- Email: [email protected]
A glimpse at chromosomal order
Jews
TIG [3] ~ January 1987
A glimpse at chromosomal order
The flow of information from the DNA of a eukaryotic nucleus is thought to be controlled by trans- and c/s-acting mechanisms and by the availability of the template. Bulk S. M. Gasser and U. K. Laemmli chromatin may serve to hide the information content of the genome (with histones playThe DNA in nuclei and chromosomes is highly organized on several different levels, from winding of the helix around histones to the clustering of hundreds of trig the role of repressors), so kilobase pairs into the banding patterns of metaphase chromosomes. Recent studies that the potential activity of on the organization of DNA into loops have b@un to shed light on the functional a genomic region is due significance of chromosomal organization. to alterations, both local and extended, of bulk chromatin structure. The main changes characteristic of 'active' or 'open' chromatin are its Structure and order in the nucleus generally higher sensitivity, and site-restricted Spatial organization of chromosomes The question of order in the interphase nucleus hypersensitivity, to nucleases like DNase I. If the repertory of active genes for a given cell line results has interested biologists for over 100 years. Rabl from these and other alterations of bulk chromatin proposed in 1885 that interphase chromosomes structure and by DNA modification (methylation), occupy fixed territories in the nucleus4, with their then there must exist systems for the maintenance, centromeres at one pole and the telomeres at the regulation and repair of such altered states and for opposite pole. This is similar to the polarized their high fidelity transmission to daughter cells. The orientation of the chromosomes during telophase. Recent papers support the notion of defined cell has developed a simple copying mechanism for methylation, but little is known about the chromatin chromosomal territories and the Rabl configuration in templating mechanisms that assure the maintenance the interphase nucleus. For example, micrographs of and duplication of variations in chromatin organ- interphase chromosomes in the nuclei of Drosophila embryos during the syncyfial blastoderm stage clearly ization1. The modifying enzymes, pelymerases, repressors establish that the telophase orientation is maintained and activators (called simply tactors in the following) through interphase s. In addition, the nuclei themrequired for chromatin tempiating and gene expres- selves appear to have a fixed relationship toward the sion must search for and bind to their specific target cytoplasm in the Drosophila embryo: the centromere sites rapidly. Studies of prokaryotic systems have poles point to the embryo exterior while the telomeres taught us that difffusion-controlled, bimolecular point to the interior. It is not clear from this work, processes are much too slow to account for the however, whether the centromere-to-telomere axis measured rates of this process. The problem would be is rotationally fixed, so that a given domain on a additionally aggravated in eukaryotic cells by their chromosome would always face the same cytoplasm. vastly increased DNA content. Therefore, two Herr Rabl would certainly have liked these recent facilitating mechanisms have been proposed to observations: 'Ordnung muss sein'. account for the enhanced rate at which specific DNA-protein association proceeds 2'~. According to Chromosomal bands and functional clustering the 'sliding' model, proteins bind non-specifically to of chromatin Genes are not randomly assembled in chromothe DNA and subsequently 'slide' along the DNA in search of the specific target site. In contrast, the somes, but are organized into clusters which can be 'direct transfer' mechanism involves the transient made visible as transverse bands by special staining formation of a complex in which the protein is bound techniques. This gene clustering relates to two to two DNA segments. Upon dissociation of the functional criteria: replication and the potential for complex, the protein remains bound to one or the transcription. other DNA segment and so migrates in jumps along Transcriptionally active genes are known to be the DNA. Both mechanisms lead to a reduction of preferentially digested by pancreatic DNase I, a the search volume within the nucleus by reducing a phenomenon that can also be observed in mitotic three-dimensional target search to one dimension. chromosomes. Visualization of the location of the active We would like to propose an additional mechanism genes by nick translation in situ demonstrates that for accelerating the search for specific binding sites: a these genes are organized into bands (called D bands), structural 'indexing' or compartmentalization of the which in general correspond to the so-called light nucleus. The 'indices' of the nucleus would be physical Giemsa bandse. The Giemsa light bands contain clusters o[ imporiznt sequences (binding sites, clusters of replicons that are activated early in S promoters, enhancers, etc.) which need to be scanned phase, while the dark bands contain clusters activated and acted on by various protein factors. The physical in late S phase. Thus, the transcriptionally competent proximity of such sequences would effectively reduce genes tend to be localized in the early-replicating the search volume for a given factor, functioning bands, while the late-replicating bands tend to be in concert with the direct transfer and/or sliding genetically inert7. mechanisms. A prerequisite for such a mechanism is a The bands observed in the compact metaphase highly structured and ordered nucleus. chromosome correspond to a coarse subdivision that © 1987, Elsevier Sam.ce Pub~shers IkV., Ams~ml~
0168 - 9525/8"//$0 2~)
review
TIC, [ 3 ] - January 1987
is known to be composed of an assembly of sub-bands, visiblein the more extended prophase chromosomes s. It will be of interest to see whether a functional clustering also occurs at this finer level of organization. These observations of transverse rather than longitudinal bands reveal an important notion: in chromosomes the chromatin fiber is organized into clusters of functionallyrelated chrornatin that form transverse disks or gyres. These apparently stack on top of each other to comprise the body of the chromosome. In situ nick translation has also been applied to interphase nuclei and has revealed cell type-specific organization of actively transcribed regions. In the nuclei of transformed mouse lymphocytes, DNase I sensitiveregions were localizedat the periphery of the interphase nucleus, whereas in mature red blood cells they were localized along 'intercfiromatin'channels that appeared to communicate with nuclear pores9.
DNA of each loop, while the ~e.~ffolding proteins organize the bases of loops. It is not known how neighboring loops are arranged with respect to one another, but given the unineme concept of chromosome structure, a helical arrangement progressing along the axis of the chromatid seems likely. It is attractive to think of chromatin loops as the higher-order, structural subunit of chromosomes not as a strictly conserved, regularly repeated structure, like the subunit protein of a virus, but as subunits with certain biochemical and structural features in common, over which modifications can be appfied. Thus one would expect to find different classes of loops defining different compartments, just as modifications of nucleosomes could define different
domains. Intel~actioo of the bases of the loops with the scaffoldingnetwork or with other subnuclear elements would maintain order in the nucleus, facilitating gene expression, chromatin templating, chromosome segregatien and orderly replication. The dynamic changes of chromosomes (condensation, decondensation, Active and heterochromatic chromatin d o ~ a i ~ The general DNase I sensitivity associated with puffing, etc.) could be driven by dynamic changes in active genes is not limited to the transcribed portion of the gene, but extends both 5' and 3' to define an (a) 'active domain'. For example, the ovalbumin domain in o hen oviduct nuclei contains three genes and extends over 100 kbp of DNA, and the active {3-giobindomain extends 6-7 khp 5' and 8 kbp 3' of the gene m'u. Recent evidence suggests that heterochromatin is also organized into domains. Studies of variegating position effects that result from chromosome rearrangements suggested that stretches of heterochromatin are defined by sites at which the heterochromatin is initiated and sites at which it is terminatedTM. All these observations point towards the existence of domains that are subject to coordinate structural changes. If this notion is correct, mechanisms must exist that propagate the structural change along the domain, and sites must exist that define the borders of the domain. The relationship between these functional domains and the structural organization of the chromosome is discussed below• '
Chromosomal subunits: nucleosomes and loops In the basic chromatin fiber, packaging of the DNA molecule into nucleosome suhunits compresses its length roughly 30- to 40-fold. It appears, however, that nucleosomes alone do not determine the higherorder folding of the chromatin fiber in chromosomes, where the packaging ratio is roughly 1: 10000. Various models for this higher-order folding have been proposed; of these the loop model has substantial experimental support from electron microscopy, sedimentation and nuclease digestion studies (reviewed in Ref. 13). In this model, the fiber is first folded into loops consisting of about 30-100 kbp of DNA, and the loops are fastened at their bases by non-histone proteins14. In the condensed chromosome, neighboring loops are somehow held together by protein-protein or protein-DNA interactions which form an internal network or scaffolding along the chromosomal axis. The role of the histones in this model is to package the
k
m
Fig. L lmmunolocalization of t@oisomerase H in metaphase chromosomes. (a) The antibody directed against topoisomerase l l (Scl protein, 170 kDa) identifies an axial element that extends along the whole length of the chromatid, passing th~a h the kinetachore elements. A peroxidase.czoupled ~eco ry antibody teas used for the staining reactionI with both zmmune (a) and preimmune (b) sera. The kinetochore e l ~ n t s , lmt not the axial stm'ning, are seen in the control (b). Some of these micrographs provide evidence of the substructural organizatwn of the scaffold, which appears to consist of an assembly of foal, forming in places a zig-zag or coiled arrangement. Scale bar = I ttm.
Jews the scaffold that would drag along the associated chromatin loops, rather than by a synchronized modification of all the nucleosomes within a given loop. In the following we briefly review current concepts of the structure and function of the chromatin loop.
The major scaffold protein is topoisomerase II The microscopic observation of looped structures in metaphase chromosomes and interphase nuclei after the extraction of histones suggested that some of the proteins left in the extracted chromosomes or nuclei function as fasteners to constrain the DNA into looped domains. These residual structures were variously called the chromosomal scaffold or nuclear matrix or scaffold. Since harsh procedures were initially used to remove the histones, it was difficult to rule out the possibility that the observed organization was artefactual. Recent data, however, have provided strong evidence that the scaffold structure of chromosomes and nuclei is not artefactual and is of biological importance. Attempts to identify the minimal set of proteins necessary to restrain DNA loops in metaphase chromosomes led to the identification of two proteins, Sol and Sc2 (170 and 135 kDa) xs. The Sol protein is the most abundant non-histone protein found in metaphase chromosomes, it binds DNA and is present in roughly three copies per average DNA loop in human metaphase chromosomes. This number is consistent with the postulated role of these proteins as 'loop-fasteners'. With the help of a specific antiserum raised against Sol, this protein has been shown to be identical to topoisomerase II le'lT. Topoisomerase II has also been shown to be a major componeat of the residual nuclear matrix of Drosophila embryonic cellsis. Immunulocalization of topoisomerase II both by immunofluorescence and electron microscopy allowed identification of the scaffold structure directly in 'native', gently expanded chromosomes. The immunopositive reaction is found along a central, axial region, extending through the kinetochore along the entire length of the chromatid. In histone-depleted chromosomes, where the scaffold is further expanded, the scaffold appears to be an assembly of loci, that in places forms a zig-zag arrangement ~ (see Fig. 1). These foci are more closely packed in the compact, histone-containing chromosome, and some micrographs suggest a hefical progression along the chromatid. It is tempting to propose that each focus represents a scaffold 'subunit', consisting of an assembly of bases of loops. These data establish the existence of the scaffold in unextracted metaphase chromosomes, confirm that the protein Sol is indeed a component of the scaffold, and more importantly, suggest a structural as well as an enzymatic role for topnisomerase 1I. The nuclear matrix of interphase nuclei is obtained under conditions similar to that for the metaphase scaffold, but is much more complex in morphology and composition. It is composed of the peripheral lamina, an ill-defined internal network and a residual nucleolus19. Immunolocalization of topoisomerase II in nuclei reveals a diffuse general staining of the interior lumen excluding the nucleolusis. Not surprisingly, in interphase nuclei an axial, localized staining is not observed.
TIG[3]mJanm~y1987
Specific scaffold-associated DNa~regions As a test for the loop model one mighthope to find specific regions, spaced along the DNA, at which the scaffold interaction occurs. Such specific scaffoldassociated regions (SAR) have been identified with the help of a novel extraction procedure that uses lithium 3', 5-diiodosalicylate (LIS). At low concentration.,; and in physiological salt buffers, this compound extracts histones and other proteins under conditions apparently 'mild' enough to preserve a specific scaffoldDNA interaction. The LIS is removed by repeated washing and the extracted nuclei are digested to completion with various restriction enzymes. A restriction fragment that contains a SAR cosediments with the nuclear scaffold. In the Drosophila system we have mapped 18 SARs near a variety of polymerase IItranscribed genes z°-zs, extending over 400 kbp of DNA, 320 kbp of which is within a chromosomal 'walk' around the rosy locus (see Fig. 3). A number of experiments have been carried out to determine the biochemical nature and the functional significance of scaffuld-DNA binding. At present, our basic observations are as follows. (1) SARs can be mapped to fragments ranging in size from 0.6 to I kbp, containing multiple sites of scaffold-DNA interaction. (2) In Drosophila, SARs are found in nontranscribed regions. For the mouse [clight chain gene, however, a SAR was identified adjacent to the enhancer sequence in a transcribed regionzs. (3) The distance between two adjacent SARs varies between 4.5 and 112 kbp. (4) One or several genes can occur between two SARs. (5) In Drosophila, several enhancer-like elements for developmentally regulated genes cohabit with SARs. (6) No changes have been observed in scaffold attachment upon the induction of transcription. (7) The SAR interactions are similar in nuclei derived from developmentally different cells. These observations are discussed in more detail in the following sections. 5AR s contain clusters of the topoisomeraseII consensus sequence and two additional sequence motifs The best characterized SAR is found (on a 657 bp fragment) in the non-transcribed spacer between the H1 and H3 genes. This attachment is observed in all the tandemly organized histone-gene repeats, defining small 5 kbp loops (Fig. 2). Exanuclease lII digestion studies have identified two protein-binding domains within this SAR, each covering about 200 bp, as depicted in Fig. 2. Studies of other SAR fragments also reveal multiple binding sites within rather large regions (up to 1.1 kbp). Each individual binding domain is able to mediate scaffold association, albeit with a somewhat reduced affinity. The presence of topoisomerase 11in the metaphase scaffold prompted us to screen the available SAR sequences for the Dros@hila topoisomerase lI consensus sequence 2s. All SARs tested contain a strikingly large number (8-17 per fragment) of sequences related to the 15 bp topoisomerase II cleavage site (topo H box) [GTN(A/T)A(T/C)ATTNATNN(G/A)] zl'zs'z4. Although this is a weak and loosely defined consensus, two results suggest that the clustering of such sequences within the SAR fragments is of significance. Firstly, Udvardy et aL 2~ have shown that the SARs of the histone-gene cluster and of the hspT0 heat-shock genes are major targets
reviews 2
TIG [ 3 1 - - J a n u a r y 1987
H2a b
~ _ ~ _
H3
m ~ H i
~,
_ /'" ~c,RV .-
~
•
nnmmm ,
_
\/
l
.2a b
~
] [AATAAA(T/C)AAA](Fig. 2). The pattern of topo I!, ] ] T and A boxes of four different SARs is shown in Figs ~ 2 and 4. The clustering of topo II boxes in the various SAlts is impressive, but does not appear to follow a simplepattern" N°te' h°wever, that theTb°xis °lien ~ found downstream from the A box; this run of thymidine residues may be responsible for bends and ecoR: kinks in the DNA or may discourage nucleosome formation.
~
~*R~
~ ,
qlo O O g l
I~lN
SARs are often close to promoters and cohabit with upstream regulatory sequences
a 0__.._~9 L_a____z~x
For three developmentally regulated genes of
Fig. 2. Repeat of histone gene,s: one repeat, one loop. T~o repeats of the 5 kbp DNA fragment that contains all five Drosophila histone genes are shown in the upper part of the figure. The 65',7bp scaffold-o~sociated region (SAR) occurs in the non.trans cribedspacer between theH I and the l13 ger~s and is indicated by a bar containing a hook. Roughly 100 tandem histone gene repeats are present in the &rnome,forming a series of snlall loops. Sequence raohys common to a number of SARs are indicated in the enlarged map of the SAR. V represents sequences with 707b homology to the tOoisomerase 11 consensus sequence~, in either the Watson (lop) ar Crick (bottom) strand. • imticatea the lO-bp A box, and • the lO-bp T box described in the text~. Two 200-bp domains within the SAR (here enconOassed by dotted lines) were resistent to exonuclease III d~gestion in intact scaffolds, indicah'ng the presence of pro~n-DNA complexe~ z.
for topoisomersse cleavage in vitro. Secondly, the DNA regions not bound to the scaffoldgenerally do not contain such clusters of topo II boxes. However, the occurrence of the consensus alone does not appear to be sufficient to create a SAR, since in the Adh gene region we found a fragment with several topo II boxes which is not scaffold-bound2s. In vivo localization of DNA topoisomerase Il cleavage sites in the hspT0 genes has revealed multiple specific cleavage sites both at the 3' and 5' ends of the genes, but none in the 5' SAR region, unless the cells were heat-shocked zT. Thus, the presence of the topo II boxes in the SAR may represent potential sites of action for a topoisomerase H which is regulated in vivo. The SARs that have been analysed contain two additional 10 bp sequence motifs, the T box [TT(AFF)T(T/A)TT(T/A)TT] and the A box
12osy
Drosophila (Adh, Sgs-4 and ftz) the SARs 5' of the
genes have been found to cohabit with regulatory sequenceszs-~ up to 4.5 kbp upstream from the start of transcription (see Fig. 4). Remarkably, for theAdh locus, from which two transcripts are made, two upstream/enhancer-like regulatory regions were identified as well as two 5' scaffold-attached regions. The sequences required for t~ssne-specificexpression of Adh and ~z, on the other hand, are not scaffoldbound, nor are the actual coding sequences for any of the genes studied. For each of these three highly expressed, developmentally regulated loci, SARs are also found 3' of the transcription units. These could interact with the 5' SARs to form smallloops ranging in size from 4.5 to 13 kbp, each containing one gene. The term cohabitation describes our finding that the restriction enzyme fragments defining upstream/ enhancer-like elements of these genes also contain SARs. Both types of mapping data do not exclude the possibility that the two DNA elements might still be separable by the appropriate experimentation. In the case of the mouse immunoglobulin g gene, the matrixbinding site is adjacent to and separable from the enhancer sequence24. Yet for the Drosophila Sgs-4 gene, this cohabitation appears quite intimate; a 710bp region immediately upstream from the transcription start site contains the essential regulatory sequences, the SAR, and the DNase I hypersensitive sites as~mciated with gene activity~. Such a close functional link between the SAR and upstream regulatory element is not observed in the case of the major heat-shock gene hspT0. This SAR is upstream from the DNase I hypersensitive sites associated with active transcription, and is also upstream from control elements that are necessary
l
__ ~
\1 :>78 kb
/ ~ kb
...
/rn32
\1,,.1¢ "°''°'' 112 kb
26 kb
> 62 kb
Fig. 3. Loops and genes in 320 kbp ofDrosophilaDNA. The lo~ organization is shown for a 320.kbp region ~ o u . n d i ~ the~sy and Acelocifrom DrosophilaXc cell~. The va~nLs Ee~elicloci are in dicated by lines and t~eposltions of the SAR soy nooReaoars. The loop sizes in this r e g ~ are large, as indicated in the f ~ r e , and the various transcripts are o/low abundance, an obsersaffon which sw~gests an inverse correlation between the potential level of transcription and loop size.
TIG [3] --January 1987
iews
What is the importance of the loop size and/or the proximity of the 5' SAR? The data available at present fit best with the notion that in Drosophila the highly expressed genes are found in small loops of 4-13 kbp while genes with less abundant transcriptional activity are found in much larger loops of 50 kbp or more. Examples of the former class would be hspT0, actin 5C, the hlstone cluster, Adh, Sgs-4 andflz, and for the latter class, all the genes localized within the 320 kbp 'walk' around the rosy locus (Fig. 3). These observations suggest an inverse correlation between the potential level of transcription and loop size.
(a)
,
E
,
4.7kb
\\ V
II
A~
,,'-
(b)
~ "T"
~
= s
/
II.lkb V
~.-~ /~/
/'~
¢-'='
/~/
\ \
= [ ~/
\2 .V
~ /
~../
Are SAR positions and loop structure tissue-opecifw?
One can imagine that certain attachment sites are preferentially lost or created during cell differentiation, perhaps mediated by specific transcription factors, allowing the modulation of nuclear domains as required by the cell's specific pattern of gene exprescion. It follows that some SARs, those near housekeeping genes, would remain constant in all proliferating cells. In both the mouse and Drosophila systems, few significant differences in loop organization have been seen among different cell types ~'24. This may be due, however, to the character of the assay for these ~sites. Since SARs are detected in the absence of histories, any fragment with the potential to bind might be detected as scaffold-bound. Thus, it is possible that some fragments identified as SAR in our assay in vitro may actually have their scaffold-binding sites blocked by histones in the intact nucleus~. Some SARs co-map with boundaries of' adive" domains
i ,s
"..
Fig. 4. Cohabitation of SARs with upstream~enhancerelements. Loop structure for three developmentallyregulatedgenes: (a) the glue grotein gene, Sgs-429; (b) the homeobox.containing gene fushi tarazu(ftz)ZS; and (c) the alcohol dehydrogenasegene (Adh)~°. SARs arefound both 5' and3' from the codingsequences(open boxes)as indicated by the hooked bars. As in the case of the histone gene cluster, the loop sizes of these temporally and highly expressedgenes are small, at maximum 11 k~. In the case ofAdh, which has ~o gromoters and two transcriNsfrom the same coding unit, there are two upstream and ~o downstream SARs. For all three genes, the 5' SAles cohabit with enhancer-like regulatory elementsz~. Loop size measured from center to center of two adjacent SARs is given below the correepondmg loops. The positions of several sequence motifs are shown in the enlarged maps of the SARs for Sgs-4 and Adh. V representsthe tepoisomeraseH consensus, • theA box and [] the T box as discussed in F~g. 2. For orientation, a selection of relevant restriction sites is given.
Non-transcribed regions of chromatin at the borders of actively transcribed regions are often associated with highly ordered, or 'static' nucleosome organization, whereas a more 'blurred' organization of nucleosomes is seen in the nearby transcribed regions. In two cases, SARs co-map with such regions of static nucleosome organization bordering actively transcribed domains. One is the non-transcribed region of the H1-H3 spacer, in which the two proteinbinding elements of the SAR coincide quite well with nucleosome-sized particles detected by micrococcal nuclease digestion32. In addition, the SARs located 5 of the hsp?0 genes (at both loci 87C1 and 87A7) fall into regions of static, 'phased' nucleosomes. Like the SAR interaction, the static pattern is maintained during transcriptional activation of these genes. These data suggest that some SARs may be involved in defining boundaries. SARs may define a positionaUy-independent functioning unit
Do SARs define a region that will function and apparently sufficient for its complete regulation~1. independently of its position in the genome? The The observation that the SAR of the hsp?O gene lacks available data from P element transformation using the the usual series of upstream A boxes suggests to us ftz gene are consistent with this notion. Constructs that different SAR types may correlate with differently containing either the ftz gene and both its 5' and 3' regulated genes. For example, a developmentally SARs, or just the gene and 5' SAR, were used; when regulated gene may have a 'regulated' SAR fragment both SARs were present in the transforming vector containing both topo II boxes and A boxes, while a none of the eight ftz + transformants showed a position housekeeping gene may have a 'constitutive' SAR effect. Among the seven transformants obtained with without A boxes. If A boxes are diagnostic of the construct lacking the 3' SAR, four showed low 'regulated' SARs, then one would predict that the levels of expression, presumably due to the site of histone SAR is involved in transcriptional control. insertion (position effect).
TIG [3] - - J m m m y 1987
Another example concerns the expression of the Sgs-4 glue protein gene. Individual transformed flies
derived from constructs which included the 5' regulatory/SARregion, but not the 3' SAR, expressed the introduced Sgs-4 gene with great variability, and no puffing was observed at the various sites of insertion29. Both the Sgs-4 andflz genes are highly transcribed genes with closelypositionedSARs. One might expect
~g. 5. Scheme of the loop organizalion in a Mstone-depleted nucleus, The genomic DNA is organized into loops varying in size between 5 a,d 100 hbp. The loops are anchored to the residual ha!ear scaffold at highly specific DNA regions called SAR (scaffold associated re~n). SARs occur in Drosophila cells in non-transcribed regfims, e~tend over about 1 kbp of DNA, and contain usually eight or more DNA elements related to the topoisomerose II cleavage consensus, as well as hoo additio,al sequence elements. Loops can have one or several transcription units, indicated by arrows. Available data are consistent with the notion t i ~ kigidy aclive or polontially h~kly active genes are organ~d into small loops of about I0 kbp, while genes with less transcriptional aclivity are arranged in muck larger loops containing several lranscription units. It has not been established whether the SARs interact with the peripheral lamina slruclure andlor the interna! m~ork of nuclear scaffolds. While the peripheral lamina structure is known to have a simple polypeptidecomposition, the structurally and biochemically ill-defined internal network is composedof a very complexpattem of pt~ins. TopoisomeraseII is one of the components of the internal network. The loop organization mediated by the SARs is experimentally indisffnguishable in metaphase chromosomes. The protein composition of the metaphase scaffold, however, is considerably less complex than that of the nuclear equivalent. TopoisomeraseH is in this case the major protein of the metaphusescaffold. About three copies of topoisomerase H are found per average size loop both in metaphase and nuclear scaffolds, an observation consistent with the possible role of this protein as a loop-fastener.
that transformation with genes that are tran~cr~ibeVdi a t ~ ~ low levels and have no nearby SAR would be less susceptible to position effects. Indeed, this is the case for the weakly expressed rosy gene~.
Nuclearorganization" conclusion and speculation We have proposed that the genome is organized into loops that have both a structural and functional role, and that SAR sequences define the boundaries of these loops. Figure 5 shows an 'artist's impression' depicting some features of our current ideas about how these loops are organized in a histone-depleted nucleus. Loops may range in size from 5 to 112 kbp, and we find a loose, inverse relationship between loop size and the level of transcription of genes contained within the loop. Topoisomerase lI appears to be a pivotal protein in chromosomal structure and nuclear order. It is present in amounts sufficient to serve as 'loopfastener', and has been shown to be a major component of the chromosomal scaffold. The activity of this enzyme is likely to be highly regulated and may be involved in the release of stress during transcription or replication, as well as in the decatenation of replicated DNA (e.g. Ref. 34). If topoisemerase 11exerts its action at SAR ~quences at the bases of loops, it would be strategically located to control long-range order in chromatin domains during these processes. SARs may also serve as preferred sites at which DNA replication begins or ends. In Drosoph//a, SARs tend to be found close to the promoter elements, and cohabit with the upstream/ enhancer-like elements of three developmentally regulated genes (Adk, Sgs-4 and ftz), The scaffold interaction may serve to bring distant regulatory sequences close together to create functional complexes for the regulation of transcription, in analogy to the mini-loops discussed by Ptashne3s. Seen on a larger scale, the positioning of SARs may serve to 'index' those sequences which need to be acted upon by factors or polymerases, to form a subnuclear compartment. Such a compartment, rich in specific DNA binding sites, would facilitate the formationof the appropriate DNA-protein complexes required for the control of transcription, replication and for chromosome templating, by reducing a factor's search volume within the nucleus. The close pro~nity of many regulatory sequences would have a Circean effect, effectively caging DNA-binding proteins within this comparUment. Regulatory proteins could nonetheless traverse the compartment by the direct transfer mechanism, physically scanning the DNA exposed at the bases of loops, Within these compartments one might expect to find both high and weaker affinity binding sites for factors, which could be important for chromatin templating. If the assembly of active chromatin structure results from the competitive binding of either factors or histones to newly replicated DNA, then the existence of factor-rich compartments could provide a kinetic advantage for the binding of factors over histones, during replication. Thus, high fidelity transmission of the epigenetic chromatin structure to the daughter cell would he largely a consequence of the compartmentalization of the nucleus.
TIG [3] - - J a n u a r y 1987
iews The channels defined by the bases of loops of one chromosome could be linked to the periphery of the nucleus via the nuclear pores, which may direct and kinetically regubte influx of proteins to the channels. A highly organized nucleus with a fixed orientation toward the cytoplasm, as observed in the Drosophila embryo, might permit the relay of spatial information from a structured cytoplasm into the nucleus, to affect gene expression. Finally, if one assumes that the flux of proteins into the nuclear channels is kinetically dependent on nuclear pores, then dissociation of the nuclear lamina/pore complex during mitosis could lead to an automatic loss of these proteins from chromatin. In this light one would not need to propose an additional mechanism for chasing nuclear proteins into the cytoplasm din-rag metaphase, Our results suggest that the higher-order organization of the nucleus is determined by bases of DNA loops and the proteins that bind to them. If these sites change with the differentiation of a cell and specialization of its pattern of expression, then one would expect that nuclei from different cell types would have different networks and channels. Such a cell-specific three-dimensional organization ef nuclei was also discussed in the 'gating' hypothesis of BlobelaS. Although these predictions have not yet been shown to be correct, with the means to dissect the higher order chromatin cor~ormation at hand, we may soon get more than a glimpse at chromosomal order.
I0 Stadler,J. et eL (1980) Cell 19, 973-980 11 Ciejek, E. M., Tsai, M-J. and O'Malley, B. W. (1983)Nab~re 306, 607-609 12 Tarter, D. K., Hobbs, Ch. and Jones, M. (1984) Cell 97,
869-878 13 Paulson, J. L. in Chromosomes and Chromatin Structu~
(Adolph, K. W., ed.), CRC Press (in press)
14 Laemmh,U. K. etal. (1978)ColdSpringHarborSym]).QuanL Biol. 42, 109-I18 15 Lewis, C. D. and Laemmli,U. K. (1982) Cell 29, 171-181 16 Earnshaw,W, C. and Heck, M. M. S. (1985)]. Cell. Biol. 100,
1716-1725
17 Gasser, S. M. etal. (1986)]. Moi. Biol. 188, 613-629 18 Berrios, M., Osheroff, N. and Fmher, P. (1985) Proc. Natl. Acad. Sc=. USA 82, 4142-4146
19 Lewis, C. D., Lebkowski, J. D., Daly, A. and Laemmli, U. (1984) ]. Cell SoL 1, 103-122 20 Mirkovltch,J., Mirault, M. E. and Laemmli,U. K. (1984) Cell 39, 223-232 21 Gasser,S. M. andLaenmdi,U. K. (1986)EMBO]. 5, 511-518 22 Mirkovitch,J., Spierer, P. and Laermdli,U. K. (1986)]. Mol. Bwl 190, 255-258 23 Gasser,S. and Laemmli, U. K. (1986) Cell 46, 521-530 24 Cockerill, P. N. and Garrard, W. 1". (1986) Cell 44, 273-282 25 Sander, M. and Tao-Shib, H. (1985) Nucldc Acids Res. 13, 1057-1071 26 Udvardy,A., Schedl, P., Sander,M. and Tao-Shih, H. (1985) Cell 40, 933-94] 27 Rowe, T. C., Wang, J. and Lin, L. (1986) Mol. CellBioL 6, 985-992 28 Hiroml, Y., Kuroiwa, A. and Gehztag, W. J. (1985) Cell 43, 603-613 29 McNabb, S. L. and Beckendorf, S. K. (1986) EMBO ]. 5, 2331-2340 30 Posakony,J. W., Fischer, J. A., Corbin, V. and Maniatis, 1". (1985) m Current Commun~a~o=, pp. 194-200, Cold Spring [-[arbor Laboratory 31 Dudler, R. and Travers, A. A. (1984) Cell 38, 391-398 References 32 Worcel, A. et al. (1983)NucleieAddsRes. 11, 421-439 1 Weintraub,H. (1985) Cell 42, 705-711 2 Berg, O. G., Winter,R. B. andvonHippel,P. H. (1982) Trends 33 Spradlin¢ A, C. and Rubirk G. M. (1983) Cell 34, 47-57 34 Uemura, T. and Yanagida,M. (1984)EMBO]. 3, 1737-1744 Biochem. Sci. 7, 52-55 3 Fried, M. G. and Crothers, M. (1984) ]. Mol. Biol. 172, 35 Ptashne,M. (1986)Nat=re 322, 697-701 36 Binbel, G. (1985)Proc. Natl, Aead. Sd. USA 82, 8527-8529 263--282 4 Rabl, C. (1885)MorphoL]ahrb. 10, 214--330 5 Foe, V. E. and Alberts,B. (1985)]. Cell. Biol. 100, 1623--1636 S. M. Gasser and U. K. Laemmli are at the D~artr~nts of 6 Kerem, B-S. et al. (1984) Cell 38, 493-499 7 Goldman,M. A., Holmquist,G. P., Gray, M. C., Caston, L. A. Molecular Biolo£y and Biochemistry, Universi(y o/Geneva, 1211 Geneva 4, Switzerland; S. M. Gasser is presently at the and Nag, A. (1985) Sdenee 224, 686-692 8 Yunis, J-J. (1976) Science 191, 1268--1270 Swiss Institute/or Experimental Cancer Resmrch (ISREC). 9 Hutchison, N. and Weirztraub,H. (1985) Cell 43, 471-482 CH 1066 EpalingeslLausanne, Switzerland.
Coming soon, in Trends in Genetics A giant locus for the Duchenne and Backer muscular dystrophygene by Anthony P, Monaco and Louis M. Kunkel
The gene responsible for Duchenne and Becker muscular dystrophy has been assigned to band Xp21 of the human X chromosome, and the nsolation of DNA segments from this region has provided probes useful for prenatal dnagnos=s and carrier detection. It has also led to the identification and partial cloning of a large transcrnpt that is found in skeletal muscle and potentially spans over 1.0 million basepairs of DNA. A large size for the muscular dystrophy gene locus is consistent with the observed high mutatnon frequency, the heterogeneity of the deletion breakpoints that are associated with the disease and the high recombination frequency of DNA markers that Ine within the gene itself.
Pool your useful hints throughTechnical 'tips Technical Tips (see p. 2) is a place where readers can exchange information about new experimental techniques. To make this section really useful to experimental geneticists and developmental biologists we need the active participation of our readers. If you have information about methods developed in your lab or elsewhere why not share it with your colleagues through the Technical Tips section of Trend= In @mmlle=? Each month Technical Tips draws attention to such methods by presenting very brief art¢les. These do not attempt to provide all the information required to use the method but rather a clear outline of the method's claimed advantagesand present and potential applications; readers can then look to the reference for complete details. The only exception to this general policy conums descriptions of unpublished methods where more precise experimental detads should be provided, Please send all information to: Trends in (~meticg, Elsevier Publications Cambridge, 68 Hills Road, Cambridge CB2 1LA, UK, or call (0223) 315961.
TIG [3] ~ January 1987
A glimpse at chromosomal order
The flow of information from the DNA of a eukaryotic nucleus is thought to be controlled by trans- and c/s-acting mechanisms and by the availability of the template. Bulk S. M. Gasser and U. K. Laemmli chromatin may serve to hide the information content of the genome (with histones playThe DNA in nuclei and chromosomes is highly organized on several different levels, from winding of the helix around histones to the clustering of hundreds of trig the role of repressors), so kilobase pairs into the banding patterns of metaphase chromosomes. Recent studies that the potential activity of on the organization of DNA into loops have b@un to shed light on the functional a genomic region is due significance of chromosomal organization. to alterations, both local and extended, of bulk chromatin structure. The main changes characteristic of 'active' or 'open' chromatin are its Structure and order in the nucleus generally higher sensitivity, and site-restricted Spatial organization of chromosomes The question of order in the interphase nucleus hypersensitivity, to nucleases like DNase I. If the repertory of active genes for a given cell line results has interested biologists for over 100 years. Rabl from these and other alterations of bulk chromatin proposed in 1885 that interphase chromosomes structure and by DNA modification (methylation), occupy fixed territories in the nucleus4, with their then there must exist systems for the maintenance, centromeres at one pole and the telomeres at the regulation and repair of such altered states and for opposite pole. This is similar to the polarized their high fidelity transmission to daughter cells. The orientation of the chromosomes during telophase. Recent papers support the notion of defined cell has developed a simple copying mechanism for methylation, but little is known about the chromatin chromosomal territories and the Rabl configuration in templating mechanisms that assure the maintenance the interphase nucleus. For example, micrographs of and duplication of variations in chromatin organ- interphase chromosomes in the nuclei of Drosophila embryos during the syncyfial blastoderm stage clearly ization1. The modifying enzymes, pelymerases, repressors establish that the telophase orientation is maintained and activators (called simply tactors in the following) through interphase s. In addition, the nuclei themrequired for chromatin tempiating and gene expres- selves appear to have a fixed relationship toward the sion must search for and bind to their specific target cytoplasm in the Drosophila embryo: the centromere sites rapidly. Studies of prokaryotic systems have poles point to the embryo exterior while the telomeres taught us that difffusion-controlled, bimolecular point to the interior. It is not clear from this work, processes are much too slow to account for the however, whether the centromere-to-telomere axis measured rates of this process. The problem would be is rotationally fixed, so that a given domain on a additionally aggravated in eukaryotic cells by their chromosome would always face the same cytoplasm. vastly increased DNA content. Therefore, two Herr Rabl would certainly have liked these recent facilitating mechanisms have been proposed to observations: 'Ordnung muss sein'. account for the enhanced rate at which specific DNA-protein association proceeds 2'~. According to Chromosomal bands and functional clustering the 'sliding' model, proteins bind non-specifically to of chromatin Genes are not randomly assembled in chromothe DNA and subsequently 'slide' along the DNA in search of the specific target site. In contrast, the somes, but are organized into clusters which can be 'direct transfer' mechanism involves the transient made visible as transverse bands by special staining formation of a complex in which the protein is bound techniques. This gene clustering relates to two to two DNA segments. Upon dissociation of the functional criteria: replication and the potential for complex, the protein remains bound to one or the transcription. other DNA segment and so migrates in jumps along Transcriptionally active genes are known to be the DNA. Both mechanisms lead to a reduction of preferentially digested by pancreatic DNase I, a the search volume within the nucleus by reducing a phenomenon that can also be observed in mitotic three-dimensional target search to one dimension. chromosomes. Visualization of the location of the active We would like to propose an additional mechanism genes by nick translation in situ demonstrates that for accelerating the search for specific binding sites: a these genes are organized into bands (called D bands), structural 'indexing' or compartmentalization of the which in general correspond to the so-called light nucleus. The 'indices' of the nucleus would be physical Giemsa bandse. The Giemsa light bands contain clusters o[ imporiznt sequences (binding sites, clusters of replicons that are activated early in S promoters, enhancers, etc.) which need to be scanned phase, while the dark bands contain clusters activated and acted on by various protein factors. The physical in late S phase. Thus, the transcriptionally competent proximity of such sequences would effectively reduce genes tend to be localized in the early-replicating the search volume for a given factor, functioning bands, while the late-replicating bands tend to be in concert with the direct transfer and/or sliding genetically inert7. mechanisms. A prerequisite for such a mechanism is a The bands observed in the compact metaphase highly structured and ordered nucleus. chromosome correspond to a coarse subdivision that © 1987, Elsevier Sam.ce Pub~shers IkV., Ams~ml~
0168 - 9525/8"//$0 2~)
review
TIC, [ 3 ] - January 1987
is known to be composed of an assembly of sub-bands, visiblein the more extended prophase chromosomes s. It will be of interest to see whether a functional clustering also occurs at this finer level of organization. These observations of transverse rather than longitudinal bands reveal an important notion: in chromosomes the chromatin fiber is organized into clusters of functionallyrelated chrornatin that form transverse disks or gyres. These apparently stack on top of each other to comprise the body of the chromosome. In situ nick translation has also been applied to interphase nuclei and has revealed cell type-specific organization of actively transcribed regions. In the nuclei of transformed mouse lymphocytes, DNase I sensitiveregions were localizedat the periphery of the interphase nucleus, whereas in mature red blood cells they were localized along 'intercfiromatin'channels that appeared to communicate with nuclear pores9.
DNA of each loop, while the ~e.~ffolding proteins organize the bases of loops. It is not known how neighboring loops are arranged with respect to one another, but given the unineme concept of chromosome structure, a helical arrangement progressing along the axis of the chromatid seems likely. It is attractive to think of chromatin loops as the higher-order, structural subunit of chromosomes not as a strictly conserved, regularly repeated structure, like the subunit protein of a virus, but as subunits with certain biochemical and structural features in common, over which modifications can be appfied. Thus one would expect to find different classes of loops defining different compartments, just as modifications of nucleosomes could define different
domains. Intel~actioo of the bases of the loops with the scaffoldingnetwork or with other subnuclear elements would maintain order in the nucleus, facilitating gene expression, chromatin templating, chromosome segregatien and orderly replication. The dynamic changes of chromosomes (condensation, decondensation, Active and heterochromatic chromatin d o ~ a i ~ The general DNase I sensitivity associated with puffing, etc.) could be driven by dynamic changes in active genes is not limited to the transcribed portion of the gene, but extends both 5' and 3' to define an (a) 'active domain'. For example, the ovalbumin domain in o hen oviduct nuclei contains three genes and extends over 100 kbp of DNA, and the active {3-giobindomain extends 6-7 khp 5' and 8 kbp 3' of the gene m'u. Recent evidence suggests that heterochromatin is also organized into domains. Studies of variegating position effects that result from chromosome rearrangements suggested that stretches of heterochromatin are defined by sites at which the heterochromatin is initiated and sites at which it is terminatedTM. All these observations point towards the existence of domains that are subject to coordinate structural changes. If this notion is correct, mechanisms must exist that propagate the structural change along the domain, and sites must exist that define the borders of the domain. The relationship between these functional domains and the structural organization of the chromosome is discussed below• '
Chromosomal subunits: nucleosomes and loops In the basic chromatin fiber, packaging of the DNA molecule into nucleosome suhunits compresses its length roughly 30- to 40-fold. It appears, however, that nucleosomes alone do not determine the higherorder folding of the chromatin fiber in chromosomes, where the packaging ratio is roughly 1: 10000. Various models for this higher-order folding have been proposed; of these the loop model has substantial experimental support from electron microscopy, sedimentation and nuclease digestion studies (reviewed in Ref. 13). In this model, the fiber is first folded into loops consisting of about 30-100 kbp of DNA, and the loops are fastened at their bases by non-histone proteins14. In the condensed chromosome, neighboring loops are somehow held together by protein-protein or protein-DNA interactions which form an internal network or scaffolding along the chromosomal axis. The role of the histones in this model is to package the
k
m
Fig. L lmmunolocalization of t@oisomerase H in metaphase chromosomes. (a) The antibody directed against topoisomerase l l (Scl protein, 170 kDa) identifies an axial element that extends along the whole length of the chromatid, passing th~a h the kinetachore elements. A peroxidase.czoupled ~eco ry antibody teas used for the staining reactionI with both zmmune (a) and preimmune (b) sera. The kinetochore e l ~ n t s , lmt not the axial stm'ning, are seen in the control (b). Some of these micrographs provide evidence of the substructural organizatwn of the scaffold, which appears to consist of an assembly of foal, forming in places a zig-zag or coiled arrangement. Scale bar = I ttm.
Jews the scaffold that would drag along the associated chromatin loops, rather than by a synchronized modification of all the nucleosomes within a given loop. In the following we briefly review current concepts of the structure and function of the chromatin loop.
The major scaffold protein is topoisomerase II The microscopic observation of looped structures in metaphase chromosomes and interphase nuclei after the extraction of histones suggested that some of the proteins left in the extracted chromosomes or nuclei function as fasteners to constrain the DNA into looped domains. These residual structures were variously called the chromosomal scaffold or nuclear matrix or scaffold. Since harsh procedures were initially used to remove the histones, it was difficult to rule out the possibility that the observed organization was artefactual. Recent data, however, have provided strong evidence that the scaffold structure of chromosomes and nuclei is not artefactual and is of biological importance. Attempts to identify the minimal set of proteins necessary to restrain DNA loops in metaphase chromosomes led to the identification of two proteins, Sol and Sc2 (170 and 135 kDa) xs. The Sol protein is the most abundant non-histone protein found in metaphase chromosomes, it binds DNA and is present in roughly three copies per average DNA loop in human metaphase chromosomes. This number is consistent with the postulated role of these proteins as 'loop-fasteners'. With the help of a specific antiserum raised against Sol, this protein has been shown to be identical to topoisomerase II le'lT. Topoisomerase II has also been shown to be a major componeat of the residual nuclear matrix of Drosophila embryonic cellsis. Immunulocalization of topoisomerase II both by immunofluorescence and electron microscopy allowed identification of the scaffold structure directly in 'native', gently expanded chromosomes. The immunopositive reaction is found along a central, axial region, extending through the kinetochore along the entire length of the chromatid. In histone-depleted chromosomes, where the scaffold is further expanded, the scaffold appears to be an assembly of loci, that in places forms a zig-zag arrangement ~ (see Fig. 1). These foci are more closely packed in the compact, histone-containing chromosome, and some micrographs suggest a hefical progression along the chromatid. It is tempting to propose that each focus represents a scaffold 'subunit', consisting of an assembly of bases of loops. These data establish the existence of the scaffold in unextracted metaphase chromosomes, confirm that the protein Sol is indeed a component of the scaffold, and more importantly, suggest a structural as well as an enzymatic role for topnisomerase 1I. The nuclear matrix of interphase nuclei is obtained under conditions similar to that for the metaphase scaffold, but is much more complex in morphology and composition. It is composed of the peripheral lamina, an ill-defined internal network and a residual nucleolus19. Immunolocalization of topoisomerase II in nuclei reveals a diffuse general staining of the interior lumen excluding the nucleolusis. Not surprisingly, in interphase nuclei an axial, localized staining is not observed.
TIG[3]mJanm~y1987
Specific scaffold-associated DNa~regions As a test for the loop model one mighthope to find specific regions, spaced along the DNA, at which the scaffold interaction occurs. Such specific scaffoldassociated regions (SAR) have been identified with the help of a novel extraction procedure that uses lithium 3', 5-diiodosalicylate (LIS). At low concentration.,; and in physiological salt buffers, this compound extracts histones and other proteins under conditions apparently 'mild' enough to preserve a specific scaffoldDNA interaction. The LIS is removed by repeated washing and the extracted nuclei are digested to completion with various restriction enzymes. A restriction fragment that contains a SAR cosediments with the nuclear scaffold. In the Drosophila system we have mapped 18 SARs near a variety of polymerase IItranscribed genes z°-zs, extending over 400 kbp of DNA, 320 kbp of which is within a chromosomal 'walk' around the rosy locus (see Fig. 3). A number of experiments have been carried out to determine the biochemical nature and the functional significance of scaffuld-DNA binding. At present, our basic observations are as follows. (1) SARs can be mapped to fragments ranging in size from 0.6 to I kbp, containing multiple sites of scaffold-DNA interaction. (2) In Drosophila, SARs are found in nontranscribed regions. For the mouse [clight chain gene, however, a SAR was identified adjacent to the enhancer sequence in a transcribed regionzs. (3) The distance between two adjacent SARs varies between 4.5 and 112 kbp. (4) One or several genes can occur between two SARs. (5) In Drosophila, several enhancer-like elements for developmentally regulated genes cohabit with SARs. (6) No changes have been observed in scaffold attachment upon the induction of transcription. (7) The SAR interactions are similar in nuclei derived from developmentally different cells. These observations are discussed in more detail in the following sections. 5AR s contain clusters of the topoisomeraseII consensus sequence and two additional sequence motifs The best characterized SAR is found (on a 657 bp fragment) in the non-transcribed spacer between the H1 and H3 genes. This attachment is observed in all the tandemly organized histone-gene repeats, defining small 5 kbp loops (Fig. 2). Exanuclease lII digestion studies have identified two protein-binding domains within this SAR, each covering about 200 bp, as depicted in Fig. 2. Studies of other SAR fragments also reveal multiple binding sites within rather large regions (up to 1.1 kbp). Each individual binding domain is able to mediate scaffold association, albeit with a somewhat reduced affinity. The presence of topoisomerase 11in the metaphase scaffold prompted us to screen the available SAR sequences for the Dros@hila topoisomerase lI consensus sequence 2s. All SARs tested contain a strikingly large number (8-17 per fragment) of sequences related to the 15 bp topoisomerase II cleavage site (topo H box) [GTN(A/T)A(T/C)ATTNATNN(G/A)] zl'zs'z4. Although this is a weak and loosely defined consensus, two results suggest that the clustering of such sequences within the SAR fragments is of significance. Firstly, Udvardy et aL 2~ have shown that the SARs of the histone-gene cluster and of the hspT0 heat-shock genes are major targets
reviews 2
TIG [ 3 1 - - J a n u a r y 1987
H2a b
~ _ ~ _
H3
m ~ H i
~,
_ /'" ~c,RV .-
~
•
nnmmm ,
_
\/
l
.2a b
~
] [AATAAA(T/C)AAA](Fig. 2). The pattern of topo I!, ] ] T and A boxes of four different SARs is shown in Figs ~ 2 and 4. The clustering of topo II boxes in the various SAlts is impressive, but does not appear to follow a simplepattern" N°te' h°wever, that theTb°xis °lien ~ found downstream from the A box; this run of thymidine residues may be responsible for bends and ecoR: kinks in the DNA or may discourage nucleosome formation.
~
~*R~
~ ,
qlo O O g l
I~lN
SARs are often close to promoters and cohabit with upstream regulatory sequences
a 0__.._~9 L_a____z~x
For three developmentally regulated genes of
Fig. 2. Repeat of histone gene,s: one repeat, one loop. T~o repeats of the 5 kbp DNA fragment that contains all five Drosophila histone genes are shown in the upper part of the figure. The 65',7bp scaffold-o~sociated region (SAR) occurs in the non.trans cribedspacer between theH I and the l13 ger~s and is indicated by a bar containing a hook. Roughly 100 tandem histone gene repeats are present in the &rnome,forming a series of snlall loops. Sequence raohys common to a number of SARs are indicated in the enlarged map of the SAR. V represents sequences with 707b homology to the tOoisomerase 11 consensus sequence~, in either the Watson (lop) ar Crick (bottom) strand. • imticatea the lO-bp A box, and • the lO-bp T box described in the text~. Two 200-bp domains within the SAR (here enconOassed by dotted lines) were resistent to exonuclease III d~gestion in intact scaffolds, indicah'ng the presence of pro~n-DNA complexe~ z.
for topoisomersse cleavage in vitro. Secondly, the DNA regions not bound to the scaffoldgenerally do not contain such clusters of topo II boxes. However, the occurrence of the consensus alone does not appear to be sufficient to create a SAR, since in the Adh gene region we found a fragment with several topo II boxes which is not scaffold-bound2s. In vivo localization of DNA topoisomerase Il cleavage sites in the hspT0 genes has revealed multiple specific cleavage sites both at the 3' and 5' ends of the genes, but none in the 5' SAR region, unless the cells were heat-shocked zT. Thus, the presence of the topo II boxes in the SAR may represent potential sites of action for a topoisomerase H which is regulated in vivo. The SARs that have been analysed contain two additional 10 bp sequence motifs, the T box [TT(AFF)T(T/A)TT(T/A)TT] and the A box
12osy
Drosophila (Adh, Sgs-4 and ftz) the SARs 5' of the
genes have been found to cohabit with regulatory sequenceszs-~ up to 4.5 kbp upstream from the start of transcription (see Fig. 4). Remarkably, for theAdh locus, from which two transcripts are made, two upstream/enhancer-like regulatory regions were identified as well as two 5' scaffold-attached regions. The sequences required for t~ssne-specificexpression of Adh and ~z, on the other hand, are not scaffoldbound, nor are the actual coding sequences for any of the genes studied. For each of these three highly expressed, developmentally regulated loci, SARs are also found 3' of the transcription units. These could interact with the 5' SARs to form smallloops ranging in size from 4.5 to 13 kbp, each containing one gene. The term cohabitation describes our finding that the restriction enzyme fragments defining upstream/ enhancer-like elements of these genes also contain SARs. Both types of mapping data do not exclude the possibility that the two DNA elements might still be separable by the appropriate experimentation. In the case of the mouse immunoglobulin g gene, the matrixbinding site is adjacent to and separable from the enhancer sequence24. Yet for the Drosophila Sgs-4 gene, this cohabitation appears quite intimate; a 710bp region immediately upstream from the transcription start site contains the essential regulatory sequences, the SAR, and the DNase I hypersensitive sites as~mciated with gene activity~. Such a close functional link between the SAR and upstream regulatory element is not observed in the case of the major heat-shock gene hspT0. This SAR is upstream from the DNase I hypersensitive sites associated with active transcription, and is also upstream from control elements that are necessary
l
__ ~
\1 :>78 kb
/ ~ kb
...
/rn32
\1,,.1¢ "°''°'' 112 kb
26 kb
> 62 kb
Fig. 3. Loops and genes in 320 kbp ofDrosophilaDNA. The lo~ organization is shown for a 320.kbp region ~ o u . n d i ~ the~sy and Acelocifrom DrosophilaXc cell~. The va~nLs Ee~elicloci are in dicated by lines and t~eposltions of the SAR soy nooReaoars. The loop sizes in this r e g ~ are large, as indicated in the f ~ r e , and the various transcripts are o/low abundance, an obsersaffon which sw~gests an inverse correlation between the potential level of transcription and loop size.
TIG [3] --January 1987
iews
What is the importance of the loop size and/or the proximity of the 5' SAR? The data available at present fit best with the notion that in Drosophila the highly expressed genes are found in small loops of 4-13 kbp while genes with less abundant transcriptional activity are found in much larger loops of 50 kbp or more. Examples of the former class would be hspT0, actin 5C, the hlstone cluster, Adh, Sgs-4 andflz, and for the latter class, all the genes localized within the 320 kbp 'walk' around the rosy locus (Fig. 3). These observations suggest an inverse correlation between the potential level of transcription and loop size.
(a)
,
E
,
4.7kb
\\ V
II
A~
,,'-
(b)
~ "T"
~
= s
/
II.lkb V
~.-~ /~/
/'~
¢-'='
/~/
\ \
= [ ~/
\2 .V
~ /
~../
Are SAR positions and loop structure tissue-opecifw?
One can imagine that certain attachment sites are preferentially lost or created during cell differentiation, perhaps mediated by specific transcription factors, allowing the modulation of nuclear domains as required by the cell's specific pattern of gene exprescion. It follows that some SARs, those near housekeeping genes, would remain constant in all proliferating cells. In both the mouse and Drosophila systems, few significant differences in loop organization have been seen among different cell types ~'24. This may be due, however, to the character of the assay for these ~sites. Since SARs are detected in the absence of histories, any fragment with the potential to bind might be detected as scaffold-bound. Thus, it is possible that some fragments identified as SAR in our assay in vitro may actually have their scaffold-binding sites blocked by histones in the intact nucleus~. Some SARs co-map with boundaries of' adive" domains
i ,s
"..
Fig. 4. Cohabitation of SARs with upstream~enhancerelements. Loop structure for three developmentallyregulatedgenes: (a) the glue grotein gene, Sgs-429; (b) the homeobox.containing gene fushi tarazu(ftz)ZS; and (c) the alcohol dehydrogenasegene (Adh)~°. SARs arefound both 5' and3' from the codingsequences(open boxes)as indicated by the hooked bars. As in the case of the histone gene cluster, the loop sizes of these temporally and highly expressedgenes are small, at maximum 11 k~. In the case ofAdh, which has ~o gromoters and two transcriNsfrom the same coding unit, there are two upstream and ~o downstream SARs. For all three genes, the 5' SAles cohabit with enhancer-like regulatory elementsz~. Loop size measured from center to center of two adjacent SARs is given below the correepondmg loops. The positions of several sequence motifs are shown in the enlarged maps of the SARs for Sgs-4 and Adh. V representsthe tepoisomeraseH consensus, • theA box and [] the T box as discussed in F~g. 2. For orientation, a selection of relevant restriction sites is given.
Non-transcribed regions of chromatin at the borders of actively transcribed regions are often associated with highly ordered, or 'static' nucleosome organization, whereas a more 'blurred' organization of nucleosomes is seen in the nearby transcribed regions. In two cases, SARs co-map with such regions of static nucleosome organization bordering actively transcribed domains. One is the non-transcribed region of the H1-H3 spacer, in which the two proteinbinding elements of the SAR coincide quite well with nucleosome-sized particles detected by micrococcal nuclease digestion32. In addition, the SARs located 5 of the hsp?0 genes (at both loci 87C1 and 87A7) fall into regions of static, 'phased' nucleosomes. Like the SAR interaction, the static pattern is maintained during transcriptional activation of these genes. These data suggest that some SARs may be involved in defining boundaries. SARs may define a positionaUy-independent functioning unit
Do SARs define a region that will function and apparently sufficient for its complete regulation~1. independently of its position in the genome? The The observation that the SAR of the hsp?O gene lacks available data from P element transformation using the the usual series of upstream A boxes suggests to us ftz gene are consistent with this notion. Constructs that different SAR types may correlate with differently containing either the ftz gene and both its 5' and 3' regulated genes. For example, a developmentally SARs, or just the gene and 5' SAR, were used; when regulated gene may have a 'regulated' SAR fragment both SARs were present in the transforming vector containing both topo II boxes and A boxes, while a none of the eight ftz + transformants showed a position housekeeping gene may have a 'constitutive' SAR effect. Among the seven transformants obtained with without A boxes. If A boxes are diagnostic of the construct lacking the 3' SAR, four showed low 'regulated' SARs, then one would predict that the levels of expression, presumably due to the site of histone SAR is involved in transcriptional control. insertion (position effect).
TIG [3] - - J m m m y 1987
Another example concerns the expression of the Sgs-4 glue protein gene. Individual transformed flies
derived from constructs which included the 5' regulatory/SARregion, but not the 3' SAR, expressed the introduced Sgs-4 gene with great variability, and no puffing was observed at the various sites of insertion29. Both the Sgs-4 andflz genes are highly transcribed genes with closelypositionedSARs. One might expect
~g. 5. Scheme of the loop organizalion in a Mstone-depleted nucleus, The genomic DNA is organized into loops varying in size between 5 a,d 100 hbp. The loops are anchored to the residual ha!ear scaffold at highly specific DNA regions called SAR (scaffold associated re~n). SARs occur in Drosophila cells in non-transcribed regfims, e~tend over about 1 kbp of DNA, and contain usually eight or more DNA elements related to the topoisomerose II cleavage consensus, as well as hoo additio,al sequence elements. Loops can have one or several transcription units, indicated by arrows. Available data are consistent with the notion t i ~ kigidy aclive or polontially h~kly active genes are organ~d into small loops of about I0 kbp, while genes with less transcriptional aclivity are arranged in muck larger loops containing several lranscription units. It has not been established whether the SARs interact with the peripheral lamina slruclure andlor the interna! m~ork of nuclear scaffolds. While the peripheral lamina structure is known to have a simple polypeptidecomposition, the structurally and biochemically ill-defined internal network is composedof a very complexpattem of pt~ins. TopoisomeraseII is one of the components of the internal network. The loop organization mediated by the SARs is experimentally indisffnguishable in metaphase chromosomes. The protein composition of the metaphase scaffold, however, is considerably less complex than that of the nuclear equivalent. TopoisomeraseH is in this case the major protein of the metaphusescaffold. About three copies of topoisomerase H are found per average size loop both in metaphase and nuclear scaffolds, an observation consistent with the possible role of this protein as a loop-fastener.
that transformation with genes that are tran~cr~ibeVdi a t ~ ~ low levels and have no nearby SAR would be less susceptible to position effects. Indeed, this is the case for the weakly expressed rosy gene~.
Nuclearorganization" conclusion and speculation We have proposed that the genome is organized into loops that have both a structural and functional role, and that SAR sequences define the boundaries of these loops. Figure 5 shows an 'artist's impression' depicting some features of our current ideas about how these loops are organized in a histone-depleted nucleus. Loops may range in size from 5 to 112 kbp, and we find a loose, inverse relationship between loop size and the level of transcription of genes contained within the loop. Topoisomerase lI appears to be a pivotal protein in chromosomal structure and nuclear order. It is present in amounts sufficient to serve as 'loopfastener', and has been shown to be a major component of the chromosomal scaffold. The activity of this enzyme is likely to be highly regulated and may be involved in the release of stress during transcription or replication, as well as in the decatenation of replicated DNA (e.g. Ref. 34). If topoisemerase 11exerts its action at SAR ~quences at the bases of loops, it would be strategically located to control long-range order in chromatin domains during these processes. SARs may also serve as preferred sites at which DNA replication begins or ends. In Drosoph//a, SARs tend to be found close to the promoter elements, and cohabit with the upstream/ enhancer-like elements of three developmentally regulated genes (Adk, Sgs-4 and ftz), The scaffold interaction may serve to bring distant regulatory sequences close together to create functional complexes for the regulation of transcription, in analogy to the mini-loops discussed by Ptashne3s. Seen on a larger scale, the positioning of SARs may serve to 'index' those sequences which need to be acted upon by factors or polymerases, to form a subnuclear compartment. Such a compartment, rich in specific DNA binding sites, would facilitate the formationof the appropriate DNA-protein complexes required for the control of transcription, replication and for chromosome templating, by reducing a factor's search volume within the nucleus. The close pro~nity of many regulatory sequences would have a Circean effect, effectively caging DNA-binding proteins within this comparUment. Regulatory proteins could nonetheless traverse the compartment by the direct transfer mechanism, physically scanning the DNA exposed at the bases of loops, Within these compartments one might expect to find both high and weaker affinity binding sites for factors, which could be important for chromatin templating. If the assembly of active chromatin structure results from the competitive binding of either factors or histones to newly replicated DNA, then the existence of factor-rich compartments could provide a kinetic advantage for the binding of factors over histones, during replication. Thus, high fidelity transmission of the epigenetic chromatin structure to the daughter cell would he largely a consequence of the compartmentalization of the nucleus.
TIG [3] - - J a n u a r y 1987
iews The channels defined by the bases of loops of one chromosome could be linked to the periphery of the nucleus via the nuclear pores, which may direct and kinetically regubte influx of proteins to the channels. A highly organized nucleus with a fixed orientation toward the cytoplasm, as observed in the Drosophila embryo, might permit the relay of spatial information from a structured cytoplasm into the nucleus, to affect gene expression. Finally, if one assumes that the flux of proteins into the nuclear channels is kinetically dependent on nuclear pores, then dissociation of the nuclear lamina/pore complex during mitosis could lead to an automatic loss of these proteins from chromatin. In this light one would not need to propose an additional mechanism for chasing nuclear proteins into the cytoplasm din-rag metaphase, Our results suggest that the higher-order organization of the nucleus is determined by bases of DNA loops and the proteins that bind to them. If these sites change with the differentiation of a cell and specialization of its pattern of expression, then one would expect that nuclei from different cell types would have different networks and channels. Such a cell-specific three-dimensional organization ef nuclei was also discussed in the 'gating' hypothesis of BlobelaS. Although these predictions have not yet been shown to be correct, with the means to dissect the higher order chromatin cor~ormation at hand, we may soon get more than a glimpse at chromosomal order.
I0 Stadler,J. et eL (1980) Cell 19, 973-980 11 Ciejek, E. M., Tsai, M-J. and O'Malley, B. W. (1983)Nab~re 306, 607-609 12 Tarter, D. K., Hobbs, Ch. and Jones, M. (1984) Cell 97,
869-878 13 Paulson, J. L. in Chromosomes and Chromatin Structu~
(Adolph, K. W., ed.), CRC Press (in press)
14 Laemmh,U. K. etal. (1978)ColdSpringHarborSym]).QuanL Biol. 42, 109-I18 15 Lewis, C. D. and Laemmli,U. K. (1982) Cell 29, 171-181 16 Earnshaw,W, C. and Heck, M. M. S. (1985)]. Cell. Biol. 100,
1716-1725
17 Gasser, S. M. etal. (1986)]. Moi. Biol. 188, 613-629 18 Berrios, M., Osheroff, N. and Fmher, P. (1985) Proc. Natl. Acad. Sc=. USA 82, 4142-4146
19 Lewis, C. D., Lebkowski, J. D., Daly, A. and Laemmli, U. (1984) ]. Cell SoL 1, 103-122 20 Mirkovltch,J., Mirault, M. E. and Laemmli,U. K. (1984) Cell 39, 223-232 21 Gasser,S. M. andLaenmdi,U. K. (1986)EMBO]. 5, 511-518 22 Mirkovitch,J., Spierer, P. and Laermdli,U. K. (1986)]. Mol. Bwl 190, 255-258 23 Gasser,S. and Laemmli, U. K. (1986) Cell 46, 521-530 24 Cockerill, P. N. and Garrard, W. 1". (1986) Cell 44, 273-282 25 Sander, M. and Tao-Shib, H. (1985) Nucldc Acids Res. 13, 1057-1071 26 Udvardy,A., Schedl, P., Sander,M. and Tao-Shih, H. (1985) Cell 40, 933-94] 27 Rowe, T. C., Wang, J. and Lin, L. (1986) Mol. CellBioL 6, 985-992 28 Hiroml, Y., Kuroiwa, A. and Gehztag, W. J. (1985) Cell 43, 603-613 29 McNabb, S. L. and Beckendorf, S. K. (1986) EMBO ]. 5, 2331-2340 30 Posakony,J. W., Fischer, J. A., Corbin, V. and Maniatis, 1". (1985) m Current Commun~a~o=, pp. 194-200, Cold Spring [-[arbor Laboratory 31 Dudler, R. and Travers, A. A. (1984) Cell 38, 391-398 References 32 Worcel, A. et al. (1983)NucleieAddsRes. 11, 421-439 1 Weintraub,H. (1985) Cell 42, 705-711 2 Berg, O. G., Winter,R. B. andvonHippel,P. H. (1982) Trends 33 Spradlin¢ A, C. and Rubirk G. M. (1983) Cell 34, 47-57 34 Uemura, T. and Yanagida,M. (1984)EMBO]. 3, 1737-1744 Biochem. Sci. 7, 52-55 3 Fried, M. G. and Crothers, M. (1984) ]. Mol. Biol. 172, 35 Ptashne,M. (1986)Nat=re 322, 697-701 36 Binbel, G. (1985)Proc. Natl, Aead. Sd. USA 82, 8527-8529 263--282 4 Rabl, C. (1885)MorphoL]ahrb. 10, 214--330 5 Foe, V. E. and Alberts,B. (1985)]. Cell. Biol. 100, 1623--1636 S. M. Gasser and U. K. Laemmli are at the D~artr~nts of 6 Kerem, B-S. et al. (1984) Cell 38, 493-499 7 Goldman,M. A., Holmquist,G. P., Gray, M. C., Caston, L. A. Molecular Biolo£y and Biochemistry, Universi(y o/Geneva, 1211 Geneva 4, Switzerland; S. M. Gasser is presently at the and Nag, A. (1985) Sdenee 224, 686-692 8 Yunis, J-J. (1976) Science 191, 1268--1270 Swiss Institute/or Experimental Cancer Resmrch (ISREC). 9 Hutchison, N. and Weirztraub,H. (1985) Cell 43, 471-482 CH 1066 EpalingeslLausanne, Switzerland.
Coming soon, in Trends in Genetics A giant locus for the Duchenne and Backer muscular dystrophygene by Anthony P, Monaco and Louis M. Kunkel
The gene responsible for Duchenne and Becker muscular dystrophy has been assigned to band Xp21 of the human X chromosome, and the nsolation of DNA segments from this region has provided probes useful for prenatal dnagnos=s and carrier detection. It has also led to the identification and partial cloning of a large transcrnpt that is found in skeletal muscle and potentially spans over 1.0 million basepairs of DNA. A large size for the muscular dystrophy gene locus is consistent with the observed high mutatnon frequency, the heterogeneity of the deletion breakpoints that are associated with the disease and the high recombination frequency of DNA markers that Ine within the gene itself.
Pool your useful hints throughTechnical 'tips Technical Tips (see p. 2) is a place where readers can exchange information about new experimental techniques. To make this section really useful to experimental geneticists and developmental biologists we need the active participation of our readers. If you have information about methods developed in your lab or elsewhere why not share it with your colleagues through the Technical Tips section of Trend= In @mmlle=? Each month Technical Tips draws attention to such methods by presenting very brief art¢les. These do not attempt to provide all the information required to use the method but rather a clear outline of the method's claimed advantagesand present and potential applications; readers can then look to the reference for complete details. The only exception to this general policy conums descriptions of unpublished methods where more precise experimental detads should be provided, Please send all information to: Trends in (~meticg, Elsevier Publications Cambridge, 68 Hills Road, Cambridge CB2 1LA, UK, or call (0223) 315961.