Interaction in vitro of non-epithelial intermediate filament proteins with

supercoiled plasmid DNA*

S. KUHN, C. E. VORGIAS and P. TRAUB

Max-Planck-histitut fur Zellbiologte, Rosenhof, D-6802 Ladeiiburg/Heidelberg, Federal Republic of Germany

•This paper is dedicated to Professor Wohlfarth-Bottcrmann for his 65th birthday on 22 May 1987

Summary

Sucrose gradient analysis of reaction productsobtained from non-epithelial intermediate fila-ment (IF) subunit proteins and a mixture ofsupercoiled, relaxed and linearized plasmidpBR322 DNA at low ionic strength revealed thatlimited amounts of these polypeptides interactedexclusively with the supercoiled form of the plas-mid DNA. These results were corroborated byelectron-microscopic analysis of the reactionproducts, which showed that only circles ofsupercoiled pBR322 DNA were completely andsmoothly covered with vimentin. IFs reconsti-tuted from pure vimentin reacted with super-coiled pBR322 DNA only through their physicalends. The reaction of an aged preparation ofvimentin with supercoiled pBR322 DNA produced

large aggregates consisting of a central, axiallyoriented protein scaffold to which individualloops of DNA were attached at their bases in ahalo-like arrangement. The electron-microscopicappearance of such complexes was very remi-niscent of that of histone-depleted metaphasechromosomes. Together with the previous obser-vations that non-epithelial IF proteins have highaffinities for single-stranded DNA and core his-tones and that they are structurally and function-ally closely related to the nuclear lamins, theseresults were used to advance a novel hypothesison the biological role of IF proteins in eukaryoticcells.

Key words: intermediate filament proteins, vimentin,supercoiled DNA, plasmid pBR322.

Introduction

The in vitro characterization of non-epithelial inter-mediate filament (IF) subunit proteins has shown that,besides their marked propensity to polymerize into10 nra filaments (for a review, see Traub, 1985a), thesepolypeptides exhibit a strong tendency to associate withsingle-stranded nucleic acids, particularly with single-stranded DNA (Traub & Nelson, 1982, 1983; Traub etal. 1983, 1985; Vorgias & Traub, 1986), and with corehistones (Traub et al. 1987c). Because of their highaffinities for nuclear constituents, we postulated thatIF proteins eventually fulfil nuclear functions, thatthey might be involved in such activities as replication,recombination and repair of DNA, RNA transcriptionand processing and transport of nuclear RNA (Traub,1985a,6; Traub et al. 1987a). On the other hand, thestructural stability of IFs and their association with amultitude of extranuclear, cellular substructures andcomponents (reviewed by Traub, 1985a) suggest that

Journal of Cell Science 87, 543-554 (1987)Printed in Great Bntain © The Company of Biologists Limited 1987

the filaments play a cytoskeletal, coordinating role inthe cytoplasm of eukaryotic cells (Lazarides, 1980,1982). In this respect, the capability of non-epithelialIF(protein)s to interact with membrane-associatedcytoskeletal elements like ankyrin (Georgatos & Mar-chesi, 1985; Georgatos et al. 1985), spectrin (Mangeat& Burridge, 1984; Langley & Cohen, 1986), plectin(Wiche et al. 1983) etc., and with the lipid bilayer itself(Perides et al. 1986a,6; Traub et al. 19866, 19876) iscertainly of great importance. On the basis of such avariety of reactivities, it is reasonable to assume that IFsubunit proteins are multifunctional in the life cycle ofeukaryotic cells.

Concerning our postulate that IF proteins are alsoinvolved in nuclear activities (Traub, 1985a,b; Traubet al. 1987a), it is pertinent to refer to the recentfinding that IF proteins are structurally closely relatedto the family of nuclear lamins (McKeon et al. 1986;Aebi et al. 1986; Fisher et al. 1986; Parry et al. 1986;

543

Goldman et al. 1986a). The latter have been recog-nized to represent nothing else but another type of IFprotein. Thus, it is not surprising that both families ofproteins also are very similar to each other in theirfunctional properties. Exactly like IF proteins (forreferences, see above), the nuclear lamins are capableof interacting with (single-stranded)DNA (Comings &Wallack, 1978; Hancock, 1982; Lebkowski & Laemmli,19826; Boulikas, 1986) and with the nuclear membrane(Gerace et al. 1984; Lebel & Raymond, 1984). Veryprobably, these activities play a crucial role in theattachment of chromatin (DNA) to the envelope ofinterphase nuclei (Agutter & Richardson, 1980; Han-cock, 1982; Hancock & Hughes, 1982; Hancock &Boulikas, 1982; Lebkowski & Laemmli, 1982a,b;Smith et al. 1984; Bouvier et al. 1985; Bureau et al.1986; Krachmarov et al. 1986). Since IFs also associatewith the nuclear envelope (Goldman et al. 1985,1986a,6; for further references, see Traub, 1985a) andperhaps intercalate into the nuclear lamina (Fey et al.1984, 1986), it might well be that their subunit proteinssomehow change the organization of the nuclear laminathereby modulating its activities in the framework ofDNA replication and RNA transcription.

It is known that DNA replication and RNA tran-scription in interphase nuclei are activated by torsionalstress in DNA (for a brief review, see Scott, 1985).However, because the bulk of DNA supercoils ininterphase chromatin is locked into nucleosomes bycore histones, the chromatin appears to be topologicallyrelaxed. Supercoil tension can be reintroduced intochromatin by removal or at least partial dissociation ofthe core histones from the nucleosomes. Since in vitroIF proteins interact very tightly and with a distinctstoichiometry with core histones through their a-helical rod domains (Traub et al. 1987c), they mightbe able to induce decondensation and dissociation ofinterphase chromatin. Simultaneously, in a concertedreaction, they might bind to the released and re-supercoiled stretches of DNA through their non-a-helical, arginine-rich N termini and thus contribute tothe production of entry sites for the DNA replicationand RNA transcription machineries.

To demonstrate that IF proteins are indeed endowedwith such a supercoil DNA recognition capacity, weinvestigated the interaction in vitro of all major non-epithelial IF proteins with plasmid pBR322 DNA. Ourresults show that IF proteins react readily and exclus-ively with supercoils when added to a mixture ofsupercoiled, relaxed and linearized plasmid DNA. Inaddition, our results point to the potential capacity ofIF(protein)s to participate in the construction of nu-clear protein scaffolds for chromatin (DNA) attach-ment.

Materials and methods

MaterialsUnlabelled and 3H-labelled vimentin were prepared fromcultured Ehrlich ascites tumour cells (Nelson & Traub, 1982;Nelson et al. 1982), desmin from porcine stomach smoothmuscle (Vorgias & Traub, 1983a), glial fibrillary acidicprotein from bovine brain white matter (Vorgias & Traub,19836) and individual neurofilament triplet proteins fromporcine spinal cord (Traub et al. 1985), as described pre-viously. Pancreatic RNase A and cytochrome c were obtainedfrom Sigma (St Louis, MO, USA). The isolation of plasmidpBR322 DNA followed the procedure of Clewell & Helinski(1969).

Sucrose gradient centrifugation

A sample (0-05 A260 unit; =2-5 fig) of plasmid pBR322 DNAwas mixed with increasing quantities (see legend to Fig. 1) ofindividual IF subunit proteins and 10 \vg ethidium bromide in100^tl lOmM-Tris-acetate (pH7-6), 3mM-EDTA, 6mM-2-mercaptoethanol (buffer I). After standing at 0°C for 5 min,the reaction mixtures were layered on top of 4-ml 10% to30 % (w/w) sucrose gradients in buffer I and centrifuged at257 000 &v and 5°C for 3 h in the SW60 Ti rotor of theBeckman L2 65B centrifuge. The gradients were photo-graphed in front of an ultraviolet (u.v.) light box (wave-length: 366 nm) on Agfapan 400 film using u.v. and Kodakwratten gelatin 23A filters. When [3H]vimentin was used as areactant, the gradient was pumped through the flow-throughcuvette of an Aminco-Bowman spectrophotofluorometer(excitation wavelength: 340 nm; emission wavelength:590 nm) and fractionated collecting 10 drops/fraction. Eachfraction was mixed with 0-5 ml Soluene 350 (Packard Instru-ments) and 10 ml toluene-based scintillation mixture andcounted in a Packard Tri-Carb liquid scintillation spec-trometer.

The reaction of unlabelled vimentin with plasmid pBR322DNA was also carried out in the absence of ethidiumbromide. After sucrose gradient centrifugation of the reactionproducts under the conditions specified above, the DNAdistributions were determined by adding ethidium bromideto each gradient fraction (final concentration: 1 fig ml"1) andmeasurement of the fluorescence intensity.

Electron microscopyPlasmid pBR322 DNA and vimentin were mixed at differentratios in buffer I, the resulting complexes fixed according toGriffith (1978) and then spread either protein-free as de-scribed by Vollenweiderefa/. (1975) or using the cytochromec method of Kleinschmidt (1968) as modified by Davis et al.(1971). Reaction products of vimentin filaments reconsti-tuted in buffer I containing 150mM-KCl with supercoiledpBR322 DNA were mixed with glycerol (1:1, v/v), sprayedonto freshly cleaved mica and rotary shadowed with tungstenat an angle of 10° (Tyler & Branton, 1980; Henderson et al.1982). The samples were viewed in a Siemens EM IAelectron microscope.

544 S. Kiihn et al.

Results

Sucrose gradient analysis of pBR322 DNA-IF proteinaggregates

In the first set of experiments, purified non-epithelialIF subunit proteins were reacted with plasmid pBR322DNA at low ionic strength and the reaction productsanalysed by sucrose gradient centrifugation. Fig. 1depicts the results obtained when a constant amountof an ethidium bromide-labelled mixture of super-coiled, relaxed and linearized pBR322 DNA wastitrated with vimentin (Fig. 1A), desmin (Fig. IB),glial fibrillary acidic protein (Fig. 1C), and the threeneurofilament triplet proteins NFP68, NFP145,NFP200 (Fig. 1D-F), respectively. It is evident thatonly the supercoiled form of plasmid DNA progress-ively increased its sedimentation rate with increasingquantities of IF protein, whereas the relaxed andlinearized forms retained their original sucrose gradientpositions throughout titration. It is also obvious thatreaction of the various types of IF protein withsupercoiled pBR322 DNA gave rise to the formation ofcomplexes sedimenting with distinctly different rateson sucrose gradients. While the neurofilament proteinNFP145 exerted the weakest effect (Fig. IE), NFP68changed the sedimentation rate of supercoiled DNAdramatically (Fig. ID). At higher protein concen-trations, the deoxyribonucleoprotein bands were barelyvisible. This was due partly to band spreading andpartly to pelleting of the complexes. Controls wereperformed using two small, positively charged proteinspecies, pancreatic RNase A (Fig. 1G) and cytochromec (Fig. 1H). Only the single-stranded DNA-bindingprotein RNase A (Jensen & von Hippel, 1976) behavedidentically to IF proteins, while cytochrome c non-specifically shifted the sedimentation rates of all threeforms of plasmid pBR322 DNA to higher values to anequal extent. To exclude the possibility that thebinding of IF proteins to supercoiled DNA was affec-ted by ethidium bromide, titration of the pBR322DNA mixture with vimentin was also performed in theabsence of the drug. Following sucrose gradient centri-fugation of the reaction products, the DNA distri-butions were visualized by the addition of ethidiumbromide to individual fractions after gradient fraction-ation and by fluorescence measurement. Exactly thesame results were obtained as in the experimentperformed under standard conditions, except that allthree forms of plasmid DNA sedimented at a somewhatlower rate than in the presence of ethidium bromide(data not shown). Further evidence, that also in theabsence of ethidium bromide vimentin efficiently andselectively associated with supercoiled plasmid DNA,was provided by electron-microscopic examination ofthe reaction products (see below).

That the IF proteins indeed interacted only withsupercoils when offered to the plasmid DNA mixturecould be also demonstrated using radioactively labelledIF protein. Fig. 2 shows the sedimentation pattern ofthe original plasmid DNA in comparison with thatafter its reaction with [3H]vimentin. While linearizedand relaxed pBR322 DNA remained completely free ofvimentin and, therefore, did not change their sedimen-tation behaviour, supercoiled DNA bound virtually allfilament protein resulting in the formation of fastersedimenting DNA-protein complexes.

Electron microscopy of pBR322 DNA-IF proteinaggregates

In addition to sucrose gradient analysis of the associ-ation products, the deoxyribonucleoprotein particleswere also examined by electron microscopy. Providedthat the vimentin preparation used was completely freeof aggregates and only consisted of protofilaments,molecules of supercoiled pBR322 DNA were com-pletely and smoothly covered with the filament protein(Fig. 3). Occasionally, incomplete coverage of theDNA molecules with protein was observed. In suchcases, the protein appeared to bind in clusters to theDNA molecules leaving the remainder of the DNAstrands more or less free of protein. The averagethickness of the pBR322 DNA-vimentin cables was25 nm in comparison to the 6nm of naked supercoiledDNA. It should be taken into account, however, thatdue to binding of uranyl acetate to the DNA-proteincomplexes and their shadowing with tungsten thethickness of the association products appeared greaterthan natural. The length of the DNA molecules did notchange significantly in response to protein binding.

In agreement with the results of sucrose gradientanalysis (Figs 1,2), vimentin decorated supercoils onlywhen allowed to react with a mixture of supercoiled,relaxed and linearized pBR322 DNA. In Fig. 4, amolecule of supercoiled pBR322 DNA is seen sur-rounded by a circle of relaxed pBR322 DNA. While theformer was completely covered with vimentin, thelatter appeared to be totally free of the filament protein.

An interesting observation was made when a some-what aged preparation of vimentin was used for suchelectron-microscopic studies. On standing at 0°C forextended periods of time, even at low ionic strengthvimentin formed irregular aggregates that are separablefrom protofilamentous vimentin by centrifugation.These aggregates were definitely not identical to IFfragments. SDS-polyacrylamide gel electrophoresisrevealed intactness of vimentin. Fig. 5 shows theelectron-microscopic appearance of complexes ob-tained from such an aged vimentin preparation andsupercoiled plasmid pBR322 DNA. The complexesconsisted of a central, axially oriented protein scaffoldsurrounded by a halo of plasmid DNA. In the smaller

Intermediate filament protein-supercoil DNA interactions 545

Fig. 1. Sucrose gradient analysis of pBR322 DNA-IF protein aggregates: 2-5ng plasmid DNA was mixed with (from leftto right): A, 0, 1-6, 3-3, 5 5 , 11 j/g vimentin; B, 0, 0-5, 0-9, 1-8, 2-7/ig desmin; C, 0, 2-4, 3-6, 7-2, 12jUg glial fibrillaryacidic protein; D, 0, 1-5, 3, 4-5, 9, 15/ig NFP68; E, 0, 2, 4, 6, 12, Wfig NFP145; F, 0, 2-5, 5, 7-5, 15, 25 fig NFP200; G,0, 5, 10, 15 ^g RNase A; H, 0, 3 5 , 7, 105, 14/ig cytochrome c. The reaction products were analysed as described inMaterials and methods. The three DNA bands, visualized by ethidium bromide fluorescence, are from the top to thebottom: relaxed circle, linearized and supercoiled DNA.

546 5. Kiihti et al.

complex shown in the inset to Fig. 5, individual loopsof supercoiled plasmid DNA can be seen to be attachedat their bases to the central vimentin aggregate. Thestructure of such vimentin-pBR322 DNA adductsas depicted in Fig. 5 is very reminiscent of that ofhistone-depleted metaphase chromosomes (Paulson &Laemmli, 1977), with the only difference that becauseof the small size of the plasmid DNA the diameter ofthe DNA halo around the central protein scaffold wasmuch smaller in the case of vimentin-pBR322 DNAcomplexes than in the case of histone-depleted meta-phase chromosomes.

It should be emphasized here that all the exper-iments described thus far have been performed at lowsalt concentration. This was necessary because atphysiological and higher ionic strength IF proteinsshow a strong tendency to polymerize into filaments.Since under such conditions the arginine-rich, non-a'-helical N termini of non-epithelial IF subunits aredeeply involved in the formation of thermodynamicallymore stable 10 nm filaments (Traub & Vorgias, 1983;Kaufmann et al. 1985), they are no longer available forthe binding of the subunit proteins to DNA. Neverthe-less, as shown for vimentin filaments in Fig. 6, IFs

10 15Fraction no.

20

Fig. 2. Reaction of plasmid pBR322 DNA with [HJvimentin and sucrose gradient analysis of the reaction products.Ethidium bromide fluorescence distribution when plasmid DNA was analysed alone ( ) and after its reaction with

). The dotted area shows the distribution of [HJvimentin in the sucrose gradient. For experimentaldetails, see Materials and methods.

Fig. 3. Electron micrograph of plasmid pBR322 DNA-vimentin aggregates after protein-free spreading withbenzyldimethyl alkylammonium chloride. The arrow points to an incompletely covered DNA molecule. X28 000.

Intermediate filament protein-supercoil DNA interactions 547

could still interact with supercoiled plasmid DNA.Obviously, free N termini were still available to super-coiled DNA at the physical ends of the filaments. Suchinteractions could also be seen at sites along thefilaments where the polymerization of the subunitproteins was incomplete (not shown). In any event,these results demonstrate that non-epithelial IF pro-teins can also associate with supercoiled plasmid DNAat physiological ionic strength.

Discussion

Together with our previous findings, that IF proteinshave high and specific affinities for single-strandedDNA (Traub & Nelson, 1982, 1983; Traub etal. 1983,1985; Vorgias & Traub, 1986) and core histones (Traubet al. 1986a), the present results suggest that IFproteins eventually fulfil nuclear functions, in additionto the cytoskeletal roles they play in the cytoplasm ofeukaryotic cells (Lazarides, 1980, 1982). This possi-bility gains further credibility by the recent discoverythat IF proteins are structurally closely related to thenuclear lamins (McKeone/ al. 1986; Fisher et al. 1986;Parry et al. 1986; Goldman et al. 1986a) that constitutea cortical shell of IFs, the nuclear lamina, subjacent to

the inner nuclear membrane (Aebi et al. 1986). Inmany laboratories, the nuclear lamina has been shownto act as a protein scaffold to which the DNA moleculesof interphase chromatin attach (Agutter & Richardson,1980; Hancock, 1982; Hancock & Hughes, 1982;Hancock & Boulikas, 1982; Lebkowski & Laemmli,\982a,b; Smith eta/. 1984; Bouviereta/. 1985; Bureauet al. 1986; Krachmarov et al. 1986). Since thisassociation seems to play a pivotal role in intranuclearchromosome distribution and chromatin organization(Hubert & Bourgeois, 1986; Razin, 1987), it is prob-ably of paramount significance for the proper sequenceof all DNA-based nuclear events. The capability of thenuclear lamina to associate with chromatin is veryprobably due to the capacity of its subunit proteins,lamins A, B and C (Gerace et al. 1978), to bind toDNA, preferentially to single-stranded DNA (Com-ings & Wallack, 1978; Hancock, 1982; Lebkowski &Laemmli, 19826; Boulikas, 1986), and probably tosupercoiled DNA also. On the basis of these structuraland functional homologies between IF proteins andnuclear lamins it is tempting to assume that bothfamilies of proteins are in charge of similar functions inthe nuclei of eukaryotic cells. By some intercalation

mmmmmim&mmmmmmmmFig. 4. Electron micrograph of a molecule of supercoiled pBR322 DNA covered with vimentin and a molecule of relaxedplasmid DNA that is free of protein (after protein-free spreading). X70000.

548 5. Kiihn et al.

mechanism, IF(protein)s might change the architec-ture of the nuclear lamina and thereby modulate itsbasic activities in the organization and regulation ofDNA-based nuclear reactions (Razin, 1987).

In this context, it is interesting to note that in nuclearmatrices that have been prepared from Triton cytoskel-etons (Staufenbiel & Deppert, 1983; Goldman et al.19866) by deprivation of DNA, RNA and a largenumber of proteins under IF-preserving conditions,the IFs remain stably bound to the cytoplasmic side ofthe nuclear lamina (Fey et al. 1984, 1986). Whethersuch direct IF-lamina interactions also exist in intactcells is not known. Although IF(protein)s exhibit astrong tendency to interact with artificial lipid bilayers

(Peridesefa/. 1986a,6; Traube/ al. 19866, 19876), it isdifficult to imagine how IFs could directly penetratethe double membrane of the nuclear envelope.

However, another means by which IF proteinsmight come into contact or be intermingled with thenuclear lamins is through mitosis. It is known that inmitosis the nuclear lamina is totally disintegrated anddispersed throughout the cytoplasm (Ely et al. 1978;Gerace et al. 1978, 1984; Gerace & Blobel, 1982).Simultaneously, IFs also disintegrate or at least changetheir cytoplasmic distribution (speckling process)(Franke et al. 1982; Lane et al. 1982; for furtherreferences, see Traub, 1985a). Interestingly, both

Fig. 5. Electron micrograph of a complex obtained from aged, aggregated vimentin and plasmid pBR322 DNA. X30000.In the inset, individual loops of circular, supercoiled plasmid DNA are seen to be attached at their bases to a centralvimentin aggregate. X 40 000. The complexes were spread without fixation using the cytochrome c method.

Intermediate filament protein-supercoil DNA interactions 549

processes are accompanied by extensive phosphoryl-ation of the respective subunit proteins (Gerace &Blobel, 1982; Gerace et al. 1984; Evans & Fink, 1982;Ben Ze'ev, 1983; Celis et al. 1985). It is possible,therefore, that in the state of disintegration the nuclearlamins pick up IF proteins and incorporate them intothe nuclear lamina during its regeneration in telophase.Such a mechanism seems to be operative, for instance,in the incorporation of a vimentin-related protein intothe nuclear lamina of yeast as a consequence of nucleargrowth and division. Since this protein can also bindplasmid DNA, it was supposed to be functional as anessential element of a partitioning system ensuring

equidistribution of plasmid molecules to both progenycells following cell division (Wu et al. 1987).

Today, the notion prevails that the nuclear lamina issomehow involved in maintaining chromatin architec-ture (Hancock, 1982; Benavente & Krohne, 1986;Razin, 1987). In addition to chromatin-lamina associ-ations, interactions of chromatin with elements of theinternal, fibrogranular network of nuclear matrices alsoappear to occur (Lebkowski & Laemmli, 1982a,6;Dijkwel & Wenink, 1986; Bekersef al. 1986). All theseinteractions are supposed to form the operational basisfor DNA replication (Carri et al. 1986; Jackson &Cook, 1986), RNA transcription (Jackson et al. 1981;

Fig. 6. Electron micrographs of association products obtained from vimentin filaments and supercoiled pBR322 DNA. IFswere reconstituted from pure vimentin at physiological ionic strength and reacted with supercoiled plasmid DNA. Theresulting complexes were mixed with glycerol, sprayed onto freshly cleaved mica and rotary shadowed with tungsten. In theright-hand panel, some of the filament-associated DNA molecules seem to have been cleaved during preparation of thespecimens for electron microscopy. X120000.

550 5. Kiihn et al.

Robinson et al. 1983), and processing and transport ofnuclear RNA (Mariman et al. 1982). In addition, it hasbeen found that the attachment to the nuclear matrix ofgenes that are to be transcribed is under hormonalcontrol (Ciejek et al. 1983; Robinson et al. 1983). Thenuclear forms of a series of hormone receptors havebeen tentatively identified as the active mediators ofthese associations (Barrack & Coffey, 1982; Buttyane<al. 1983; Rennie et al. 1983; Kirsch et al. 1986;Kaufmann et al. 1986; Kumara-Sirie/a/. 1986). In thiscontext, it is of great interest that in vitro steroidhormone receptors and vimentin (desmin) have ex-tremely similar gross nucleic acid-binding properties(for references, see Traub & Nelson, 1982, 1983;Traub et al. 1983). Because of these functional homolo-gies, non-epithelial IF proteins and their post-trans-lational derivatives might well be considered as activeprinciples determining the attachment of certain genesto the nuclear matrix in the scope of specific geneexpression and DNA replication. By binding throughtheir non-ar-helical N termini to regulatory DNAsequences (possibly promoter and/or enhancer se-quences) at the 5' end of genes to be transcribed(Zehnbauer & Vogelstein, 1985), they could structur-ally alter these DNA regions in such a way that they arerecognized by accessory transcription factors and RNApolymerase. Since transcription and DNA replicationappear to be activated by torsional stress, the bulk ofDNA loops, however, seems to be relaxed by corehistones, such conformational changes in DNA struc-ture might be jointly brought about by the transientremoval or at least partial dissociation of the corehistones from the nucleosomes by the ar-helical, centraldomains of the IF proteins (Traub et al. 1987c). Thispossibility would be supported by the observation ofnucleosome-free, DNase-hypersensitive DNA regionsin transcriptionally active chromatin (for reviews, seeWeisbrod, 1982; Weintraub, 1985; Yaniv & Cereghini,1986; Pederson ei a/. 1986).

This concept of IF protein function might also behelpful in understanding the phenomenon of cell ortissue specificity of IF protein expression duringembryogenesis (for a review, see Traub, 1985a). Thesynthesis and thus availability of distinct types of IFproteins during differentiation might determine theattachment to the nuclear matrix of certain regulatoryDNA sequences that control the transcription of wholesets of structural genes whose expression is character-istic of the respective cells or tissues. In this sense, IFproteins might act as commitment proteins. Togetherwith other regulatory proteins, they might function inthe limitation of the total potential for gene expressionexhibited by early embryonic cells and, on the basisof the model for replication-transcription coupling(Zehnbauer & Vogelstein, 1985), in the restriction of

the multitude of potential replication origins collec-tively initiated at early developmental stages (Zehn-bauer & Vogelstein, 1985; Carri et al. 1986; Dijkwel etal. 1986). Future experiments should show whether IFproteins indeed participate in the attachment of repli-cation origins to the nuclear matrix. Because thenuclear lamins are structurally and functionally closelyrelated to IF proteins and, in addition, also exhibit celltype-specific expression during embryonic develop-ment and terminal differentiation (Stick & Hausen,1985; Krohne & Benavente, 1986), they should beincluded in these considerations.

Thus, IF proteins might act at two (hierarchicallyand topologically) different levels of gene expression.At a superordinate level, they could induce globalchanges in chromatin structure as a prerequisite for theaction of tissue or cell-specific, positive transcriptionfactors. These changes in commitment might be lim-ited to the nuclear lamina as the site of direct lamin-IFprotein interference. At a subordinate level, on theother hand, IF proteins could operate, after post-translational processing (for a discussion of this model,see Traub, 1985a), in a similar way to the cytoplasmicactivation of hormone receptors, as inducers of theexpression of individual genes; such activities would bepreferentially localized in the internal nuclear skeleton.

We thank Mrs Brigitte Burger and Miss Sabine Hotten-trager for excellent technical assistance and Mrs HeidiKlempp for typing the manuscript.

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(Received 20 January 1987 -Accepted 9 February 1987)

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