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Chromatin and histones in mealy bug spermatogonia
Prinied in Sweden Copyright 0 1974 by Academic Press, Inc. AIt rights of reproduction in any form reserued
Experimental
ROMATIN
Cell Research 85 (1974) 205-21 I
AND HISTONES IN MEALY In Situ Alteration
IA
of Template
in Hetero- and Euchromatin M. A. TUBBERT
A~~~~~~~~~ty by a Synthetic ~~~y~~i~~
and L. BERLOWITZ
Department of BioIogy, State University of New York, Buffdo, N.3’. 14214, USA
SUMMARY Polystyrene sulfonate (PSS) was used to determine the relationship between histones and the transcriptional availability of facultative heterochromatin and euchromatin in mealy bug cell explants. PSS decondenses facultative heterochromatin, activates RNA synthesis in goniai cells and removes histones in a specific sequence. The removal of histone makes template available for the binding of actinomycin D (AMD). In metabolically active gonial cells, PSS (2 mg/ml) treated heter6chromatin i&orporated 11.3 times more
Although little is known about the specific mechanisms regulating RNA synthesis in eukaryotic cells, such mechanisms must involve, in part, a regulation of the interactions between DNA and its associated proteins, histones and the non-h&one chromosomal proteins. Actinomycin D (AMD), an inhibitor of DNA-directed RNA synthesis [I], is a useful molecule to determine the accessibility of DNA for transcription. There is a large body of evidence implicating histones as natural inhibitors of transcription (fcr review see [Z]). In addition it has been shown istones block potential actinomycin D binding sites in chromatin [3-61, and that the availability of chromatin binding sites to ~ctiuomyci~ often parallels their RNA synthetic activity [5, 7-151.
In t paper we exploit tritiate (3H-AMD) as a cytochem mycin to determine the localization o transcribable template after hist been removed by a synthetic poly laboratory has determined that t polyanion, polystyrene sulfonat tivates RNA synthesis in mealy bug gsnial cells, and, in low concentrations, leaves these cells structurally intact (1161 an Sheeley & Thomas, unpublished exposure to high concentrations of PSS results in the structural ondensaticm of heterochromati~ in situ. resolution autoradiography of mealy bug testes treated wit’ sH-PSS indicated that the p~~ya~~~~ entere the nucleus and was associated with the chromatin. Studies on calf thymus nuclei [17]
206
Tubbert and Berlowitz
have demonstrated that PSS removes histones from chromatin, preferentially extracting the arginine-rich fractions. In calf thymus nuclei maximum RNA synthesis occurred when the arginine-rich fractions were extracted by PSS
WI. In present investigation we utilized the mealy bug, Planococcus citri. The mealy bug gonial cell contains two structural and functional states of chromatin which may be directly compared. In male mealy bugs, the paternally derived haploid set of five chromosomes undergoes heterochromatization during early embryogenesis and remains heterochromatic in most tissues (e.g., germ line tissue of the testes), while the maternally derived chromosome set appears euchromatic. It has been shown that mealy bug heterochromatin is genetically inactive [19], and that this inactivity resides at the level of DNA-directed RNA synthesis [20]. The decreased ternplating activity of mealy bug heterochromatin is also correlated with a reduced capacity to bind AMD [I I]. Unlike constitutive heterochromatin, the facultative heterochromatin of mealy bugs does not differ in base ratio [21] or quantity of DNA [22] from its euchromatic counterpart and represents a highly condensed, but potentially transcribable chromatin. Since 3H-AMD will bind specifically to deoxyguanosine residues [I] the similarity of base ratio between the hetero- and euchromatic sets prevents distortion of the data. The aim of the present investigation was to discover differences in the DNA-histone binding relationship between transcriptionally active heterochromatin and inactive euchromatin when they are exposed to PSS, a stimulator of RNA synthesis. MATERIALS
AND
METHODS
Testis explants from premeiotic second instar males of the mealy bug Plunococc~s citri (Risso) were used Exptl Cell Res 85 (1974)
in these experiments. The testes were isolated in well slides containing 0.05 ml of 15 ,&i 3H-AMD/ml Ringer (Schwarz BioResearch, Orangeburg, N.Y., spec. act. 6.5 Ci/mmole) for 15 min. 0.05 ml of PSS (polystyrene sulfonate, average mol. wt 18 000; Lustrex X-710 Lot S-811; Monsanto Chemical Corp.) was added resulting in final concentrations of 4 mg PSS/ml Ringer and 2 mg PSSjml Ringer. Controls received equal volumes of Ringer alone. Both groups were incubated in their respective solutions for 60 min at 25°C and washed for 30 min in three changes of Ringer. The testes were transferred to gelatinized slides and squashed in 10 % neutral buffered formalin. Coverslips were removed after rapid freezing on dry ice and all slides were post-fixed for 5 min in 10% neutral buffered formalin followed by two min washes in distilled water. One set of slides was immersed in deoxyribonuclease (Worthington, 160 ,ug/ml 0.01 M phosphate, 0.003 M MgC12, pH 6.2), while a replicate set was immersed in the phosphate buffer-MgCl, solution without DNase. Both sets were incubated at 37°C for 2 h, washed in distilled water three times and air-dried for autoradiography. In order to determine the effect of increasing concentrations of PSS on 3H-AMD binding by spermatogonia, a PSS concentration profile was carried out. The testis explants were treated as outlined above with the following changes: (1) PSS was used at final concentrations of 10 g/ml, 5 mg/ml, 2 mg/ml, 1 mg/ml, 0.5 mg/ml and 0.1 mg/ml Ringer; (2) the incubation time was decreased from 60 to 30 min so that the number of grains over nuclei treated at higher concentrations of PSS would be countable. The effect of pretreatment with unlabelled AMD on PSS-induced 3H-AMD binding by euchromatin and heterochromatin was studied. Based on pilot experiments using various concentrations of unlabelled AMD to saturate AMD binding sites before exposure to 3H-AMD (10 &i/ml), a final cont. of 5 pg/ml was chosen. Although this concentration of unlabelled AMD did not saturate completely the binding sites present in control spermatogonia, it did result in a low level of aH-AMD binding by these cells. Testes were incubated in well slides containing 0.05 ml unlabelled AMD at 5 pg/ml for 30 min and washed three times in Ringer for 30 min. The testes were then exposed to 0.1 ml of $H-AMD (15 @/ml) for 15 min. Then 0.1 ml of PSS in a series of concentrations was added and the testes were incubated in this solution for 60 min at 25°C. Fixation and preparation for autoradiography was the same as in the previous experiments except that occasionally acetic acid was used as the squashing medium. Autoradiography was performed according to standard technique using NTB-2 nuclear track emulsion (Eastman Kodak, Rochester, N.Y.). After an 8 day exposure, slides were developed and stained with Wright’s stain.
RESULTS The data from fig. 1 shows the influence of PSS on the uptake of 3H-AMD by gonial cell nuclei. After 60 min incubation in PSS
Chromatin
and histones in medy bug $perrn~togo~~~
Table 1. incorporation of tritiated-actinomycin D (3H-AMD) increasing concentrations of polystyrene sulfonate (PSS)
20’7
by mealy bug ~perrnatog~~~~ with
Fifteen minutes’ exposure to 3H-AMD at 15 /Xi/ml before addition of PSS (v/v) for an additional 30 min incubation. H, heterochromatin; E, euchromatin; S.E., standard error; fc, final concentration. Fixation. 10 % Neutral Formalin Celis n
Grains/nucleus (av. No. +S.E.)
0
a 126 b 135
3.67 20.24
0.1
a IO2
7.11 i0.32
.5 1.0 2.0
5.0 10.0
b a b a b a b a b a
405 116 130 100 107 191 124 207 57 213
11.37+_0.33 16.67&0.59 8.89 iO.32 16.89 kO.55
%
Ratio of
Grains/H (av. No. &SE.)
Grains H
0.50 i-o.09
11.6
3.82 50.21
7.65
1.83 40.12
22.4
6.33 kO.23
3.45
3.16kO.16
25.1
9.44 kO.37
2.99
4.84kO.24
27.7
12.62&0.52
2.61
5.14&0.21
52.8
4.59 &0.28
0.89
7.1810.41
53.4
626 *0.59
0.87
33.08 $_0.84
Grains/E (av. No. &SE.)
grains E/H
--
Grain counts were made on two classes of nuclei in testis explants to serve as an internal check on autoradiographic efficiency: a, nuclei in which heterochromatin was indistinguishable from euchromatin; b, nuclei in which heterochromatin was clearly visible. At PSS (10 mg/ml) the H set is no longer visible. Cells from three or four testes were counted for each treatment.
(2 mg/ml) and 3H-AMD (7.5 ,&X/ml), there was 1A.3 times as much label over heterochromatin and only 1.1 times as much label over e~chromati~ when compared to controls incubated without PSS. This disproportionate increase in 3H-AMD binding to heterochromatin also occurred with incubations in PSS (4 mg/ml). Under these conditions there were 20.9 times as many grains over heterochromatin and only 1.85 times as many grains over euchromatin when compared to controls. DNase completely removed the bound 3Hon control slides. The replicate samples hate buffer-MgC1, soluimmersed in tion without e exhibited a pattern of Iabelling consistent in both number and dis~ribu~~o~ of grains to that observed in comparable slide samples without this treatment. Table 1 illustrates the effect of increasing
concentrations of PSS on the uptake of a AMD by gonial cell nuclei. Under t ditions, the incubation time was reduced to 30 min to allow for a more accurate grain counting at higher PSS concentrations, an to minimize the deco~de~sa~~ou of heterochromatin which occurs with increased of incubation in this polyanion [I6 for controls (i.e. cells not treated wit from table 1 are more indicative of difference in the availability of act binding sites between eucbromat~~ an terochromatin than those found in earlier reports [lf]. In those earlier experiments the testes explants were incubated in 3 for from 2 to 4 h. In the present inv control testes were incubated in 3 for not more than 45 min (see La Studies on the kinetics of AM living cells 19, 231 indicated that the early
208
Tubbert and Berlowitz
I
o-
I:.98
H E CONTROL
H E PSS 2mg/ml
164
275
H E PSS 4mq/ml 165
Fir. I. Abscissa: cells (no.): ordinate: mean no. of silver grains + S.E. . ‘. The effect of polystyrene sulfonate (PSS) on the incorporation of tritiated actinomycin D (7.5 &i/ ml) by heterochromatin (H) and euchromatin (E) within mealy bug spermatogonia. Mean grain density + S.E.
rapid phase of AMD binding was correlated with gene activity. It can be seen from table 1 that increasing the concentration of PSS from 0.1 mg/ml to 5.0 mg/ml resulted in a progressive increase in the uptake of 3H-AMD by heterochromatin. After 30 min in PSS at 2 mg/ml and 3H-AMD at 7.5 &i/ml there were 10.3 times as much label over heterochromatin and 1.2 times as much label over euchromatin
when compared to controls incubated without PSS (see table 1). This enhanced incorporation is in agreement with data for the longer incubation time (fig. 1). The effect of increasing concentrations of PSS on the ratio of mean grain density over euchromatin (E) to grain density over heterochromatin (H) can be seen in table 1. The E/H ratio decreased from 7 665 for controls (i.e. cells not treated with PSS) to a value of 2.61 for cells incubated in PSS (1 mg/ml) for 30 min. At PSS (2 mg/ml) the heterochromatic actually incorporated somewhat more 3H-AMD than the euchromatic set. No significant change took place with PSS (5 w/ml>. When the concentration of PSS was increased from 5 to 10 mg/ml (see table l), the average number of grains per nucleus doubled. However, it was not possible to determine whether this increase reflected a disproportionate uptake of 3H-AMD by heterochromatin, since the heterochromatic set decondensed and became indistinguishable from euchromatin. Fig. 2 represents a comparison of heterochromatin and euchromatin from table 1 in which PSS-stimulated binding of 3H-AMD is expressed as a percent increase over control levels. PSS-stimulated heterochromatin shows a steeper initial rise in grain
Fig. 2. Abscissa: PSS mg/ml; ordinate: silver grains (% of controls). The effect of increasing concentrations of PSS on the incorporation of 3H-AMD in the entire nucleus (O-----O), heterochromatin (A-----A), and euchromatin (O-----O) of mealy bug spermatogonia. 1
Exptl Cell Res 85 (1974)
2
3
4
5
Chromatin
and histones in mealy bug ~~errna~ogo~~~ 209
Table 2. The effect ofpreincubation with unlabelied actinomycin D (30 min) on the i~~or~or~t~~~~ QJ”tritiated actinomycin D (3H-AMD) into mealy bug spermatogonia Fifteen minute exposure to 3H-AMD at 15@/ml beforeadditionof equalvolumesof polystyrenesuitonate (PSS) for additiona 60 min. H, heterochromatin;E, euchromatin;SE., standard error; fc, final concen-
tration. Fixation: 4.5% acetic acid. 4-8 testesper treatment Preincubation with AMD Grains/H 0 0 4 4
178 202 172 224
(cont. pug/ml)
Grains/E (ave. No. +S.E.) (ave. No. +S.E.)
0 5 0 5
0 0 6.83+0.?4 5.60t0.22
density, followed by a slower rise in the amount of “H-AMD bound at PSS concentrations greater than 1 mg/ml. A comparable progression but of lessermagnitude was seen for PSS-stimulated euchromatin, but only at PSS concentrations up to 1 mg/ml. The grain density over euchromatin appears to plateau or rise slowly for PSS between 2 and 5 mg/ml. Table 2 describes the effect which results from pre-treating gonial cells with unlabelled D before incubation with 3H-AMD and . Under these conditions the unlabelled AMD partially saturated available DNA binding sites, so that,the mean grain density was reduced. In the euchromatin of control cells (0 mg/ml PSS) the decrease was 6-fold over cells not preincubated with AMD. Cells either partially saturated with AMD or not so-treated, and which were subsequently incubated in PSS (4 mg/ml), showed large increases in their capacity to incorporate 3H~etero~hromati~ and euchromatin incorporated 3 -AMD equally. DISCUSSION It has been reported that the polyanion, SS, extracts histones from calf thymus [17] and chicken erythrocyte nuclei [24] in a specific sequence, and enhances RNA synthesis in calf thymus auclei [18]. Consistent with
1.65iO.?7 0.3210.08 6.35t0.40 5.53kO.30
ata is the loss of alkaline fast green staining by mealy bug spermatogonial nuclei after PSS treatment [16]. Previous work in this laboratory [l I] showed that acid-fixation of squashed mealy bug gonial cells could result in a differential increase incorporation into the heterocbr mosome set. In the present pa utilized PSS to remove histones from cells still metabolically active by virtue of their ability to synthesize RNA [is]; we have, thereby, confirmed that the extraction of histone exposes the repressed, highly condensed template of facultative heterochromatin rendering it potentially available for
NA sitesfor AM exposed by histone extraction are especially suggested by the experiments (table 2) in which large increases in 3H-A ation occur in cellsin which existing 3 binding sites have been partially saturated by cold and then treated with PSS. These -binding experiments correlate well with our RNA synthesisstudies on goniai cells [16] in which increasing concentrations of PSS activate both beterochromatin and. euchromatin with differentially greater reactivation of the heterochromatin. These experiments taken toge r strongly suggest that the differences in synthetic activity of Exptl
Cell Res 85 (1974)
210
Tubbert and Berlowitz
facultative heterochromatin and euchromatin result from differences in the binding relationships between histone and DNA in the two states of chromatin. It is interesting to inquire if the mode of action of PSS (its binding to histone) thereby activating and decondensing heterochromatin mimics the action of naturally occurring polyanions which may be involved in the modulation of transcription. One such class of polyanions are the non-histone chromosomal proteins. We observed (table 1) that at higher PSS concentrations, the ratio of grains over euchromatin to those over heterochromatin was almost unity. This observation is in agreement with data on the activation of the template activity of rat liver condensed and diffused chromatin by naturally occurring polyanions, the non-histone proteins. Kamiyama & Wang [25] reported that the RNA synthetic activity of both chromatin fractions approached almost the same level by the addition of non-histone protein. It is difficult to account for the initial increase and subsequent decrease in 3H-AMD binding by the euchromatic set with low concentrations of PSS (see fig. 2). One possible explanation would involve changes in the localized ionic milieu. The availability of DNA sites to AMD has been shown to depend upon the ionic milieu [4,23]. We have observed that PSS is neutral in Ringer until 2 mg/ml. At higher concentrations it becomes acidic. The heterochromatic set, being more compact than the euchromatic set, would be less susceptible to slight ionic change. Despite the acidity of PSS at 4 mg/ml Ringer, RNA synthesis continues to be enhanced in gonial cells and the chromatin structure is not detectably altered. Facultative and constitutive heterochromatin differ in their AMD binding characteristic as they do in many other ways. Facultative heterochromatin within mealy bug spermatoExptl
Cell Res 85 (1974)
gonia is, for the most part, transcriptionally inactive [16, 291. The low level of 3H-AMD binding reported earlier [lo] for mealy bug heterochromatin even at long incubations and the lower levels of AMD binding reported here (table 1) are in accord with a number of investigations which have suggested a correlation between transcriptional inactivity and reduced numbers of AMD binding sites [8, 10, 12,13, 151. On the other hand, constitutive heterochromatin in Micvotus agrestis bound 3H-AMD to the same degree, per unit DNA, as did euchromatin [27]. The acid extraction of histone in Microtus agrestis brought about an increase in AMD binding in the entire chromatin, with the number of free binding sites being independent of heteropycnosis. Unlike facultative heterochromatin the constitutive heterochromatin of Microtus agrestis is composed of highly repetitive nucleotide sequences, is part of both homologous giant sex chromosomes, and never transcribes RNA [28]. This accessibility of constitutive heterochromatin, as opposed to the relative inaccessibility of facultative heterochromatin, probably reflects a difference in the DNA-histone binding relationships between these superficially similar, but structurally and functionally divergent forms of chromatin. This work was supported by USPHS grant HD 06282 to L. B.
REFERENCES 1. Sobell, H M, Jam, S G, & Sakore, T D, Nature new biol 231 (1971) 200. 2. Stellwagen, R H, & Cole, R D, Ann rev biochem 38 (1969)
951.
Desai, L’& Tencer, R, Exptl cell res 52 (1968) 185. Ringertz, N R & Bolund, L. Biochim biouhvs _acta-174’(1969) 147. ’ 5. Beato, M, Seifart, K H & Sekeris, C E, Arch biochem biophys I38 (1970) 272. 6. K$iman, L & Huang, R C, J mol biol 55 (1971) 3. 4.
7. Dariynkiewicz, Z, Bohmd, L & Ringertz, N R, Exptl cell res 55 (1969) 120. 8. Dariynkiewicz, Z, Gledhill, B L & Ringertz, N R, Exptl cell res 58 (1969) 435. 9. Ringertz, N R, Dariynkiewicz, Z & Bolnnd, L, Exptl cell res 56 (1969) 411. 10. Bra&et, J & Hulin, N, Nature 222 (1969) 481. 11. Berlowitz, L, Pallotta, D & Sibley, C H, Science 164 (1969) 1527. 12. Dariynkiewicz, Z & Andersson, J, Exptl cell res 67 (1971) 39. 13. Dariynkiewicz, Z & Jacobson, B, Exptl cell res 67 (1971) 49. 14. Wong, K Y, Patei, 4 & Krause, M 0, Exptl cell res 69 (1971) 456. 15. Andersson, J & Dariynkiewicz, Z, Exptl cell res 75 (1972) 410. 15. - Ibid. 75 (1972) 410. 16. Milier, 6, Beriowitz, L & Regelson, W, Chromosoma 32 (1971) 251. erlowitz, L, Kitchin, R & Pallotta, D, Biochim biopbys acta 262 (1972) 160.
14 -
741812
18. Miller, 6, & Berlowitz, I+ J cell bioE 59 (1973) 226a. 19. Brown. S W & Nelson-Rees. I W., Genetics 46 (1961) $83. 20. Berlowitz, L, Proc natl acad sci US 53 (1965) 68. 21. Loewus, M W. Brown. S W & McLaren. , A D.i Nature ‘203 (1964) 104: 22. Lorick, 6, Chromosoma (Berl) 32 (1970) 11. 23. Bohmd, L, Expti cell res 63 (1970) 171. 24. Miller, G, Berlowitz, L & cell res 71 (1972) 409. 25. Kamiyama, M & Wang, T Y, Biochim biophys acta 228 (1971) 563. 26. Sieger, x/1, Garweg, G & Schwarzacher, H G, Chromosoma 35 (1971) 84. 27. Lee J C 2%Yunis, J 5, Chromosoma 32 (1971) 237.
Received October 1, 1973 Revised version received November 26, 1973
Exprl
Cell Res 82 (19749
Experimental
ROMATIN
Cell Research 85 (1974) 205-21 I
AND HISTONES IN MEALY In Situ Alteration
IA
of Template
in Hetero- and Euchromatin M. A. TUBBERT
A~~~~~~~~~ty by a Synthetic ~~~y~~i~~
and L. BERLOWITZ
Department of BioIogy, State University of New York, Buffdo, N.3’. 14214, USA
SUMMARY Polystyrene sulfonate (PSS) was used to determine the relationship between histones and the transcriptional availability of facultative heterochromatin and euchromatin in mealy bug cell explants. PSS decondenses facultative heterochromatin, activates RNA synthesis in goniai cells and removes histones in a specific sequence. The removal of histone makes template available for the binding of actinomycin D (AMD). In metabolically active gonial cells, PSS (2 mg/ml) treated heter6chromatin i&orporated 11.3 times more
Although little is known about the specific mechanisms regulating RNA synthesis in eukaryotic cells, such mechanisms must involve, in part, a regulation of the interactions between DNA and its associated proteins, histones and the non-h&one chromosomal proteins. Actinomycin D (AMD), an inhibitor of DNA-directed RNA synthesis [I], is a useful molecule to determine the accessibility of DNA for transcription. There is a large body of evidence implicating histones as natural inhibitors of transcription (fcr review see [Z]). In addition it has been shown istones block potential actinomycin D binding sites in chromatin [3-61, and that the availability of chromatin binding sites to ~ctiuomyci~ often parallels their RNA synthetic activity [5, 7-151.
In t paper we exploit tritiate (3H-AMD) as a cytochem mycin to determine the localization o transcribable template after hist been removed by a synthetic poly laboratory has determined that t polyanion, polystyrene sulfonat tivates RNA synthesis in mealy bug gsnial cells, and, in low concentrations, leaves these cells structurally intact (1161 an Sheeley & Thomas, unpublished exposure to high concentrations of PSS results in the structural ondensaticm of heterochromati~ in situ. resolution autoradiography of mealy bug testes treated wit’ sH-PSS indicated that the p~~ya~~~~ entere the nucleus and was associated with the chromatin. Studies on calf thymus nuclei [17]
206
Tubbert and Berlowitz
have demonstrated that PSS removes histones from chromatin, preferentially extracting the arginine-rich fractions. In calf thymus nuclei maximum RNA synthesis occurred when the arginine-rich fractions were extracted by PSS
WI. In present investigation we utilized the mealy bug, Planococcus citri. The mealy bug gonial cell contains two structural and functional states of chromatin which may be directly compared. In male mealy bugs, the paternally derived haploid set of five chromosomes undergoes heterochromatization during early embryogenesis and remains heterochromatic in most tissues (e.g., germ line tissue of the testes), while the maternally derived chromosome set appears euchromatic. It has been shown that mealy bug heterochromatin is genetically inactive [19], and that this inactivity resides at the level of DNA-directed RNA synthesis [20]. The decreased ternplating activity of mealy bug heterochromatin is also correlated with a reduced capacity to bind AMD [I I]. Unlike constitutive heterochromatin, the facultative heterochromatin of mealy bugs does not differ in base ratio [21] or quantity of DNA [22] from its euchromatic counterpart and represents a highly condensed, but potentially transcribable chromatin. Since 3H-AMD will bind specifically to deoxyguanosine residues [I] the similarity of base ratio between the hetero- and euchromatic sets prevents distortion of the data. The aim of the present investigation was to discover differences in the DNA-histone binding relationship between transcriptionally active heterochromatin and inactive euchromatin when they are exposed to PSS, a stimulator of RNA synthesis. MATERIALS
AND
METHODS
Testis explants from premeiotic second instar males of the mealy bug Plunococc~s citri (Risso) were used Exptl Cell Res 85 (1974)
in these experiments. The testes were isolated in well slides containing 0.05 ml of 15 ,&i 3H-AMD/ml Ringer (Schwarz BioResearch, Orangeburg, N.Y., spec. act. 6.5 Ci/mmole) for 15 min. 0.05 ml of PSS (polystyrene sulfonate, average mol. wt 18 000; Lustrex X-710 Lot S-811; Monsanto Chemical Corp.) was added resulting in final concentrations of 4 mg PSS/ml Ringer and 2 mg PSSjml Ringer. Controls received equal volumes of Ringer alone. Both groups were incubated in their respective solutions for 60 min at 25°C and washed for 30 min in three changes of Ringer. The testes were transferred to gelatinized slides and squashed in 10 % neutral buffered formalin. Coverslips were removed after rapid freezing on dry ice and all slides were post-fixed for 5 min in 10% neutral buffered formalin followed by two min washes in distilled water. One set of slides was immersed in deoxyribonuclease (Worthington, 160 ,ug/ml 0.01 M phosphate, 0.003 M MgC12, pH 6.2), while a replicate set was immersed in the phosphate buffer-MgCl, solution without DNase. Both sets were incubated at 37°C for 2 h, washed in distilled water three times and air-dried for autoradiography. In order to determine the effect of increasing concentrations of PSS on 3H-AMD binding by spermatogonia, a PSS concentration profile was carried out. The testis explants were treated as outlined above with the following changes: (1) PSS was used at final concentrations of 10 g/ml, 5 mg/ml, 2 mg/ml, 1 mg/ml, 0.5 mg/ml and 0.1 mg/ml Ringer; (2) the incubation time was decreased from 60 to 30 min so that the number of grains over nuclei treated at higher concentrations of PSS would be countable. The effect of pretreatment with unlabelled AMD on PSS-induced 3H-AMD binding by euchromatin and heterochromatin was studied. Based on pilot experiments using various concentrations of unlabelled AMD to saturate AMD binding sites before exposure to 3H-AMD (10 &i/ml), a final cont. of 5 pg/ml was chosen. Although this concentration of unlabelled AMD did not saturate completely the binding sites present in control spermatogonia, it did result in a low level of aH-AMD binding by these cells. Testes were incubated in well slides containing 0.05 ml unlabelled AMD at 5 pg/ml for 30 min and washed three times in Ringer for 30 min. The testes were then exposed to 0.1 ml of $H-AMD (15 @/ml) for 15 min. Then 0.1 ml of PSS in a series of concentrations was added and the testes were incubated in this solution for 60 min at 25°C. Fixation and preparation for autoradiography was the same as in the previous experiments except that occasionally acetic acid was used as the squashing medium. Autoradiography was performed according to standard technique using NTB-2 nuclear track emulsion (Eastman Kodak, Rochester, N.Y.). After an 8 day exposure, slides were developed and stained with Wright’s stain.
RESULTS The data from fig. 1 shows the influence of PSS on the uptake of 3H-AMD by gonial cell nuclei. After 60 min incubation in PSS
Chromatin
and histones in medy bug $perrn~togo~~~
Table 1. incorporation of tritiated-actinomycin D (3H-AMD) increasing concentrations of polystyrene sulfonate (PSS)
20’7
by mealy bug ~perrnatog~~~~ with
Fifteen minutes’ exposure to 3H-AMD at 15 /Xi/ml before addition of PSS (v/v) for an additional 30 min incubation. H, heterochromatin; E, euchromatin; S.E., standard error; fc, final concentration. Fixation. 10 % Neutral Formalin Celis n
Grains/nucleus (av. No. +S.E.)
0
a 126 b 135
3.67 20.24
0.1
a IO2
7.11 i0.32
.5 1.0 2.0
5.0 10.0
b a b a b a b a b a
405 116 130 100 107 191 124 207 57 213
11.37+_0.33 16.67&0.59 8.89 iO.32 16.89 kO.55
%
Ratio of
Grains/H (av. No. &SE.)
Grains H
0.50 i-o.09
11.6
3.82 50.21
7.65
1.83 40.12
22.4
6.33 kO.23
3.45
3.16kO.16
25.1
9.44 kO.37
2.99
4.84kO.24
27.7
12.62&0.52
2.61
5.14&0.21
52.8
4.59 &0.28
0.89
7.1810.41
53.4
626 *0.59
0.87
33.08 $_0.84
Grains/E (av. No. &SE.)
grains E/H
--
Grain counts were made on two classes of nuclei in testis explants to serve as an internal check on autoradiographic efficiency: a, nuclei in which heterochromatin was indistinguishable from euchromatin; b, nuclei in which heterochromatin was clearly visible. At PSS (10 mg/ml) the H set is no longer visible. Cells from three or four testes were counted for each treatment.
(2 mg/ml) and 3H-AMD (7.5 ,&X/ml), there was 1A.3 times as much label over heterochromatin and only 1.1 times as much label over e~chromati~ when compared to controls incubated without PSS. This disproportionate increase in 3H-AMD binding to heterochromatin also occurred with incubations in PSS (4 mg/ml). Under these conditions there were 20.9 times as many grains over heterochromatin and only 1.85 times as many grains over euchromatin when compared to controls. DNase completely removed the bound 3Hon control slides. The replicate samples hate buffer-MgC1, soluimmersed in tion without e exhibited a pattern of Iabelling consistent in both number and dis~ribu~~o~ of grains to that observed in comparable slide samples without this treatment. Table 1 illustrates the effect of increasing
concentrations of PSS on the uptake of a AMD by gonial cell nuclei. Under t ditions, the incubation time was reduced to 30 min to allow for a more accurate grain counting at higher PSS concentrations, an to minimize the deco~de~sa~~ou of heterochromatin which occurs with increased of incubation in this polyanion [I6 for controls (i.e. cells not treated wit from table 1 are more indicative of difference in the availability of act binding sites between eucbromat~~ an terochromatin than those found in earlier reports [lf]. In those earlier experiments the testes explants were incubated in 3 for from 2 to 4 h. In the present inv control testes were incubated in 3 for not more than 45 min (see La Studies on the kinetics of AM living cells 19, 231 indicated that the early
208
Tubbert and Berlowitz
I
o-
I:.98
H E CONTROL
H E PSS 2mg/ml
164
275
H E PSS 4mq/ml 165
Fir. I. Abscissa: cells (no.): ordinate: mean no. of silver grains + S.E. . ‘. The effect of polystyrene sulfonate (PSS) on the incorporation of tritiated actinomycin D (7.5 &i/ ml) by heterochromatin (H) and euchromatin (E) within mealy bug spermatogonia. Mean grain density + S.E.
rapid phase of AMD binding was correlated with gene activity. It can be seen from table 1 that increasing the concentration of PSS from 0.1 mg/ml to 5.0 mg/ml resulted in a progressive increase in the uptake of 3H-AMD by heterochromatin. After 30 min in PSS at 2 mg/ml and 3H-AMD at 7.5 &i/ml there were 10.3 times as much label over heterochromatin and 1.2 times as much label over euchromatin
when compared to controls incubated without PSS (see table 1). This enhanced incorporation is in agreement with data for the longer incubation time (fig. 1). The effect of increasing concentrations of PSS on the ratio of mean grain density over euchromatin (E) to grain density over heterochromatin (H) can be seen in table 1. The E/H ratio decreased from 7 665 for controls (i.e. cells not treated with PSS) to a value of 2.61 for cells incubated in PSS (1 mg/ml) for 30 min. At PSS (2 mg/ml) the heterochromatic actually incorporated somewhat more 3H-AMD than the euchromatic set. No significant change took place with PSS (5 w/ml>. When the concentration of PSS was increased from 5 to 10 mg/ml (see table l), the average number of grains per nucleus doubled. However, it was not possible to determine whether this increase reflected a disproportionate uptake of 3H-AMD by heterochromatin, since the heterochromatic set decondensed and became indistinguishable from euchromatin. Fig. 2 represents a comparison of heterochromatin and euchromatin from table 1 in which PSS-stimulated binding of 3H-AMD is expressed as a percent increase over control levels. PSS-stimulated heterochromatin shows a steeper initial rise in grain
Fig. 2. Abscissa: PSS mg/ml; ordinate: silver grains (% of controls). The effect of increasing concentrations of PSS on the incorporation of 3H-AMD in the entire nucleus (O-----O), heterochromatin (A-----A), and euchromatin (O-----O) of mealy bug spermatogonia. 1
Exptl Cell Res 85 (1974)
2
3
4
5
Chromatin
and histones in mealy bug ~~errna~ogo~~~ 209
Table 2. The effect ofpreincubation with unlabelied actinomycin D (30 min) on the i~~or~or~t~~~~ QJ”tritiated actinomycin D (3H-AMD) into mealy bug spermatogonia Fifteen minute exposure to 3H-AMD at 15@/ml beforeadditionof equalvolumesof polystyrenesuitonate (PSS) for additiona 60 min. H, heterochromatin;E, euchromatin;SE., standard error; fc, final concen-
tration. Fixation: 4.5% acetic acid. 4-8 testesper treatment Preincubation with AMD Grains/H 0 0 4 4
178 202 172 224
(cont. pug/ml)
Grains/E (ave. No. +S.E.) (ave. No. +S.E.)
0 5 0 5
0 0 6.83+0.?4 5.60t0.22
density, followed by a slower rise in the amount of “H-AMD bound at PSS concentrations greater than 1 mg/ml. A comparable progression but of lessermagnitude was seen for PSS-stimulated euchromatin, but only at PSS concentrations up to 1 mg/ml. The grain density over euchromatin appears to plateau or rise slowly for PSS between 2 and 5 mg/ml. Table 2 describes the effect which results from pre-treating gonial cells with unlabelled D before incubation with 3H-AMD and . Under these conditions the unlabelled AMD partially saturated available DNA binding sites, so that,the mean grain density was reduced. In the euchromatin of control cells (0 mg/ml PSS) the decrease was 6-fold over cells not preincubated with AMD. Cells either partially saturated with AMD or not so-treated, and which were subsequently incubated in PSS (4 mg/ml), showed large increases in their capacity to incorporate 3H~etero~hromati~ and euchromatin incorporated 3 -AMD equally. DISCUSSION It has been reported that the polyanion, SS, extracts histones from calf thymus [17] and chicken erythrocyte nuclei [24] in a specific sequence, and enhances RNA synthesis in calf thymus auclei [18]. Consistent with
1.65iO.?7 0.3210.08 6.35t0.40 5.53kO.30
ata is the loss of alkaline fast green staining by mealy bug spermatogonial nuclei after PSS treatment [16]. Previous work in this laboratory [l I] showed that acid-fixation of squashed mealy bug gonial cells could result in a differential increase incorporation into the heterocbr mosome set. In the present pa utilized PSS to remove histones from cells still metabolically active by virtue of their ability to synthesize RNA [is]; we have, thereby, confirmed that the extraction of histone exposes the repressed, highly condensed template of facultative heterochromatin rendering it potentially available for
NA sitesfor AM exposed by histone extraction are especially suggested by the experiments (table 2) in which large increases in 3H-A ation occur in cellsin which existing 3 binding sites have been partially saturated by cold and then treated with PSS. These -binding experiments correlate well with our RNA synthesisstudies on goniai cells [16] in which increasing concentrations of PSS activate both beterochromatin and. euchromatin with differentially greater reactivation of the heterochromatin. These experiments taken toge r strongly suggest that the differences in synthetic activity of Exptl
Cell Res 85 (1974)
210
Tubbert and Berlowitz
facultative heterochromatin and euchromatin result from differences in the binding relationships between histone and DNA in the two states of chromatin. It is interesting to inquire if the mode of action of PSS (its binding to histone) thereby activating and decondensing heterochromatin mimics the action of naturally occurring polyanions which may be involved in the modulation of transcription. One such class of polyanions are the non-histone chromosomal proteins. We observed (table 1) that at higher PSS concentrations, the ratio of grains over euchromatin to those over heterochromatin was almost unity. This observation is in agreement with data on the activation of the template activity of rat liver condensed and diffused chromatin by naturally occurring polyanions, the non-histone proteins. Kamiyama & Wang [25] reported that the RNA synthetic activity of both chromatin fractions approached almost the same level by the addition of non-histone protein. It is difficult to account for the initial increase and subsequent decrease in 3H-AMD binding by the euchromatic set with low concentrations of PSS (see fig. 2). One possible explanation would involve changes in the localized ionic milieu. The availability of DNA sites to AMD has been shown to depend upon the ionic milieu [4,23]. We have observed that PSS is neutral in Ringer until 2 mg/ml. At higher concentrations it becomes acidic. The heterochromatic set, being more compact than the euchromatic set, would be less susceptible to slight ionic change. Despite the acidity of PSS at 4 mg/ml Ringer, RNA synthesis continues to be enhanced in gonial cells and the chromatin structure is not detectably altered. Facultative and constitutive heterochromatin differ in their AMD binding characteristic as they do in many other ways. Facultative heterochromatin within mealy bug spermatoExptl
Cell Res 85 (1974)
gonia is, for the most part, transcriptionally inactive [16, 291. The low level of 3H-AMD binding reported earlier [lo] for mealy bug heterochromatin even at long incubations and the lower levels of AMD binding reported here (table 1) are in accord with a number of investigations which have suggested a correlation between transcriptional inactivity and reduced numbers of AMD binding sites [8, 10, 12,13, 151. On the other hand, constitutive heterochromatin in Micvotus agrestis bound 3H-AMD to the same degree, per unit DNA, as did euchromatin [27]. The acid extraction of histone in Microtus agrestis brought about an increase in AMD binding in the entire chromatin, with the number of free binding sites being independent of heteropycnosis. Unlike facultative heterochromatin the constitutive heterochromatin of Microtus agrestis is composed of highly repetitive nucleotide sequences, is part of both homologous giant sex chromosomes, and never transcribes RNA [28]. This accessibility of constitutive heterochromatin, as opposed to the relative inaccessibility of facultative heterochromatin, probably reflects a difference in the DNA-histone binding relationships between these superficially similar, but structurally and functionally divergent forms of chromatin. This work was supported by USPHS grant HD 06282 to L. B.
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Desai, L’& Tencer, R, Exptl cell res 52 (1968) 185. Ringertz, N R & Bolund, L. Biochim biouhvs _acta-174’(1969) 147. ’ 5. Beato, M, Seifart, K H & Sekeris, C E, Arch biochem biophys I38 (1970) 272. 6. K$iman, L & Huang, R C, J mol biol 55 (1971) 3. 4.
7. Dariynkiewicz, Z, Bohmd, L & Ringertz, N R, Exptl cell res 55 (1969) 120. 8. Dariynkiewicz, Z, Gledhill, B L & Ringertz, N R, Exptl cell res 58 (1969) 435. 9. Ringertz, N R, Dariynkiewicz, Z & Bolnnd, L, Exptl cell res 56 (1969) 411. 10. Bra&et, J & Hulin, N, Nature 222 (1969) 481. 11. Berlowitz, L, Pallotta, D & Sibley, C H, Science 164 (1969) 1527. 12. Dariynkiewicz, Z & Andersson, J, Exptl cell res 67 (1971) 39. 13. Dariynkiewicz, Z & Jacobson, B, Exptl cell res 67 (1971) 49. 14. Wong, K Y, Patei, 4 & Krause, M 0, Exptl cell res 69 (1971) 456. 15. Andersson, J & Dariynkiewicz, Z, Exptl cell res 75 (1972) 410. 15. - Ibid. 75 (1972) 410. 16. Milier, 6, Beriowitz, L & Regelson, W, Chromosoma 32 (1971) 251. erlowitz, L, Kitchin, R & Pallotta, D, Biochim biopbys acta 262 (1972) 160.
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18. Miller, 6, & Berlowitz, I+ J cell bioE 59 (1973) 226a. 19. Brown. S W & Nelson-Rees. I W., Genetics 46 (1961) $83. 20. Berlowitz, L, Proc natl acad sci US 53 (1965) 68. 21. Loewus, M W. Brown. S W & McLaren. , A D.i Nature ‘203 (1964) 104: 22. Lorick, 6, Chromosoma (Berl) 32 (1970) 11. 23. Bohmd, L, Expti cell res 63 (1970) 171. 24. Miller, G, Berlowitz, L & cell res 71 (1972) 409. 25. Kamiyama, M & Wang, T Y, Biochim biophys acta 228 (1971) 563. 26. Sieger, x/1, Garweg, G & Schwarzacher, H G, Chromosoma 35 (1971) 84. 27. Lee J C 2%Yunis, J 5, Chromosoma 32 (1971) 237.
Received October 1, 1973 Revised version received November 26, 1973
Exprl
Cell Res 82 (19749