The Association of the Interspersed Repetitive KpnI Sequences with ...

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Aug 5, 2016 - From the Department of Biochemistry, Quillen-Dishner College of Medicine, East Tennessee State Uniuersity,. Johnson City ... California, Davis, CA 95616. To whom ... After gel photography, the DNA was transferred to.
Vol. 260. No. 16, Issue of August 5. pp. 9373-9379,1985 Printed in U. S.A

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

The Association of the Interspersed RepetitiveKpnI Sequences with the Nuclear Matrix* (Received for publication, December 6, 1984)

Joseph A. Chimera3 and PhillipR. Musichg From the Department of Biochemistry, Quillen-Dishner College of Medicine, East Tennessee State Uniuersity, Johnson City, Tennessee, 37614

The KpnI sequences constitute the dominant, long, replication, transcription, and/orregulation occur at the prointerspersed repetitive DNA families in primate ge- teinaceous scaffold of the nuclear matrix (reviewed by Hancock, 1982a, 1982b). It remains unclear whether the repeating nomes. These families contain related, but nonidentical sequence subsets, some of which border functional gene loop structures are anchored to the matrix scaffold viaspecific domains and are transcribed intoRNA. To test whether attachment sequences or whether the anchoring is random these sequences perform an organizational function with in respect to DNAsequences. the nucleus, their association with the nuclear matrix Likely candidates for DNA structural elementsinvolved in has been examinedinAfricangreenmonkey cells. the attachmentof DNA loops are thosewhich contain families of most of long interspersed repeats, now collectively referred to as DNase I treatment depleted the residual matrix of the KpnI 1.2- and 1.5-kilobase pair family se- LINES (Singer, 1982). The LINE sequences in primate gequences although significant amounts of each family nomes are the KpnI families which are cleaved into discrete remained in theloop attachment DNA fragments. HyDNA fragments by KpnI endonuclease (Maio et al., 1981). bridization analysis of the KpnI and RsaI cleavage patterns of matrix loop attachment DNA indicate that The various families of KpnI sequences account for a subsome sequence subsets of these KpnI families are rel- stantial part (-4%) of the human and African green monkey atively less depleted than others. The nuclear matrix (AGM) genomes and consist of related but nonidentical seassociation of subpopulations of KpnI 1.2- and 1.5- quence subsets (Maioet al., 1981; Shafit-Zagardo et al., 1982a; kilobase pair families was also shownby metrizamide Chimera, 1984). These primate KpnI families are related to gradient centrifugation of nuclear matrix complexes the mouse LINE family sequences denoted BamHI or MIF-1. cleaved by KpnI endonuclease. The gradientsdemon- Thisrelatedness includes shared sequence homologies alet al., 1983). In addition, strate that some KpnI segments are differentially as- though they are not identical (Singer mouse MIFsociated with nuclear matrix proteins. Moreover, the individual membersof the primate KpnI and the procedures permit the preparative isolation and puri- 1 families have been found to be transcribed into RNA and fication of the DNA-proteincomplexescontaining to be present both at the boundaries and within P-globin gene these KpnI 1.2-and 1.5-kilobase pair sequence fami- domains(Shafit-Zagardo et al., 1982b, 1983; Heller et al., lies. Speculations on the relationship between the ma- 1984). This relatedness of the different LINE sequences totrix association of these KpnI family sequences and gether with their sequence conservation indicates that they their possible roles in gene organization and expression perform one or more important functions in genome organiare presented anddiscussed. zation. The observed microheterogeneity in the KpnI family subsets suggests that individual sequence subsets of these familiesmay performdifferentnuclearfunctions.Theobserved transcription of some KpnI sequencesintonuclear RNAs suggests their possible involvement in the control of The nuclear matrix has been proposed as an attachment transcription and/or RNA processing (Shafit-Zagardo et al., site for the anchoring of higher order chromatin structures referred to as DNA loops. These loops, with 60-100 kb’ of 1983). Their location at theboundaries of gene domains and DNA, may contain functional domainsof related genes whose their transcription suggest that these KpnI families, or specific sequence subsets, may participate in the nuclear matrix attachment and regulation of particular gene domains. Deter* This investigation was supported by a grant from the National mining if there is a role forthe KpnIsequences in the nuclear Institutes of Health (to P. R. M.). The costs of publication of this matrix structure will also increase our understanding of the article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- functional importance of the evolutionary conserved LINE sequence families. ance with 18 U.S.C. Section 1734 solely to indicate this fact. The KpnI families have not been examined for a possible $ Present address: Department of Plant Pathology, University of California, Davis, CA 95616. DNA loop anchoring function. For this reason, we have inTo whom correspondence should be addressed. vestigated thenuclear matrix associationof the KpnI 1.2- and ’ The abbreviations used are:kb,kilobase pairs; AGM,African 1.5-kb families in AGM cells. While their relative content in green monkey; att-DNA, matrixloop attachment DNA;SDS, sodium some memdodecyl sulfate;EGTA,ethylene glycol bis((3-aminoethyl ether)- residual matrix is reduced, the data indicate that bers (specific sequence subsets) of both families are present N,N,N’,N’-tetraacetic acid; DBM, diazobenzyloxymethyl; SSC, standard sodium citrate (0.15 M NaCI, 0.015 M Na citrate, pH 7.0); in the nuclear matrix and are differentially associated with EBr, ethidium bromide. matrix proteins. 9373

Matrix Associatiol of KpnI DNAs

9374 A B C

D

E

F

G

TABLE I Relative content of Kpnl-sensitive segments in att-DNA DNA-rich matrix was mildly digested with DNase I (80 units/ml). At the timesindicated, equal aliquots of the digest were removed for residual matrixandatt-DNA preparation. KpnI digests of equal amounts of att-DNA were electrophoresed on 0.7% agarose gels containing EBr. After gel photography, the DNA was transferred to diazobenzyloxymethyl paper for multiple hybridization analysis with KpnI 1.2- and 1.5-kb family probes. Incubation time

FIG. 1. Comparison of KpnI and RsaI cleavage patterns of AGM total DNA and the KpnI 1.2-and 1.5-kb families. Equal amounts of AGM DNA were digested with KpnI (lane B ) or RsaI (lane C) electrophoresed through a 1.0% agarose gel containing EBr, and the gel was photographed. Lane A contains phage T 3 DNA digested with MboI as a size standard (Sarkaret al., 1980). The DNA in lanes B and C was transferred toZeta-Probe membrane for hybridization with either KpnI 1.2- (lanes D and E ) or 1.5-kb family probes (lanes F and C). The numbers refer to the DNA fragment sizes in kilobases. EXPERIMENTAL PROCEDURES~

min

RFc size”

kb >40 35 30 10 64

DNA in matrixb

% of KmI-sensitive sementsC

1.2 kb

EBr

Probe

1.5 kb

EBr

Probe

%

100 100 100 100 100 0 53 43 93 75 70 1.5 12 85 26 64 48 5.0 2.8 18 35 20 20.0 Sizes of att-DNA fragments were determined by 0.6% agarose gel electrophoresis. The estimated sizes represent the mean length of a heterogeneous collection of fragments. * % = A2W residual matrix DNA/AZW undigested matrix DNA X 100. e % = band intensity of att-DNA fragmentband intensityof control DNA fragment X 100.

RESULTS

KpnI 1.2- and 1.5-kb Sequence Families and Subpopula- population), and less than 20% remained longer than 6.4 kb tions-Cleavage of purified AGM nuclear DNA by KpnI re- (KpnI-resistant population).As with the KpnI1.2-kb family, leased three distinct fragments of 1.2, 1.5, and 1.9 kb (Fig. 1, RsaI reducedthe KpnI1.5-kb family to a simpler set of smaller lane B ) . These discrete fragments are representatives of the fragments of 1.5, 0.927, 0.795, and 0.702 kb (Fig. 1, lane G). KpnI 1.2-, 1.5-, and 1.9-kb interspersed, repetitive sequence Less prominent, minor RsaI fragments which hybridize to the families in the AGM genome. Analysis of this restriction KpnI 1.5-kbprobe are visible on longerautoradiographic pattern by blot hybridization using cloned human 1.2- and exposures (data not shown). 1.5-kbsequence probes revealssequenceheterogeneity in These results indicate that although 60% of the 1.2- and these two KpnI families. The spacing andfrequency of KpnI 1.5-kb family sequences in AGM DNA have KpnI sites a t sites describe three populations of KpnI 1.2-kbfamilysespacings of 1.2 and 1.5 kb, respectively, other sequence popquences.Over 60% of the hybridizableAGM KpnI 1.2-kb ulations do exist.Moreover, in studying subfractions of AGM sequences were reduced to fragmentsof 1.2 kb and are denotedDNA or different preparations of nuclease-treatedmatrix the KpnI-sensitive population. Hybridization also occurred to DNA, it will be possible to analyze the three populations of segments of 2.7-6.4 kb (intermediate population) and toseg- each family either separately using KpnI restriction analysis ments greater than 6.4 kb (KpnI-resistant population) (Fig. or together by RsaI cleavage analysis. A fuller description of 1, lane D).These results indicate that in the KpnI 1.2-kb the shokt- and long-range organization of these KpnIsequence family, approximately 5% (intermediate population) and 25% families will be presented elsewhere (Chimera, 1984).3 (resistant population) of the sequences remained as longer Isolation and Characterization of Nuclear Matrices-RelaKpnI fragments. tively mild digestion of DNA-rich nuclear matrix with low RsaI restriction endonuclease recognizes the four internal concentrations of DNase I (80 units/ml) resulted in thegradnucleotides(5’-GTAC-3’) of the KpnI restriction site (5’- ual cleavage and release of DNAloopsfrom the matrix. GGTACC-3’). RsaI cleaved AGM DNA toproduce a contin- Depending on the extentof digestion, from 67 to 2.8% of the uum of fragments between 0.2 and 13 kb(Fig. 1, lane C). RsaI total DNA remained attached to theresidual nuclear matrix reduced the KpnI 1.2-kb family to a simpleset of three (Table I). Thisresidual DNA represents portionsof the loops fragments of 1.2, 0.835, and 0.408 kb (Fig. 1,lane E Chimera, that occur progressively nearer to the sites of loop attachment 1984). to the proteinaceous nuclearscaffold. Those DNA fragments Hybridization analysis with a cloned KpnI 1.5-kb family remaining with the residual matrix will bereferred to as probe reveals that this family’s KpnI cleavage products can attachment DNA (att-DNA)sequences. also be described as three fragment populations (Fig. 1, lane Analysis of att-DNA fragment lengths indicated that un0. Over 60% of the family sequences were reduced to 1.5-kb digested loops (>40 kb) were reduced to a mean length of 10 fragments (KpnI-sensitive population), about 15% were kb after mild digestion (Table I). It is possible that only a cleaved into fragments between 2.7 and 6.4 kb (intermediate portion of the 10-kb att-DNA fragments is actuallyinvolved in anchoring a DNA loop to the proteinaceous scaffold. To examine this possibility, DNA-rich nuclear matrix was diPortions of this paper (including “Experimental Procedures”and gested extensively with DNase I (160 units/ml). If a portion Figs. 3 and 4) are presented in miniprint at the end of this paper. of the 10-kb att-DNA isindeed anchored to protein, then it Miniprint is easily read with the aid of a standard magnifying glass. should be partially protected from DNase I cleavage and, as Full size photocopies are available from the Journal of Biological a result, exhibit larger fragment sizes than unprotected conChemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Doc- trol DNA. The data in Table I1 indicate that att-DNA reument No. 84M-3694, cite the authors, andinclude a check or money order for $3.40 per set of photocopies. Full size photocopies are also mained consistently larger than similarlydigestedcontrol included in the microfilm edition of the Journal thatis available from Waverly Press.

J. A. Chimera and P. R. Musich, manuscript in preparation.

Matrix Association of KpnZ DNAs TABLE I1 Comparison of DNA fragment sizes of att-DNA and control DNA after treatment with DNaseI DNA-richnuclear matrix and control (protein-free) DNA were digested extensivelywithDNase I (160 units/ml). At thetimes indicated, aliquots were removed, the DNA was purified, and the fragment sizes were determined. DNA fragment size DNase I incubation time

att-DNA Control >40 9.7

0 10 30 90

RESIDUAL DNA SIZE (kb)

24

DNA kb

rnin

0.4

I .2 kb FAMILY Film % Hybrid

>4 0

0

100

9.7

0

40

2.4

24

0.4

17

>40 7.7 1.3 co.1

I .5 kb FAMILY Film % Hybrid

-

0

100 44

25

FIG. 2. Hybridization of KpnI probes to residual att-DNA. DNA-rich nuclear matrix was extensively digested with DNase I. At various times, residual matrix was isolated and the att-DNA was purified. Equal amounts of att-DNA samples were denatured and slot-blotted onto Zeta-Probemembrane for hybridizationanalysis using KpnI 1.2- and 1.5-kb family probes. The per centhybridization (% Hybrid) value equals the ratio of the amount of hybridization to the att-DNA divided by the hybridization to untreated matrix DNA.

DNA. The most extensive DNase I treatment reduced attDNA to a mean fragment length of 0.4 kb, compared to~ 0 . 1 kb for control DNA. Thus, some portions of the 10-kb attDNA fragments appear to be more intimately associated with matrix proteins than do other portions. Presence of KpnI DNAs in the NuclearMatrix-To determine the relative amountsof KpnI sequences present in attDNA, DNA-rich nuclear matrix was digested with DNase I to various degrees. The extensive digestion conditions were used todeterminethecontent of KpnI sequences in the fragments most intimately associated with matrix proteins. Fig. 2 illustrates a gradual decrease in the total hybridizable 1.2- and 1.5-kb family sequences in att-DNA as the extent of degradation increases. The content of hybridizable 1.2-kb family sequences in att-DNA fragments of0.4 kb was less than 20% of the untreated DNA value. Similarly, att-DNA fragments of0.4 kb were depleted inhybridizable 1.5-kb family sequences to 25% of the initial value. The above analysis could not discriminate among the different sequence populations(KpnI-sensitive,intermediate, and KpnI-resistant)which constitute a KpnI family (see Fig. 1, lanes Dand F). This ambiguity was resolved by mildDNase I digestion of DNA-rich matrix, followed by KpnI treatment of the purified att-DNA. Mild DNase I conditions were required, since KpnI segments are greater than 1kb; they would be severely nicked under the extensive conditions used above and not resolvable as distinct restriction fragments.

9375

att-DNA fragments of 10 kb were reduced in their content of EBr-stained 1.2-kb segments to64%of the amount present in undigested DNA (Table I). A depletion in EBr-stained 1.5kb segments to 35% of the control amountwas also observed. Blot hybridization of the same KpnI segmentswith 1.2- and 1.5-kb family probes indicated that hybridizable 1.2-kb segments were reduced to 18% and 1.5-kb segments to 20% of their respective contents in undigested DNA (Table I). To determine if mild DNase I treatment preferentially released particular sequence subsets from the nuclear matrix, the RsaIcleavage patterns of isolated att-DNA were analyzed. The results of this analysis are summarized inTable 111. Hybridization with a KpnI 1.2-kb family probed revealed a reduction in all threeRsaI fragments in the att-DNA. When greater than 97% of the matrix DNA was released, the attDNA was depleted in 1.2-, 0.835-, and 0.405-kb fragments to 31, 45, and 23%,respectively. For the KpnI1.5-kb family, all four major RsaI fragments were depleted 1 5 , 0.927-, 0.795-, and 0.702-kb fragments were reduced to 49, 43,23, and 23%, respectively. The differences in the calculated amounts of fragment depletion are not due to variations in hybridization efficiencies due to fragments of different size. The control DNA in each measurement consisted of fragments of the same size as the experimental fragments. Therefore, the different depletions for various RsaI fragment mustreflect differential protection by the nuclear matrix proteins. Asignificantdifferencewasobserved in the amount of hybridizable 1.2-kb fragments detected in the KpnI digest uersus the RsaI digest of att-DNA (Tables I and 111). This difference (18 uersus 31%, respectively) reflects the contribution of fragments from the RsaIcleavage of the intermediate and KpnI-resistant populations in the att-DNA preparation (Fig. 1, lune E; Chimera, 1984). The hybridizable segments found in RsaI digests of att-DNA, therefore, represent the cumulative contributionsfrom the three sequence populations in the 1.2-kb family. Likewise, the different amounts of hybridizable 1.5-kb (KpnI-sensitive) segments detected in the KpnI and RsaIdigests of att-DNA have a similar explanation. Metrizamide Gradient Fractionation of KpnI-digested Matrix-We have demonstrated that the DNA component of the TABLE111 Content of RsaI segments of KpnI families in att-DNA DNase I-treated matrix, containing 2.8% of the input DNA, was isolated. Equal amounts of purified att-DNA were digested with RsaI, separated on a1.0% agarose gel, and blotted to Zeta-Probe membrane for hybridization analysis with KpnI 1.2- and 1.5-kb family probes. Control DNA" Att-DNAb ?6

1.2-kb family RsaI fragment size (kb)' 1.2 0.835 0.408 1.5-kb family RsaI fragment size (kb)' 1.5 0.927 0.795 0.702

100 100 100

31 45 23

100 100 100 100

49 43 23 23

The control DNA was prepared from undigested DNA-rich matrix. The contentof the RsaI fragments in the att-DNAwas calculated as theper cent value relative to the contentof respective fragment in the control DNA. The per centvalues were calculated by densitometric quantitation of the bands in autoradiograms of the appropriate blot hybridizations. These RsaI fragments contain sequences from all three populations (KpnI-sensitive, intermediate, and KpnI-resistant).

9376

Matrix Association DNAs of KpnI

residual matrix contains members from the KpnI 1.2- and 1.5kb families. To gain an understanding of the possible role(s) of KpnI DNAs in the anchoringof DNA loops to the matrix, methods were developed to isolate KpnI DNA-protein complexes. Nuclei were subjected to a series of high salt (2 M NaC1) bufferextractionsanddifferentialcentrifugationstepsto removeionically bound histones and nonhistone proteins. Those proteins remaining are the nuclear matrix proteins. The DNA-rich nuclear matrix was digested with KpnI, and the digestion products were fractionated by isopycnic centrifugation inmetrizamidegradients.The bulk of theDNA banded a t a density of 1.14-1.16 g/cm3 and was contained in fractions 9, 10, and 11. Lesser amounts of DNA were found in fractions with higher densities (Fig. 3). This distribution represents a continuum of DNA complexed with increasing amounts of bound protein. In metrizamide gradients in low salt buffers, protein-free DNA bands at a density of approximately 1.12 g/cm3, while DNA-protein complexes band at densities ranging from 1.185 to 1.247 g/cm3 (Rickwood, 1976; Monahan and Hall, 1974). The densities reported here are 0.02-0.04 g/cm3 higher due to the1 M NaCl in the gradients, which decreases the hydration of the macromolecules, resulting in an increased buoyant density (Rickwood, 1976). The two smaller peaks, observed in fractions 16-18 and 19-21, do not appear to be DNA andmay represent ribonucleoprotein contaminants. The majorityof the 1.2-kb family sequences were found in fractions 9, 10, and 11, which contained greater than 65% of the DNA loaded on the gradient (Fig. 4A). Some of these fragments represent protein-free sequences cleaved from the DNA loops of the matrix by KpnI endonuclease. Relative to the intermediate and KpnI-resistant populations there is an enrichment for the KpnI-sensitive population of segments (1.2 kb) in fractions with increasing density (see Table IV). For example, the DNA in fraction 13 exhibited an approximate 2-fold relative enrichment for1.2-kbsegments. The majority of 1.5-kbfamilysequences were also presentin fractions 9, 10, and 11 (Fig. 4B),althoughthere was an approximateiy 1.7-fold enrichment for the 1.5-kb segments in fraction 13 (Table IV). The higher buoyant densities in this portion of the metrizamide gradients (-1.19 g/cm3) indicate that these 1.2- and 1.5-kb fragments were associated with matrix proteins. Thus, certain membersof both the 1.2- and 1.5-kbfamilies appeartoremainassociatedwithgreater amounts of matrixproteinsand, by implication,mustbe differentially associated with these proteins. The amountsof protein in thehigher density regions of the gradientswere too low for their characterization by sodium dodecyl sulfate gel electrophoresis using the sensitive silver staining procedure (Wray, 1981). It should be noted that the DNA in gradient fraction 9 was depleted almost 10-fold in 1.2-kb segments and greater than 3-fold in 1.5-kbsegments(TableIV).These decreases are consistent with the shift of some 1.2- and 1.5-kb segments to the higher density portion of the gradients. In control studies, bacteriophageT 3 DNA, either intact or after cleavage with MboI (Sarkar et al., 1980), was incubated in the nuclear matrix preparation before loading in the metrizamide gradient. All the T3 DNA banded at a density of 1.14-1.15 g/cm3 (data not shown), indicating that there had been no redistribution of matrix proteins onto the bacteriophage DNA. In another gradient containinga KpnI digest of purified AGM DNA, all the KpnI fragments were found at a density of 1.14-1.15 g/cm3 (data not shown). Thus, the observed banding of KpnI 1.2- and 1.5-kb fragments a t densities of -1.19 g/cm3 does not appear to result from unusual density

TABLE IV Distribution of KpnZ sequence families in metrizamide gradients DNA-rich nuclear matrix wascleaved with KpnI endonuclease, and the totaldigest was sedimented to equilibrium in a metrizamide density gradient. The DNA in each of the fractions was purified and redigested with KpnI before electrophoresis on a 0.7% agarose gel. The DNAwas blotted to diazobenzyloxymethyl paper andhybridized with radioactive probes to either theKpnI 1.2- or the 1.5-kb family. Fraction No.

Total DNA 9 10 11 12 13 14-16

1.2-kb fragments"

1.5-kb fragments"

total 1.2-kb family

total 1.5-kbfamily

0.42 0.05 0.48 0.70 0.72 0.80 NDb

0.45 0.10 0.36 0.54 0.73 0.77 NDb

The hybridization distribution patterns in the autoradiograms shown in Fig. 4 were quantitated by densitometric scanning. The ratios listed represent the relative amountof probe hybridization to the 1.2- or 1.5-kb segments relative to the total hybridization to the DNA in that fraction. * ND, not determined; the intensity of hybridization to the 1.2- and 1.5-kb fragmentsin fractions 14-16, although detectable, was too low for accurate quantitation.

properties of these fragments, their size, or the formation of nonspecific DNA-protein complexes during gradient preparation. DISCUSSION

The structural organization of the eukaryotic nucleus has been the subject of intense investigation. DNAis arranged in the nucleus as loops of 60-100 kb, which are anchored to a proteinaceous nuclear matrix (Cook and Brazil, 1975, 1976; Hancock, 1982b). The nature of the DNA attachment sequences which anchor the loops and their binding to the matrix proteins has not yet been established. The data presented here suggest that 2.8% of the AGM nuclear DNA, in DNA fragments of about 10 kb, is associated with nuclear matrix proteins. Thisvalue presumes that the nuclear matrix proteins afforded protection to att-DNA againstmild DNase I digestion. More extensive DNase treatments indicate that within the 10-kb att-DNA fragments, oneor more regions of about 0.4 kb appear to be more intimately associated with matrix proteins than other regions. Thus, when examining att-DNA for specific DNA sequence classes, it is important to consider how experimental conditions may influence the size and composition of the att-DNA fragments in the residual matrix. Mild nuclease digestion conditions may allow extraneous DNA in the loops to remain associatedwith the matrix, while extensive digestion conditions could release less protected but integralregions of the att-DNA fragments. In this investigation, both mildly and extensively digested nuclear matrix preparations were examined for their relative content of the long, interspersed, repetitive KpnI DNAs. If KpnI families serve as att-DNAs for the anchoringof all DNA loops, one would expect them to be highly enriched in the population of att-DNA fragments. Conversely, if none of the KpnI sequences are involved in loop anchoring, then they should be absent in att-DNA. Our results indicate that althoughpresentingreatly reduced amounts,theKpnIsequences persist in att-DNA. The relative content of hybridizable 1.2-kb familymembers was depleted in att-DNA isolated from matrix subjected to extensive DNase I digestion. A similar depletion of hybridizable 1.5-kb family sequences was also detected. It should be emphasized that the matrixwas extensively digested t o frag-

Matrix Associationof KpnI DNAs

9377

hnRNA transcripts, in addition to a more detailed analysisof ments of approximately 0.4 kb, and the resulting att-DNA AGM must be operationally defined as those fragments that resist the matrix att-DNA and matrix-associated RNAs in cells. DNase I cleavage under these extremedigestion conditions. The differential association of some KpnI segments with In order to examine the content of the individual KpnI sequence populations (KpnI-sensitive u e r s u KpnI-resistant) matrix proteins was also demonstrated by metrizamide grain att-DNA, mild DNase I digestion conditions were chosen. dient centrifugation. Although the bulk of the hybridizable sequences were in the protein-free DNA fractions, some KpnI Both general (EBr) and specific (blothybridization)techniques revealeda reduction in the amount of KpnI 1.2-kb fragments banded as protein-DNA complexes.Specifically, segments. However, a greater depletion was detected by hy- the KpnI-sensitive populationsof the 1.2- and 1.5-kb families of bridization analysis thanby EBr staining. Thissuggests that were enrichedinfractionswithdensitiescharacteristic some fragments in the 1.2-kb stained band represent a sub- DNA-protein complexes. It should be noted that the continpopulation of the 1.2-kb sequence familythat lackshomology uum of the KpnI fragment distributions in the gradient has to the cloned human probe used here. Furthermore, the dif- two possible interpretations. Onepossibility is that the DNAs of intermediate density contain fragments withdecreasing ference between the staining and the hybridization results suggests that populations of the nonhomologous 1.2-kb seg- amounts of bound protein per unit fragment length (i.e. the gradual shift of 1.2- and 1.5-kb fragments). Alternatively, a ments may beenrichedintheatt-DNA relative tothose studied here. To address this possibility, these non-homolo- particular sequence may maintain a constantamount of gous 1.2-kb KpnI fragments are being cloned from the att- bound protein per unit length, but may be linked in longer amount of DNApreparations for direct analysis: Analysis of 1.5-kb DNA fragments which,overall,haveareduced segments also revealed that they were present in att-DNA in bound protein. Thispossibility may apply to the intermediate reduced amounts, indicating that the bulk of the 1.5-kb se- and KpnI-resistant populations of the KpnI 1.2- and 1.5-kb families, which were found in att-DNA. quences are not matrix-associated. This methodof digesting DNA-rich matrixwith restriction IndividualKpnI family subsegments, releasedfrom attDNA by RsaI, were also examined. All segments belonging to enzymes and fractionating the DNA-proteincomplexes from both the 1.2- and 1.5-kbfamilies were depletedin mildly the protein-free DNA has numerous applications to the study digested matrix. Someof the RsaI fragmentsin att-DNA were of regulatory andstructuralDNA-proteininteractions.In depleted more than other fragments, suggesting that RsaI repetitive sequence families, the members exhibitingdiffering cleavage analysis can resolve different sequence subsets in degrees of (matrix) protein association can be separated on att-DNA preparations. The data in Table I11 indicate that in metrizamide gradients according to the amountof associated the KpnI 1.2-kb family the sequences in the 0.835-kb RsaI protein. Thus, particular sequence fragments with their asfragment are most protected and those in the 0.408-kb frag- sociated proteins can be isolated for further detailed study. ment are least protected against DNase I cleavage by the Relative to the present study, scaling up of the procedure will matrix proteins. The nature of these differences in matrix permit the isolationof the KpnI 1.2- and 1.5-kb family memassociation with the various sequence subsets remains to be bersactuallypresentinDNA-protein complexes. The seresolved. However, these and other observationssuggest sev- quence subset composition of the DNA and the proteincomeral interesting speculations about the possible function(s) of position of the complexes canthen be determined.Such the KpnI sequences in the nuclear matrix. Nuclear transcrip-experimental approaches are necessary to further our undertion occurs at the matrix (Jacksonet al., 1981) to which both standing of the DNA sequence and polypeptide specificities active genes (Robinson et al., 1982; Hentzen et al., 1984) and of the DNA-protein interactions for gene regulation and the heterogeneous nuclear RNA (hnRNA) (van Eekelen and van dynamic structural organizationof the nucleus. Venrooij, 1981) have been found associated. Shafit-Zagardo Acknowledgments-We would like to thank Rhesa Dykes for her et al. (1982b, 1983) have observed an interspersion of KpnI sequences with gene domains and the transcription of KpnI technicalassistance and Dr. Lee Pike for critical reading of this manuscript. families intohnRNA. Moreover,sequence analysis of a cloned, representative member of the KpnI 1.2-kb family of REFERENCES AGM DNA has revealed a copy of the hexanucleotide poly- Birnhoim, H. C., and Doly, J. (1979) Nucleic Acids Res. 7 , 1513-1523 adenylationsignal sequence(5’-AATAAA-3’) withinthe Cook, P. R. and Brazell, I. A. (1975) J . Cell Sci. 19, 261-279 0.835-kb RsaI subfragment (Chimera, 1984). This signal se- Cook, P. R., Brazell, I. A., and Jost, E. (1976) J . Cell Sci. 22, 303324 quence, characteristic of DNA transcribed by RNA polymerase 11, implies that this cloned DNA may represent the KpnI Chimera, J. A. (1984) Ph.D. dissertation, East Tennessee State University 1.2-kb family sequences actually transcribed into hnRNA in Christophe, D., Brocas, H., and Vassart, G . (1982) Anal. Biochern. the cell. This possibility is consistent with our observation 120,259-261 that in the nuclear matrix the0.835-kb RsaI sequence subset Hancock, R. (1982a) Int. Reu. Cytol. 7 9 , 165-214 is relativelymore resistanttoDNase I thanother 1.2-kb Hancock, R. (198213) Biol. Cell 4 9 , 105-122 family sequence subsets. A similar relationship between the Heller, D., Jackson, M., and Leinwand, L. (1984) J. Mol. Biol. 1 7 3 , 419-436 transcriptionallyactiveandthe relativelymore DNaseIHentzen, P. C., Rho, J. H., and Bekhor, I. (1984) Proc. Natl. Acad. resistant RsaI subfragments in theKpnI 1.5-kb family is also sci. U. S. A . 81, 304-307 possible but more speculative. Some support for these specu- Jackson, D. A., McCready, S. J., and Cook, P. R. (1981) Nature 292, lative proposals is in the finding of long open translational 552-555 reading frames in the human KpnI families whose members Kurnit, D. M., and Maio, J. J. (1973) Chrornosorna (Berl.) 4 2 , 23-36 are also represented in hnRNA (Potter, 1984; Shafit-Zagardo Maio, J. J., Brown, F. L., McKenna, W . G., and Musich, P. R. (1981) Chrornosorna (Berl.)83, 127-144 et al., 1983). Verification of these proposed functional-strucManiatis, T., Fritsch, E. F., and Samhrook, J. (1982) Molecular tural relationships will require further sequence characteriCloning, pp. 468-469, Cold Spring Harbor Laboratory,Cold Spring zation of genomic and cloned KpnI family members and their Harbor, NY P. R. Musich, work in progress.

Monahan, J. J., and Hall, R. H. (1974) Nucleic Acids Res. 1, 13591370 Musich, P. R., Brown, F. L., and Maio, J. J. (1980) Chromosorna

Matrix Association of KpnI DNAs

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(Bed.) 80,331-348 Potter, S. S. (1984) Proc. Natl. Acud. Sci. U.S. A. 81, 1012-1016 Rickwood, D. (1976) in Biological Separations in Iodinated Density GradientMedia (Rickwood, D.,ed) pp. 27-40, Information Retrieval Limited, Washington, D. C. Robinson, S. I., Nelkin, B. D., and Vogelstein, B. (1982) Cell 28, 99106 Sarkar, P., Adhya, S., Musich, P. R., and Maitra, U. (1980) Gene (Amst.) 12, 161-163 Shafit-Zagardo, B., Brown, F. L., Maio, J. J., and Adams, J. W.

MINIPAINT SUPPLEMENT T h eA s s o c i a t i o n

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D N a s e I D i g e s t i o n g P u r i f i e d DNA--In c o n t r o l e x p e r i m e n t s p u r i f i e d ,h i g hm o l e c u l a rw e i g h t CV-1 D N A w a s a d j u s t e d t o HS b u f f e r c o n d i t i o n sa n d a t a f i n e 1 DNA c o n c e n t r a t i o n o f 0.5 m g / m l . The s o l u t i o n was p r e w a r m e d t o 3 7 ° C f o p 5 m i n u t e s a n d D N a s e I a d d e d t o A t v a ~ i o u stimes d u P i n gt h ed i g e s t i o n equal a l i 1 6 0u n i t s l m l . 4 v o l u m e s o f HS b u f f e r q u o t s were r e m o v e d f ~ o mt h es a m p l ea n d a d d e da n dt h e D N A p r e c i p i t a t e dw i t h2 . 5v o l u m e s o f 9 5 %e t h a n o l . D N A was r e s u s p e n d e da n dp u r i f i e d as d e s c r i b e d T h ep r e c i p i t a t e d above.

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(1982b) Gene (Amst.) 20,397-407 Shafit-Zagardo, B., Brown, F. L., Zavodny, P. J., and Maio, J. J. (1983) Nature 304, 277-280 Singer, M. F. (1982) Cell 28,433-434 Singer, M.F., Thayer, R. E., Grimaldi, G., Lerman, M. I., and Fanning, T. G . (1983) Nucleic Acids Res. 11,5739-5745 Southern, E. M. (1975) J. Mol. Biol. 98,503-517 van Eekelen, C. A., and van Venrooij, W. J. (1981) J. Cell Biol. 88, 554-563 Wray, W. (1981) Anal. Biochern. 118, 197-203

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DNA Preparations--AGH total n u c l e a Dr N A w a s p ~ e p a r e d from train C V - 1 c e l l s as d e s c r i b e db yK u r n i at n dM a i o( 1 9 7 3 ) C. o n t r o l m a t r i x D N A w a s p ~ e p a ~ e d s i m i l a r l fyr o m u n d i g e s t e dD N A - r i c hn u c l e a r m a t r i xl y s a t e s( d e s c r i b e db e l o w ) . D N A w a s i s o l a t e df r o m n u c l e a s ed i g e s t e d m a t r i xs a m p l e s b yt r e a t m e n tw i t h Protease K i n e x t r a c t e d and a n SDS-EDTA b u f f e r a t 37OC f o r 1 h o u r t, h e np h e n o l e t h a n o lp r e c i p i t a t e d T . op r e p a r e D N A f r o mm e t r i z a m i d eg p a d i e n t Of f ~ a c t i o n s t h e m e t r i z a m i d e w a s f i r s tr e m o v e db yp r e c i p a t a t i o n t h e D N A w i t he t h a n o l T . h ep r e c i p i t a t e dD N A - p r o t e i nc o m p l e x e ~ w e r e t h e nt p e a t e d a s d e s c r i b e da b o v e f o r t h en u c l e a z ed i g e s t e d m a t r i xs a m p l e s .

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B a c t e r i o p h a g eT 3 D N A w a s a g i f tf r o mU m a d a sM a i t r a . The were c a r r i e d in 20" s t p a i n p l a ~ m i dD N A s u s e di nt h i s t u d y F O Pp r e p a r a C 6 0 0 c e l l s a n d w e r e o b t a i n e d from J o s e p h J. H a i o . were a m p l i f i e d u s i n g c h l o r a m p h e n i t i o n of t h e i r D N A t h ep l a s m i d s c o la n di s o l a t e du s i n gt h ea l k a l i n el y s i sp r o c e d u r e( B i r n b o i ma n d D o l y 1, 9 7 9 ) .

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of D N A - r i c hN u o l e a rM a t r i x - - R a n d o m l yc y c l i n g CV-1 c e l l s w e r e i s o l a t e d f v o m s u b c o n f l u e n ct e l cl u l t u r e s A . p p r o x i m a t e l y5 x 1 0 ' c e l l s w e r e h y p o t o n i c a l l ys w o l l e n f o r 1 0 m i n u t e s in l o wm a g n e s i u m b u f f e(rL H : 1 0 m M T r i s - H C 10, . 2 mM MgCl , pH 1 . 4 ) T . h i sa n da l l s u b s e q u e n ts t e p s i n t h ei s o l a t i o n o f th:DNA-rich n u c l e a rm a t r i x w e r e p e r f o r m e d a t O - Z O C a n d in b u f f e r s c o n t a i n i n g 1 mH p h e n y l m e T h es w o l l e n c e l l s WePe t h y l s u l f o n y lf l u o r i d ea n d 2 m M EGTA. l y s e d b y a d d i t i o n o f ~ o n i d e t p - U O t o 1 % a n d h o m o g e n i z a t i o n i na D o u n e eh o m o g e n i z e rw i t h 1 0 s t r o k e s o f t h el o o s e - f i t t i n gp e s t l e . N u c l e i w e r e c o l l e c t e db yc e n t r i f u g a t i o na t 8 0 0 x g f o r 1 0m i n u t e s release t h e a n dr e s u s p e n d e di n LM b u f f e r T . ol y s et h en u c l e ai n d from t h ec h p o m a t i np r o t e i n sa ne q u a l D N A - r i c hn u c l e a rm a t r i x was v o l u m e o f 4 H N a C l , 2 m W H s C l , 1 0 m H T r i s - H C 1( p H7 . 4 ) s l o w l ya d d e dw i t hg e n t l es t i r z i n gw i t h a T e f l o nr a d T . h es a m p l e a t t h i s s t a g e , r e f e r r e d t o as t h eD N A - r i c hn u c l e a rm a t r i xl y s a t e , w a s t h e ~ t a r t i n gm a t e r i a l f o r t h ee x p e r i m e n t so u t l i n e d below. DNase ""-

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I "T "_ r e a t m e n t o f D N A - r i c hN u c l e a rm a t r i x - - T h eD N A - r i c h nuclea; m a t r i x l y s s t e w a 9 a d j u s t e dt o a D N A c o n c e n t r a t i o n O f 0.5 mg/mlwith a high salt (HS) buffer containing 2 H NaC1, 1 mH H g C1l0 m M T r i s - H C(1p H 1 . 4 )T. hley s a t e w a s p r e w a r m e tdo 31'C f o r ? ' m i n u t e sb e f o r ea d d i t i o n of D N a s e I . F O P m i l dd i g e s t i o n s t u d i e s ~ ~ a Is was e a d d e dt o 8 0 u n i t s / m lw h i l et h ee n z y m e was a d d e dt o1 6 0u n l t s l m fl o re x t e n s i v ed i g e s t i o nc o n d i t i o n s b. t v a p i o u s t i m e i n t e r v a l se q u a la l i q u o t s of t h e d i g e s t i o n s o l u t l o n were r e m o v e d a n d a d d e d t o 4 v o l u m e s o f i c e c o l d HS b u f f e r t o s t o p t h e DNase I r e a c t i o n .T h er e s i d u a ln u c l e a rm a t r i x was c o l l e c t e d b y c e n t r i f u g a t i o n a t 5 0 0 0 x g in a s w i n g b u c k e t r o t o r ( B e c k m a n J S - 1 3 ) f o r 2 5m i n u t e s a t 4OC. T h e DNA w a s p u r i f i e d f r o m t h e r e s i d u a lm a t r i x as d e s c r i b e d above.

D i g e s t i o n o f D N A - r i c hN u c l e a rM a t r i x - - T o remove t h en u c l e a r p r o t e i n s r e l e a s e d i n t o t h e l y s a t e b y t h e h i g h s a l t from t h e D N A r i c hn u c l e a rm a t r i x , a s e r i e s of HS D u f f e r w a s h e sa n dd i f f e r e n l y s a t e was t i a l c e n t r i f u g a t i o ns t e p s w e r e u s e d . T h en u c l e a r d i l u t e d t o 2 5 0 m l w i t h HS b u f f e r a n d c e n t p i f u g e d a t 1 0 , 0 0 0 x g f o r 2 5m i n u t e s a t 4'C i n a s w i n g b u c k e pt o t o r( B e c k m a nJ S - 1 . 5 ) . w a s r e p e a t e dt w l c et oe n s u r ec o m p l e t er e m o v a l of T h ew a s hs t e p a l l n u c l e a rp ~ o t e i n sd i s s a o i a t e d in t h e 2 H NaCl c o n d i t i o n s T. h e was d i a l y z e da g a i n s t1 0 0 0v o l r e s u l t i n gD N A - r i c hn u c l e a rm a t r i x L m e s o f K p n Ir e s t r i c t i o nb u f f e r( 6 mH T r i s - H C 1( p H1 . 5 ) , 6 mM NaC1, 6 mH MgCl 6 m H 2 - m e r c a p t o e t h a n o l ) f o r 1 6h o u r sa t4 ° C . t%e s a m p l e w a 3 w a r m e dt o 37'C, K p n Ir e s t r i c t i o n A f t e rd i a l y s i s e n z y m e( B e t h e s d aR e ~ e a r c hL a b s . G , aithersburg, MD) a d d e dt o 1 u n i t l u g D N A a n dt h e= a m p l ei n c u b a t e d f o r 1 2h o u r s a t 31'C. A s e c o n da l i q u o t o f e n z y m e( 0 . 5 u n i t / u g DNA) was a d d e da n dt h e f o r t h r e ea d d i t i o n a lh o u r s .T h ed i g e s t was i n c u b a t i o nc o n t i n u e d t h e n c h i l l e d t o 4OC a n d m a d e 1 M i n N a C l - L M b u f f e r a n d 45% i w l v ) in m e t v i z a m i d e( A e e u r a t eC h e m i c a l a n d S c i e n t i f i cC o r p . ,W e s t b u r y , N.Y.). Metrizamide Gradient vltracentrifu~tion--The KpnI d i g ne us -t e d c l e a rm a t r i xc o m p l e x e s w e ~ es u b j e c t e d t o m e t r i z a m i d e g p a d i e n t centrifugationT . h eg r a d i e n t s w e r e p r e p a r e di nt h ec e n t r i f u g a i n 2 0 m l o f l H NaC1-LH t i o nt u b e sb yd i s s o l v i n gm e t r i z a m i d e a final c o n c e n t r a t i o n of 4 0 % (w/v). T h i ss o l u t i o n was b u f f e rt o u n d e r l a y e dw i t h 5.0 m l o f K p n Im a t r i xd i g e s tp r e p a r e d as d e s at c p i b e da b o v e T . h eg r a d i e n t sf o r m e dd u r i n gt h ec e n t r i f u g a t i o n 3 0 , 0 0 0 ppm a n d 5OC f o rI 2h o u r si n a B e c k m a nT i - I Or o t o r T. h e i n 1 m l f r a c t i o n sb yP u m p g r a d i e n t s w e r e collected from t h et o p i n gF l u o r i n e r t RF C - 4 0( I n s t r u m e n tS p e c i a l t i e 3 Co., L i n c o l n , N e b r . i) n t ot h eb o t t o m o f t h et u b et od i s p l a c et h eg r a d i e n t T . he w e r e c a l c u l a t e df r o mt h e refracd e n s i t i e s ( p ) o f t h ef r a c t i o n s t i v ei n d e x (q) u s i n g t h ef o l l o w i n gf o r m u l a : p z 0 o : 3.350?200 3 . 4 6(2R i c k w o o d1,9 1 6 ) . To m e a s u r e i t s d i s t r i b u t i o ni nt h e remove t h e g r a d i e n t s D N A was f i ~ s t e t h a n o l p r e c i p i t a t e d t o m e t r i z a m i d ea n dt h e na s s a y e d u s i n g t h ee t h i d i u mb r o m i d e flUOFeS~ e n c e t e c h n i q u (e M a n i a t i s e t al., 1982). " " " "

-

_" DNA

"_"

G e l E l e c t r o p o y e q t p and B3ot-hybridization A n a l x j s i s - - D N A f r a g m e n t s w e ~ eseparated b y electrophoresis i n h o p i z o n t a l a g a r o s e g e l sc o n t a i n i n g 0.5 u g l molEf B rT. h e r u n n i n g b u f f e ?c o n t a i n e d 5 0 m M T r i s , 50 mH b o p i c a c i d a n d 2 m M EDTA. D N A s i z e s t a n d a r d s f o r t h ec a l c u l a t i o no ff r a g m e n tl e n g h t sc o n s i s t e d of b a c t e r i o p h a g eT 3 D N A c l e a v e dw i t hM b o I( S a r k a re t al., 1 9 8 0 ) o r p B R 3 2 2 D N A c l e a v e dw i t h H i n f I ( M a n i a t i s e t a l .1, 9 8 2 )A. f t e P electroD N A i n t h eg e l s w a s d e n a t u r e da n d p h o r e s i sa n dp h o t o g r a p h yt h e o f S o u t h e r n( 1 9 1 5 ) t r a n s f e r r e dt os o l i ds u p p o r t sb yt h em e t h o d u s i n g 12X SSC a s t h et r a n s f e r Duffer. T h es o l i ds u p p o r t s Were 8 8 5n i t r o c e l l u l o s e( S c h l e i o h e r k S c h u e l lK , eene, N.H.) a n d Z e t a P P o b em e m b r a n e (s B i o - R a dR, o c k v i l l eC e n t e r , N.Y.) OII DBH p a p e r s yhristophe e t a l . (1982)). Slot( p r e p a r e d a s d e s c r i b e bd C b l o t t i n g v a s d o n eo n t oZ e t aP r o b em e m b r a n e sb yg r a v i t yf i l t r a t i o n o fd e n a t u r e d D N A t h p o u g h a m a n i f o l d (SAC 0 1 2 / 0 ,S c h l e i c h e r b S c h u e l 1 , K e e n e , N.H.). "

Matrix Association DNAs of KpnI The hybvidization probes used in this study were the r e c o r n binant plasmids pBK(1.2)12 and pBK(1.5)54. These plasmids containhumanDNAinsert3representingtheKpnI 1.2 kbandtheKpnI 1.5 kb families, respectively, o f t h e p ~ i m a t e i n t e r s p e r s e dr e p e titive DNAs (Shafit-Zagardo et a l . , 1982a). The plasmids were radioactively labelled in the following manner. Plasmid DNA (100 uglrnl) was partially cleaved with 0.23 unitslml of micrococcal n u c l e a s e ( W o r t h i n g t o n , F ~ e e h o l d ,N.J.) in 5 m H Tris-HC1 (pH 7.4), 1.5 m H C a C l b u f f e r a t 2 5 O C f o r1 m i n u t e a n d t h e D N A p u r i f i eby d phenol extrzotion and ethanol precipitation. This tPeatment reduced the plasmid DNA to a h e t e r o g e n e o u s c o l l e ~ t i o no f fragments between 1 0 0 and 1000 bp in length with 5"OH termini. These termini were labelled with ATP g a m m a 32p (crude) (ICN, Irvine, CA) using TU polynucleotide kinase (Bethesda Research Labs, Gaithersburg. YD) a s described by Haniatis et al. (1982). The minimum specificities obtained w e r e 2 x lo7 c p m l u g DNA. Thehybridizationoftheprobe tothemembranescontaining DNA were Derformed in a 3 X SSC-10X Denhardts buffer and autoradioKPaPhed a s described p~eviously (Musich et a l . , 1980). The probe was stripped off the Zeta Probe membranes or the DBM papers UainR 0 . 0 N NaOH at 4 2 O C for 30 minutes. The filters could then be rehybridired with andthe? probe.

9379

A 2.7-

I .2-

B 2.7-

I .5IO

12

14

I 6 Total

GRADIENTFRACTIONNUMBER Fraction Number Figure 3. Isopycnic centrifugation o f nuclear matrix complexes In a HetrIzamide gradient. DNA-rich matrix was digested with KpnI and subjected to density gradient centrifugation in metrizamide. Fractions were collected from the top and their refractive index measured to determine the density (-8.m). Equal aliquots were removed, the DNA was purified and its concentration (-) determined by the Pelative EBr fluorescence assay.

Figure 4. Distribution of KpnI 1.2 kb and 1.5 kb family sequenc e s i n metrizamide gradient fractions. The DNA In equal volume aliquots of the gpadient fractions ( s e e Fig. 3) was purified, redigested with KpnI to e n s u r e complete restriction cleavage, and electrophoresed in a 0.71 a g a r o s e gel. The gel Included a KPnI digest of total ACM n u c l e a ~DNA a s a n internal standard. The DNA was tPansfePred from the gel to DBM paper for hybridization with e i t h e r a K p n I 1.2 k b ( P a n e l A) o r a K p n I 1.5 k b ( P a n e l B ) f a m i l y pvobe.