E R N T R O D U C T I O N

The functional organization of chromosomes and the nucleus ~ a s p e c i a l i s s u e ~

ALISON STEWART

EDITOR,

TRENDS IN GE.~ICS

This month's issue of T/G highlights aspects of the way in which chro- mosomes and the nucleus are or- ganized for efficient and regulated gene expression, for recombination and for progression through the cell cycle. The functional conse- quences of chromosomal organiz- ation can be viewed at many levels, ranging from the molecular archi- tecture of individual genes and sets of genes, to the three-dimensional disposition of the chromosomes in the nucleus.

Chromatin and gene expression At the molecular level, there are

now many examples of regulation of gene activity by the combinatorial action of transcription factors. But does chromatin structure play a role in establishing and regulating patterns of gene expression? There is substantial evidence that it does, and some of the most interesting new developments are reviewed in this issue in articles by Renato Paro, Steven Henikoff and Michael Grunstein. Renato Paro discusses a mechanism by which a cell's devel- opmental decision can be clonally inherited by 'imprinting' a particu- lar structure on the chromatin, in this case the homeotic gene com- plexes of Drosopbfla, The proposal is that, depending on whether or not transcription has been activated by early acting maternal or zygotic transcription factors, the chromatin of the complex is subsequently maintained in an 'open' or 'closed' state by other sets of genes.

Significantly, the Polycomb pro- tein, which is one of a group of proteins proposed to maintain chromatin in the 'closed' state, is related to the protein HP1, a known component of Drosophila hetero- chromatin. The differentiation of chromatin into euchromatin and heterochromatin in Drosophila has long been known to be associated with different characteristics of gene expression. Particular genes appear

to be adapted for expression in one chromatin environment or the other, and moving a gene from one region to another can cause altered expression in some cells, as dis- cussed by Steven Henikoff. When inactivation occurs, this 'decision' is then clonally inherited. For the brown locus, inactivation can occur in tram, as a result of chromosome pairing between alleles. Position- effect variegation has been mysteri- ous for many years, and the de- tailed mechanism by which clonal inactivation occurs has yet to be approached experimentally. How- ever, a provocative new clue has come from the finding that a num- ber of loci that can modify position- effect variegation encode known components of heterochromatin - one such locus encodes the Polycomb-related HP1.

The heritable propagation of an inactive chromosomal state in cis is observed not only in classical position-effect variegation in Drosophila, but also in X chromo- some inactivation in mamn'lals, and in genomic imprinting in a variety of creatures from mealy bu.~s to mouse. While clearly not the sole explanation, in many examples DNA methylation participates in these heritable inactivation processes1.

The histones are also con- stituents of heterochromatin. Apart from such relatively nonspecific functions, individual histone pro- teins can also participate in specific regulatory processes. The most prominent role of the histones is, of course, to package DNA into nudeosomes and .higher order chromatin structures. The nucleo- some has often been ignored in studies on control of gene expression, but Michael Grunstein marshals evidence that it plays an importam role. Spec,:.qcally, when histones are depleted or mutated genetically, a large activation of transcription occurs that is inde- pendent of specific trans-acting

TIG DECEMBER 1990 VOL. 6 NO. 12

factors, suggesting that such factors not only activate transcription by directly activating the initiation sys- tem, but also by displacing his- tones. Since mutagenesis of partic- ular histone lysine residues, which have the potential to be modified by acetylation, leads to altered patterns of gene expression, there is now clear reason to believe that histone modifications initiate the nucleosome unfolding that is a pre- requisite for active transcription. Unfolding may occur within func- tional domains, bordered by sites of attachment to the nuclear scaf- fold z and/or by 'locus control regions' (also called locus activa- tion regions and dominant control regions) such as have been found in the far-upstream region of the [3- globin locus (reviewed in 7IG by Townes and Behringer3). These new types of genetic elements seem to insulate associated DNA from re- pressive effects of adjacent, inactive chromatin. It may be interactions of proteins with targets such as these that lead to the histone modifi- cations that in turn trigger unfolding.

Histone-like proteins have also been found in the 'chromatin' of bacteria. Although they are only distantly related to the histones of eukaryotes, they do appear to package DNA in a way that may have the potential to influence gene expression, for example by bringing distantly spaced sites near to each other. Specifically, the HU proteins bind to DNA in such a way that the DNA is wound around a protein core. In the topologically constrained circular bacterial chro- mosome, HU binding affects super- coiling. Topoisomerases that can introduce or relax supercoils nor. maUy act to maintain an overall status quo in the level of supercoil- ing, but it is possible that local changes in supercoiiing may have repercussions for gene expression. In fact, there is ample evidence that supercoiling and gene expression

@1990 Elsevier Science Publishers Lid (UK) 0168 - 9479/90/$02.00 3 " "

~ R N T R O D U C T I O N

are interrelated in bacteria, as discussed by Karl Drlica. For example, transcription can bring about local topological changes that may in turn influence neigh- bouring regions of the DNA mol- ecule. Conversely, factors such as the cell's energy status and some environmental stimuli may influence supercoiling, perhaps with conse- quences for gene expression, as re- flected in RNA polymerase binding, isomerization and clearance.

Transcription and beyond The role of the nucleus in

eukaryotic gene expression extends beyond transcription, to splicing of transcripts and their export to the cytoplasm. For the latter functions, there is evidence for the par- ticipation of specialized nuclear 'organelles'. The importance of the nucleolus in rRNA gene expression was first recognized almost three decades ago, and this month sees the 25th anniversary of the first isolation of rRNA genes (in fact the first isolation of any gene); Max Birnstiel reminisces about this landmark achievement in a Comment article in this issue. The molecular organization and control of expression of the rDNA gene array in the nucleolar organizer region has been revealed by more recent research, discussed by Ron Reeder, but still many questions remain about the composition of the nucleolus and the molecular mechanisms by which this organelle brings about the syn- thesis and processing of rRNA molecules, their packaging with other ribosomal constituents and export to the cytoplasm.

Recent evidence suggests that the nucleolus may not be the only nuclear organelle with a function in the assembly of ribonucleoproteins. Structures called 'spheres' that are routinely observed in oocyte nuclei of amphibians and a variety of invertebrates have now been found to contain the Sm antigen and trimethylguanosine, both character- istic components of small nuclear ribonucleoprotein#. Some spheres are free in the nucleoplasm, while others appear to be associated with specific chromosomal loci that, in analogy to the nucleolar organizers, have been dubbed 'sphere organ- izers'. Although tlie evidence is still

very preliminary, the intriguing suggestion has been made that spheres represent assembly precur- sors of spliceosomes, and that the sphere organizers either participate in or provide (RNA) components for this assembly.

Chromosome structure and cell division

Chromosomal organization is important not only for gene ex- pression during interphase, but also for efficient replication of DNA and its distribution to the daughter cells during cell division. Bacterial ori- gins of replication are well defined, but replication origins have proved more elusive to characterize in eukaryotes. Bruce Stillman and John Diffley discuss evidence that eukaryotic chromosomes have dif- ferent classes of replication origins whose level of use and time of in- itiation during S phase are regu- lated in different cell types. Origin function involves recruitment of initiation factors, which initiate unwinding at the site and assembly of the replication apparatus. How elongating polymerases (both RNA and DNA polymerases) copy DNA associated with nucleosomes is still unclear. Nucleosomes might be transiently displaced, they may open up, or they may be designed to allow polymerase to copy with minimum structural perturbation. After the passage of the replication fork, active and inactive chromatin must be assembled, at least on one daughter double helix. Aspects of this process were discussed in T/G earlier this year by Svaren and Chalkley5. In this issue, Ronald Laskey and Gregory Leno take the analysis in a different direction, dealing in particular with the question of the relationship be- tween the replication-dependent and replication-independent path- ways of nucleosome assembly that have been observed in different systems.

The replication of telomeres, the physical ends of chromosomes, has been a puzzling process for many years, but substantial pro- gress has now been made in un- ravelling the enzymology of telo- mere maintenance and replication. Unlike centromeres, telomere se- quences seem to be very similar in a wide range of eukaryotic

11G DECEMBER 1990 VOL. 6 NO. 12

organisms. A telomerase enzyme has been identified in a number of different ciliated protozoa and more recently in human cells. Remarkably, this telomerase is a ribonucleoprotein, its RNA compo- nent containing a sequence that in ciliates has been shown to act as a template for telomere synthesis, both in vitro and in viv06. However, this type of telomerase has not so far been detected in yeast, which Zakian and colleagues have argued may have both a telomerase-dependent and a recombinational mode of telomere maintenance 7.

As the cell progresses from DNA replication through the subse- quent stages of either mitotic or meiotic division, the chromosomes undergo profound structural changes in preparation for their interaction with the spindle appar- atus. However it is also during this period that recombinational pro- cesses occur. Meiotic recombi- nation leads to new assortments of genetic material and evolutionary variation. It has long been thought that crossing over, a microscopi- cally visible manifestation of genetic exchange between synapsed homologues, marked the time when all recombinational events occurred, but Shirleen Roeder argues that an earlier phase of recombination also occurs, as part of the process by which chromosomes search for their homologous counterparts. The stabilization of these homologous chromosome pairs at the meta- phase I plate, and their segregation during anaphase I, involve dynamic interactions between the kineto- chore and the spindle apparatus. It appears that chiasmata can physi- cally stabilize homologous pairs, but members of the kinesin family, encoding microtubule 'motor' proteins, also play a part. In Drosophila, two loci (ncd and nod), mutations at which lead to meiotic nondisjunction, have been found to encode kinesin- related proteins 8--10. The nod locus may be required in particular for correct segregation of nonchias- mate homologues 10. Intriguingly though, ncd (also called ca ha+) product expressed in bacteria has been shown to have motor activity directed towards the minus end of

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microtubules, a property usually associated with dyneins rather than kinesins, and one that would be consistent with a role in the pole- ward movement of chromosomes at anaphase H (also see Monitor in this issue).

Both mitotic and m,,~.iotic div- isions bring into play the centro- mere, the chromosomal region that together with the kinetochore or- ganizes the attachment of the spindle apparatus. Yeast centro- meres have been characterized at the molecular level, as described in a recent T/G review by Clarke 12. Mammalian centromeres are dis- cussed by Hunt Willard in this issue: at the sequence level, there is evidence that they consist at least partly of repeat-sequence satellite DNA; what singles out a particular subset of satellite DNA in the region of the primary constriction to complex with specific proteins to form a functional centromere is as yet unclear.

Nudear architecture In order to re-establish the cor-

rect environment for interphase gene expression, the newly divided eukaryotic cell must assemble a nuclear envelope. Although aspects of this process appear to vary in different organisms, a common feature is that nuclear pores are constructed first and then mem- brane vesicles form over the remaining surface of the chromatin, thus excluding any proteins that do not have the correct targeting sig- nals for entry through the pores, and establishing the functional dis- tinction between nucleus and cyto- plasm (see article by Laskey and Leno). The process by which specific transcription factors enter the nucleus has i~ecendy emerged as an important regulatory feature in development and gene acti- vation, with the study of NF-KB and Dorsal/rel ~3,14. These factors have homologous DNA-binding domains and also appear to share a regulatory mechanism that works via control of their subcellular localization. They are retained in inactive form in the cytoplasm (in the case of NF-KB by complexing with an inhibitor protein; analogous inhibitors have been postulated for Dorsal and rel), and are only active when release from this form allows

them access to the nucleus, pre- sumably by unmasking or creating a nuclear localization signal.

Nuclear assembly after cell div- ision must involve the re-formation of interphase chromatin domains in the decondensed chromosomes. But is there a still higher order of interphase chromosome organiz- ation, such that particular chro- mosomes have specific domains of residence and positions with respect to each other? For example, do the homologous pairs of chro- mosomes that are so evident at metaphase I of meiosis have any association with each other in cells other than those of the germ line? Attempts to answer questions such as this in recent years have employed sophisticated light microscopy and computing tech- niques to reconstruct into a three- dimensional image a set of staged optical sections through cells. Such analyses are difficult, and conflict- ing results have been reported. For plants, Pat Heslop-Harrison and Michael Bennett conclude that the balance of evidence favours individual chromosomes occupying distinct nuclear territories (perhaps with consequences for gene ex- pression), but that it is unlikely that there is any somatic association of homologues.

This is in contrast to the situ- ation in Drosophila, where somatic association of homologues is well established, and indeed is the basis for both transvection effect# 5 and for dominant positic, n-effect variegation, as discussed by Henikoff. Sedat, Agard and their colleagues have recently extended their pioneering studies on the organization of chromosomes in the syncytial blastoderm nuclei of Drosophila embryos, by developing a three-dimensional time-lapse

fluorescence microscopy technique that allowed them to follow the condensation and decondensation of chromosomes as nuclei pro- gressed through the cell cycle 16. Remarkably, specific chromosomal regions attached to the nuclear membrane appeared to act as foci for condensation and deconden- sation. Adding the dimension of time to studies of nuclear order is clearly a breakthrough in studying the dynamics of nuclear behaviour.

References 1 Holliday, R. (1987) Science 238,

163-170 2 Gasser, S.M. and Laemmli, U.

(1987) Trends Genet. 3, 16-22 3 Townes, T.M. and Behringer, R.R.

(1990) Trends Genet. 6, 219-223 4 Gall, J.G. and Callan, H.G. (1989)

Proc. Natl Acad. Sci. USA 86, 6635-6639

5 Svaren, J. and Chalkley, R. (1990) Trends Genet. 6, 52-56

6 Yu, G-L., Bradley, J.D., Attardi, L.D. and Blackburn, E.H. (1990) Nature 344, 126-132

7 Zakian, V.A., Runge, K. and Wang, S-S. (1990) Trends Genet. 6, 12-16

8 Endow, S.A., Henikoff, S. and Soler-Niedziela, L. (1990) Nature 345, 81--83

9 McDonald, H.B. and Goldstein, L.S.B. (1990) Cell 61,991-1000

10 Zhang, P., Knowles, B.A., Goldstein, L.S.B. and Hawley, R.S. (1990) Cell62, 1053-1062

11 Walker, R.A., Salmon, E.D. and Endow, S.A. (1990) Nature 347, 780-782

12, Clarke, L. (1990) Trends Genet. 6, 150-154

13 Kieran, M. et al. (1990) Cell 62, 1007-1018

14 Ghosh, S. et al. (1990) Cell 62, 1019-1029

15 Wu, C-t. and Goldberg, M.L. (1990) Trends Genet. 5, 189--194

16 Hiraoka, Y. et al. (1989) Nature 342, 293-296

T/G Issue on Chromosomal and Nuclear Organization

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TIG DECEMBER 1990 VOL. 6 NO. 12