. J. and Matthews, H. R. (1974) Nature 247, 257-261 10 Inglis, R. J. et al. (1976) Exptl Cell Res. 97, 418-425 11 Murray, A. W. and Kirschner, M. W. (1989) Science 246, 614-621 12 Langan, T. A. et al. (1989) Mol. Cell Biol. 9, 3860-3868 i 3 Kuang, J. et al. (1991) Dev. Biol. 144, 54-64 14 Gorovsky, M. A. (1986)in Molecular Biology of Ciliated Protozoa (Gall, J. G., ed.), pp. 227-261, Academic Press 15 Roth, S. Y. et al. (1988) J. Cell Biol. 107, 2473-2482 16 Lin, R., Cook, R. G. and Allis, C. D. (1991) Genes Dev. 5, 1601--1610 17 Roth, S. Y. etal. (1991) EMBOJ. 10, 2069-2075 18 Sung, M. T. (1977) Biochemistry 16, 286-290 19 Bergman, M. G., Wawra, E. and Winge, M. (1988) J. Cell Sci. 91, 201-209 20 Sun, J-M., Wiaderkiewicz, R. and Ruiz-Carillo, A. (1989) Science 245, 69-71 21 Sun, J-M., All, Z., Lurz, R. and Ruiz-Carillo, A. (1990) EMBO J. 9, 1651-1658 22 Aubert, D. et al. (1991) J. Cell Biol. 113, 497-506 23 Green, G. R. and Poccia, D. L. (1985) Dev. Biol. 108, 235-245 24 Poccia, D. (1989) in The Molecular Biology of Fertilization (Schatten, H. and Schatten, G., eds), pp. 115-135, Academic Press 25 Suzuki, M. (1989) EMBO J. 8, 797-804 26 Hill, C. S., Packman, L. C. and Thomas, J. O. (1990) EMBO J. 9, 805-813 27 Green, G. R. and Poccia, D. L. (1988) Biochemistry 27, 619-625 28 Hill, C. S. et al. (1991) EMBO J. 10, 1939-1948 29 Lamb, N. J. C., Labbe, J. C., Mauer, R. and Fernandez, A. (1991) EMBO J. 10, 1523-1533 30 Sternglanz, R. (•989) Curr. Opin. Cell Biol. 1, 533-535 31 Adachi, Y., Luke, M. and Laemmli, U.K. (1991) Cell 64, 137-148 32 Wood, E. R. and Eamshaw, W. C. (1990) J. Cell Biol. 111, 2839-2850 33 Grunstein, M. (1990) Annu. Rev. Cell Biol. 6, 643-678 34 Smith, M. M. (1991) Curr. Opin. Cell Biol. 3, 429-437 35 Zhang, D-E. and Nelson, D. A. (1986) Biochem. J. 240, 857-862 36 Hills, C. S. and Thomas, J. O. (1990) Eur. J. Biochem. 187, 143-153 37 Perry, C. A. and Annunziato, A. T. (1991) Exp. Cell Res. 196, 337-345
11 Eigen, M., ed. (1991) Report on the Intemational Workshop 'Selection - Natural and Unnatural - in Biotechnology', Max-Planck-lnstitut
12 13 14 15 16 17 18
fur Biophysikalische Chemie, Gottingen, Germany Horwitz, M. S. Z. and Loeb, L. (1986) Proc. Natl Acad. Sci. USA 83, 7405-7409 Oliphant, A. R. and Struhl, K. (1987) Methods Enzymol. 155, 568-582 Gronostajski, R. M. (1987) Nucleic Acids Res. 15, 5545-5559 Kaiser, C. A., Preuss, D., Grisafi, P. and Botstein, D. (1987) Science 235, 312-317 Ma, J. and Ptashne, M. (1987) Cell 51, 113-119 Dube, D. K. and Loeb, L. (1989) Biochemistry 28, 5703-5707 Perkins, A. S., Fishel, R., Jenkins, N. A. and
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20 21 22 23 24 25
26 27
Copeland, N.G. (1991) Mol. Cell. Biol. 11, 2665-2674 Chittendon, T., Livingston, D. M. and Keilin, Jr, W. G. (1991) Cell 65, 1073-1082 Andrake, M. et al. (1988) Proc. Natl Acad. Sci. USA 85, 7942-7946 Barrel, D., Zapp, M., Green, M, and Szostak, J. W. (1991) Cell 67, 529-536 Kruger, K. et al. (1982) Cell 31, 147-157 Kramer, F. R. et al. (1974) J. Mol. Biol. 89, 719-736 Miele, E. A., Mills, D. R. and Kramer, F. R. (1983) J. Mol. Biol. 171, 281-295 Guatelli, J. C. et al. (1990) Proc. Natl Acad. Sci. USA 87, 1874-1878 Abelson, J. (1990) Science 249, 488-489 Riordan, M. L. and Martin, J. C. (1991) Nature 350, 442-443
TALKINGPOINT EUKARYOTIC DNA is packaged with histone proteins into chromatin 1. Core histone proteins (H2A, H2B, H3 and H4) form an octameric complex around which is wound approximately two turns of DNA. This basic nucleosomal unit is highly conserved and is compacted into higher-order structures such as the 10 nm thin filament and the 30 nm thick fiber. How these structures are formed or how chromatin decondenses and recondenses to facilitate biological processes such as transcription, replication and recombination is unclear. A fifth histone, H1, binds to the nucleosome core particle, sealing the entry and exit points of the DNA. In vitro studies strongly suggest that electrostatic interactions between H1 and DNA play a paramount role in chromatin folding5. Most Hls have a tripartite structure consisting of a globular central domain flanked by lysine-rich, highly charged amino-terminal and carboxytermin~ tails 2. The central domain appears to interact with the nucleosome core particle while the positively charged tails interact with the linker DNA and are important in condensation 3,4. Post-translational phosphorylation of these tails may well influence their interaction with DNA. Changing levels of H1 phosphorylation could
s. Y. Roth is at the Laboratoryof Cellular and Developmental Biology, NIDDK,NIH, Bethesda, MD 20892, USA.C. D. Allis is at the Department of Biology, Syracuse University,Syracuse, NY 13244, USA. © 1992,ElsevierSciencePublishers, (UK)
In this article we describe three distinct biological systems where histone H1 phosphorylation is uncoupled from mitosis and highly condensed chromatin is enriched in dephosphorylated forms of HI: t,he amitotic macronucleus of Tetrahymena, terminally differentiated avian erythrocytes and sea urchin sperm. Each system offers informative contrasts to the idea that H1 hyperphosphorylation is causally related to mitotic chromosome condensation. Assuming that higher order chromatin folding is primarily driven by electrostatic interactions between H1 and DNA, an alternative model is presented for the role of H1 phosphorylation in chromatin condensation.
then, in turn, modulate chromatin condensation/decondensation.
a number of consistent observations have been made:
H1 phosphorylationand mitotic chromosome condensation:the original correlation
(1) Phosphorylation of H1 is increased in rapidly dividing cells and decreased in non-proliferating or quiescent cells. (2) Levels of H1 phosphorylation are usually lowest in the G1 phase of the cell cycle, and rise during both S phase (in which DNA replication also occurs) and mitosis. (3) During mitosis, phosphorylation of H1 and H3 becomes maximal just before or at metaphase and sharply decreases thereafter, reaching a minimum during anaphase or telophase. (4) Extracts from mitotic cells containing an 'H1 kinase' activity can stimu-
Phosphorylation of H1 during the cell cycle has been examined in a number of different organisms and cell types. Pioneering work was performed in two systems: the slime mold, Physarum polycephalum, which undergoes naturally synchronous mitotic divisions, and artificially synchronized Chinese hamster ovary cells in culture. Many other mammalian and non-mammalian systems have been studied subsequently ~ . Although the exact timing of events varies somewhat in different systems,
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late premature chromatin condensation when added to non-mitotic cells. (5) Inhibition of intracellular H1 kinase activity and subsequent dephosphorylation of H1 can cause partial chromosome decondensation. These correlations imply a role for H1 phosphorylation in mitotic chromosome
~
condensation and lead to a proposal that H1 hyperphosphorylation itself might serve as a mitotic 'trigger 'gJ°. Our understanding of the factors involved in regulation of the cell cycle has increased dramatically in the past few years with the characterization of various cyclin proteins and p34 cdc2
(a) Tetrahymena Micronuclei. ~ inactive ~
,.
Old macronuclei inactive condensed H1 dephosphorylated
active amitotic H1 phosphorylated
(b) Avian erythropoiesis
v
Erythroblast active H1 phosphorylated
Erythrocyte inactive condensed H5 dephosphorylated
Immature erythrocyte active H5 phosphorylated
(c) Sea urchin spermatogenesis and fertilization
Spermatocyte active H1 phosphorylated
Mature sperm inactive condensed H1 dephosphorylated
Male pronucleus reactivated decondensed H1 rephosphorylated
Figure 1 Exceptions to the long-held correlation between H1 phosphorylation and chromatin condensation are depicted schematically. In each case, the transcriptional activity (active or inactive), the condensation state of chromatin (condensed or decondensed), and the phosphorylation state of H1 (or H5) within particular nuclei are indicated. (a) Tetrahymenacontain germ-line micronuclei, which are transcriptionally inactive and divide mitotically, and macronuclei, which are transcriptionally active and divide amitotically. Macronuclei contain an Hl-like histone whose phosphorylation is modulated under different physiological conditions by a cdc2-1ike kinase activity. During conjugation (double cell figures), old macronuclei become highly pycnotic and transcriptionally inactive, as new macronuclei develop from micronuclei. Highly condensed chromatin in the old macronucleus contains only dephosphorylated H1. (b) During avian enjthropoiesis, nuclei are retained but become progressively more condensed. Another lysine-rich histone, H5, is synthesized and deposited in the immature erythrocyte in a highly p~sphonjlated form. In the mature erythrocyte, H5 is dephosphorylated as chromatin is compacted into a transcriptionally inactive state. (c) During sea urchin spermatogenesis, nuclei are compacted and chromatin becomes highly condensed, concomitant with the dephosphorylation of HI. These events are reversed upon fertilization, with decondensation of chromatin in the male pronucleus (white) and phosphorylation of HI.
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kinase homologues u. H1 serves as an important in vitro substrate for cdc2 kinase with phosphorylation occurring exclusively on the tail domains at conserved (K[S/T]PXK) or (K[S/T]PK) motifs ~2. However, it is not clear whether H1 is phosphorylated in vivo by cdc2 kinase ~3or what role H1 hyperphosphorylation plays in the condensation of chromosomes or the progression of mitosis. Direct evidence linking these processes is limited. In considering the 'effect' of H1 phosphorylation on mitotic chromosome condensation, we point out that addition of negatively charged phosphate groups to the tail domains of H1 should weaken interactions of H1 with DNA. Furthermore, in some systems H1 phosphorylation is uncoupled from mitosis and chromosome condensation; in these systems condensed chromatin is enriched in dephosphorylated forms of H1. In this article, we examine some of these 'exceptions' which are frequently overlooked. These include the amitotic macronucleus of Tetrahymena, the terminally differentiated nuclei of avian erythrocytes, and the highly condensed chromatin of sea urchin sperm. Each of these systems exhibits remarkable transitions between decondensed and condensed chromatin in the absence of mitosis (see Fig. 1). Each offers an informative contrast to the long-held notion that H1 phosphorylation is causally related to chromosome condensation during mitosis and suggests other functions that might be regulated by H1 phosphorylation/dephosphorylation.
Phosphorylation of H1 in the amitotic macronucleus of Tetrahymena . Ciliated protozoa such as Tetrahymena pr6vide a unique system in which to study H1 phosphorylation and transcriptional regulation outside mitosis. These organisms contain two distinct nuclei, differing both in function and mode of division (Fig. la) TM. The somatic macronucleus is transcriptionally active and divides amitotically without overt chromatin condensation. By contrast, the germ-line micronucleus is transcriptionally silent under most physiological conditions and undergoes normal mitotic divisions including the formation of mitotic chromosomes. The composition of histones differs significantly in these two nuclei. In particular, macronuclei contain an H1 histone which is highly phosphorylated during vegetative growth. In micronuclei, H1 is
TIBS 1 7 - MARCH 1992 missing and is replaced by specialized linker polypeptides which are also highly phosphorylated ~4. The level of macronuclear H1 phosphorylation changes dramatically in response to varying physiological conditions ~s.A high level of phosphorylation occurs in exponentially growing cells, and hyperphosphorylation is observed upon heat shock and induction of conjugation, the sexual cycle. Moderately phosphorylated H1 is observed upon starvation and in highdensity, stationary phase cultures. Most interesting, however, is the recent finding that H1 is rapidly and completely dephosphorylated during conjugation when the macronucleus ceases transcription and becomes highly condensed, prior to its eventual elimination from the cell (see Fig. la and Ref. 16). Since the highest levels of H1 phosphorylation are observed under conditions in which no division of the cell or macronucleus occurs (heat shock and conjugation), it appears that H1 phosphorylation is not, by itself, uniquely linked to either mitotic or amitotic division. Indeed, changes in the levels of H1 phosphorylation in Tetrahymena appear to occur whenever there are marked changes in gene expression due to altered physiological conditions, supporting a role for H1 phosphorylation in transcriptional regulationlS,16. Despite the lack of correlation between H1 phosphorylation and mitosis in macronuclei, macronuclear H1 is nonetheless phosphorylated by a cdc2-1ike kinase activity~7. This activity is highest in rapidly growing cells, and declines steadily as cells reach stationary phase. Importantly, the Tetrahymena kinase activity displays several hallmark features of cdc2 kinase in other systems, including binding to Suc1/p13 beads and recognition by polyclonal antibodies to fission yeast cdc2, indicating~hat it shares some conserved structural features with the yeast kinase. Macronuclear H1 is phosphorylated in vitro by mammalian cdc2 at sites remarkably similar to those phosphorylated by the Tetrahymena kinase in vivo and in vitro, suggesting it is a bona fide substrate for this activity in vivo. Phosphorylation of H1 by a cdc2-1ike kinase activity in amitotic macronuclei suggests that although H1 phosphorylation may be required for chromosome condensation, it is not sufficient to induce the condensation process. Macro-
nuclear H1 differs from other H1 molecules in that it lacks some features of the central globular domain, and this structural difference may underlie a different function for macronuclear H1 phosphorylation. However, phosphorylation of motifs believed to be involved in mitotic chromosome condensation is conserved in Tetrahymena H1, arguing against this possibility.
chromatin compaction during spermatogenesis does not represent a terminally differentiated state but instead is reversible upon fertilization (Fig. lc). Thus, changes in H1 phosphorylation can be studied both in regards to condensation of chromatin as spermatids develop into mature sperm and to decondensation of chromatin in the male pronucleus following fertilization 23.24. Unlike sperm development in many other species, that occurring in sea Phosphorylationof histone H5 in avian erythrocytes urchins does not involve replacement During the final stages of erythrocyte of core histones with protamines; development into mature red blood cells, somatic variants of histones H2A, H3 transcriptional inactivation occurs and H4 are maintained. Sperm-specific along with the formation of highly con- forms of H1 and H2B are deposited densed chromatin. In mammalian cells, early in the differentiation of the male nuclei are eventually discarded from germ line. These sperm-specific hismature erythrocytes. In avian species, tones contain extended amino-termini however, nuclei are retained and several consisting largely of repeats of the specialized proteins associate with the sequence SPKK24,25, the target phoshighly condensed chromatin (Fig. lb). phorylation site of cdc2 kinase24.2E H1 One of these proteins, H5, another type and H2B are highly phosphorylated in of lysine-rich histone, is present in spermatids, and are dephosphorylated low levels early in erythropoiesis and in the highly condensed chromatin of reaches maximal levels only in fully dif- mature sperm 24. Further, chromatin ferentiated red blood cells2E Newly syn- isolated from spermatids condenses thesized H5 is highly phosphorylated, less readily at increasing ionic strength but during the last stages of red cell dif- than does sperm chromatin, and ferentiation, when transcription ceases phosphorylation inhibits self-associand nuclei condense, H5 is quantitatively ation of oligonucleosomes27,28. One dephosphorylatedTM. Hence, dephos- explanation for these observations is phorylation of H5 rather than phos- that phosphorylation effectively neuphorylation is correlated with the com- tralizes the positive charge in the tail domain, perhaps lowering the affinity of paction of chromatin in these nuclei. Recent experiments further support H1 termini for DNA thereby uncovering a direct correlation between dephos- linker DNA phosphates and inhibiting phorylation of H5, cessation of tran- chromatin condensation. Following fertilization, sperm H1 and scription, and chromatin condensation. Introduction of H5 protein 19or the gene H2B are re-phosphorylated, prior to the encoding iF°-22into normal cells typically decondensation of chromatin and reaccauses nuclear condensation similar to tivation of gene expression in the male that observed in mature red blood pronucleus 2s and sperm H1 is replaced cells. However, Aubert et al.22 have with a phosphorylated, egg-specific shown that phosphorylation of H5 is (CS) form of H1. Thus, dephosphorylminimal in normal cells, but is more ation of sperm H1 appears necessary pronounced in transformed cells, which for the stabilization of highly condo not undergo nuclear compaction. densed chromatin within mature Thus, it appears that in cycling, trans- sperm, and rephosphorylation preformed cells, expression of a kinase cedes decondensation of chromatin in (perhaps cdc2) allows constitutive the male pronucleus and loss of sperm phosphorylation of H5, and that this H1 from chromatin. phosphorylation inhibits rather than Alternatives and explanations promotes chromatin condensation. In all three systems described above, phosphorylation of H1 correlates best Phosphorylationof H1 in sea urchin spermatogenesis and fertilization with decondensation of chromatin Compaction of chromatin during sea while H1 dephosphorylation is associurchin spermatogenesis in some ways ated with the formation or stabilization resembles that which occurs during of highly condensed structures. This is erythropoiesis. However, in contrast to exactly the opposite of the expectation erythropoiesis and the elimination of based on the correlation between parental macronuclei in Tetrahymena, hyperphosphorylation of H1 and mitotic
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(a) Decondensed
P-~1~(~H l ~ p H1 phosphorylated core histones acetylated
(b) Condensed
(c) Mitosis
P - ~ p -
_
H1 dephosphorylated core histories deacetylated
H1 phosphorylated core histones deacetylated
Figure2 Possible involvement of histone post-translational modifications in chromatin condensation. The positively charged tails of the core histones within the nucleosome (N) and histone H1 are shown in close association with linker DNA. (a) Upon acetylation (Ac) of the core histones and phosphorylation (P-) of H1, positive charges on the tails are reduced, favoring chromatin decondensation. (b) Conversely, shielding of charge in the DNA backbone through interactions with the positively charged (dephosphorylated, deacetylated) tails could facilitate chromatin condensation. (c) During mitosis, a unique configuration of modifications may exist wherein the core histones are deacetylated and histone H1 is phosphorylated. Partial shielding of linker DNA in this case may allow access to secondary factors involved in mitotic condensation (see Fig. 3). In addition, opposite charges on the core histone and H1 tails could facilitate interactions between these tails (within or between nucleosomes), allowing a high degree of compaction.
chromosome condensation. How might this apparent contradiction be reconciled? Firstly, it might be argued that all of the examples cited above involve variant forms of H1 which might have specialized roles in chromatin condensation/ decondensation. The altered globular domain in Tetrahymena H1, well known differences in structure and composition between H5 and H1, and the extended amino-terminal and carboxyterminal tails of sperm H1 could each alter the interaction of these molecules with DNA, with themselves, or with other components of chromatin. These changes, in turn, could affect consequences of phosphorylation on the formation or maintenance of higher order chromatin structures. Secondly, even if restricted domains within these Hls are functionally equivalent to more typical Hls, phosphorylation of different domains within them may affect their association with chromatin. Recently, Hill et al. 28 examined the effects of phosphorylation on the DNA binding affinity of intact H1 and of isolated amino-terminal and carboxyterminal H1 tails. Phosphorylation of tandemly repeated sites in the aminoterminal tail severely inhibited its interaction with DNA in vit;o, while phosphorylation of dispersed gites in the carboxy-terminal tail had little effect on its DNA binding capability. Surprisingly, phosphorylation of both amino-terminal and carboxy-terminal
96
sites did not affect the association of intact H1 with DNA but did weaken the binding of H1 to chromatin. These workers propose that different domains within H1 have separate functions which are differentially affected by phosphorylation. In particular, they suggest that the H1 tails are important in establishing intermolecular contacts between condensed chromatin filaments, and that such interactions are inhibited by phosphorylation. The idea that phosphorylation of different sites in H1 has different functional consequences can be taken a step further by supposing the involvement of multiple kinase activities which might phosphorylate specialized, individual sites. Many Hls, for example, contain sites recognized by cAMP-dependent protein kinase (PKA) 7. Interestingly, microinjection of a stable peptide inhibitor of PKA into mammalian cells rapidly induces chromatin condensation, regardless of the phase of the cell cycle29. Both the inactivation of PKA and the activation of cdc2 kinase, then, may be necessary for the full induction of mitotic chromosome condensation. A third possibility is that the dynamic nature of H1 phosphorylation/ dephosphorylation in chromatin might then influence the accessibility of other factors involved in condensation. Phosphorylation facilitates exchange of H1 in chromatin and so changes in the steady state level of H1 phosphoryl-
ation could result in changes in the level of association of other proteins with DNAo The differences observed between mitosis and the examples reviewed here might thus reflect a different composition of non-histone proteins present in different nuclei. For example, genetic and biochemical experiments indicate that topoisomerase II (topo II) is required for mitotic chromosome condensation ~. Interestingly, chicken erythrocyte nuclei contain low levels of endogenous topo 1I and are not condensed upon addition of topo lIdepleted mitotic extracts prepared from Xenopus eggs. HeLa cell nuclei, on the other hand, are well condensed in topo lI-depleted extracts, and contain high levels of endogenous topo I131,32. Thus, H1 may be able to regulate the topology of looped chromosomal domains indirectly by modulating the accessibility of topo I1 for its consensus sequences. The binding of other non-histone chromosomal proteins could also be modulated by H1 phosphorylation. Finally, we point out that other postsynthetic modifications, such as core histone acetylation, are known to modulate the structure of chromatin dramatically. As shown in Fig. 2, the overall level of specific sites of core histone acetylation/deacetylation may play an important, perhaps pivotal, role in the final outcome of the chromatin folding process (condensed or decondensed states). For example, when levels of core histone acetylation and H1 phosphorylation are both high (Fig. 2a), numerous histone-DNA contacts would be weakened, leading to an overall decondensed chromatin conformation. Under this set of conditions there may be exchange of free and chromatinbound H1 explaining numerous reports of reduced "(or missing) H1 in hyperacetylated transcriptionally competent chromatinL In contrast, stronger H1DNA interactions may occur when core histones are deacetylated and H1 is dephosphorylated (Fig. 2b). That core histones are largely deacetylated in highly condensed, mature avian erythrocytes 35 and in sea urchin sperm 24 is consistent with this idea. Interestingly, when macronuclei in Tetrahymena condense and undergo transcriptional inactivation, core histone amino termini are removed proteolytically and H1 is dephosphorylated 16. Removal of amino termini from core histones during this interval may mimic the reduction in charge which occurs upon acetylation and could promote H1-DNA interac-
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MARCH 1 9 9 2
tions by exposing regions of linker DNA previously engaged with core histone amino terminP6. Although only speculation at this point, a unique configuration of posttranslational modifications may exist during the transition into mitosis. In addition to phosphorylation of H1, deacetylation of the core histones appears to be required for mitotic progression, as suggested by genetic studies in yeast and biochemical studies in other sytems34. Under the set of conditions depicted in Fig. 2c, phosphorylated H1 tails might dissociate from the linker DNA while the positively charged tails of the core histones would continue to shield charges in the DNA phosphate backbone. In addition, the opposite charges on the tails of H1 and the core histones could conceivably facilitate interactions between the tails of neighboring nucleosomes (or within a nucleosome), further promoting condensation. Although a direct association in chromatin of H1 and core histone termini has not been reported, amino-terminal domains of the core histories are in contact with linker DNAs6 and the binding of H1 to nascent chromatin is influenced by core histone acetylation37. Whether direct linkage of H1 phosphorylation and core histone deacetylation occurs during mitotic chromosome condensation remains to be determined.
(a) Decondensed
Condensed H1 phosphorylated
H 1 dephosphorylated
Reversible f
S/T phosphorylation
(b) Condensed H1 dephosphorylated
• J~'~ ~
~ll
Decondensed H1 phosphorylated
II
Reversible
~,,
~..
S/T phosphorylation
Figure 3 Models for the involvement of H1 phosphorylation/dephosphorylation in chromatin condensation. (a) Adapted from Bradbury et al.9 Dephosphorylated H1 is depicted as having a stronger interaction with DNA than with other Hls favoring decondensation of chromatin. Upon phosphorylation, at serine or threonine (S/T) residues, H1 is less tightly bound to DNA and is in a conformation which favors H1-H1 interactions. Interactions between H1 molecules on the same, or different, chromatin fibers results in condensation of chromatin. (b) In an alternative model, the positive charge of the lysine-rich tails of H1 shields the negative charge of the phosphate backbone of DNA, allowing interactions between neighboring chromatin fibers and, ultimately, chromatin condensation. Target sequences for trans-acting factors (black boxes) would be inaccessible in this state. Upon phosphorylation, the increased negative charge of the H1 tails causes repulsion of the tails from DNA, unshielding the charged DNA backbone. Interactions between chromatin fibers would not be favored under these conditions, allowing local decondensation. Target sequences for trans-acting factors would be more accessible in this state, and upon binding, such factors could promote further condensation (as during mitosis) or decondensation (as during transcription) of chromatin. Particular phosphorylation sites (shown as larger Ps) might have specialized roles in chromatin condensation/decondensation. N represents a single nucleosome associated with a single molecule of H1.
A revised model for the role of H1 phosphorylation in chromatin condensation A model proposed by Bradbury and colleagues to explain how phosphorylation of H1 might result in chromosome condensation is summarized in Fig 3a9. During interphase, non-phosphorylated H1 present in decondensed chromatin was assumed to be loosely bound to DNA through interactions involving the amino-terminal and carboxy-terminal tails, as well as the central globular domaitr. Phosphorylation of H1 tails during mitosis was proposed to weaken H1-DNA interactions, thereby promoting H1-H1 interactions necessary for for- ated tails of H1. This situation is conmation of higher order chromatin sistent with the dephosphorylation of H1 (or H5) observed during chromatin structures. Taking into consideration features of condensation in old Tetrahymena macrothe systems described in this review, nuclei, avian erythrocytes and mature we propose an alternative model for the sperm. In keeping with the original role of H1 phosphorylation in chro- Bradbury model, we envisage that matin condensation outside of mitosis. In phosphorylation of the H1 tails should this model (Fig. 3b) much of the nega- weaken H1-DNA interactions, perhaps tive charge from linker DNA phosphates in a domain-specific manner. Unlike the is shielded in condensed chromatin by original model, however, we suppose the positively charged, non-phosphoryl- that the resulting increase in negative
charge and/or the exposure of negative charge in the DNA phosphate backbone would cause a repulsion of adjacent DNA fibers, leading to chromatin decondensation. Consistent with this idea is the finding that chromatin folding in vitro is largely electrostatic in nature and is governed by repulsions between DNA regions which are reduced upon binding of H1s. One attractive feature of this model is that it would provide a means where-
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by the cell could, through reversible H1 phosphorylation, 'reprogram' the DNA template with non-histone chromosomal proteins. For example, a transacting regulatory factor (black box, Fig. 2b) might be excluded from its target binding site in condensed chromatin. Transient phosphorylation of H1 could result in increased accessibility of the target sequence through chromatin decondensation. Once bound, such a factor might either promote further decondensation, as during transcription, or condensation, as during mitosis. Cycles of H1 phosphorylation/dephosphorylation could thus be viewed to function similarly to core histone acetylation/deacetylation (see Fig. 2). Both acetylation, and phosphorylation effectively decrease the amount of net positive charge able to interact with DNA, and acetylation has been proposed to 'open' the chromatin fiber by negating histone-DNA interactions. Is the model shown in Fig. 3b consistent with the correlation between H1 hyperphosphorylation and chromosome condensation observed during mitosis? H1 tail repulsion from DNA via phosphorylation may be an essential first step for mitotic chromosome condensation. In this regard, our model is similar to the original Bradbury model in which HI1 phosphorylation was proposed to initiate chromosome condensation. However, rather than play a direct, physical role in the condensation process, we propose that H1 phosphorylation actually allows a transient decondensation of chromatin which allows specific chromosome condensation factors (for example, topo II) to gain access to specific DNA sequences. Cell cycle control of the steady-state amount of such factors, coupled with H1 hyperhphosphorylation driven by an increase in cdc2 kinase activity, would insure that chromosome condensation occurs at the appropriate time. Thus, systems like macronuclei in Tetrahymena may not undergo mitotic chromosome condensation because they lack appropriate amounts of such factors necessary for the actual second step of the condensation process. The apparent lack of an Hl-like histone in yeast33'34may obviate the need for the first step, ~since the more open configuration of yeast chromatin may allow ready access to such condensing factors. Two features of this alternative model are worth emphasizing. First, there may be two distinct types of chro-
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matin condensation: (1) a type as in macronuclear degeneration, avian red blood cell maturation and sea urchin sperm formation which directly reflects the dephosphorylation of H1 (and possibly other chromosomal proteins) and (2) a factor-mediated process as in mitosis, requiring H1 phosphorylation for factor accessibility to DNA. Second, H1 phosphorylation may be a general first step mechanism for inducing chromatin decondensation throughout the cell cycle enabling access of specific DNA binding factors for processes such as gene activation or replication as well as chromosome condensation. In conclusion, the Bradbury model has provided a very useful framework for studies of H1 phosphorylation, but it does not account for the examples of non-mitotic chromatin condensation described here. The alternative ideas we present attempt to accommodate these unique systems. Perhaps a consideration of such 'exceptions' will provide a basis for the design of future experiments which will allow a more complete definition of the rules for chromatin condensation and of the role of H1 phosphorylation/dephosphorylation in this process.
Acknowledgements The authors acknowledge past and present associates whose work has made much of this review possible. In particular, we appreciate the critical review and valuable comments of Drs A. Annunziato, D. Clark, R. Green, M. Gorovsky, R. Morse, D. Poccia, R. Simpson and A. Wolffe. We also thank T. Hunt for his suggestion to write this article for TIBS and M. Fagliarone for her help in preparing the manuscript. Published work by the authors was supported by grants from NIH.
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