Journal of Neuroscience Research 33:112-121 (1992)
Changes in Chromatin Proteins During Optic Nerve Regeneration in the Goldfish J.M. Gossels, S.E. Lewis, N.I. Perrone-Bizzozero, and L.I. Benowitz Mailman Research Center (J.M.G., S.E.L., N.1.P.-B., L.I.B.), McLean Hospital, Belmont , and Departments of Psychiatry (J.M.G., N.1.P.-B., L.I.B.) and Neurobiology (S.E.L.), Harvard Medical School, Boston, Massachusetts
Regeneration of the goldfish optic nerve involves mas- ON, (Quitschke and Schechter, 1983a), the microtubulesive changes in the structure and pattern of macro- associated tau protein (Neumann et a]. , 1983), and others molecular synthesis in the retinal ganglion cells. To (Giulian et al., 1980; Benowitz et al., 1981; Perry et al., explore the mechanisms that underlie these events, 1987). At least some of these cellular alterations may be we investigated the changes in chromatin proteins during the course of regeneration. Three major reti- determined by changes in the constellation of proteins nal chromatin proteins, two with apparent molecular that are bound to the nuclear DNA. For many genes, weights of 58 kDa (C1 and C2) and one at 51 kDa RNA transcription is regulated by the binding of specific (C3), all having isoelectric points around 5.5, showed proteins to enhancer and promoter regions. These proa fourfold increase in their synthesis and/or accumu- teins include steroid receptors (reviewed by Beato, lation by 14 days of regeneration. Synthesis of C1 and 1989), proto-oncogene products such as cJun (BohC3 decreased by day 32, the time at which the axons mann et al., 1987), a class of mammalian and Drosohave grown back to the optic tectum and have formed phila proteins known as POU proteins (Herr et al., 1988; many of their synapses; synthesis of C2 remained Sturm and Herr, 1988), and others. In addition to these, high through day 32. All three proteins bound to proteins which affect the extent of chromatin condensaDNA-cellulose and required high salt concentrations tion could be important in determining the accessibility (0.2-0.5 M KCI) to be eluted. C1 and C2 had similar of particular regions of the genome for transcription. proteolytic digestion patterns and reacted with mono- Such a role has been suggested for intermediate filament clonal antibodies that recognize the goldfish interme- proteins in the nucleus (Traub, 1985). diate filament proteins of the ON complex. The proIn an effort to explore some of the molecular mechteins identified here could be involved in structural anisms that underlie optic nerve regeneration, we have alterations in the chromatin, or might serve as tran- investigated the sequence of changes in nonhistone scription factors to regulate gene expression during acidic chromatin proteins, proteins which are associated nerve regeneration. 0 1992 Wiley-Liss, Inc. with nuclear DNA. We have used two-dimensional gel electrophoresis, DNA-cellulose chromatography, proteKey words: retina, DNA binding, two-dimensional olytic digestion, and immunoblotting to identify and gel electrophoresis characterize three retinal chromatin proteins whose synthesis and accumulation increase during the course of optic nerve regeneration (Gossels et al., 1989). INTRODUCTION Regeneration of the goldfish optic nerve is associated with a number of changes in the retinal ganglion cells (reviewed by Grafstein, 1986) that include dramatic increases in cell volume and nucleolar size (Murray and Grafstein, 1969; Murray and Forman, 1971; Whitnall and Grafstein, 1983) and in the synthesis and transport of RNA and protein (Ingoglia et al., 1975; Grafstein, 1986). Specific proteins that increase during the course of this process include GAP-43 (Benowitz and Lewis, 1983; Perrone-Bizzozero and Benowitz, 1987), ptubulin (Heacock and Agranoff, 1976, 1982; Giulian et al., 1980), the intermediate filament proteins ON, and 0 1992 Wiley-Liss, Inc.
Received January 13, 1992; revised April 14, 1992; accepted April 15, 1992. Address reprint requests to Dr. Lany I. Benowitz at his current address: Laboratory for Neuroscience Research in Neurosurgery, Children’s Hospital/Enders 250, 300 Longwood Avenue, Boston, MA 021 15. Abbreviations used: IEF, isoelectric focusing; mAb, monoclonal antibody; MEM, minimal essential medium; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; SAP, Staphylococcus uureus V8 protease; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid.
Chromatin Proteins in the Goldfish Retina
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MATERIALS AND METHODS Optic Nerve Crush Common goldfish (3.5-4 in. total length; Mt. Parnell Fisheries, Mercersburg, PA) were anesthetized in tricaine methanesulfonate (Finquel; Ayerst Co. and Sigma Chemical Co., St. Louis, MO). Fish were held in place with water circulating through their gills and the optic nerve was crushed 1-2 mm behind the eye as described previously (Yoon, 1971). Controls were derived from the same batches as operated animals. In Vitro Labeling At times varying from 5 to 32 days after crushing the optic nerve, fish were dark-adapted for 2 hr, sacrificed, and their retinas dissected as described previously (Landreth and Agranoff, 1976). To study protein synthesis, retinas were pre-equilibrated for 1 hr in methionineand cysteine-free minimal essential medium (MEM; Gibco Laboratories, Grand Island, NY) containing 10% horse serum (dialyzed against 25 mM HEPES; Sigma) and 20 pg/ml gentamicin (Sigma), then incubated for 2
Centrifuge 2 h 100,000 g
hr in this same medium containing 250 pCiiml 13’S]methionine + cysteine (Tran~-~’Slabel; ICN Biomedicals, Costa Mesa, CA). To study protein phosphorylation, retinas were pre-equilibrated for 15 min in phosphate-free MEM containing 10% dialyzed horse serum and 20 pg/ml gentamicin, then incubated for 1 hr in this medium containing 100-125 pCi/rnl [32P]orthophosphate (New England Nuclear/Dupont, Boston, MA). For all experiments, groups of 10-12 retinas were incubated in 2 ml of medium at room temperature on a rotating table in 24-well dishes. Labeled retinas were rinsed twice in phosphate-buffered saline (PBS) and frozen on dry ice.
Subcellular Fractionation Chromatin. The retinal chromatin fraction was isolated essentially as described by Bonner et al. (1968). Groups of 10-12 frozen retinas were thawed quickly and homogenized [ 10 strokes in a teflodglass motorized homogenizer in 10 ml solution A: 75 mM NaCl, 25 mM EDTA, 25 mh4 NaF, 0.4 mM phenylmethylsulfonyl flu-
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Fig. 2. Comparison of 14 day postcrush retinal chromatin proteins to proteins in other subcellular fractions. [35S]-methionine+ cysteine labeled retinal chromatin (A), non-nuclear particulate (B), nuclear soluble (C), and non-nuclear soluble (D) proteins were separated by two-dimensional gel electrophoresis and visualized by fluorography . Fluorograms were exposed for 9-14 days. The basic end of each gel is to the left. oride (PMSF); pH 8.01. The homogenate was spun at 1,500g for 10 min at 4°C and the supernatant (S 1) further separated into non-nuclear soluble and particulate fractions (see below and Fig. 1). The pellet (Pl) was resuspended in 10 ml buffer A and spun at 6,OOOg for 10 min at 4°C.The resulting pellet (P2) was suspended in 2.5 ml lysis buffer (10 mM Tris-HCI, pH 7.5, 1 mM EGTA, 25
mM NaF, 0.4 mM PMSF) and spun at 12,OOOg for 10 min at 4°C. The supernatant (S3) was the nuclear soluble fraction. The lysis buffer extraction was repeated twice on the pellet (P3). The resulting pellet was resuspended in approximately 4 ml lysis buffer, layered over 8 ml 1.7 M sucrose in lysis buffer, and spun at 100,OOOg for 2 hr. The resulting pellet was the chromatin fraction. (In some
Chromatin Proteins in the Goldfish Retina
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Fig. 3. The labeling of several retinal chromatin proteins increases by 14 days postcrush. Retinas from control (intact) fish and those undergoing axonal regeneration for 5 or 14 days were labeled with [35S]-methionine cysteine. Chromatin was prepared, proteins were separated by two-dimensional gel electrophoresis, and visualized by fluorography. Fluorograms were exposed for 28 days. The basic end of each gel is to the left.
The region in each gel corresponding to the boxed area has been enlarged to show the increases in synthesis of proteins C1, C2, and C3 by 14 days of regeneration. The increase in synthesis of C1 is statistically significant at the level of P < 0.02, C2 at P < 0.01, and C3 at P < 0.001. C4 is an example of a protein whose labeling did not change during this time period.
experiments the gradient was spun at a lower speed for a longer time, but in all experiments the g force times t was held constant at 200,000.)In preparation for two-dimensional gel electrophoresis, the chromatin fraction was lyophilized, dissolved in isoelectric focusing (IEF) lysis buffer (40 pketina) (O'Farrell, 1975), sonicated 3 times, 10 sec each, and spun for 2 min at 12,OOOg in a microfuge. Non-nuclear soluble and particulate fractions. The S1 supernatant was spun at 100,OOOg for 1 hr. The resulting pellet, the non-nuclear particulate fraction, was lyophilized, dissolved in IEF lysis buffer (70 plhetina), and prepared for two-dimensional gel electrophoresis in the same way as the chromatin. The supernatant (i.e., the non-nuclear soluble fraction) was treated with 10% trichloroacetic acid (TCA) on ice for 1 hr to precipitate proteins. This fraction was spun for 15 min at 12,OOOg at 4°C. The pellet was suspended in double distilled water
and also spun for 15 min at 12,OOOgat 4°C. This pellet was dissolved in IEF lysis buffer (70plhetina) for twodimensional gel electrophoresis. Nuclear soluble protein fraction. The nuclear soluble protein fraction (S3 from the chromatin preparation) was precipitated with TCA in the same way as was the non-nuclear soluble fraction. The washed pellet was dissolved in IEF lysis buffer (17.5plhetina). Triton X-100fractionation. The total retinal homogenate was extracted with Triton X-100 to separate intermediate filament proteins (Triton X- 100 insoluble) from other components. Extraction was carried out in solution A according to the method of Chiu et al. (1981). The pH was adjusted to 6.8 with HCl and Triton X-100 was added to a final concentration of 0.5%. After a 10 min incubation at 4"C, the homogenate was spun for 10 min at 12,OOOg at 4°C. The pellet was lyophilized, solubilized in IEF lysis buffer (100 plhetina), and spun at
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12,OOOg for 2 min in preparation for two-dimensional gel electrophoresis.
Gel Electrophoresis and Fluorography Proteins were separated by two-dimensional gel electrophoresis (O’Farrell, 1975; with modifications as described by Benowitz and Lewis, 1983). The ratio of ampholytes used was 2:2:1 of ampholytes of pH 3.5-5, 5-8, and 3.5-10. This mixture produced a gradient of pH 4.5-6.8. In the IEF (first) dimension, 150-175 p1 (approximately 100 pg) of protein was loaded onto each tube. Samples were run at 300 V for 16 hr, followed by 400 V for 4 hr. In the second dimension [sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis], 5 15% linear gradients of acrylamide were used. Gels were stained in Coomassie brilliant blue, prepared for fluorography (Autofluor; National Diagnostics, Manville, NJ), and exposed to Kodak XAR-5 film. DNA-Cellulose Column Chromatography The chromatin fraction was applied to a DNA-cellulose column and proteins were eluted with increasing concentrations of KCl as described by Sambucetti and Curran (1986). The lyophilized chromatin fraction (from 30 retinas) was suspended in 500 p1 of 0.8 M KC1 in column buffer [20 mM KPO,, pH 7.0, 1 mM EDTA, 10% glycerol, 0.5% NP-40 (Particle Data Laboratories Ltd., Elmhurst, IL), 2 pg/ml leupeptin (Sigma), 3 pg/ml aprotinin (Sigma), and 0.1 mM PMSF]. The suspension was sonicated 4 times, 10 sec each, incubated on ice for 30 min and centifuged at 80,OOOg for 30 min at 4°C. Column buffer without salt (2.8 ml) was added to the supernatant to a final concentration of 0.1 M KCl. The sample (approximately 2.5 ml) was loaded onto a 2.5 ml column of calf thymus native DNA-cellulose (Pharmacia, Piscataway, NJ) at 4°C. The column was first washed with 2 column volumes of buffer without salt. Proteins were then eluted with 2 column volumes each of 0.2 , 0.5, 1.O, and 2.0 M KCl. Fractions of one column volume were collected, dialyzed against 50 mM ammonium acetate, lyophilized, and dissolved in IEF lysis buffer in preparation for two-dimensional gel electrophoresis. Proteolytic Digestion Staphylococcus aureus V8 protease (SAP; Sigma) was used to digest proteins essentially according to the procedure of Cleveland et al. ( 1977). [35S]-labeled gel spots of retinal chromatin proteins were cut from twodimensional gels which had been lightly stained with Coomassie blue. The spots were boiled for 2 min in 1% SDS, equilibrated for 30 min in equilibration buffer (125 mM Tris-HC1, pH 6.8, 0.1% SDS, 1 mM EDTA) at room temperature, and stored frozen at -20°C. For pro-
teolytic digestion, gel spots were thawed, placed in lanes containing the equilibration buffer of a 15% polyacrylamide gel, and overlaid with V8 protease diluted in 125 mM Tris-HC1, pH 6.8, 10% glycerol, 0.5% SDS, 1 mM EDTA, 0.002% bromphenol blue. The gel was run normally except that the current was turned off for 30 min while the dye front was in the stacking gel.
Immunoblots Gels were transferred to nitrocellulose and immunoblotted essentially as described previously (Gossels and Ingram, 1989). Unblocked sites on the nitrocellulose membrane were blocked with 3% bovine serum albumin in 0.01 M Tris-HCL, pH 7.5, 0.15 M NaCl, and all washes were in 0.02 M Tris-HC1, pH 7.5,0.15 M NaC1, 0.5% Triton X- 100. Monoclonal antibodies against the neurofilament ON proteins (Jones et al., 1989) were diluted 1:lO. Antibody binding was visualized using a biotin-conjugated goat anti-mouse IgM antibody and the Vectastain ABC-peroxidase method (Vector Laboratories, Burlingame, CA), with 4-chloro- 1-naphthol as the substrate for the peroxidase. Densitometric Analysis of Two-Dimensional Gels Fluorograms were scanned with a two-dimensional densitometry system (Visage 110, Bio Image). The integrated intensity of proteins of interest relative to the total intensity of all spots was determined and results of several experiments were averaged. Experiments were replicated 2-6 times and each sample in each experiment was run on two gels. Statistical significance of differences in relative intensities of various spots was evaluated for different time points of regeneration using the Student’s t-test. RESULTS Enrichment of Proteins in the Chromatin Fraction In order to study changes in goldfish retinal chromatin proteins during optic nerve regeneration, retinas were removed from intact fish or at 5 , 14, or 32 days following bilateral optic nerve crush. Retinas were incubated in the presence of [35S]-methionine cysteine to label newly synthesized protein and were biochemically fractionated. Labeled proteins of each fraction were visualized on fluorograms of two-dimensional gels and compared. The protein pattern detected in the chromatin fraction was different from those of the non-nuclear soluble and particulate fractions and from that of the nuclear soluble fraction, indicating that the chromatin fraction is a distinct entity (Fig. 2). The major chromatin proteins designated C1, C2, and C3 in Figure 2A, e.g., are at most only minor components in the other fractions.
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Fig. 4. Decreases in C1 and C3 by 32 days postcrush. Proteins synthesized in retinas 14 or 32 days postcrush were radiolabeled with [3sS]-methionine + cysteine. Proteins in the chromatin fraction were then separated by two-dimensional gel electrophoresis and visualized by fluorography. Fluorograms
were exposed for 6-8 days. The basic end of each gel is to the left. The decrease in labeling of protein C1 is significant at P < 0.01 and protein C3 at P < 0.05. The labeling of protein C2 did not change significantly between days 14 and 32. (The increase in labeling of protein C4 is significant at P < 0.02.)
Changes in Chromatin Proteins During Optic Nerve Regeneration Densitometric analysis of two-dimensional gel fluorograms of chromatin proteins during optic nerve regeneration revealed a fourfold, statistically significant increase in [35S]-labelingfor three retinal chromatin proteins, C1, C2, and C3, by day 14 of optic nerve regeneration (Fig. 3). This could indicate either an increase in synthesis of these proteins and/or an increase in their association with chromatin. A comparison of Coomassie blue stained gels indicates that the total accumulation of each of these proteins in the chromatin fraction was also greater at 14 days of regeneration than in intact retinas or those undergoing axonal regeneration for only 5 days (data not shown). C1 and C2 have apparent molecular weights of 58 kDa, while C3 is approximately 51 kDa. Each has a slightly different isoelectric point (PI) in the range of 5.5. In contrast to C1, C2, and C3, labeling of a reference protein, designated C4, was constant during this period of regeneration (Fig. 3). Between days 14 and 32 of regeneration, labeling of C1 decreased 3-fold and C3 decreased 2.5-fold; however, the level of labeled C2 remained elevated (Fig. 4). Labeling of C4 increased almost fourfold between 14 and 32 days. Among experiments, several other proteins variably showed changes in labeling during regeneration but these did not prove to be statistically significant.
applied to a column of native calf thymus DNA-cellulose, and eluted stepwise with increasing concentrations of KCl (Fig. 5). C1, C2, and C3 were retained on native DNA-cellulose and were eluted with 0.2-0.5 M KCI. Similar results were obtained using denatured, rather than native, calf thymus DNA-cellulose (data not shown). Although these proteins were also detected in the first no-salt wash fraction (Fig. 5B), they were not enriched relative to the other proteins as they were in the higher salt fractions. Their presence in the no-salt fraction probably reflects an overloading of the column since no additional proteins were eluted with increased washing (Fig. 5C).
Binding of Chromatin Proteins C1, C2, and C3 to DNA-Cellulose In order to examine whether the identified chromatin proteins bind directly to DNA, chromatin-associated proteins were released from this fraction by sonication,
In Vitro Phosphorylation of Chromatin Proteins In vitro labeling of retinas with [32P]-orthophosphate indicated that C1, C2, and C3 are all phosphoproteins (Fig. 6). The degree to which these proteins were labeled did not appear to change appreciably with optic nerve regeneration (data not shown). Proteolytic Digestion of Chromatin Proteins C1, C2, and C3 The interrelationship of C1, C2, and C3 was investigated by subjecting the three proteins to partial digestion with SAP. C1 and C2 had identical SAP digestion patterns, and these in turn were different from that of C3 (Fig. 7). Relationship of Chromatin Proteins C1 and C2 to ON Proteins The pattern of migration of C1 and C2 on twodimensional gels was reminiscent of that seen for the
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Fig. 5. Binding of chromatin proteins to native calf thymus DNA-cellulose. [35S]-methionine + cysteine labeled chromatin proteins from retinas 14 days postcrush were solubilized and applied to a column of native calf thymus DNA-cellulose. This starting material is the “chromatin extract” (A). The column was washed with a no-salt buffer (see Materials and Methods), proteins were eluted stepwise with 2 column volumes each of 0.2, 0.5, 1.0, and 2.0 M KCl, and fractions of 1 column volume were collected. Proteins from each fraction were analyzed by two-dimensional gel electrophoresis and fluorography. The basic end of each gel is to the left. Fluorograms of two fractions (the first and third column volumes) of the
no-salt wash (B,C), and one fraction each of 0.2 M (D), 0.5 M (E), and 1 .0 M (F) KCI eluted proteins are shown. The fluorogram of the 2.0 M KCl fraction, like that for 1.O M KCl, is blank and is therefore not shown. One half of the column fraction was loaded onto gels B and E. For all other fluorograms, the entire fraction was loaded. All fluorograms were exposed for 21 days. The chromatin extract (A) is 1/16 of the starting material loaded onto the column. Additional protein was also eluted with a second column volume of no-salt wash and a second column volume of 0.2 M KCl. The gel patterns of these eluted proteins are not shown because they are qualitatively similar to B and D, respectively.
goldfish intermediate filament proteins, the ON complex (Quitschke and Schechter, 1983a). We used monoclonal antibodies (mAbs) specific for the ON proteins (mAbs 187 and 84) (Jones et al., 1989) to determine whether C1 and C2 are immunologically related to the ON proteins. Anti-ON antibodies stained several spots, including those that comigrate with C1 and C 2 , on a two-dimensional gel Western blot of [35S]-labeled chromatin pro-
teins (Fig. 8). The spots designated C1 and C2 on the immunoblot correspond exactly to C1 and C2 on a fluorogram of the nitrocellulose filter when the film is overlaid onto the filter. Spots with the same migrations as Cl and C2 were also stained with the anti-ON antibodies on a blot of a two-dimensional gel of Triton X- 100 insoluble retinal proteins, a protein fraction that is enriched in intermediate filaments (Chiu et al., 1981).
Chromatin Proteins in the Goldfish Retina 32
P- Intact
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Fig. 6 . Retinal chromatin proteins C1, C2, and C3 are phosphoproteins. [32P]-Iabeledchromatin proteins from intact retinas were separated by two-dimensional gel electrophoresis and visualized by fluorography. The basic end of each gel is to the left. The levels of phosphorylation of these proteins did not appear to change appreciably during regeneration.
31* 21*
DISCUSSlON In order to understand better the cascade of molecular events that are associated with nerve regeneration, we have investigated the changes in proteins of the retinal chromatin fraction which accompany regeneration of the goldfish optic nerve. The fractionation method used to prepare the chromatin produces a fraction which was a distinct entity, without gross contamination by the proteins of other fractions. During optic nerve regeneration, labeling of three phosphoproteins in the retinal chromatin fraction, C1, C2, and C3, increased fourfold by 14 days postcrush. By 32 days postcrush, labeling of C1 and C3 decreased 2.5-fold and 3-fold, respectively; labeling of C2 remained elevated. C1 and C2 have similar SAP peptide maps and similar molecular sizes on SDS-polyacrylamide gels, but differ slightly in PI. This difference could be attributed to post-translational modifications which affect the charge (PI) of the proteins. C3 cannot as yet be related to any other proteins. The enrichment of proteins C1, C2, and C3 in the chromatin fraction, along with their high affinity binding to DNA-cellulose, points to a possible association with DNA in vivo. It is possible that the interaction is nonspecific, since it occurs equally with native and denatured DNA. It is interesting to note, however, that c-Fos is also retained on both native and single-stranded DNAcellulose, and is likewise eluted at 0.5-1 .O M KC1 (Sambucetti and Curran, 1986). This protein has been shown to increase the affinity of another proto-oncogene, c-Jun, for a specific DNA site, and thus to amplify the gene activating effect of c-Jun (Halazonetis et al., 1988). FOS itself, though a transcription factor, has not so far been shown to bind to DNA in a sequence-specific manner.
Fig. 7. Staphylococcus aureus V8 protease (SAP) digests of C1, C2, and C3. Gel slices containing [35S]-methionine + cysteine labeled C l , C2, and C3 were digested with 0.015 or 0.25 p g SAP in a 15% polyacrylamide gel. Proteolytic fragments were separated by SDS-polyacrylamide gel electrophoresis and visualized by fluorography .
Whether C1, C2, and C3 bind directly to DNA, or to other proteins in the chromatin, is also unknown. It is of interest to note that C1, C2, and C3 are all phosphorylated both in the control retina and in retinas 5 and 14 days postcrush. This is potentially significant in that many transcription factors are phosphoproteins and, in some cases, their phosphorylation state may affect the DNA binding or transcriptional activation (Sorger and Pelham, 1988; Tanaka and Herr, 1990). Based on the time course of their labeling changes, C1, C2, and C3 are not likely to be invoIved in events which occur very early during regeneration, at the time of axonal outgrowth (at around 1 week). However, these
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the intermediate filament-like ON proteins and have similar molecular weights and PIS(Quitschke and Schechter, 1983a). However, the time course of labeling and accumulation of C1 and C2 differs from that of the ON proteins (Quitschke and Schechter, 1983b); in addition, the conditions under which the ON proteins are phosphorylated are also different from those of C1 and C2 (Quitschke and Schechter, 1984). One possible interpretation of these data is that C1 and C2 represent a pool of ON proteins that accumulates and undergoes phosphorylation in the nucleus with a time course that is different from that which has been described in the retina as a whole. Alternatively, C1 and C2 could be intermediate filament proteins related to, but distinct from, the ON proteins. C1 and C2 might, like nuclear lamins which share DNA sequence homology with intermediate filaments, provide structure in the nuclear envelope which surrounds the chromosomes (Fisher et al., 1986; Hoger et ai., 1988; McKeon et al., 1986). C1 and C2 could also be involved in the decondensation of compacted chromatin to make it available for transcription and replication. This hypothesis for intermediate filament function in the nucleus has been suggested by Traub (1985). Further studies are required to determine the functions of C1, C2, and C3.
ACKNOWLEDGMENTS This work was supported by NIH EY05690. J.M.G. was supported by NIH postdoctoral fellowship EY06 152.
Fig. 8. Reactivity of the retinal chromatin fraction and the retinal Triton X-100 insoluble fraction to anti-ON antibodies. Retinal chromatin proteins (A) and retinal Triton X-100 insoluble proteins (B) were separated by two-dimensional gel electrophoresis. The gels were transferred to nitrocellulose and the nitrocellulose was immunoblotted with a 1: 10 dilution of antiON antibody supernatants. Reactivity was visualized using the Vectastain ABC-peroxidase method and 4-chloro- I-naphthol as a substrate for the peroxidase. The basic ends of each gel is to the left. The molecular weight and pH indications are interpolations from markers which are not in the field shown.
three proteins could influence the expression of proteins involved in later events of regeneration such as synaptogenesis. Peak synthesis and transport of many proteins in the optic nerve occurs at 2 weeks or later postcrush (Perry et al., 1987), and it is conceivable that Cl-C3 could play a role in the expression of some of these proteins. C1 and C2 appear to be immunologically related to
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Chromatin Proteins in the Goldfish Retina Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK (1977): Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem 252: 1102-1 106. Fisher DZ, Chaudhary N, Blobel G (1986): cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filaments. Proc Natl Acad Sci USA 83~6450-6454. Giulian D, Des Ruisseaux H, Cowburn D (1980): Biosynthesis and intra-axonal transport of proteins during neuronal regeneration. J Biol Chem 2556494-6501, Gossels JM, Ingram VM (1989): A tubulin identified by a monoclonal antibody is not present in mitotic spindles. Exp. Cell Res 184: 471-483. Gossels JM, Lewis SE, Perrone-Bizzozero NI, Benowitz LI (1989): Changes in the synthesis of chromatin proteins during goldfish optic nerve regeneration. Neurosci Abstr 15:3 18. Grafstein B (1986): The retina as a regenerating organ. In Adler R, Farber DB (eds): “The Retina: A Model for Cell Biology Studies, Part 11.” New York: Academic Press, pp 275-335. Halazonetis TD, Georgopoulos K, Greenberg ME, Leder P (1988): c-Jun dirnerizes with itself and with c-Fos, forming complexes of different DNA binding affinities. Cell 55:917-924. Heacock AM, Agranoff BW (1976): Enhanced labeling of a retinal protein during regeneration of optic nerve in goldfish. Proc Natl Acad Sci USA 733328-832. Heacock AM, Agranoff BW (1982): Protein synthesis and transport in the regenerating goldfish visual system. Neurochem Res 7: 77 1-788. Herr W, Sturm RA, Clerc RG, Corcoran LM, Baltimore D, Sharp PA, Ingraham HA, Rosenfeld MG, Finney M, Ruvkun G, Horvitz HR (1988): The POU domain: A large conserved region in the mammalian pit-1, oct-1, oct-2 and c. elegans unc-86 gene products. Genes Dev 2:1513-1516. Hoger TH, Krohne G, Franke W (1988): Amino acid sequence and molecular characterization of murine lamin B as deduced from cDNA clones. Eur J Cell Biol 47:283-290. Ingoglia NA, Weis P, Mycek J (1975): Axonal transport of RNA during regeneration of optic nerves in goldfish. Brain Res 112: 37 1-38 1. Jones PS, Tesser P, Borchert J, Schechter N (1989): Monoclonal antibodies differentiate neurofilaments and glial filament proteins in the goldfish visual pathway: Probes for monitoring neurite outgrowth from retinal explants. J Neurosci 9:454465. Landreth GE, Agranoff BW (1976): Explant culture of adult goldfish retina: Effect of prior optic nerve crush. Brain Res 118:299303. McKeon FD, Kirschner MW, Caput D (1986): Primary and secondary
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structural homology between the major nuclear envelope and cytoplasmic intermediate filament proteins. Nature 319:463468. Murray M, Forman DS (1971): Fine structural changes in goldfish retinal ganglion cells during axonal regeneration. Brain Res 32:287-298. Murray M, Grafstein B (1969): Changes in the morphology and amino acid incorporation in regenerating goldfish optic neurons. Exp Neurol 2 3:544 -560. Neumann D, Scherson T, Ginzburg I, Littauer UZ, Schwartz M (1983): Regulation of mRNA levels for microtubule proteins during nerve regeneration. FEBS Lett 162:270-276. O’Farrell PH (1975): High resolution two dimensional gel electrophoresis of proteins. J Biol Chem 250:4007-4021. Perrone-Bizzozero NI, Benowitz LI (1987): Expression of a 48kilodalton growth associated protein in the goldfish retina. J Neurochem 48:64&652. Perry GW, Burmeister DW, Grafstein B (1987): Fast axonally transported proteins in regenerating goldfish optic axons. J Neurosci 71792-806. Quitschke W, Schechter N (1983a): Specific optic nerve proteins during regeneration of the goldfish retinotectal pathway. Brain Res 258 :69-78. Quitschke W, Schechter N (1983b): In vitro protein synthesis in the goldfish retinotectal pathway during regeneration: Evidence for specific axonal proteins of retinal origin in the optic nerve. J Neurochem 41:1137-1142. Quitschke W, Schechter N (1984): Fifty-eight thousand dalton intermediate filament proteins of neuronal and nonneuronal origin in the goldfish visual pathway. J Neurochem 42569-576. Sambucetti LC, Curran T (1986): The Fos protein complex is associated with DNA in isolated nuclei and binds to DNA cellulose. Science 234: 1417-141 9. Sorger PK, Pelham HRB (1988): Yeast heat shock factor is an essential DNA-binding protein that exhibits temperature-dependent phosphorylation. Cell 54855 -864. Sturm RA, Herr W (1988): The POU domain is a bipartite DNAbinding structure. Nature 336:601-604. Tanaka M, Herr W (1990): Differential transcriptional activation by oct-1 and oct-2: Interdependent activation domains induce oct2 phosphorylation. Cell 60:375-386. Traub P (1985): “Intermediate Filaments: A Review .” Berlin: Springer-Verlag, pp 170-195. Whitnall MH, Grafstein B (1983): Changes in perikaryal organelles during axonal regeneration in goldfish retinal ganglion cells: An analysis of protein synthesis and routing. Brain Res 272: 49-56. Yoon MG (1971): Reorganization of the retinotectal projection following surgical operations on the optic tectum in goldfish. Exp Neurol 33:395-411.
Changes in Chromatin Proteins During Optic Nerve Regeneration in the Goldfish J.M. Gossels, S.E. Lewis, N.I. Perrone-Bizzozero, and L.I. Benowitz Mailman Research Center (J.M.G., S.E.L., N.1.P.-B., L.I.B.), McLean Hospital, Belmont , and Departments of Psychiatry (J.M.G., N.1.P.-B., L.I.B.) and Neurobiology (S.E.L.), Harvard Medical School, Boston, Massachusetts
Regeneration of the goldfish optic nerve involves mas- ON, (Quitschke and Schechter, 1983a), the microtubulesive changes in the structure and pattern of macro- associated tau protein (Neumann et a]. , 1983), and others molecular synthesis in the retinal ganglion cells. To (Giulian et al., 1980; Benowitz et al., 1981; Perry et al., explore the mechanisms that underlie these events, 1987). At least some of these cellular alterations may be we investigated the changes in chromatin proteins during the course of regeneration. Three major reti- determined by changes in the constellation of proteins nal chromatin proteins, two with apparent molecular that are bound to the nuclear DNA. For many genes, weights of 58 kDa (C1 and C2) and one at 51 kDa RNA transcription is regulated by the binding of specific (C3), all having isoelectric points around 5.5, showed proteins to enhancer and promoter regions. These proa fourfold increase in their synthesis and/or accumu- teins include steroid receptors (reviewed by Beato, lation by 14 days of regeneration. Synthesis of C1 and 1989), proto-oncogene products such as cJun (BohC3 decreased by day 32, the time at which the axons mann et al., 1987), a class of mammalian and Drosohave grown back to the optic tectum and have formed phila proteins known as POU proteins (Herr et al., 1988; many of their synapses; synthesis of C2 remained Sturm and Herr, 1988), and others. In addition to these, high through day 32. All three proteins bound to proteins which affect the extent of chromatin condensaDNA-cellulose and required high salt concentrations tion could be important in determining the accessibility (0.2-0.5 M KCI) to be eluted. C1 and C2 had similar of particular regions of the genome for transcription. proteolytic digestion patterns and reacted with mono- Such a role has been suggested for intermediate filament clonal antibodies that recognize the goldfish interme- proteins in the nucleus (Traub, 1985). diate filament proteins of the ON complex. The proIn an effort to explore some of the molecular mechteins identified here could be involved in structural anisms that underlie optic nerve regeneration, we have alterations in the chromatin, or might serve as tran- investigated the sequence of changes in nonhistone scription factors to regulate gene expression during acidic chromatin proteins, proteins which are associated nerve regeneration. 0 1992 Wiley-Liss, Inc. with nuclear DNA. We have used two-dimensional gel electrophoresis, DNA-cellulose chromatography, proteKey words: retina, DNA binding, two-dimensional olytic digestion, and immunoblotting to identify and gel electrophoresis characterize three retinal chromatin proteins whose synthesis and accumulation increase during the course of optic nerve regeneration (Gossels et al., 1989). INTRODUCTION Regeneration of the goldfish optic nerve is associated with a number of changes in the retinal ganglion cells (reviewed by Grafstein, 1986) that include dramatic increases in cell volume and nucleolar size (Murray and Grafstein, 1969; Murray and Forman, 1971; Whitnall and Grafstein, 1983) and in the synthesis and transport of RNA and protein (Ingoglia et al., 1975; Grafstein, 1986). Specific proteins that increase during the course of this process include GAP-43 (Benowitz and Lewis, 1983; Perrone-Bizzozero and Benowitz, 1987), ptubulin (Heacock and Agranoff, 1976, 1982; Giulian et al., 1980), the intermediate filament proteins ON, and 0 1992 Wiley-Liss, Inc.
Received January 13, 1992; revised April 14, 1992; accepted April 15, 1992. Address reprint requests to Dr. Lany I. Benowitz at his current address: Laboratory for Neuroscience Research in Neurosurgery, Children’s Hospital/Enders 250, 300 Longwood Avenue, Boston, MA 021 15. Abbreviations used: IEF, isoelectric focusing; mAb, monoclonal antibody; MEM, minimal essential medium; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; SAP, Staphylococcus uureus V8 protease; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid.
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MATERIALS AND METHODS Optic Nerve Crush Common goldfish (3.5-4 in. total length; Mt. Parnell Fisheries, Mercersburg, PA) were anesthetized in tricaine methanesulfonate (Finquel; Ayerst Co. and Sigma Chemical Co., St. Louis, MO). Fish were held in place with water circulating through their gills and the optic nerve was crushed 1-2 mm behind the eye as described previously (Yoon, 1971). Controls were derived from the same batches as operated animals. In Vitro Labeling At times varying from 5 to 32 days after crushing the optic nerve, fish were dark-adapted for 2 hr, sacrificed, and their retinas dissected as described previously (Landreth and Agranoff, 1976). To study protein synthesis, retinas were pre-equilibrated for 1 hr in methionineand cysteine-free minimal essential medium (MEM; Gibco Laboratories, Grand Island, NY) containing 10% horse serum (dialyzed against 25 mM HEPES; Sigma) and 20 pg/ml gentamicin (Sigma), then incubated for 2
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hr in this same medium containing 250 pCiiml 13’S]methionine + cysteine (Tran~-~’Slabel; ICN Biomedicals, Costa Mesa, CA). To study protein phosphorylation, retinas were pre-equilibrated for 15 min in phosphate-free MEM containing 10% dialyzed horse serum and 20 pg/ml gentamicin, then incubated for 1 hr in this medium containing 100-125 pCi/rnl [32P]orthophosphate (New England Nuclear/Dupont, Boston, MA). For all experiments, groups of 10-12 retinas were incubated in 2 ml of medium at room temperature on a rotating table in 24-well dishes. Labeled retinas were rinsed twice in phosphate-buffered saline (PBS) and frozen on dry ice.
Subcellular Fractionation Chromatin. The retinal chromatin fraction was isolated essentially as described by Bonner et al. (1968). Groups of 10-12 frozen retinas were thawed quickly and homogenized [ 10 strokes in a teflodglass motorized homogenizer in 10 ml solution A: 75 mM NaCl, 25 mM EDTA, 25 mh4 NaF, 0.4 mM phenylmethylsulfonyl flu-
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Fig. 2. Comparison of 14 day postcrush retinal chromatin proteins to proteins in other subcellular fractions. [35S]-methionine+ cysteine labeled retinal chromatin (A), non-nuclear particulate (B), nuclear soluble (C), and non-nuclear soluble (D) proteins were separated by two-dimensional gel electrophoresis and visualized by fluorography . Fluorograms were exposed for 9-14 days. The basic end of each gel is to the left. oride (PMSF); pH 8.01. The homogenate was spun at 1,500g for 10 min at 4°C and the supernatant (S 1) further separated into non-nuclear soluble and particulate fractions (see below and Fig. 1). The pellet (Pl) was resuspended in 10 ml buffer A and spun at 6,OOOg for 10 min at 4°C.The resulting pellet (P2) was suspended in 2.5 ml lysis buffer (10 mM Tris-HCI, pH 7.5, 1 mM EGTA, 25
mM NaF, 0.4 mM PMSF) and spun at 12,OOOg for 10 min at 4°C. The supernatant (S3) was the nuclear soluble fraction. The lysis buffer extraction was repeated twice on the pellet (P3). The resulting pellet was resuspended in approximately 4 ml lysis buffer, layered over 8 ml 1.7 M sucrose in lysis buffer, and spun at 100,OOOg for 2 hr. The resulting pellet was the chromatin fraction. (In some
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Fig. 3. The labeling of several retinal chromatin proteins increases by 14 days postcrush. Retinas from control (intact) fish and those undergoing axonal regeneration for 5 or 14 days were labeled with [35S]-methionine cysteine. Chromatin was prepared, proteins were separated by two-dimensional gel electrophoresis, and visualized by fluorography. Fluorograms were exposed for 28 days. The basic end of each gel is to the left.
The region in each gel corresponding to the boxed area has been enlarged to show the increases in synthesis of proteins C1, C2, and C3 by 14 days of regeneration. The increase in synthesis of C1 is statistically significant at the level of P < 0.02, C2 at P < 0.01, and C3 at P < 0.001. C4 is an example of a protein whose labeling did not change during this time period.
experiments the gradient was spun at a lower speed for a longer time, but in all experiments the g force times t was held constant at 200,000.)In preparation for two-dimensional gel electrophoresis, the chromatin fraction was lyophilized, dissolved in isoelectric focusing (IEF) lysis buffer (40 pketina) (O'Farrell, 1975), sonicated 3 times, 10 sec each, and spun for 2 min at 12,OOOg in a microfuge. Non-nuclear soluble and particulate fractions. The S1 supernatant was spun at 100,OOOg for 1 hr. The resulting pellet, the non-nuclear particulate fraction, was lyophilized, dissolved in IEF lysis buffer (70 plhetina), and prepared for two-dimensional gel electrophoresis in the same way as the chromatin. The supernatant (i.e., the non-nuclear soluble fraction) was treated with 10% trichloroacetic acid (TCA) on ice for 1 hr to precipitate proteins. This fraction was spun for 15 min at 12,OOOg at 4°C. The pellet was suspended in double distilled water
and also spun for 15 min at 12,OOOgat 4°C. This pellet was dissolved in IEF lysis buffer (70plhetina) for twodimensional gel electrophoresis. Nuclear soluble protein fraction. The nuclear soluble protein fraction (S3 from the chromatin preparation) was precipitated with TCA in the same way as was the non-nuclear soluble fraction. The washed pellet was dissolved in IEF lysis buffer (17.5plhetina). Triton X-100fractionation. The total retinal homogenate was extracted with Triton X-100 to separate intermediate filament proteins (Triton X- 100 insoluble) from other components. Extraction was carried out in solution A according to the method of Chiu et al. (1981). The pH was adjusted to 6.8 with HCl and Triton X-100 was added to a final concentration of 0.5%. After a 10 min incubation at 4"C, the homogenate was spun for 10 min at 12,OOOg at 4°C. The pellet was lyophilized, solubilized in IEF lysis buffer (100 plhetina), and spun at
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12,OOOg for 2 min in preparation for two-dimensional gel electrophoresis.
Gel Electrophoresis and Fluorography Proteins were separated by two-dimensional gel electrophoresis (O’Farrell, 1975; with modifications as described by Benowitz and Lewis, 1983). The ratio of ampholytes used was 2:2:1 of ampholytes of pH 3.5-5, 5-8, and 3.5-10. This mixture produced a gradient of pH 4.5-6.8. In the IEF (first) dimension, 150-175 p1 (approximately 100 pg) of protein was loaded onto each tube. Samples were run at 300 V for 16 hr, followed by 400 V for 4 hr. In the second dimension [sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis], 5 15% linear gradients of acrylamide were used. Gels were stained in Coomassie brilliant blue, prepared for fluorography (Autofluor; National Diagnostics, Manville, NJ), and exposed to Kodak XAR-5 film. DNA-Cellulose Column Chromatography The chromatin fraction was applied to a DNA-cellulose column and proteins were eluted with increasing concentrations of KCl as described by Sambucetti and Curran (1986). The lyophilized chromatin fraction (from 30 retinas) was suspended in 500 p1 of 0.8 M KC1 in column buffer [20 mM KPO,, pH 7.0, 1 mM EDTA, 10% glycerol, 0.5% NP-40 (Particle Data Laboratories Ltd., Elmhurst, IL), 2 pg/ml leupeptin (Sigma), 3 pg/ml aprotinin (Sigma), and 0.1 mM PMSF]. The suspension was sonicated 4 times, 10 sec each, incubated on ice for 30 min and centifuged at 80,OOOg for 30 min at 4°C. Column buffer without salt (2.8 ml) was added to the supernatant to a final concentration of 0.1 M KCl. The sample (approximately 2.5 ml) was loaded onto a 2.5 ml column of calf thymus native DNA-cellulose (Pharmacia, Piscataway, NJ) at 4°C. The column was first washed with 2 column volumes of buffer without salt. Proteins were then eluted with 2 column volumes each of 0.2 , 0.5, 1.O, and 2.0 M KCl. Fractions of one column volume were collected, dialyzed against 50 mM ammonium acetate, lyophilized, and dissolved in IEF lysis buffer in preparation for two-dimensional gel electrophoresis. Proteolytic Digestion Staphylococcus aureus V8 protease (SAP; Sigma) was used to digest proteins essentially according to the procedure of Cleveland et al. ( 1977). [35S]-labeled gel spots of retinal chromatin proteins were cut from twodimensional gels which had been lightly stained with Coomassie blue. The spots were boiled for 2 min in 1% SDS, equilibrated for 30 min in equilibration buffer (125 mM Tris-HC1, pH 6.8, 0.1% SDS, 1 mM EDTA) at room temperature, and stored frozen at -20°C. For pro-
teolytic digestion, gel spots were thawed, placed in lanes containing the equilibration buffer of a 15% polyacrylamide gel, and overlaid with V8 protease diluted in 125 mM Tris-HC1, pH 6.8, 10% glycerol, 0.5% SDS, 1 mM EDTA, 0.002% bromphenol blue. The gel was run normally except that the current was turned off for 30 min while the dye front was in the stacking gel.
Immunoblots Gels were transferred to nitrocellulose and immunoblotted essentially as described previously (Gossels and Ingram, 1989). Unblocked sites on the nitrocellulose membrane were blocked with 3% bovine serum albumin in 0.01 M Tris-HCL, pH 7.5, 0.15 M NaCl, and all washes were in 0.02 M Tris-HC1, pH 7.5,0.15 M NaC1, 0.5% Triton X- 100. Monoclonal antibodies against the neurofilament ON proteins (Jones et al., 1989) were diluted 1:lO. Antibody binding was visualized using a biotin-conjugated goat anti-mouse IgM antibody and the Vectastain ABC-peroxidase method (Vector Laboratories, Burlingame, CA), with 4-chloro- 1-naphthol as the substrate for the peroxidase. Densitometric Analysis of Two-Dimensional Gels Fluorograms were scanned with a two-dimensional densitometry system (Visage 110, Bio Image). The integrated intensity of proteins of interest relative to the total intensity of all spots was determined and results of several experiments were averaged. Experiments were replicated 2-6 times and each sample in each experiment was run on two gels. Statistical significance of differences in relative intensities of various spots was evaluated for different time points of regeneration using the Student’s t-test. RESULTS Enrichment of Proteins in the Chromatin Fraction In order to study changes in goldfish retinal chromatin proteins during optic nerve regeneration, retinas were removed from intact fish or at 5 , 14, or 32 days following bilateral optic nerve crush. Retinas were incubated in the presence of [35S]-methionine cysteine to label newly synthesized protein and were biochemically fractionated. Labeled proteins of each fraction were visualized on fluorograms of two-dimensional gels and compared. The protein pattern detected in the chromatin fraction was different from those of the non-nuclear soluble and particulate fractions and from that of the nuclear soluble fraction, indicating that the chromatin fraction is a distinct entity (Fig. 2). The major chromatin proteins designated C1, C2, and C3 in Figure 2A, e.g., are at most only minor components in the other fractions.
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Fig. 4. Decreases in C1 and C3 by 32 days postcrush. Proteins synthesized in retinas 14 or 32 days postcrush were radiolabeled with [3sS]-methionine + cysteine. Proteins in the chromatin fraction were then separated by two-dimensional gel electrophoresis and visualized by fluorography. Fluorograms
were exposed for 6-8 days. The basic end of each gel is to the left. The decrease in labeling of protein C1 is significant at P < 0.01 and protein C3 at P < 0.05. The labeling of protein C2 did not change significantly between days 14 and 32. (The increase in labeling of protein C4 is significant at P < 0.02.)
Changes in Chromatin Proteins During Optic Nerve Regeneration Densitometric analysis of two-dimensional gel fluorograms of chromatin proteins during optic nerve regeneration revealed a fourfold, statistically significant increase in [35S]-labelingfor three retinal chromatin proteins, C1, C2, and C3, by day 14 of optic nerve regeneration (Fig. 3). This could indicate either an increase in synthesis of these proteins and/or an increase in their association with chromatin. A comparison of Coomassie blue stained gels indicates that the total accumulation of each of these proteins in the chromatin fraction was also greater at 14 days of regeneration than in intact retinas or those undergoing axonal regeneration for only 5 days (data not shown). C1 and C2 have apparent molecular weights of 58 kDa, while C3 is approximately 51 kDa. Each has a slightly different isoelectric point (PI) in the range of 5.5. In contrast to C1, C2, and C3, labeling of a reference protein, designated C4, was constant during this period of regeneration (Fig. 3). Between days 14 and 32 of regeneration, labeling of C1 decreased 3-fold and C3 decreased 2.5-fold; however, the level of labeled C2 remained elevated (Fig. 4). Labeling of C4 increased almost fourfold between 14 and 32 days. Among experiments, several other proteins variably showed changes in labeling during regeneration but these did not prove to be statistically significant.
applied to a column of native calf thymus DNA-cellulose, and eluted stepwise with increasing concentrations of KCl (Fig. 5). C1, C2, and C3 were retained on native DNA-cellulose and were eluted with 0.2-0.5 M KCI. Similar results were obtained using denatured, rather than native, calf thymus DNA-cellulose (data not shown). Although these proteins were also detected in the first no-salt wash fraction (Fig. 5B), they were not enriched relative to the other proteins as they were in the higher salt fractions. Their presence in the no-salt fraction probably reflects an overloading of the column since no additional proteins were eluted with increased washing (Fig. 5C).
Binding of Chromatin Proteins C1, C2, and C3 to DNA-Cellulose In order to examine whether the identified chromatin proteins bind directly to DNA, chromatin-associated proteins were released from this fraction by sonication,
In Vitro Phosphorylation of Chromatin Proteins In vitro labeling of retinas with [32P]-orthophosphate indicated that C1, C2, and C3 are all phosphoproteins (Fig. 6). The degree to which these proteins were labeled did not appear to change appreciably with optic nerve regeneration (data not shown). Proteolytic Digestion of Chromatin Proteins C1, C2, and C3 The interrelationship of C1, C2, and C3 was investigated by subjecting the three proteins to partial digestion with SAP. C1 and C2 had identical SAP digestion patterns, and these in turn were different from that of C3 (Fig. 7). Relationship of Chromatin Proteins C1 and C2 to ON Proteins The pattern of migration of C1 and C2 on twodimensional gels was reminiscent of that seen for the
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Fig. 5. Binding of chromatin proteins to native calf thymus DNA-cellulose. [35S]-methionine + cysteine labeled chromatin proteins from retinas 14 days postcrush were solubilized and applied to a column of native calf thymus DNA-cellulose. This starting material is the “chromatin extract” (A). The column was washed with a no-salt buffer (see Materials and Methods), proteins were eluted stepwise with 2 column volumes each of 0.2, 0.5, 1.0, and 2.0 M KCl, and fractions of 1 column volume were collected. Proteins from each fraction were analyzed by two-dimensional gel electrophoresis and fluorography. The basic end of each gel is to the left. Fluorograms of two fractions (the first and third column volumes) of the
no-salt wash (B,C), and one fraction each of 0.2 M (D), 0.5 M (E), and 1 .0 M (F) KCI eluted proteins are shown. The fluorogram of the 2.0 M KCl fraction, like that for 1.O M KCl, is blank and is therefore not shown. One half of the column fraction was loaded onto gels B and E. For all other fluorograms, the entire fraction was loaded. All fluorograms were exposed for 21 days. The chromatin extract (A) is 1/16 of the starting material loaded onto the column. Additional protein was also eluted with a second column volume of no-salt wash and a second column volume of 0.2 M KCl. The gel patterns of these eluted proteins are not shown because they are qualitatively similar to B and D, respectively.
goldfish intermediate filament proteins, the ON complex (Quitschke and Schechter, 1983a). We used monoclonal antibodies (mAbs) specific for the ON proteins (mAbs 187 and 84) (Jones et al., 1989) to determine whether C1 and C2 are immunologically related to the ON proteins. Anti-ON antibodies stained several spots, including those that comigrate with C1 and C 2 , on a two-dimensional gel Western blot of [35S]-labeled chromatin pro-
teins (Fig. 8). The spots designated C1 and C2 on the immunoblot correspond exactly to C1 and C2 on a fluorogram of the nitrocellulose filter when the film is overlaid onto the filter. Spots with the same migrations as Cl and C2 were also stained with the anti-ON antibodies on a blot of a two-dimensional gel of Triton X- 100 insoluble retinal proteins, a protein fraction that is enriched in intermediate filaments (Chiu et al., 1981).
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Fig. 6 . Retinal chromatin proteins C1, C2, and C3 are phosphoproteins. [32P]-Iabeledchromatin proteins from intact retinas were separated by two-dimensional gel electrophoresis and visualized by fluorography. The basic end of each gel is to the left. The levels of phosphorylation of these proteins did not appear to change appreciably during regeneration.
31* 21*
DISCUSSlON In order to understand better the cascade of molecular events that are associated with nerve regeneration, we have investigated the changes in proteins of the retinal chromatin fraction which accompany regeneration of the goldfish optic nerve. The fractionation method used to prepare the chromatin produces a fraction which was a distinct entity, without gross contamination by the proteins of other fractions. During optic nerve regeneration, labeling of three phosphoproteins in the retinal chromatin fraction, C1, C2, and C3, increased fourfold by 14 days postcrush. By 32 days postcrush, labeling of C1 and C3 decreased 2.5-fold and 3-fold, respectively; labeling of C2 remained elevated. C1 and C2 have similar SAP peptide maps and similar molecular sizes on SDS-polyacrylamide gels, but differ slightly in PI. This difference could be attributed to post-translational modifications which affect the charge (PI) of the proteins. C3 cannot as yet be related to any other proteins. The enrichment of proteins C1, C2, and C3 in the chromatin fraction, along with their high affinity binding to DNA-cellulose, points to a possible association with DNA in vivo. It is possible that the interaction is nonspecific, since it occurs equally with native and denatured DNA. It is interesting to note, however, that c-Fos is also retained on both native and single-stranded DNAcellulose, and is likewise eluted at 0.5-1 .O M KC1 (Sambucetti and Curran, 1986). This protein has been shown to increase the affinity of another proto-oncogene, c-Jun, for a specific DNA site, and thus to amplify the gene activating effect of c-Jun (Halazonetis et al., 1988). FOS itself, though a transcription factor, has not so far been shown to bind to DNA in a sequence-specific manner.
Fig. 7. Staphylococcus aureus V8 protease (SAP) digests of C1, C2, and C3. Gel slices containing [35S]-methionine + cysteine labeled C l , C2, and C3 were digested with 0.015 or 0.25 p g SAP in a 15% polyacrylamide gel. Proteolytic fragments were separated by SDS-polyacrylamide gel electrophoresis and visualized by fluorography .
Whether C1, C2, and C3 bind directly to DNA, or to other proteins in the chromatin, is also unknown. It is of interest to note that C1, C2, and C3 are all phosphorylated both in the control retina and in retinas 5 and 14 days postcrush. This is potentially significant in that many transcription factors are phosphoproteins and, in some cases, their phosphorylation state may affect the DNA binding or transcriptional activation (Sorger and Pelham, 1988; Tanaka and Herr, 1990). Based on the time course of their labeling changes, C1, C2, and C3 are not likely to be invoIved in events which occur very early during regeneration, at the time of axonal outgrowth (at around 1 week). However, these
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the intermediate filament-like ON proteins and have similar molecular weights and PIS(Quitschke and Schechter, 1983a). However, the time course of labeling and accumulation of C1 and C2 differs from that of the ON proteins (Quitschke and Schechter, 1983b); in addition, the conditions under which the ON proteins are phosphorylated are also different from those of C1 and C2 (Quitschke and Schechter, 1984). One possible interpretation of these data is that C1 and C2 represent a pool of ON proteins that accumulates and undergoes phosphorylation in the nucleus with a time course that is different from that which has been described in the retina as a whole. Alternatively, C1 and C2 could be intermediate filament proteins related to, but distinct from, the ON proteins. C1 and C2 might, like nuclear lamins which share DNA sequence homology with intermediate filaments, provide structure in the nuclear envelope which surrounds the chromosomes (Fisher et al., 1986; Hoger et ai., 1988; McKeon et al., 1986). C1 and C2 could also be involved in the decondensation of compacted chromatin to make it available for transcription and replication. This hypothesis for intermediate filament function in the nucleus has been suggested by Traub (1985). Further studies are required to determine the functions of C1, C2, and C3.
ACKNOWLEDGMENTS This work was supported by NIH EY05690. J.M.G. was supported by NIH postdoctoral fellowship EY06 152.
Fig. 8. Reactivity of the retinal chromatin fraction and the retinal Triton X-100 insoluble fraction to anti-ON antibodies. Retinal chromatin proteins (A) and retinal Triton X-100 insoluble proteins (B) were separated by two-dimensional gel electrophoresis. The gels were transferred to nitrocellulose and the nitrocellulose was immunoblotted with a 1: 10 dilution of antiON antibody supernatants. Reactivity was visualized using the Vectastain ABC-peroxidase method and 4-chloro- I-naphthol as a substrate for the peroxidase. The basic ends of each gel is to the left. The molecular weight and pH indications are interpolations from markers which are not in the field shown.
three proteins could influence the expression of proteins involved in later events of regeneration such as synaptogenesis. Peak synthesis and transport of many proteins in the optic nerve occurs at 2 weeks or later postcrush (Perry et al., 1987), and it is conceivable that Cl-C3 could play a role in the expression of some of these proteins. C1 and C2 appear to be immunologically related to
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