Cell, Vol. 18, 1079-l

090. December

1979,

Copyright

0 1979

by MIT

Isolation and Characterization of the Nuclear Matrix in Friend Erythroleukemia Cells: Chromatin and hnRNA Interactions with the Nuclear Matrix Byron H. Long, Chen-Ya Huang and A. Oscar Laboratory of Cell Biology Lindsley F. Kimball Research Institute of The New York Blood Center 310 East 67th Street New York, New York 10021

Pogo

Summary Nuclear matrices from undifferentiated and differentiated Friend erythroleukemia cells have been obtained by a method which removes DNA in a physiological buffer. These matrices preserved the characteristic topographical distribution of condensed and diffuse “chromatin” regions, as do nuclei in situ or isolated nuclei. Histone Hl was released from the nuclear matrix of undifferentiated cells by 0.3 M KCI; inner core histones were released by 1 M KCI. Nuclear matrix from differentiated cells did not maintain Hl , and histone cores were fully released in 0.7 M KCI. KCI removed the core histones as an octameric structure with no evidence of preferential release of any single histone. Electron microscopy of KCI-treated matrix revealed no condensed regions but rather a network of fibrils in the whole DNA-depleted nuclei. When nuclear matrices from both types of cell were exposed to conditions of very low ionic strength, inner core histones and condensed regions remained. These observations support the contention that inner core histones are bound to matrix through natural ionic bonds or saline-labile elements, and that these interactions are implicated in chromatin condensation. hnRNA remained undegraded and tenaciously associated to the matrix fibrils, and was released only by chemical means which, by breaking hydrophobic and hydrogen bonds, produced matrix lysis. Very few nonhistone proteins were released upon complete digestion of DNA from either type of nuclei. The remaining nonhistone proteins represent a large number of species of which the majority may be matrix components. The molecular architecture in both condensed and diffuse regions of interphase nuclei appears to be constructed of two distinct kinds of fibers; the thicker chromatin fibers are interwoven with the thinner matrix fibers. The latter are formed by a heteropolymer of many different proteins. Introduction The first evidence for the existence of a highly ordered structure within the nucleus, aside from chromatin and nucleoli, was obtained when Mirsky and Ris (1951) introduced high salt treatment and DNAase digestion into cell nucleus studies. These procedures yielded a structure, composed primarily of proteins, which the

investigators interpreted as “residual chromosomal proteins.” The first electron microscopic observation of such a residual nuclear structure was made by Georgiev and Chentsov (1960), again using high salt treatment and DNAase digestion of rat liver nuclei. Several groups have recently reported the existence of this highly ordered structure, the nuclear matrix or nuclear skeleton, in a variety of nuclei (Shankara Narayan et al., 1967; Berezney and Coffey, 1974; Riley, Keller and Hyeres, 1975; Scheer et al., 1976; Wunderlich and Herlan, 1977; Herman, Weymouth and Penman, 1978; Miller, Huang and Pogo, 1978a). Our earlier studies on rat liver nuclei indicated that heterogeneous RNA (hnRNA) molecules do not exist as ribonucleoprotein particles but are in intimate association with a network (Faiferman and Pogo, 1975). More recently we have demonstrated that this network of thin fibrils is a matrix structure (Miller et al., 1978a). hnRNA is not an integral component of the matrix, since RNAase digestion can release the majority of hnRNA without altering the matrix architecture. We have observed that all the small molecular weight nuclear RNAs also are matrix components, and have suggested that some of them may play a structural role since they are metabolically stable and highly resistant to RNAase (Miller, Huang and Pogo, 1978b). Furthermore, we have anticipated that matrix fibrils, which have a zigzag configuration, may be in close contact with chromatin fibers and perhaps follow the contours of these fibers (Miller et al., 1978a). We now believe that a new experimental approach is necessary to determine the molecular architecture of the nuclear matrix and its interactions with both chromatin and hnRNA. This line of investigation is essential for the elucidation of the matrix role in the organization of DNA-histone complexes, primary transcripts and processing and transport of mRNA, as well as for clarification of function in the processes that lead to the formation of chromosomes in the prophase stage of the mitotic cycle. We decided to perform this study with undifferentiated and differentiated Friend erythroleukemia cells because they have two useful features (Friend, Preisler and Scher, 1974). One is the modification of interphase nuclei as a result of differentiation; undifferentiated cells have nuclei in which most of the chromatin is diffuse, while differentiated cells contain nuclei with mostly condensed chromatin. The other feature is the possibility of studying, in future experiments, the organization of specific primary transcripts such as the globin pre-mRNA molecules. Results Conditions for the Isolation and DNAase Digestion of Nuclei To investigate the architecture of the nuclear matrix and its interactions with both chromatin and hnRNA,

Cell 1080

nuclei must be isolated under the mildest conditions. Thus the cells must never be exposed to hypotonic media but must be lysed gently with a mixture of nonionic detergents in an isotonic buffer, and in the presence of a proteolytic inhibitor (see Experimental Procedures). Stronger detergents such as sodium deoxycholate (DOC) are not used because they have been shown to remove proteins from hnRNA-protein complexes (Faiferman, Hamilton and Pogo, 1971; Stevenin and Jacob, 1972) and to disorganize the nuclear matrix from rat liver (Miller et al., 1978a) and erythroleukemia cells (see below). Almost complete removal of DNA from nuclei was accomplished by digestion with 500 pg/ml of DNAase I in a physiological buffer at low temperatures. Digestion of DNA in nuclei from undifferentiated cells at 0” and 10°C occurred at a greater rate than that from differentiated cells (Figure 1). Near complete digestion (-95%) was obtained by a 80 min incubation of nuclei isolated from undifferentiated cells and by a 120 min incubation of nuclei isolated from differentiated cells at 10°C (Figures 1 A and 1 B). This difference is attributed to the different states of chromatin condensation,

2 40 20

0'

I

I

40

80

1I 120 MINUTES

I

I

I

40

80

120

Figure 1. Effect of DNAase Digestion at 0’ and 10°C on DNA and hnRNA in Nuclei Isolated from Undifferentiated and Differentiated Cells The 3H-thymidin~ and “C-uridine-labeled nuclei were incubated as explained in Experimental Procedures; aliquots were removed at the indicated intervals and purified through 1 M sucrose buffer. The nuclear pellets were suspended and washed twice with cold 10% TCA. resuspended in 1 ml of 10% TCA and heated in boiling water for 15 min. The suspensions were cooled in ice before centrifugation at 500 x g for 30 min, and the radioactive TCA-soluble material was counted in a Packard Scintillation Counter with a built-in program for counting dual-labeled samples. The radioactivities in each sample were related to that of an aliquot of undigested nuclei sedimented through 1 M sucrose buffer. (A) DNA from nuclei obtained from undifferentiated cells; (B) DNA from nuclei obtained from differentiated cells: (C) hnRNA from nuclei obtained from undifferentiated ceils; (D) hnRNA from nuclei obtained from differentiated cells. f-) incubation at O’C; (O- - - -0) incubation at 10°C.

as suggested by Weintraub and Groudine (1978). Essentially all of the hnRNA was found in the nuclear matrix structures (Figures 1C and 1 D), a situation similar to that in rat liver nuclei (Miller et al., 1978a). Ultrastructure of Isolated Nuclei and DNA-Depleted Nuclei Nuclei obtained by very mild detergent lysis of cells show an ultrastructure similar to that observed in situ (Figure 2). Undifferentiated cells contain nuclei with mostly diffuse chromatin while differentiated cells have nuclei with mostly condensed chromatin (Figures 2A and 28). These condensed and diffuse chromatin regions were maintained in the respective nuclear preparations (Figures 2C and 2D). However, the unexpected and remarkable fact was that condensed and diffuse regions were retained in DNA-depleted nuclei (Figures 2E and 2F). Moreover, DNA-depleted nuclei from either undifferentiated or differentiated cells were observed to maintain the same proportion and distribution of condensed and diffuse regions as did whole cells or isolated nuclei. The condensed regions in DNA-depleted nuclei appeared to be more tightly packed than those in undigested nuclei. The ultrastructure of the diffuse regions in the two types of nuclei appeared very similar, but the total quantity of matrix per nucleus seemed to be much less in differentiated than in undifferentiated cells (Figures 2E and 2F). DNA-depleted nuclei from differentiated cells were generally smaller than either nuclei in situ or isolated nuclei. Protein Composition and hnRNA in DNA-Depleted Nuclei Electrophoresis of proteins from DNA-depleted nuclei revealed the unanticipated information that the nuclear matrix from both types of cells retained histones in the absence of DNA (Figure 3, lanes 3 and 6). The most prominent difference between these two types of matrix is that histone Hl was not retained by DNAdepleted nuclei obtained from differentiated cells. Furthermore, these DNA depleted nuclei did not retain an additional histone species that was present in undigested nuclei (Figure 3, lanes 5 and 6). This histone variant, designated Hl O, is a characteristic feature of nondividing mammalian cells (Panyim and Chalkley, 1969) and appears to be unrelated to the phenomenon of cell differentiation (A. Zweidler, B. H. Long and A. 0. Pogo, unpublished results). Polyacrylamide gel electrophoresis also revealed similar nonhistone protein composition in undigested and DNA-digested nuclei. This was true for both undifferentiated (Figure 3, lanes 2 and 3) and differentiated (Figure 3, lanes 5 and 6) cells. In addition, few nonhistone proteins were released from both nuclear preparations when the DNA was fully digested (Figure 3, lanes 4 and 7). It is not possible at this time to

Nuclear 1081

Figure

Matrix

in Friend

2. Electron

Erythroleukemia

Microscopy

of Nuclei

Cells

in Situ, Isolated

Nuclei

and DNA-Depleted

Nuclei

from Undifferentiated

and Differentiated

Cells

(A) Undifferentiated and (8) differentiated ceils; isolated nuclei from (C) undifferentiated and (D) differentiated cells; and DNA-depleted nuclei from (E) undifferentiated and (F) differentiated cells. All samples were fixed in isotonic buffer. Notice that isolated nuclei and DNA-depleted nuclei from undifferentiated and differentiated cells still preserve their characteristic topographical distribution of condensed and diffuse regions, Magnification 13.500x.

Cdl 1062

8

Figure 3. SDS-Polyacrylamide Proteins from Undifferentiated

Slab Gel Electrophoresis and Differentiated Cells

of Nuclear

Samples prepared for electrophoresis according to the method of O’Farrel (1975) were layered over a linear 7.5-15% polyacrylamide gradient gel. (Lane 1) Ribosomal proteins obtained from the monosomal fraction of polysomes isolated from undifferentiated cells; (lane 2) undigested nuclei from undifferentiated cells; (lane 3) DNA-depleted nuclei from undifferentiated cells; (lane 4) proteins in the supernatant following sedimentation at 500 x g for 10 min of a complete DNAase digest of nuclei from undifferentiated cells; (lane 5) undigested nuclei from differentiated cells: (lane 6) DNA-depleted nuclei from differentiated cells; (lane 7) proteins in the supernatant following sedimentation at 500 x g for 10 min of a complete digest of nuclei from differentiated cells; (lane 6) commercial preparation of DNAase. The migration of proteins with known molecular weights is shown on the lefl and that of DNAase and histone is on the right.

establish the differences or similarities between nonhistone matrix proteins obtained from undifferentiated and differentiated cells. When proteins from an equal number of undigested or DNA-digested nuclei from both cell types were run on a polyacrylamide gel, however, a much smaller amount of nonhistone protein was found in differentiated nuclei (data not shown). It is doubtful that ribosomal proteins which migrated below 45,000 daltons are major nonhistone proteins in these nuclear matrix preparations since they corresponded to minor bands in the lower half of the gel (Figure 3, lane 1). Finally, it is important that hnRNA extracted from DNA-depleted nuclei has the same sedimentation velocity in a formamide-sucrose gradient and thus the same size as hnRNA extracted from whole cells (data not shown). Effect of High Salt Concentrations upon Proteins and Ultrastructure of DNA-Depleted Nuclei When DNA-depleted nuclei from undifferentiated cells were exposed to high KCI concentrations, the major changes observed were in the histone content. Thus 0.5 M KCI caused complete loss of Hl with only a partial loss of the histone cores (Figure 4). Moreover, a lower salt concentration (0.3 M KCI) was sufficient to release Hl histone fully (data not shown). On the other hand, the KCI concentration had to be raised to 1 M before an almost complete removal of histones

Figure 4. SDS-Polyacrylamide Slab Gel Electrophoresis Treated, DNA-Depleted Nuclei from Undifferentiated tiated Ceils

of High Saltand Differen-

DNA-depleted nuclei were sedimented through 25 ml of 1 M sucrose buffer containing 0.1, 0.5, 0.7, or 1 .O M KCI at 10,000 x g for 15 min. The pellets were divided and washed by suspension in buffer C and sedimentation at 500 x g for 10 min. Ihe washed pellets were dissolved in sample buffer and run in a linear 7.5-15% polyacrylamide gradient gel containing 0.1% SDS. (Lane 1) Undigested nuclei from undifferentiated cells; undifferentiated, DNA-depleted nuclei treated with (lane 2) 0.1 M, (lane 3) 0.5 I.4 and (lane 4) 1 .O M KCI; (lane 5) undigested nuclei from differentiated cells: differentiated, DNA-depleted nuclei treated with (lane 6) 0.1 M. (lane 7) 0.5 M and (lane 6) 0.7 M KCI.

H2A, H2B, H3 and H4 was accomplished. In DNAdepleted nuclei from differentiated cells a somewhat different salt effect was observed. Thus Hl was released in 0.1 M KCI, a partial loss of core histones was observed in 0.5 M KCI and almost complete loss of these cores was obtained in 0.7 M KCI (Figure 4). As shown in Figure 4, KCI treatment of DNA-depleted nuclei removed H2A, H28, H3 and H4 with no evidence of a preferential extraction of any single histone, suggesting that the histones were released as intact cores. A similar nonhistone protein pattern remains in both types of DNA-depleted nuclei after treatment with high concentraUons of KCI. Electron microscopy of salt-treated, DNA-depleted nuclei produced a striking correlation with the protein patterns observed above. Although 0.5 M KCI removed Hl from DNA-depleted nuclei from undifferentiated cells, the condensed regions remained (Figure 5A). In the case of differentiated cells, this salt concentration also preserved the condensed regions (Figure 58). A higher salt concentration (0.7 or 1 .O M) removed the condensed regions in nuclei from both cell types. yielding similar nuclear matrix structures (Figures 5C and 5D). It is important that removal of Hl, in the case of undifferentiated cells, does not affect the condensed regions of DNA-depleted nuclei. When both types of erythroleukemia cell nuclei were partially digested with DNAase I and then exposed to 0.5 M KCI, the electron microscopy of such prepared structures

showed

aged peripheral

a matrix

regions

having

fewer

fibrils.

and, in some cases,

dam-

an ab-

Nuclear 1083

Figure

Matrix

in Friend

5. Electron

Erythroleukemia

Microscopy

Cells

of High Salt-Treated,

DNA-Depleted

Nuclei

from Undifferentiated

and Differentiated

The salt-treated nuclear pellets (from the preparation described in the legend to Figure 4) were fixed in isotonic undifferentiated cell nuclei and (B) DNA-depleted, differentiated cell nuclei treated with 0.5 M KCI; (0 DNA-depleted, treated with 1 .O M KCI: (D) DNA-depleted, differentiated cell nuclei treated with 0.7 M KCI. Magnification 13,500X.

sence of nuclear lamina (data not shown). These observations indicate that parts of the matrix fibrils have been pulled away with the unwound chromatin. Our experience with these treatments indicates that the released chromatin was contaminated by matrix proteins and may account for most of the observed nonhistone proteins in these chromatin preparations. A similar situation exists when nuclei with partially digested DNA are exposed to a hypotonic buffer, the current method of preparing nucleosomes (Peterson and McConkey, 1976a, 1976b; Bakayev, Bakayeva and Varshavsky, 1977; Neumann et al., 1978). Effect of Hypotonic Buffer upon Protein Content and Ultrastructure of DNA-Depleted Nuclei Conditions of very low ionic strength have been shown to cause the release of chromatin as nucleosomes

Cells buffer. (A) DNA-depleted, undifferentiated cell nuclei

after partial digestion of DNA in either nuclei or isolated chromatin (Olins and Olins, 1974; Sahasrabuddhe and Van Holde, 1974; Griffith, 1975; Woodcock, Safer and Stanchfield, 1976; Kornberg, 1977; Muller et al., 1978). If the condensed regions observed following complete digestion of DNA are caused by precipitation of aggregated histones onto the matrix due to their low solubility (Crampton, Lipshitz and Chargaff, 19541, then exposure of DNAdepleted nuclei to a hypotonic buffer should release the intranucleosomal core histones from the matrix. This effect was not observed. DNA-depleted nuclei exposed to 2 mM Tris-HCI buffer showed identical morphology (Figure 6) and protein content (Figure 7), including all histones. These results confirm the suggestion that the core histones are bound to the matrix through ionic bonds or saline-labile elements.

Cell 1084

Figure

6. Electron

Microscopy

of 2 mM Tris-HCCTreated.

DNA-Depleted

Nuclei from

Undifferentiated

and Differentiated

Cells

DNA-depleted nuclei were washed twice (40 vol of the pellet) with 2 mM Tris-HCI (pH 7.7, 23°C) and collected by sedimentation 10 min. Pellets were fixed in hypotonic buffer as explained in Experimental Procedures. (A) DNA-depleted, 2 mM Tris-HCI-treated undifferentiated cells and (6) DNAdepleted, 2 mM Tris-HCI-treated nuclei from differentiated cells. Magnification 13,500X.

Resistance of DNA-Depleted Nuclei to Different Chemical Treatments and Their Effect upon hnRNA Release, Protein Composition and Ultrastructure DNA-depleted nuclei from undifferentiated and differentiated cells were exposed to different chemicals to investigate the possibility of reducing the matrix to a few basic polypeptides while retaining the highly ordered structure, to identify the intermolecular bonds maintaining the matrix and to investigate the interactions existing between matrix and hnRNA. These treatments were carried out with nuclei digested with iodoacetic acid-treated and untreated DNAase. The latter contains enough RNAase activity to digest matrix hnRNA partially. Table 1 shows that hnRNA was released from nuclei digested with iodoacetic acid-treated enzyme when these nuclei were treated with 1% LDS, a mixture of 4 M urea and 0.1% LDS, or a mixture of 8 M urea and 10 mM EDTA. All the other treatments were unable to release hnRNA or solubilize the matrix. When nuclei were digested with untreated DNAase, DOC, heparin and 2 M KCI, treatments produced a partial hnRNA release without matrix lysis. On the other hand, when the RNAase was fully inactivated (treated DNAase), these chemicals were unable to release hnRNA molecules. Matrix lysis occurs with a mixture of 8 M urea and 10 mM EDTA, suggesting that the matrix proteins are held together by hydrogen and hydrophobic bonds as well as divalent cations. Polyacrylamide gel electrophoresis of the proteins remaining in DNA-depleted nuclei following the above treatments is shown in Figure 8. Since DNA-depleted nuclei from either cell type did not seem to respond differently to any treatment, only DNA-depleted nuclei from undifferentiated cells are shown. None of these

at 500 x g for nuclei from

drastic methods of treatment produced a gross simplification of the nonhistone protein pattern. However, each treatment did release a distinct subset of proteins. Although no assignment of protein bands with specific subnuclear structures has been attempted, this selectivity allows us to make the following interpretations. Heparin removed mainly the proteins migrating between Hl and core histones (Figure 8, lane 2) and caused a partial release of Hl. 4 M urea removed proteins all along the gels but did not remove Hl and core histones (Figure 8. lane 3). DOC, like heparin, removed Hl and some other nonhistone proteins but retained core histone proteins (Figure 8, lane 4). This detergent also removed two proteins of about 66,000 and 62,000 daltons which became prominent after the 2 M KCI treatment (Figure 8, lane 5). DOC removed the nuclear lamina as well (see below). The effects of 10 mM EDTA (not shown in these gels) did not alter the pattern of either histone or nonhistone proteins. Most of these treatments produced gross morphological changes in the ultrastructure of the DNA-depleted nuclei. Figure 9 shows undifferentiated cells, but similar results were observed in differentiated cells. Electron microscopy demonstrated that 4 M urea caused some disorganization of both condensed and diffuse regions (compare Figures 1C and 9A). The disruption was much more prominent when 10 mM EDTA was added (compare Figures 9A and 9B). However, 10 mM EDTA in 100 mM KCI did not alter the ultrastructure of DNA-depleted nuclei (not shown). The mixture of 4 M urea and EDTA appeared to produce disaggregation of the condensed regions without any significant loss of histones. Heparin and DOC also produced rearrangements of components, mainly disaggregation of the condensed regions and

Nuclear 1085

Matrix

in Friend

Erythroleukemia

Figure 7. SDS-Polyacrylamide Tris-HCI-Treated. DNA-Depleted

Slab Gel Nuclei

Cells

Electrophoresis

of 2 mM

DNA-depleted nuclei were washed twice (40 vol of the pellet) with either buffer C or 2 mM Tris-HCI (pH 7.7, 23°C) and collected by sedimentation at 500 X g for 10 min. Pellets were dissolved in SDS sample buffer, and samples were run in a linear 7.5-l 5% polyacrylamide gradient gel containing 0.1% SDS. DNAdepleted nuclei from undifferentiated cells exposed to (lane 1) buffer C and (lane 2) 2 mM Tris-HCI fpH 7.7, 23W: DNA-depleted nuclei from differentiated cells exposed to (lane 3) buffer C and (lane 4) 2 mM Tris-HCI (pH 7.7, 23°C).

a dramatic alteration of nucleoli (Figures 9C and 9D). DOC also appears to have caused a loss of the lamina. Discussion The complete digestion of nuclear DNA at low temperature in the presence of a proteolytic inhibitor and in a quasi-physiological environment yields a nuclear matrix that maintains the inner core histones in highly organized structures (that is, a supranucleosome-like structure) and preserves native hnRNA with a high degree of organization. High voltage electron microscopy indicates that this nuclear matrix consists of highly coiled and interconnected thin fibers (C.-Y.

Huang. 6. F. Long and A. 0. Pogo, manuscript in preparation). Interphase nuclei therefore appear to be made of two distinct kinds of fibers; the thicker chromatin cables are interwoven with the thinner matrix network. The thicker chromatin cables consist of highly ordered DNA-histone complexes; the thinner matrix fibers are formed by the interactions of a large number of heterogeneous nonhistone proteins. In our previous work, interaction between DNA-histone complexes and the network had been anticipated although a high degree of organization of histone proteins in the absence of DNA was not foreseen (Miller et al., 1978a). A simple explanation for these remarkable observations is that in the absence of DNA the very negatively charged histone proteins establish fortuitous ionic bonds with some acidic matrix proteins. Many of the results obtained, however, dispute this artificial rearrangement and support the concept of specific binding between histone and nonhistone proteins. First, DNA-depleted nuclei from undifferentiated and differentiated cells still preseve their characteristic topographical distribution of condensed and diffuse regions. Second, in a hypotonic buffer there is no release of the inner core histones and the ultrastructural characteristics are well preserved. Third, removal of the full complement of intranucleosomal histones (that is, the octameric structure of H2A, H2B, H3 and H4) is obtained only by high salt treatments. This suggests that core histones have strong and specific binding sites (through ionic bonds or saline-labile elements) with the matrix fibrils. On the other hand, Hl histone, which is assumed to be bound to the linker region between adjacent nucleosomes (Varshavsky, Bakayer and Georgiev, 1976; Noll and Kornberg, 1977) has a much weaker and nonspecific interaction with the matrix fibrils. Thus Hl is released in either isotonic buffer or at 0.3 N KCI in differentiated and undifferentiated cells, respectively. Finally, heparin, which removes the last traces of DNA, is unable to remove the inner core histones but removes Hl , the only histone subject to rearrangements (Gaubatz et al., 1977; Grebanier and Pogo, 1979). This indicates that (with the exception of Hl) heparin, a Strong anionic molecule, is unable to compete with the elements that affix the core histones to the matrix. These observations support the contention that spurious interactions between the inner core histones and the matrix are highly improbable in the absence of DNA. In general, there are two levels of chromatin organization:

the

nucleosomes

and

the

supranucleosomal

structure. It is assumed that Hl histone is necessary for maintaining the supranucleosomal structure (Bradbury, Carpenter and Rattle, 1973; Finch and Klug. 1976; Worcel and Benyajati, 1977; Muller et al., 1978). It has been postulated that condensed and diffuse chromatin regions represent different degrees of compaction of the supranucleosomal structure. The

Cell 1066

Table 1. Effect

of Chemical

Treatment

on the Release

of hnRNA

Undifferentiated

Nuclei

Treated % hnRNA Released Control

and the Structural

DNAase

Untreated

Lysis

EDTA

of DNA-Depleted

Nuclei Differentiated

% hnRNA Released

0.3

+ 10mM

Integrity

ND

DNAase

Untreated

Lysis

% hnRNA Released

0.2

-

0.4

0.2

-

0.4

6.0

-

5.0

-

18.0

+ 1% DOC

6.0

-

40.0

-

40.0

+ 10 M formamide

0.2

-

0.2

-

+2MKCI

9.0

-

16.0

+ 4 M urea

0.2

+ 1 mg/ml

heparin

+4Murea+lOmMEDTA + 4 M urea

+ 0.1%

0.4

ND LDS

+ 8 M urea +BMurea+lOmMEDTA

1.3

+

-

ND

+ 0.1%

LDS

2.0

+ 1.0%

LDS

100.0

Lysis

0.2

0.3 0.3

ND

-

DNAase

28.0

1.2

100.0

Nuclei

ND 6.0

-

13.0

100.0

+

96.0

*

ND

ND

-I-

ND

ND

+

Nuclei were incubated with either iodoacetate-treated or untreated DNAase as described in Experimental Procedures. DNA-depleted nuclei, washed twice in buffer C. were suspended in 10 vol of the same buffer containing the designated chemical. After 10 min of incubation at 0°C. the suspensions were centrifuged at 500 X g for 10 min. Supernatants were diluted 10 fold with cold 10% TCA and pellets were suspended in cold 10% TCA. The resulting cold TCA precipitates in both fractions were washed twice with 10% TCA and heated in boiling water. Radioactivities in the hot TCA-soluble fractions were counted as described in the legend to Figure 2. Radioactivities in the supernatant fractions were expressed as the percentage of combined radioactivities in the pellets and supernatants. Lysis was determined by viewing under the light microscope. (ND) Not determined; (-) no lysis; (+I lysis; (*I partial lysis.

Figure Meted ments

8. SDSPolyacrylamide Undifferentiated Nuclei

Slab Gel Electrophoresis of DNA-DeExposed to Different Chemical Treat-

DNA-depleted nuclei from undifferentiated cells were washed twice in buffer C. Pellets were suspended in 10 vol of (lane 1) buffer C: (lane 2) buffer C containing lmg/ml heparin: (lane 3) 4 M urea containing 10 mM Tris-HCI (pH 7.7. 23°C) and 1.5 mM MgCI?: (lane 4) buffer C containing 1% DOC; (lane 5) 2 M KCI, 10 mM Tris-HCI (M-l 7.7. 23°C) and 1.5 mM Mg&.

fact that condensed and diffuse regions can exist in the absence of both DNA and Hl , however, indicates that matrix fibrils participate in chromatin organization in interphase nuclei. This role is illustrated by the increase of condensed regions during the process of erythrodifferentiation. Since the amount of DNA and histones is constant, condensation must occur at the expense of the matrix fibrils. The significant diminution of nonhistone proteins in differentiated cells is strong support for this assumption. The phenomena of chromatin condensation and diffusion are also related to gene activity since hnRNA synthesis takes place exclusively in the diffuse regions (Hsu, 1962; Bouteille, Lava1 and Dupuy-Coin, 1974; Bachellerie, Puvion and Zalta, 1975; Zentgraf, Scheer and Franke, 1975; Fakan. Puvion and Spohr, 1976). Thus the dispersion of chromatin at the site of gene activation implies accumulation of matrix material by filament engrossment, increase in the number of fibrils or increase in the length of the fibrils. It is not yet known which of these processes takes place, but our observations indicate that hnRNA is affixed to the surface of the matrix fibrils by forces of a different nature (hydrophobic) than those which interact with histones. Since hnRNA has a high turnover and gene activation occurs in different regions of the chromatin, it follows that on the surface of the filaments the hnRNA-matrix interactions must be transient and occur at different positions. The matrix fibrils may also participate in the orga-

Nuclear 1087

Figure

Matrix

9.

in Friend

Electron

Erythroleukemia

Microscopy

Cells

of DNA-Depleted,

Undifferentiated

Nuclei

Exposed

to Different

Chemical

Treatments

DNA-depleted nuclei were prepared and treated as described in the legend to Figure 8. (A) 4 M urea containing 10 mM Tris-HCI (pH 7.7, 23°C) and 1.5 mM MQCIL (B) 4 M urea containing 10 mM Tris-HCI (pli 7.7,23”C) and 10 mM EDTA; (C) buffer C containing 1 mg/ml heparin; (D) buffer C containing 1% DOC. Notice the absence of lamina and nuclear envelope in (D).

nization of the chromosomes during the prophase stage of the mitotic cycle. The degree of compaction of chromatin in chromosomes is not greatly different from that of condensed chromatin. The role of the matrix fibrils in the organization of chromosomes gains support with the observation by Laemmli and his collaborators that in metaphase chromosomes a protein core exists which is sufficient to maintain the chromatid structure (Paulson and Laemmli, 1977; Adolph, Cheng and Laemmli, 1977). Very few nonhistone proteins are released when DNA is completely digested. As a subgroup of nonhistones, they have to be considered truly DNA-associated proteins. The remaining proteins represent a large number of nonhistone proteins which have not yet been assigned to specific structures. Some of these nonhistone proteins are nuclear envelope and lamina proteins. The two prominent proteins with molecular weights of 66,000 and 62,000 that remain in DNA-depleted nuclei following 2 M KCI treatment may

correspond to two of the three proteins (molecular weights 69K, 66K and 62K daltons) described by Berezney and Coffey (1974) as components of the rat liver matrix. Aaronson and Blobel (1975) later described three major proteins with molecular weights of 69K, 66K and 66K in rat liver nuclear lamina preparations. It appears probable that the proteins in these three different cases represent laminar proteins, since we have shown that DOC treatment removes both the lamina and this subgroup of proteins. All methods of chemical treatment have failed to produce a gross simplification of the nonhistone protein pattern and, since no predominant polypeptides have been observed, it is improbable that a simple repetitive subunit structure exists within the matrix. On the contrary, the picture emerging is that of a complex heteropolymer. With the presence of small molecular weight RNAs which have been demonstrated to be exclusive matrix components (Miller et al., 1978b), the matrix fibrils turn out to be a network

Cell 1088

of ribonucleoprotein components of a much higher degree of complexity than that described by Berezney and Coffey (1974). This is expected for a structure believed to have the following functions: limiting the diffuse and condensed regions (that is, the active and inactive chromatin domains); determining chromosome positions in interphase nuclei; forming the chromosomes in the prophase stage of the mitotic cycle; maintaining the necessary degree of organization for splicing the primary transcripts; and establishing contacts with the cytoskeleton via the nuclear pore for the orderly transport of mRNA and ribosomal subunits. The elucidation of the molecular mechanisms of these processes should be challenging subjects for future studies. Experimental

Procedures

Cell Culture and Radioactive Labeling Friend murine erythroleukemia cells, line 745 (provided by C. Friend), were maintained by suspension in roller tubes in Dulbecco’s minimal essential medium (MEM) containing 4.5 gm/l of glucose, 10% fetal calf serum and 0.1 me/l of kanamycin, supplemented with 25 mM HEPES (pli 7.45, 23°C). For experimental preparations, the cells were seeded at 5 X 10’ cells per ml and grown for 2 days in this medium at 37’C in sealed spinner flasks gassed with 5% CO? in air. Differentiated erythroleukemia cells were obtained by seeding at 5 x 10’ cells per ml in this medium containing 1.8% DMSO and harvesting after 7 days (Friend et al., 1971). DNA in both cell types was labeled by the addition of 5 @/I of 8-3H-thymidine at the beginning of the culture. hnRNA was labeled by harvesting the thymidine-labeled cells and incubating at a density of 5-6 x 10’ cells per ml in fresh Dulbecco’s MEM containing 0.04 Fg/ml of actinomycin D for 30 min prior to the addition of 5 rCi/lOO ml of 2-“C-uridine. The culture was poured into frozen saline slush (0.15 M NaCI) after 15 min of incubation and centrifuged for 10 min at 250 x g (undifferentiated cells) and 500 x g (differentiated cells). Preparation of DNA-Depleted Nuclei Cells were washed twice with 40 vol of buffer A containing 146 mM sucrose, 100 mM KCI. 10 mM PIPES (pH 7.0, 23°C) and 1.5 mM MgCI*. The washed cells were lysed in 20 vol of buffer A containing 0.5 mM phenylmethylsulfonylfluoride (PMSF) by addition of a Triton X-lOO-Saponin mixture to a final concentration of 0.25% each. The suspension was gently pipetted lo-20 times with a plastic pipette before centrifugation. Undifferentiated nuclei were harvested by centrifugation at 250 x g for 10 min and differentiated nuclei by centrifugation at 500 x g for 10 min. Nuclei were washed twice in 20 vol of buffer B containing 146 mM sucrose, 100 mM KCI. 10 mM Tris-HCI (pH 7.7. 23’C). 5 mM MgC12. 0.5 mM CaCI, and 0.5 mM PMSF. Nuclei suspended to 1.5 X 10’ nuclei per ml in buffer B were incubated with 500 Ag DNAase/ml at 10°C for 60 or 120 min for undifferentiated or differentiated cell nuclei, respectively, unless stated otherwise. Following digestion, nuclei were washed either by sedimentation at 6000 X g for 10 min through 10 or 25 ml of a 1 M sucrose solution containing 100 mM KCI. 10 mM Tris-HCI tpH 7.7, 23°C) and 1.5 mM MgCI*, or by direct sedimentation at 250 x g for 10 min (nuclei from undifferentiated cells) and 500 x g for 10 min (nuclei from differentiated cells). The pellets ware suspended in buffer C containing 146 mM sucrose, 100 mM KCI, 10 mM Tris-HCI (pH 7.7, 23°C) and 1.5 mM MgCl*, unless otherwise specified. DNAase I Treatment to Inactive Proteases and RNAase Commercial preparations of pancreatic deoxyribonuclease I (EC. 3.1.4.5) (Worthington and/or Boehringer) contain trypsin, chymotrypsin and RNAase as contaminants. We developed a simple method which fully inactivates proteases as well as RNAase without any loss

of DNAase activity. Our method is a modification of the Zimmerman and Sandeen (I 966) procedure. 5 mg of DNAase I were dissolved in 1 ml of a solution containing 150 mM NaCI. 5 mM EDTA and 0.5 mM EGTA. PMSF was added to a final concentration of 0.5 mM. 0.1 ml of a solution containing 1.5 M iodoacetic acid (Sigma) recrystallized twice with petroleum ether, an I 1 .O M Na acetate adjusted to pH 5.65 was added. The mixture was heated at 48-5O’C for 2 hr. It was then dialyzed overnight in the cold against 500 ml of 0.15 M NaCI. Under these conditions, 100% recovery of DNAase I activity was obtained and was fully stable for several months. DNAase I activity was measured by the hyperchromicity assay of Kunitz (1950). The temperature and pH are critical to the reproducibility of the method. hnRNA Extraction and Sedimentation on FormamideSucrose Gradients hnRNA was extracted from whole cells and DNA-depleted nuclei using the following procedure. Whole cells were harvested, washed once with a buffer solution containing 10 mM Tris-HCI (pH 7.5). 1.5 mM MgCI? and 300 mM sucrose (TMS). The cells were resuspended in 10 vol of TMS and kept at - 2O’C. They were thawed rapidly, and 0.1 vol of a solution containing 100 mM Tris-HCI (pH 8.2, 23°C) and 20 mM EDTA (1 OXTE) and 1 vol of a solution containing 10 mM TrisHCI (pH 8.2, 23°C) and 2 mM EDTA (1XTE) ware added. Lithium dodecylsulfate was added to a final concentration of 0.5%. Finally, an equal volume of freshly distilled phenol saturated with 1 XTE buffer was added and the mixture was agitated vigorously for 20 min at 55°C. The aqueous phase was removed after centrifugation and the phenol phase was reextracted with 1 vol of 1XTE. The combined aqueous phases were deproteinized by the addition of an equal volume of a phenol:chloroform:isoamyl alcohol mixture (100:100:1) and agitated vigorously at room temperature. This treatment was repeated until the interphase was clear. 0.1 vol of 1 M NaCl was added to the aqueous phase. RNA was precipitated by the addition of 2.5 vol of ethanol and the solution was stored overnight at 4°C. The precipitated RNA was washed several times with a mixture of ethanol and 0.1 M NaCI. air-dried and resuspended in double-distilled and deionized water. This method of extraction yielded 70-90% of the hnRNA without contaminating DNA, as determined by the Burton reaction (1956). It was found that freezing and thawing are required for a high hnRNA yield. DNA-depleted nuclei were washed as indicated and hnRNA was extracted as for whole calls. The phenol-extracted hnRNAs were disaggregated in 57% formamide (freshly distilled and deionized) containing 10 mM Tris (pH 7.5. 23°C) and 1 mM EDTA. The mixture was heated at 45°C for 2.5 min and layered on a discontinous 80% formamide-sucrose gradient. The gradients were prepared in 5 ml polyallomer tubes by three 1.2 ml layers of 20. 16 and 12% sucrose and a fourth 1 ml layer of 8% sucrose. They were centrifuged in the SW50Ti or SW65Ti Spinco Rotor at 50K x g for 20 hr. Treatment of Digested Nuclei For high salt treatment, digested nuclei were layered over 1 M sucrose containing 10 mM Tris-HCI (pH 7.7, at 23’C), 1.5 mM MgCI. and KCI at 100, 500, 700 and 1000 mM, and pelleted at 10,000 X g for 15 min. DNA-depleted nuclei, to be treated with different chemicals or very low ionic strength solutions, were first washed in buffer C by direct sedimentation and then suspended in 10 vol of the same buffer containing the chemical or 50 vol of 2 mM Tris-HCI (pH 7.7, 23°C) for the very low ionic strength treatment. The DNA-depleted nuclei were incubated under these conditions for 10 min on ice before centrifugation at 500 X g for 10 min. Sodium Dodecylsulfate (SDStPolyacrylamide Gel Electrophoresis DNAdepleted nuclei were dissolved directly in the SDS sample buffer without prior treatment according to O’Farrell’s modifications of Laemmeli’s method (O’Farrell, 1975). In undigested nuclear samples, DNA was sheared by sonication after addition of the SDS buffer. Supernatants to be analyzed were adjusted to 20% TCA by the addition of 100% TCA. and the resulting pellets were washed with 100% ethanol before being dissolved in sample buffer. Linear poly-

Nuclear 1089

Matrix

in Friend

Erythroleukemia

Cells

acrylamide slab gel gradients were prepared essentiatty according to the method of O’Farrell (I 975). The gels were poured and run using a Buchler slab gel electrophoresis apparatus for 14 X 17 x 0.15 cm gels. Electrophoresis was at a constant current of 25 mA per gel for 5-6 hr until the bromophenol tracking dye approached the bottom. Electron Microscopy Pelleted cells, nuclei and nuclear matrix were fixed on ice for 60 min with about 100 vol of a solution containing 4% glutaraldehyde and 3.6% formaldehyde in 0.2 M cacodylate buffer (pH 7.2,23”C). Pellets of digested nuclei exposed to very low ionic strength buffers were fixed for 90 min in a solution of 1% glutaraldehyde and 0.8% formaldehyde in 10 mM PIPES buffer, (pH 7.2. 23°C). All pellets were post-fixed with 1% osmium tetroxide buffered with 0.1 M Na cacodylate (pti 7.2) at 4°C for 1 hr. Specimens were dehydrated in acetone and embedded in epon 612 mixture. Sections cut with diamond knives were stained further with uranyl acetate and lead citrate. All electron micrographs were taken with a Philips 201 electron microscope at an initial magnification of 4500X. Acknowledgments

action of dissociating

agents.

Biochim.

Fakan. S.. Puvion. E. and Spohr. acterization of newly synthesized tocytes. Exp. Cell Res. 99, 155-I

Biophys.

Acta 232, 665-695.

G. (1976). Localization nuclear RNA in isolated 64.

and charrat hepa-

Finch. J. T. and Ktug. A. (1976). Solenoidal model for superstructure in chromatin. Proc. Nat. Acad. Sci. USA 73, 1897-I 901. Friend, C.. Preisler. H. D. and Scher. W. (1974). Studies on the control of differentiation of murine virus-induced erythroleukemia cells. In Current Topics in Developmental Biology, A. A. Moscona and A. Monroy. eds. (New York: Academic Press), pp. 81-101. Friend, C., Scher, W., Holland, J. G. and Sate. T. (I 971). Hemaglobin synthesis in murine virus-induced leukemia cells in vitro: stimulation of erythroid differentiation by dimethyl sulfoxide. Proc. Nat. Acad. Sci. USA 68, 378-382. Gaubatz. J.. Hardison. R., Murphy, J.. Eichner. M. E. and Chalkley. R. (1977). The role of HI in the structure of chromatin. Cold Spring Harbor Symp. Quant. Biol. 42, 265-271. Georgiev. G. P. and Chentsov. Y. S. (I 960). On the structure of cell nucleus. Experimental electron microscopic studies on the isolated nuclei. Proc. Nat. Acad. Sci. USSR 132. 199-201.

We express our gratitude to Ms. Valerie Zbrzezna for her excellent technical assistance, and to Ms. Vivian lsenberg for preparing the samples for electron microscopy. We also thank Dr. Charlotte Friend for helpful instruction of growth of these cells, Dr. Luis Cornudella for his valuable comments and cristicism and Ms. Nancy Rips for assistance in preparing the manuscript. This work was supported by grants from the NSF and the NIH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. Received April 26. 1979; revised July 6. 1979

Grebanier, A. and Pogo, nuclei and DNA-depleted Cell 18. 1091-1099.

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