Chromatin of the developing chick oviduct: Changes in the acidic proteins

Biochimica et Biophysica Acta, 312 (1973) 765-778

© Elsevier ScientificPublishingCompany,Amsterdam- Printed in The Netherlands BBA 97734

CHROMATIN OF THE DEVELOPING CHICK OVIDUCT: CHANGES IN THE ACIDIC PROTEINS T. C. SPELSBERGa'*, W. M. MITCHELLc'd, F. CHYT1Lb'c, E. M. WILSONb and B. W. O,MALLEY**.a.b.c Departments ofaObstetrics and Gynecology, bBiachemistry, CMedicine and aMicrobiology, Vanderbilt University, Nashville, Tenn. 37232 (U.S.A.)

(Received February 12th, 1973)

SUMMARY 1. The chromatin of the oviducts of immature chicks undergoing estrogeninduced differentiation was studied. Both the species and quantitative level of the histones remained relatively unaltered throughout development. In contrast, the level of the acidic proteins displayed a biphasie pattern, increasing during the first few days of differentiation followed by a decrease during the final stages of maturation. The level of chromatin-associated RNA and the capacities of chromatin to serve as template in in vitro DNA-dependent RNA synthesis showed a similar biphasic pattern. 2. Fractionation of the acidic chromatin proteins into four groups followed by sodium dodecyl sulfate-polyacrylamidegel electrophoresis demonstrated a marked heterogeneity of the acidic chromatin proteins. The majority of protein bands were associated with two of the four groups (API and AP2). Quantitative analysis revealed that variations primarily in the level of one fraction (APz) were responsible for the changes observed in the total chromatin acidic proteins. Amino acid composition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical analysis demonstrated that alterations in the molecular species of acidic proteins occurred during development. Using circular dichroism to probe the secondary structure of whole and dehistonized chromatin, a gradual conformational change was observed in both the DNA and protein components during oviduct development. 3. The observed changes in chromatin structure and composition are suspected to be involved in the changing pattern of gene expression during estrogen-mediated development of the chick oviduct.

INTRODUCTION The concept of cell differentiation which is based on programmed selective expression of genetic information has existed for many years. In studies of embryonic * Present address: Department of Endocrine Research, Mayo Clinic, Rochester, Minn. 55901 U.S.A. ** Present address: Department of Cell Biology,Baylor College of Medicine, Houston, Texas 77025, U.S.A.

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T.C. SPELSBERGet al.

development during cleavage in sea urchins 1-3 as well as during cleavage and early organogenesis in frogs 4'5, the capacity of chromatin isolated from these cells to serve as a template for RNA synthesis in vitro increases as development progresses. Similarly, the content of acidic proteins but not histones in chromatin increases. Changes in the newly synthesized species of RNA, as demonstrated by DNA • RNA hybridization, are also noted to occur during early differentiation of these cells. Estrogen treatment of immature chicks produces marked growth and differentiation of primitive oviduct cells6-1 ~. Morphological differentiation and biochemical specialization occur coordinately with estrogen treatment resulting in the differentiation of a homogeneous population of primitive mucosal cells into three distinct types of epithelial cells (tubular gland, goblet, and ciliated cells). Qualitative changes in the RNA species synthesized, as analyzed by nearest-neighbor frequency analysis and competitive DNA • RNA hybridization (repeating species), also occur during estrogen mediated differentiationaS. A changing composition of nuclear RNA species suggests that a new pattern of gene transcription is generated during hormonally directed differentiation in the chick oviduct. Preliminary studies in these laboratories ~6 have demonstrated that quantitative changes in the chemical composition and in the capacity to bind steroid hormones occur in the oviduct chromatin during this differentiation. The experiments to be described in this study utilize a variety of analytical techniques to demonstrate complex and dramatic alterations in the chromatin of chick oviduct during estrogen-mediated development and differentiation. MATERIALS AND METHODS

Isolation, purification and analysis of chromatin Oviducts were obtained from untreated 7-dayoold Rhode Island Red chicks which were treated for various periods with diethylstilbestrol. Stimulated chicks received injections (subcutaneous) of 5 mg of diethylstilbestrol in oil and oviducts were removed following 0, 4, 7, 12, 14 or 19 daily injections. The tissues were rinsed with cold buffer (0.01 M Tris-HC1 buffer (pH 7.5)) and stored at --20 °C. The isolation and analysis of chromatin have been previously described 17'xa. Chromatin was analyzed for DNA, histone, acidic protein, and template capacity (in vitro DNAdependent RNA synthesis) as previously described 18. Isolation of acidic proteins Total acidic protein was isolated from oviduct chromatin using acidified phenol. Chromatin (2-5 mg DNA in 5 ml) in 12-ml conical glass centrifuge tubes was dehistonized by adding 2.2 M H2SO4 to make a 0.2 M solution and incubated for 15 min at 4 °C with stirring. Following centrifugation at 2000 × g for 10 rain, the pellet was resuspended in 5 ml of 0.2 M H2SO4 and centrifuged again. The acid supernatant, containing histones and about 5 % of the acidic proteins, was discarded. The acidic protein-DNA complex was resuspended in 5 ml of 0.2 M HzSO4 at 25 °C. 2.5 ml of distilled phenol saturated with 0.2 M H2SO4 were added. The mixture was vigorously shaken for 20 min, centrifuged, the upper (aqueous) layer containing DNA fragments discarded, and 5 ml of 0.2 M H2SO, added to the interphase and lower (phenol) phase. The solutions were shaken, centrifuged, and the

CHROMATIN OF THE DEVELOPING CHICK OVIDUCT

767

aqueous layer again removed. (The preparation has remained in the same conical glass centrifuge tube up to this step.) Then a total of 25 ml of a solution containing 0.1 M acetic acid and 0.14 M 2-mercaptoethanol was added to the lower phase and inlerphase, while these phases were transferred to a beaker. The mixture was stirred for 30 rain at room temperature. Centrifugation in a Sorvall RC-2B at 2000 × g for 10 min sediments the protein, which was then resuspended directly into 1-2 rnl of an sodium dodecyl sulfate solution for polyacrylamide electrophoresis as described below. For the analysis of DNA and protein, the protein pellets had to be further treated since protein-bound phenol interferes with the chemical analysis. To remove the phenol, the solution was dialyzed overnight at 4 °C against 100 vol. of 5.0 M urea in 0.01 M Tris-HCl buffer (pH 8.0). The urea was then removed by dialysis against 100 vol. of 0.01 M Tris-HC1 buffer (pH 8.0) for a further 8 h with several changes of the dialysis buffer. The fractionation of the acidic proteins from the DNA has been previously reported 19. Briefly, the histones along with fractions AP1 and AP2 are dissociated from chromatin with a solvent of 2.0 M NaC1 and 5 M urea in phosphate buffer (pH 6.0). Ultracentrifugation for 24 h sediments AP2 and the DNA complexed with AP3 and AP4. "Ihe histones are separated from AP1 by dialysis against water and acidification. The DNA-AP3-AP4 complex is solubilized in dilute Tris buffer, and the AP 2 removed by sedimentation in a clinical centrifuge. The AP3 is removed from the DNA-AP 4 complex using the same high salt-urea solvent as described above, except that Tris buffer is added to raise the pH to 8.5.

Electrophoresis in sodium dodecyl sulfate-acrylamide gels The protein from the total acidic protein isolation or from the lyophilized samples (AP1, AP2, AP3, and AP4) were resuspended in a solution containing 3.0 % sodium dodecyl sulfate, 0.14 M mercaptoethanol, 0.01 M sodium phosphate buffer (pH 7.5)to a concentration of 1-2 mg protein/ml and dialyzed overnight at room temperature against 100 vol. of a similar solution except that it contained only 1% sodium dodecyl sulfate and had 10 % glycerol. The polyacrylamide gel system is essentially that described by Laemmli2° with the addition of 4 M urea to the upper and lower gel system as recommended by MacGillivray et al. 21. Immunization procedure The acidic protein-DNA complex containing AP a and AP 4 (nucleoacidic protein) from oviduct of chicks treated with diethylstilbestrol for 15 days was used to immunize rabbits. The preparation of the immunogen and antisera, and the determination of antigenicity have been described previously22. Circular dichroism of treated and untreated oviduct chromatin Conformational changes of oviduct chromatin and nucleoacidic protein in response to diethylstilbestrol stimulation were studied using circular dichroism. Measurements were made using a Cary 60 recording spectropolarimeter with a Model 6002 circular dichroism attachment and a programmed band pass of 15 A. A thermostated cell holder enabled measurements to be made at 2 °C in a 1-mm cell. Only solutions with minimal turbidity were analyzed in order to reduce light scattering artifacts. The ellipticity of the instrument was routinely standardized with (q-)-10-

768

T . C . S P E L S B E R G et al.

camphor-sulfonic acid 23. Results are reported in terms of the mean residue ellipticity [O'] in units of degrees • cm 2 • dmole -~ using the expression o 0 ~r r [O']

-

IO / c '

where O ° is the observed eltipticity in degrees, c is the concentration in g/ml, l is the cell path length in cm, and ~r r is the mean residue molecular weight. In the 250-310-nm region, a mean residue molecular weight for DNA of 309 was used for calculations. For the region of 200-250 nm, a mean residue molecular weight was utilized based on the percent composition of DNA and protein (309 and 115 _~rr, respectively). RESULTS

Chromatin composition and template capacity Quantitative analysis of the chromatin from various stages of oviduct differentiation demonstrates that while the level of histones remained essentially unchanged, the level of the total acidic chromatin proteins increases during the first 4 days of differentiation and then decreases gradually until completion of oviduct development (14-19 days of diethylstilbestrol treatment) (Table I A). The level of chromatinassociated RNA follows a pattern similar to that of the acidic proteins. The capacity of the intact chromatins to serve as template for in vitro RNA synthesis in the presence of bacterial polymerase also follows a similar pattern, increasing during the first few days of development (0-4 days) and then gradually decreasing during final stages of development (5-15 days). Table I B shows that the removal of the AP1 fraction results in a slight reduction of total acidic protein. Removal of the AP2 fraction, however, results in a major decrease in acidic protein bound to DNA (Table I C). The nucleoacidic protein preparations (devoid of AP 1 and AP2) display a slight increase in acidic protein content during early oviduct development (0--4 days) but little change is noted during thelatter stages of development (4--19 days). The capacity of the nucleoacidic proteins (Table I C) to serve as a template for RNA synthesis markedly increases when the histones together with AP~ and AP2 are removed from the chromatin DNA. However, noticeable restriction of the DNA compared to deproteinized DNA (Table I D) or to pure DNA (Table I E) remains apparent in the nucleoacidic protein of untreated (0 day) and early (4 days) estrogen-treated chicks. Occasionally, the template activity of the 7-19 days nucleoacidic protein complex is greater than that of pure DNA. The small amounts of tightly bound protein in Table I D (AP4 bound to DNA) show. no restriction of the DNA nor any quantit~itive fluctuation during developments Fig. 1 graphically demonstrates the levels of the four acidic protein fractions in the oviduct chromatin at various stages of development. The AP2 and AP3 represent the major fractions of acidic protein present in developing oviduct chromatin. However, the level of AP2 in chromatin of 19 days-treated chicks is greatly reduced Of the four fractions examined throughout development, AP2 reveals the most striking quantitative variation. Repetition of this fractionation procedure for quantitation of the acidic protein subfractions yields similar results. The protein to DNA ratios of each of the subfractions vary by 10-15 ~ between the two experiments. The recovery of protein in the

C H R O M A T I N OF THE DEVELOPING CHICK OVIDUCT

769

TABLE I CHEMICAL COMPOSITION A N D TEMPLATE CAPACITY OF TREATED AND UNTREATED C H R O M A T I N PREPARATIONS F R O M THE CHICK OVIDUCT AT VARIOUS STAGES OF DEVELOPMENT The range of two replications of analysis of each preparation is given. The in vitro RNA synthesis was performed under conditions where the template was the rate limiting factor in the reactions. Each reaction contained 0.5/zg pure D N A or dehistonized chromatin or 4/~g untreated chromatin D N A and 5 units a 5 of RNA polymerase.

Chromatin type

Days of diethylstilbestrol treatment

Histone

Acidic protein

RNA

(A) Intact chromatin DNA-histoneA P r A P z - A P a-AP4

0 4 7 12 14 19

1.064-0.04 0.994-0.08 1.004-0.09 1.104-0.15 0.944-0.05 1.104-0.16

0.874-0.01 1.184-0.03 1.084-0.04 0.824-0.01 0.764-0.04 0.534-0.03

0.115i0.013 0.145±0.008 0.1804-0.015 0.0854-0.010 -0.0814-0.012

9.14-1.6 14.94-0.7 14.04-1.4 11.44-1.5 -11.04-0.2

(B) Dehistonized chromatin DNA-AP2-APa-AP4

0 4 7 12 19

------

0.724-0.05 1.024-0.04 0.93 4-0.06 0.70:[:0.05 0.454-0.04

------

------

(C) Nucleoacidic protein D N A - A P a-AP4

0 4 7 12 19

------

0.33:[:0.02 0.504-0.04 0.494-0.08 0.434-0.05 0.434-0.04

------

15904- 20 19504-110 22704- 30 22004- 50 20604- 10

(D) Deproteinized Chromatin

0 4 7 12 19

------

0.064-0.02 0.064-0.02 0.04i0.03 0.05-+-0.03 0.084-0.03

------

23404- 60 22104-100 23234-177 23454- 95 20704-110

--

--

--

22504- 90

(E) Pure DNA

Chemical composition (my/my DNA)

Template capacity (nmoles [I~C]UMP incorporated per my DNA)

s u b f r a c t i o n s is a b o u t 50 % o f t h e a c i d i c p r o t e i n i n t h e s t a r t i n g c h r o m a t i n . F r a c t i o n a t i o n o f t h e a c i d i c p r o t e i n s w a s p e r f o r m e d a n a d d i t i o n a l 4 t i m e s o n all s t a g e s o f o v i d u c t d e v e l o p m e n t in o r d e r to p r o v i d e material for repeat e x p e r i m e n t s for the i m m u n o c h e m i s t r y , a m i n o a c i d a n a l y s i s a n d gel e l e c t r o p h o r e s i s .

Amino acid compoMtion o f the acidic protein fraction Amino acid analyses were performed once on the histones and each of the acidic subfractions (except AP4) at each stage of development. The composition of the histones reveals a large quantity of basic residues with an acidic to basic amino a c i d r a t i o o f 0.67 f o r i m m a t u r e o v i d u c t a n d 0.65 f o r t h e m a t u r e o v i d u c t . I n c o n t r a s t ,

770

T.C. SPELSBERGet al. API

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DAYS OF DES T R E A T M E N T

Fig. 1. Levelof each of the acidicprotein fractionsin mg/rngDNA in developingoviductchromatin DES, diethylstilbestrol. the acidic protein subfractions contain a large quantity of acidic residues. The ratio of the acidic to basic amino acids are as follows: AP t -----1.64 for the immature and 1.39 for the mature oviduct; AP2 - 1.57 for the immature and 1.48 for the mature oviduct; and AP3 = 2.34 for the immature and 1.75 for the mature oviduct. DNA is not detected in any of the subfractions except, of course, for AP4. The RNA content represent about 10 % (w/w) for AP1 and AP 2 and about 5 % (w/w) for AP a at all stages of development. These nucleic acids could contribute to certain amino acid levels, e.g. glycine, but the effect of the RNA on the ratios of acidic to basic amino acids would be minimal. The analysis suggests that the acidic proteins in AP1, AP 2 and AP a but not the histones undergo changes during oviduct development. In addition, the compositions are different between the subfractions indicating that the proteins in the various subtractions are different. Isolation of the total acidic proteins of oviduct chromatin using phenol and acetic acid The phenol-acid procedure allows recovery of over 60 % of the total acidic protein of the starting chromatin. Since this represents the recovery after extensive dialysis against urea and Tris buffer to remove the protein bound phenol for quantitation, the actual recovery of proteins which are placed directly in sodium dodecyl sulfate for electrophoresis is probably significantly higher than 60 %. Comparison of the phosphoproteins extracted from chick oviduct chromatin according to Shelton and Allfrey 2'~(Fig. 2, Gel I) with the present method (Gel 2) indicates a marked increase in the number of bands on the gels which parallels the increased recovery of protein. Particularly, higher molecular weight proteins seem to be present using our method of isolation. The possibility of proteolytic breakdown of the acidic proteins during isolation was checked by adding two proteases to the preparation at two different steps. When trypsin or pronase are added before the phenol step, no change in the gel pattern is observed, suggesting an inactivation of the two enzymes. When the proteases are added after the phenol step but before dialysis against the sodium dodecyl sulfate solution, trypsin continues to have little or no effect on the patterns. Pronase, however,

CHROMATIN OF THE DEVELOPING CHICK OVIDUCT

771

Fig. 2. Comparison of the gel electrophoresis patterns of the total acidic proteins with the phosphoproteins of chromatin. Oviduct chromatin from chicks injected with diethylstilbestrolfor 15 days was used to obtain (2) total acidic proteins isolated by the phenol-acetic acid procedure, and (I) phenol-soluble proteins isolated according to Shelton and Allfreyz4. Sodium dodecyl sulfategels containing7 % acrylamidewere used. remains sufficiently active to eliminate all high molecular weight bands. Consequently since the protease activities are eliminated when exposed to the phenol-acid conditions it is highly probable that all endogenous protease activity would be inactivated as well. This is supported by the fact that 3H-labeled ovalbumin, added to the oviduct chromatin after the phenol-acid treatment but before the dialysis step, remains intact. This is shown by counting the radioactivity in 2-mm slices of polyacrylamide gels containing acidic protein and labeled ovalbumin. Both the number of counts and the relative mobility of 3H-labeled ovalbumin are unaffected. It is thus likely that no proteolytic breakdown of the acidic proteins occurs during isolation.

Electrophoresis of the acidic proteins in sodium dodecyl sulfate-polyacrylamide yels Fig. 3a represents gel pattern of the total acidic proteins of the chick oviduct (Column I) as well as the patterns of each of the subfractions of the acidic proteins (AP1, AP2, APa) (Columns 2-5). The species of total acidic proteins range from about 160 000 to less than 25 000 tool. wt. based on standard marker proteins and synthetic polypeptides. For the partially or fully mature oviduct, Fraction AP2 is clearly the most heterogeneous fraction containing a whole spectrum of molecular weight species of protein. This fraction is followed by AP1 and then AP 3 and AP4 in the degree of molecular weight heterogeneity of the protein species. Fig. 3b shows the gel patterns of the total acidic proteins isolated from various stages of development of the chick oviduct. Although some protein species remain throughout all stages of development, others show specificity for a particular stage. Fig. 3c-3f represent the gel patterns of acidic protein Fractions AP1, AP2, AP3, and AP 4, respectively, at various developmental stages. Each fraction undergoes some alteration in banding pattern during oviduct development with Fractions AP2 and AP3 showing the greatest changes. Thus these electrophoretic patterns support the amino acid composition results in that qualitative changes occur in each of the acidic protein fractions during development. Repeat experiments with other preparations of oviduct chromatins at various stages of development resulted in similar gel pattern

772

T . C . SPELSBERG e t al.

Fig. 3. Polyacrylamide gel electrophoresis of the acidic chromatin proteins of the chick oviduct. (a) Patterns of (1) total acidic proteins, (2) Fraction AP~, (3) AP2, (4) APa and (5) AP4 from the oviduct of chicks injected for 7 days with diethylstilbestrol (b) Patterns of the total acidic proteins from the oviducts of chicks injected with diethylstilbestrol for (1) 0 day, (2) 4 days, (3) 7 days, (4) 12 days, and (5) 19 days. (c) Patterns of Fraction AP~ from oviduct of chicks injected with diethylstilhestrol for (1) 0 day, (2) 4 days, (3) 7 days, (4) 12 days, and (5) 19 days. (d) Patterns of Fraction AP2 from oviduct of chicks injected with diethylstibestrol for I1) 0 day, (2) 4 days, (3) 7 days, (4) 12 days, and (5) 19 days. (e) Patterns of Fraction AP3 from oviduct of chicks injected with diethylstilbestrol for (1) 0 day, (2) 4 days, (3) 7 days, (4) 12 days, and (5) 19 days. (f) Patterns of Fraction AP4 from oviduct of chicks injected with diethylstilbestrol for (1) 0 day, (2) 4 days, (3) 7 days, (4) 12 days, and (5) 19 days. Each gel received about 150/tg protein estimated by absorption at A2ao n,~ using bovine serum albumin as standard in the same solvent (1.0 700 sodium dodecyl sulfate, 0.14 M 2mercaptoethanol, 10 % glycerol, 0.01 M sodium phosphate buffer (pH 7.4). The proteins were run on 5 % acrylamide gels (5 cm long) with a 3.0 % spacer gel (0.5 cm long) at 3 mA/gel until the bromophenol blue marker band reached the bottom of the gels. Migration was towards the anode. After electrophoresis, the gels were removed and placed in a solution containing 50 % (v/v) methanol and l0 ~ (v/v) acetic acid overnight to remove sodium dodecyl sulfate. They were stained with coomassie blue in the same solvent and destained and stored in 7 ~,, (v/v) acetic acid.

CHROMATIN OF THE DEVELOPING CHICK OVIDUCT

773

for each of the acidic protein fractions demonstrating the repeatability of the fractionation method.

Immunochernistry of chromatin acidic proteins Another technique for the analysis of the changing species of acidic chromatin proteins during development is the immunochemical method of microcomplement fixation. The antigenic properties of the acidic protein-DNA complexes (nucleoacidic protein, consisting of DNA, AP3 and AP4) isolated from oviduct and spleen are shown in Fig. 4. The nucleoacidic protein from fully differentiated oviducts (chicks treated for 15 days with diethylstilbestrol) shows a strong reactivity with the antiserum prepared against an nucleoacidic protein from oviducts of similarly treated chicks. The nucleoacidic protein from the spleens (Fig. 4) as well as other organs 22 of the same chicks show limited affinity for this antibody. Fig. 5 shows the changes in antigenicity of the nucleoacidic proteins which occur during estrogen-mediated growth and differentiation of the chick oviduct. The nucleoacidic protein from undifferentiated oviduct (0-day diethylstilbestrol treatment) show very few antigenic sites in common with nucleoacidic protein of fully developed oviduct (15-days diethylstilbestrol treatment). However, complement fixation values generated by nucleoacidic proteins which were isolated from oviducts of chicks injected with diethylstilbestrol for increasing amounts of time demonstrate a gradual appearance of antigenicity which correlate with the degree of oviduct development. The preparations of 12 day-stimulated chick oviduct fix complement to approximately the same extent as those from 15 or 19 days of estrogen treatment. Thus the degree of antigenicity of the nucleoacidic proteins show a gradual estrogen-dependent transition which coincides with the morphological and biochemical development of this organ. 100-

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Fig. 4. Complement fixation by varying quantities of nucleoacidic protein ( D N A complexed with APa and AP4) from the (O) oviduct and ( A ) spleen of chicks which were treated for 15 days with diethylstilbestrol. Fig. 5. Changes in antigenicity of nucleoacidic protein of the developing chick oviduct. Nucleoacidic protein (10/~g DNA/assay) from each stage of development (0, 4, 12, 14, and 19 days of diethylstilbestrol (DES) treatment) were added to the complement fixation assays containing the antisera prepared against fully differentiated oviduct nucleoacidic protein (15 days of diethylstibestrol treatment).

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T.C.

SPELSBERG

et aL

Circular dichroism ( CD) and template analysis qf chromatin Composite data concerning the effects of estrogen on several properties of oviduct chromatin and nucleoacidic protein are shown in Fig. 6. The values for template capacity and acidic protein content of both are shown in the top and middle panels respectively, and appear to change in a correlative manner during estrogen treatment. The lower panels demonstrate results of CD studies carried out at 275 nm. The magnitude of the ellipticity for chromatin is much less than that for pure D N A at all stages of development. However, the ellipticity of chromatin D N A increases gradually after 8 days of diethylstilbestrol-stimulated oviduct development. The nucleoacidic protein shows greater ellipticity at 275 nm than chromatin at all stages of oviduct development, suggesting that chromatin-bound protein influences D N A conformation. NUCLEOACIDIC PRO]'EI N

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Fig. 6. The percent open template (available for transcription), acidic protein levels, and circular dichroism ellipticity at 275 nm of chick oviduct chromatin and nucleoacidic protein are plotted as a function of days of diethylstilbestrol stimulation (DES). Details for the quantitation of acidic protein, analysis of template capacity, and circular dichroism measurements are described in Materials and Methods. It is known that the far ultraviolet region (190-240 nm) of CD spectra is predominated by large ellipticity values for protein relative to smaller contributions due to D N A 25. Chromatin, which has approximately twice as much protein as D N A on a weight basis, and nucleoacidic protein, which has a reduced amount of protein (Table I), both accordingly display an ellipticity in the far ultraviolet which primarily reflects protein conformation, with slight perturbations due to D N A . It was found that marked shifts in the negative 210-nm absorption band occur throughout development of diethylstilbestrol-stimulated oviduct chromatin, as represented in Fig. 7. These

CHROMATIN OF THE DEVELOPING CHICK OVIDUCT

775

changes may be interpreted as significant alterations in protein conformation. With nucleoacidic protein the negative shift in ellipticity is reduced, though still significant (Fig. 7). The DNA contribution to the spectra, which in this case would be expected to be enhanced due to reduced protein content, may account for the reductions in amplitude. Although part of these results are somewhat complicated by light scattering effects due to the particulate nature of chromatin 26, precautions were taken to maintain minimal scattering values for all samples studied. The readily soluble nucleoacidic protein samples were practically free of any scattering effects. +4

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Fig. 7. Far-ultraviolet circular dichroic spectra of chromatin and nucleoacidic protein of chick oviduct as a function of days of diethylstilbestrol stimulation. The patterns represent chromatin or nucleoacidic protein of oviduct from chicks which were either ( 0 ) u n t r e a t e d or were injected with diethylstilbestrol for ( O ) 4 days, ( × ) 7 days, ( I ) 12 days or (Fq) 19 days, See Materials and Methods for the procedure used for obtaining spectra. DISCUSSION

In many instances where the deoxyribonucleoprotein of differentiating cells has been studied, significant changes in the pattern of restriction, template capacity, and acidic protein content have been reported 27'2s. In particular, estrogen-induced differentiation of immature chick oviduct has been characterized by marked qualitative changes in the composition of newly synthesized RNA 15,29. Further, it is reported that the template capacity and chromatin protein composition of the developing oviduct undergo quantitative changes 16. The acidic chromatin proteins are of particular interest because of their suggested role in the regulation of gene function. The fractionation procedure for obtaining AP1, AP2, AP3 and AP4 is a rather crude method. However, it is effective in that it is applicable to large batch preparation and results in relatively high yields of protein. The technique is fairly reproducible as measured by quantitative analysis of protein, immunochemistry, and polyacrylamide gel electrophoresis. Repeat isolations of each of the subfractions gave similar quanti-

776

T.C. SPELSBERG

et al.

tative levels (-4- 7 % variation) and the band patterns on the acrylamide gels were identical. The DNA-APa-AP 4 complexes from repeated isolations displayed similar antigenicity, the extent of the variation was within ~ 6 ~. The acidic protein fractions also appear to be free of histones since they failed to migrate as histones in the acidic gel system of Panyim and Chalkley 3°. Other techniques for fractionating the acidic proteins, such as column chromatography using ion-exchange and molecular sieve resins with urea or sodium dodecyl sulfate solutions, results in poorer protein yields and are applicable only to smaller quantities of chromatin. The isolation of the total acidic protein of oviduct chromatin using phenol and acetic acid, as described in Materials and Methods, results in high yields, no protease activity and more protein species, especially those of higher molecular weight, than the phenol procedure for phosphoprotein isolation 24. This study has demonstrated that the variations in Fraction APz is largely responsible for the quantitative changes observed in the total acidic proteins of chromatin throughout estrogen-mediated oviduct development (Fig. 1). Fractions AP2 and AP3 quantitatively represent the major portion of the total acidic proteins of chromatin prior to 19 days of estrogen treatment. Fractions APa and AP2 at most stages of development contain the greatest number of protein species by polyacrylamide gel electrophoresis (Figs 3c and 3d). Interestingly, the relative template capacities correlate with the level of the total acidic protein in chromatin of developing oviduct (Table I). Since a role in tissue-specific restriction of DNA transcription has been assigned with supporting evidence to the acidic proteins of chromatin, it would seem that these molecules might themselves be tissue specific and possibly even specific for certain stages of differentiation within a given tissue. The amino acid composition, sodium dodecyl sulfate--polyacrylamide gel electrophoresis and immunochemistry of the acidic protein fractions are all consistent with changing protein species through development. Other studies have emphasized the tissue specificity and heterogeneity of the AP3 subfraction among various tissues of a given animal species using similar immunochemical techniques 22. The polyacrylamide gel patterns of fraction APa (Fig. 4e) show a marked transformation during oviduct development which suggests that the species of protein are changing. Recent investigations in these laboratories using higher percent acrylamide gels have shown that the marker band in the migrating front of the gel patterns of AP3, and to a lesser extent, those of AP~, AP2 and AP4, are composed of a multitude of small molecular weight protein species (less than 25 000 tool. wt.). Analysis of circular dichroism data of chromatin in both the near and far ultraviolet regions suggests that significant conformational changes occur during oviduct development. The gradual increase in ellipticity of chromatin and nucleoacidic protein at 275 nm (Fig. 6) is indicative of changing DNA conformation. It is probable that the observed ellipticity changes at 275 nm, which reflect ad altered geometry of the nucleotide bases relative to the DNA helix axis, are initiated by associated protein. Although an inherent alteration in DNA structure cannot be ruled out. It has been previously observed that when DNA is complexed with protein, as it is in chromatin, a reduced positive absorption band results at 275 nm (refs 31-33). It may be that the observed increase in ellipticity thus reflects a weakened interaction between chromatin protein and DNA.

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Spectral data in the far ultraviolet region largely represents conformational changes of chromatin associated protein. Though the optical scattering problems mentioned previously tend to complicate the work, utilization of chromatin with minimal, but equivalent, turbidity reduces the effects of artifacts. It has been suggested that such artifacts tend to reduce the intensity of CD absorption bands 26'29 thus implying that the experimental results show minimum values for secondary structure. Although CD spectroscopy is inherently limited in determining the absolute degree of various conformational states, approximations are possible using the observations of Greenfield and Fasman a 4 on homopolypeptide models at [O']208. We can estimate 10 % s-helix of the chromatin proteins at the early stages of oviduct differentiation with a progressive increase to 20 % in the fully developed oviduct. It remains unclear whether the conformation of single or multiple protein species are altered during development. Thus, the CD spectra of chromatin and nucleoacidic protein implicate changes in both DNA and protein conformation during oviduct development. The changes in CD spectra, template activity, and amount and types of acidic protein in nucleoacidic protein and chromatin during oviduct development are multiple parameters which attempt to define the molecular events which occur during the modulation of gene activity. It is possible that they all project the chemical and steric changes which are necessary to direct endogenous RNA polymerase to transcribe selective regions of the DNA of chromatin. These studies provide strong evidence that differentiation involves progressive alterations in chromatin structure and composition which ultimately result in changes in gene expression and consequently cell morphology and function. At present the exact biological meaning of these changes remains obscure simply because there are not well defined functions assigned to acidic proteins or to changes in chromatin structure. ACKNOWLEDGEMENTS The excellent technical assistance of Mrs Lucie Chytil, Mrs Irene Chrastil, Mrs Gayle Cashion, Miss Sherri Willis and Miss Ella Stitt is deeply appreciated. This research was supported by U.S. Public Health Service Grants CA 13065-01, HD 05384, HD 05797, HD 04473-01 and AM 10833, American Cancer Society Grant 1-25-L-11, Ford Foundation Grant 630-0141A, and National Science Foundation Grant BG 27936. The authors are members of the Center for Population Research at Vanderbilt University School of Medicine. T. C. S. is a Fellow of the National Genetics Foundation ,W. M. M. is a recipient of a Career Development Award from the N.I.H. and E.M.W. is a N.D.E.A. predoctoral Fellow. REFERENCES 1 Marushige, K. and Ozaki, H. (1967) Deo. BioL 16, 474--488 2 Johnson, A. W. and Hnilica, J. S. (1970) Biochim. Biophys. Acta 224, 518-530 3 Chetsanga, C. J., Poccia, P. L, Hill, R. J. and Dory, P. (1970) Cold Sprin# Harbor Syrup. Quant. Biol. 35, 629-634 4 Flickinger,R. A., Coward, S. J., Miyagi, M., Moser, C. and Rollins, F. (1965)Proc. NatL Acad. Sci. U.S. 53,783-790 5 Kohl, D. M., Greene, R. F. and Flickinger, R. A. (1969) Biochim. Biophys. Acta 179, 28-38

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