Histones of chick embryonic lens nuclei

DEVELOPMENTAL

BIOLOGY

41, 72-76 (1974)

Histones

of Chick Embryonic

Lens Nuclei

NELSON N. H. TENG, JORAM PIATIGORSKY, AND VERNON M. INGRAM Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, Maryland 20014 Accepted July l&l974 Histones from nuclei of nondividing lens fiber and lens epithelial cells have been compared with histones from nondividing erythroid cells, and from dividing fibroblasts; all cells were derived from chick embryos. Lens fiber and lens epithelial nuclei contain a very large amount of a characteristic fl histone, but they do not have the specific f2c histone characteristic of the chick erythroid nucleus. Neither the lens fiber fl histone nor the f2c histone are found in fibroblast nuclei. INTRODUCTION

Rapid progress has been made in the characterization of histones, but their biological functions are still unknown. In recent investigations several temporal features of histone development have been observed in embryogenesis of many organisms including plants (Fambrough et al., 1968) and animals (Byrd and Kasinsky, 1973; Vorobyev, et al., 1369; Bentinnen and Comb, 1971; Asao, 1972; Moss et al., 1973; Ruderman and Gross, 1974. See also Johns and Diggle, 1969; Appels et al., 1972; for histone changes in adult animals during erythroid maturation). The most significant developmental changes, both quantitative and qualitative, seem to occur in the “very lysine rich” histone, designated fl or histone I. In sea urchin embryogenesis (Ruderman and Gross, 1974), the synthesis of the fI histone characteristic of the morula stage gives way to synthesis of a new and electrophoretically distinct fl at the gastrula stage. In chick embryonic erythropoiesis (Johns and Diggle, 1969; Moss et al., 1973) a significant increase of a specific “lysine rich” histone (f2c or histone V) during maturation is observed. The increase corresponds in time to the transition from the primitive to the definitive erythroid cell series (Bruns and Ingram, 1973), and also the final division of the Copyright All rights

Q 1974 by Academic Press, Inc. of reproduction in any form reserved.

primitive erythroid cells. A similar though not identical histone is present in fish and frog erythrocytes (Edwards and Hnilica, 1968), frog liver (Nelson, Yunis, 1969) and may also be present in the frog lens (Johnson and Rothstein, 19’70). We wonder whether these specific f2c histones appear in the erythroid cell as part of the transition from dividing to a nondividing cell, perhaps as a signal (Moss et al., 1973; Seligy et al., 1973). For this reason it seemed worthwhile to look at another nondividing cell type in the chick embryo-the lens fiber cell. In the present communication we will describe a study of the histone pattern in the chick embryonic lens. The lens system provides a cell type which has characteristic features of differentiation and is conveniently free of erythrocyte contamination. During development, the lens epithelial cells differentiate into lens fibers. The lens fiber nuclei are apparently inactive in both DNA and RNA synthesis (Reeder and Bell, 1965; Modak et al., 1966; Persons and Modak, 1970; Modak and Persons, I97I), a situation which is similar to that of the mature chick erythrocyte. It is, therefore, of interest to compare the histone pattern between the lens and the erythrocytes, particularly with reference to f2c or related histones. 72

TENG, PIATIGORSKY AND INGRAM MATERIALS

AND

METHODS

Chick

Lens Histones

73

broth. The secondary culture was free of erythroid cells and the confluent culture Preparation of the Chick lens. Prelimiwas harvested by scraping. The cells were nary tests were conducted with about 1000 washed in RSB and resuspended in 9 vol of intact lenses explanted from 15-day-old RSB with 0.5% NP-40 (Shell Chemical chick embryos and stored in liquid N, imCo., London), then vortexed for 30 set and mediately. For the final experiments, the Dounce hofibers and epithelia were separated from transferred to a tight-fitting mogenizer. The cells were lysed with six approximately 1300 lenses before immergentle strokes and the nuclei collected by sion into liquid N 2. The epithelia contained The nuclei were then the cubsidal, central epithelial cells and centrifugation. the longer, peripheral annular pad cells. washed as described above and used for histone extraction. The frozen fiber masses were transferred Preparation of chick erythrocytes. Adult to a sterile loose-fitting Dounce homogenizer with 10 ml of buffer, consisting of hen red blood cells were obtained from 10 mM Tris, 1 mM EDTA (pH 7.5) and SPAFAS and used directly for nuclei prepallowed to thaw and swell in this buffer for aration. The procedure was the same as described by Moss et al. (1973), except 20 min. They were then homogenized gently that 0.5% Triton X-100 was used to solu(four strokes). The epithelia were treated bilize the erythrocyte membranes. in the same way except that 5 ml of buffer Preparation of histones. The histones was used instead of 10. A 1:lO dilution of were prepared by acid extraction either each preparation into Trypan Blue was with 0.25 N HCl or 5% w/v perchloric acid examined microscopically for cell lysis. with 20% TCA The nuclei appeared whole, but cells were and then precipitated (Moss et al., 1973). In some experiments, broken. Both homogenates were centrifuged at 10,000 rpm (Sorvall-SS34) for 8 when small amounts of histones were inhistones were min. The supernatant fractions were volved, the acid-extracted removed. The pellets were then gently re- recovered by dialysis against deionized instead of precipisuspended in 10 ml of the same buffer and water and lyophilized tated with 20% trichloroacetic acid. spun at low speed (1500 rpm) for 5 min. Polyacrylamide At this stage, the nuclear pellets were ek?CtFOphOFesis. gel Electrophoresis was performed as defrozen in liquid N, until further analyzed. scribed by Panyim and Chalkley (1969) and The nuclear pellets were then resuspended modified by Moss et al. (1973). However, in RSB (10 mM NaCl, 10 mM Tris, 3 mM unlike Moss et al., who carried out the MgC12, pH 7.4), with sodium deoxycholate, and Tween 40 was added to clean the electrophoresis at 4°C for 17 hr at 130 V (constant voltage), electrophoresis here nuclei (Penman, 1966). The purified nuclei were used directly for acid extraction of was carried out at 22°C at 130 V for 10 hr. histones. RESULTS Preparation of chick fibroblasts. Chick embryo fibroblasts were prepared from llUnder our modified conditions, histone day-old embryos (SPAFAS, Norwich, CN), f2c migrates slower than histone f3. This as described by Rein and Rubin (1968). agrees with previous reports (Sanders and Primary cells were seeded at a density of McCarty, 1972; Appels and Wells, 1972; 1 x lo7 cells per 100 mm Falcon plastic Sotirov and Johns, 1972) but disagrees Petri dish in Medium 199 supplemented with Moss et al. (1973), who found that with 10% calf serum, 1% heat-inactivated histone f2c migrated faster than histone f3. chick serum, and 2% tryptose phosphate The discrepancy is due to temperature

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VOLUME 41, 1974

differences in the two experimental conditions rather than to any other factors in the electrophoresis gel system. Furthermore, we find that the histone fl components and histone f2c all migrate more slowly relative to the other histones at a higher temperature (Table 1). This may indicate that the lysine-rich histone fl and the serine-rich histone f2c are either conformationally more sensitive to temperature or more sensitive to certain temperature dependent factors in the gel system. Further investigation is needed to elucidate the mechab c d e nism of the temperature effect on electrophoretic mobility. Whatever the mechaFIG. 1. Comparison of electrophoretic pattern of nism may be, the finding suggests that 5% w/v perchloric acid extractable histones on 15% histone f2c is more closely related to the acrylamide gels stained with 1% amido black. The slow moving band which appears in lens fibers histone fl group than to the other histones. (marked by arrow) but not in lens epithelium is of The results of Fig. 1 and 2 show clearly unknown origin; it is not affected by incubation with that in the chick embryo the erythroid 0.1 M 2-mercaptoethanol at 37°C for 1 hr. Perchloric specific histone f2c is not present at all in extmcts: (a) lens fibers (equivalent to approximately the lens fiber, nor can we find it in lens 1.5 x lOa nuclei used in the perchloric acid extract); epithelium cells or fibroblasts. We also do (b) lens epithelium (1.6 x lOa nuclei); (c) chick embryonic fibroblasts (1.1 x lo8 nuclei); (d) red blood not observe the tissue specific histone cells from adult chicken (approximately 28 pg of prowhich has been described for epithelial tein is loaded, equivalent to 1.1 x lo8 nuclei); Total and fiber cells of the frog lens (Johnson h&ones: (e) from red blood cells from adult chicken and Rothstein, 1970). However, the lens (1.1 x lOBnuclei). fiber cells and the lens epithelial cells do show that the fl” component of the fl group show little change among the tissues exis very greatly increased in quantity rela- amined (Fig. 3). tive to fibroblasts or erythroid cells. This DISCUSSION major and characteristic histone also reThe present experiments show that lens sembles histone f2c in being extractable by perchloric acid. The remaining histones nuclei from l&day-old chick embryos have an unusually high proportion of the fl” TABLE 1 component (Fig. 2) of the fl histones. This RELATIVE ELECTROPHORETICMOBILITIES OF HISTONE characteristic histone pattern is found in FRACTIONS AT 22°C AND 4°C the nuclei of cells from the epithelium and Histone 4°C 22°C Difference the fibers, indicating that it is not due to fraction the presence of the pyknotic and degenerfl” 0.69 ating nuclei present only in the fiber mass 0.65 0.04 fl’ 0.71 0.67 0.04 (Modak and Perdue, 1970). It is not posfl” 0.72 0.68 0.04 sible to determine from the present data fl”’ 0.74 0.70 0.04 whether the histone pattern found in the t2C 0.82 0.78 0.04 embryonic chick lens nuclei is associated f3 0.80 0.80 0.0 f2b 0.85 0.85 0.0 with the early stages of fiber differentiat2a2 0.86 0.86 0.0 tion, since the annular pad cells repref2al 0 0.98 0.98 0.0 sented a significant proportion of the epif2al’ 1.06 1.00 0.0 thelia which were examined. 0

Chick

TENG, PIATIGORSKY AND INGRAM

Chuck Lem

Epthelium

FIG. 2. Microdensitometer

Chick

scans (615 nm, Gilford

Adult Chicken Erymocyte

Chick

Chack Lrns

Chck Lens F,ber

FIG. 3. Comparison of electrophoretic patterns of histones f3, f2b, f2a2, f2al. After removal of histone fl and f2c by perchloric acid extraction from various nuclei, the remaining histones were isolated by further extraction in 0.25 N HCI for 16 hr.

Possibly, the novel histone pattern of the lens cell nuclei relates to the arrest of cell division, which has occurred in all of

Lens

75

Histones

Lens Fiber

model 2420 Spectrophotometer)

of gels in Fig. I

the cells of the fiber mass and those of the most peripheral annular pad region of the epithelium. Moreover, a high proportion of the cells of the central region of the epithelium are no longer dividing by this stage of development (Persons and Modak, 1970). Such an interpretation is of special interest when considering the appearance of the unique histone f2c at the time cell division ceases during erythroid cell maturation in chick embryos (Moss et al., 1973; Seligy et al., 1973). Both the fl histone in the lens cells and the f2c histone in definitive erythroid cells share the properties of being extractable with perchloric acid and of possessing a distinct temperaturemobility relationship; electrophoretic both appear to be specific for their respective tissues and both correlate with the absence of cell division. It would be of considerable interest to examine the histone contents of other cells which have entered an arrested state of cell division during development, and to follow the possible change in the pattern of histone synthesis during the decrease in cell division which occurs during lens fiber formation in tissue culture (Piatigorsky and Rothschild, 1972; Piatigorsky et al., 1973).

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This work was supported by a grant from the National Institutes of Health (AM 13945). We thank Dr. Peggy Zelenka and Ms. Sonia S. Rothschild for help in the collection and preparation of the lens cell nuclei. REFERENCES APPELS, R., and WELLS, J. R. E. (1972). Synthesis and turnover of DNA-bound histone during maturation of avian red blood cells. J. Mol. Biol. 70,425-434. APPELS, R., WELLS, J. R. E., and WILLIAMS, A. F. (1972). Characterization of DNA-bound histone in the cells of the avian erythropoietic series. J. Cell sci. 10,47-59. ASAO, T. (1972). Origin of histones, and relation between nuclear histones and cytoplasmic basic proteins in early development of Japanese newt, Trituruspyrrhogaster. Exp. Cell Res. 73,73-80. BENTINNEN, L. C., and COMB, D. G. (1971). Early and late histones during sea urchin development. J. Mol. Biol. 57.355-358. BRUNS, G. A. P., and INGRAM, V. M. (1973). The erythroid cells and hemoglobins of the chick embryo. Philos. Trans. Royal Sot. London 266, 225-305. BYRD, E. W., and KASINSKY, H. E. (1973). Histone synthesis during early embryogenesis in Xenopus laeois (South African Clawed Toad). Biochemistry 12,246-253. EDWARDS, L. J., and HNILICA, L. S. (1968). The specificity of histones in nucleated erythrocytes. Experientia 24,228-229. FAMBROUGH, D. M., FUJIMURA, F., and BONNER, J. (1968). Quantitative distribution of histone components in the pea plant. Biochemistry 7.575-585. JOHNS, E. W., and DIGGLE, J. H. (1969). A method for the large scale preparation of the avian erythrocyte specific histone f2c. Eur. J. Biochem. 11,495-498. JOHNSON, A., and ROTHSTEIN, H. (1970). Amphibian lens histones and their relation to the cell cycle. J. Gen. Phys. 35,688-702. MODAK, S. P., and PERDUE, S. W. (1970). Terminal lens cell differentiation. I. Histological and microspectrophotometric analysis of nuclear degeneraation. Exp Cell Res. 59,43-56. MODAK, S. P., MORRIS, G., and YAMADA, T. (1966). DNA synthesis and mitotic activity during early development of chick lens. Deuelop. Biol. 17, 544-561. MODAK, S. P., and PERSONS, B. J. (1971). RNA syn-

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