Biochemical controls of the G1 phase of a mammalian cell cycle. II synthesis of H1 histones in temperature sensitive mouse cells, arrested in G1 phase

Cell Biology hlternational Reports, VoL 5, No. 12, December ~98!

1105

SIOCSZMZC~L C O l O n S OF TS~ GI P ~ S ~ OF A ~ n i ~ W c E n n CYCLZ.* xI SYNTB~SIS OF HI HISTONES IN TEMPERATURE SENSITX%~ MOUSE CELLS, ARRESTED IN Sl p ~ S E . K. Gooderhaml, S and P.M. Naha 2 IMRC Clinical and POpUlation Oytogenetics Unit, w e s i e ~ General Hospital, Crewe Road, Edinburgh EH4 2XU, U.K.

2Paterson Laboratories, Christie Hospital and Holt Radium Instltu~e, Ma~ncbester, M20 9BX, U.K.

STo whom correspondence should be addressed. ABS~C~ The synthesis of Hi histories has been studied ~n two temperature-sensitive mutants, A83 ~ d AS, of the Ba!b/c-3T3 cell llne. At the non-pe~issive temperature (38=C) both clones A83 and A8 are arrested in the ~i phase of the cell cycle, their execution points being located at the two ends of the G1 phase (Naha, ~979). SDS polyacrylamide gel electrophoresis of H1 histones extracted by 5% [w/v) perchloric acid from these cells has demonstrated that histories HIA and HIB are synthesised throughout the GI phase of tht) cell cycle. These proteins are, however, s h o ~ to be synthesised at a reduced rate during G1 and the synthesis of the HIB subfraction predominates in both the A83 ~ d AS mutant clones after 3H-~eucine labBlling of the cells at 38:C. In addltiQn the minor histone HI fraction, historic HI o , has been s h o ~ to be enriched in the G1 arrested cells of A83 which ~ p s close to the mitotic phase, while no c h u g @ in the concentration of this protein is seen in either the A8 cells or in the wild type 3T3 cells g r o ~ at 38~ INTRODUCTION The HI hlstones are the most structurally diverse of the five classes of histones They display not only a large degree of species specific v ~ i a b i l i t y but mlso extensive tissue and cell specific variations (Bustin ~ d Cole, 1968; K i n k a d e a n d Cole Ig66i Hohmann, 198q). This variety Of 6 t ~ c t u r e is correlated with the u n l q u e ~ o s i t i o n o f the Hi histones in the Chromatin, situated as they are~in the I n t e ~ e n i n g lisker region rather t h ~ w l e ~ i n t h e nucleosome Core pa~ticle(McGhee and Fels~feld, 1980).

0309-1651~1/121105-10~02.0~0

,~ 1981 Academic Presslnr (London) Ltd,

1106

CeEBiologvIntemationalReports, VoL5, No. 12, December 7981

Closely related t= the HI histones are t ~ other proteins, histone H5 and histose HI ~ . These two histones, while clearly p r e s a g i n g much of the overall architecture of the }11 histone molecule, show a degree of evolutionary divergence far greater than that encountered in any of the histone HI subfraetions (Yaguchl et al., 1977; Briand et el., 1980; and Smith et el., 1980). ~ s ~ o n e sl ~, llke hist~ne~--H1 and HS, is also ~ooca~ed in the linker region of the ehromatin (smith and $ohns, 1980). Histone HIo has been f 0 ~ d in a variety of m ~ a l i ~ tissues and has been s h o ~ t o be most abundant in cells displayin~ little or no cell division IPenylm ~ d Chalkley, 1969~ Pehrson'and Cole, 1980). This correlation has led to the proposal that histone H[ ~ is involved either in the suppression of eel i :ePlication (Panyi= and Chalkley, 1969) or of DNA synthesis {Marsh and Fitzgerald, 1973). In this present investlgat• we have studied the accumulatmon of hlstone H1 ~ in the Balb/e-3T3cell l~ne and its temperature sensitive variants A8 and A83. Both these cell lines are arrested in the Gi phase of the cell cycle when g r Q ~ at the non-permissive temperature (3B~C). In addition, the synthesis of the S] h i s t ~ e s during G1 arrest was examined in the light of ~e Cent ~ePorts t ~ t histone synthesis is not tightly coupled to DNA synthesis (Tarnowaka et el., 1978~ Groppi and Coffino, 1980) in contrast to the traditi~na~-view of histone and DNA synthesis being tightly coupled events restricted to S phase (Elgin and Weintraub, ~975)~t~TERIALSAND~THODS Cell Lines The cell lines used in these experiments were Balb/C-3T3 and two temperature sensltive variants, A8 ~nd A83 derive~ from it. Both A8 and A83 cells grow n o . a l l y at the p e ~ i s s i v e temperature (33oC) b~t when incubated at the non-pe~isslve temperature (38~ they are arrested i~ the G1 phase of the cell cycle (Naha et al., 1975). Cell Culture Cells were no~ally,maintained at 33~ in L15 m e d i ~ containing I0% foetal calf s e r ~ fortified with p~nici!lin and streptomycin. For experi=ental purposes cultures g r o ~ a t 33~C were s and plated out at a cell density of I x 10 s per ml in leur and isoleucine free m e d i ~ . Replicate flasks were incubated at elther 337C or 38~ s 24 h. (approxi~tely one cell qeneration 61m@). Cultures were labelled with 3H-leucine (10~C/ml, 30C/~o~; aad~ochemical.centre, Amersh~) in Hanks BSS during the ~ast K h. of t~e incubation, after which time the cells were washed twice with DulbeCco'm p B S ( F l O w Laboratories), scraped Qff t~e culture vessels, centrlfugedand frozen.

CeUBiologyln~rnationalRepo~s, VoL ~No. 1~ December1981 5%(w/v) Perchloric A c i d

1107

Extractio_~n

All operations were carried out at 0-4~ and frozen cell pellets (2xi07 cells) were extracted directly with 3 volumes of 5% (w/v) pe=chloric acid (PCA) ~ssentially as desczlbed by Sanders and Johns (1974). Each pellet was extracted three times by vortex~ng vigorously for 20 seconds and then centrifuged at 2080x g for 10 minutes. The supernatants were pooled and made 0.1N with respect to HCl and the proteins precipitated overniqht at -20~ with 6 volu,es of acetone. Precipitated proteins were washed ~wiee with acetone, 0.1N HCI (6:1, v/v) and three times with acetone before drying in a vacuum desiccator. Ele~tro~hores~ Proteins were analysed by SDS polyncrylamide gel electrophoresis (Lae~li, 1970); a 20% acrylamlde separating gel was used and equal ~ o u n t s of protein were analysed from each extract. Proteins ~ r e detected by staining with 0.05% (w/v) Coomassie Brilliant Blue R250 i n 5 0 % {v/v) methanol, 10% (v/v) acetic acid for 2 her followed by severainhanges of 10% (v/v) methanol, 7% (V/v) acetic acid ~ n t i ~ t h e blekgrolnd had cl~ered. The gels were ~hon phot6graphed and thenegas s Scanned u s i n q a Joyce L0bei ~
d a y s and dev6i6ped~ih Di9 ~vei~ger~9s

;!theh sehihed]'a{d peak

~SU~S 5% p C A e x t r a c t i o n ~ r o v l d e s a remarkabiy specs and simpl~ m~thod for the quantitltiveisolas Of the ~[ hiSt0nes Johhs, 1977). Co-e•163 with thes~proteins a r e a number of other lysine-rich proteins, including the High MobilityGroup (BMG) nonhistone chromosomal proteins; however, these proteins are readily distinguished from the HI histones upon polyacrylamide gel electrophoresis (Fig. I). In Figure I the Hi subfractions HIA and }lib ar~ c l ~ r l y seen as a doublet, slightly ahead of which migrates histone HI ~ Also seen are proteins HMGI and 2 and a group of 3 low molecular weiqht proteins designated X. This latter group includes proteins HMGI4 and 17 and will be discussed in detail elsewhere (Gooderham and Naha, manuscript in p~e~aration). When 5% PCA ~ t r a c t s of all three cell types, 3T3, A8 and A83, grown in the pres4nce of coid leucine at both 33 and 38~ were analysed by SDS polyacrylamide gel electrophoresis, the same qualitative pattern of HI histones was seen in all six samples. Scanning mlcrodensltometry (Fig. 2),however, showed ~ a h the concentration of histone Hl ~ varied Ielative to that of histones HIA and HIB and the HMG proteins HMGI and 2. In th~ 3T3 cells

1108

Cell Biology InternationaI Reports, VoZ 5, No, 12, December 1981

+

a

b

c

Fiq. I . SDS polyacryl~ide gel elec~ro~horesis of 5% PCA ex~racte~ protelns f r ~ the Balb/c-3T3 cell line g r ~ at 38~ (~) =nd f r ~ the temperature-sensitive v a r l e t A83 i n e ~ a t e d at 38~C (b). Pig thymus histones (c) are also included for c~parlson. Bistone S~ 9 was identified Dy the foll~,ing criteria:- solubility in 5% PCA, ap~azent molecular weight of 27,400 daltoms, nuclear orig/n, elect=ophoretlc mobility with respect to Sl and oo-electropho=esis wash a u t h ~ t i e rat liver histone E I % only s ~ l l concentratiolts (,5.8%) of histone ~ were f o ~ d , while in the temperatuze sensitive variant A8 higher (~13.2%) but equal ~ o ~ n t S of histone HI" were detected at both t~peretures. In contrast the A83 c e l l S s h c w e d a s ~ l l but slqnlficant inczeas, in blstone HI" concentratlon, inozeasing f r ~ ~8.3~ at 33~ to ~14.0% at 38~C. Neither hist6~es HIA and HIB, nor the mMG1 and 2 proteins s h o w e d ~ y ss changes in concentzation at ~he t ~

Cell Biology International Reports, VoL 5, No. 12, December 1981

a

,IA

b

d

+

q,2

\

Microd e n s i ~ m e t e r traces of 5% PCA soluble p=oteins of the Balb/o-3T3 cell line (Figs. 2a and d) ~ d its t ~ p e r a t u r e sensitive v a r i ~ t s A83 (Figs. 7b and e) ~nd A8 (F~gs. 2c ~ d f} gro~l at 33~C (Figs. 2a, b and c) and ~ e replicate flask i n c ~ a t e d for 24 h. at 38~ (Figs. 2d, e ~ d f) after SDS polyacrylamide qel electrophoresis. The ~ s i t i o n of histone ~i ~ is indicated by an arrow.

CeEBio~gylnternationa/Re~o~s, VoL ~ No, I~ December1981

1110

~empe~atu~

~n ~ny of th~ c~iz

H o w e v e r , when p r o t e i n

:~n~

sy~thesiz

studied.

a s measured by 3 [ ] - l e u e i ~ e

l~cozpozatio~ into the Hi histones w ~ examined, a different picture emerged. Scanning microdensitometry (Fig, 31 fas to show any significant difference in hlstoDe HI ~ Concentration when the cells were grown at either the p ~ t s s i v e te,~perature or the non-pcr~nissive teTnperature. ~rom the gel scans presented in Fig. 3 it isz however, clear ~hat the synthesis of both histories HIA and HIB continues, though at a r~4uced re:e, both in the A83 a ~ A8 cells when arrested in G1 a t 38~ Furtbe~Gore the synthesis of =b~se two subfzncaions is seen to shift in f~vour of hist~ne HIB. During G~ arrest, histone HIA synthesis is 6*% atld 29% o~ that seen in the A83 and A~ cells respectively when grown at ~be permissive temperature; while the values for histone HIB synthesis are 72% and 50% of n o ~ a l synthesis in ~he A83 and A8 cells respectively.

OZSCUSSIO~ 'I~ tempe~atur~ sensitive variants A8 and A83 derived from the Balb/C-3T3 cell li~*~ pro~ide a powerful u ~ l fo~ th~ analysis o~ the GI phase Of the cell cycle. ?~o properties of thes~ cells are considered particularly important in this regard: (i) Cell populations containing a large percentage of G1 ~rrested cells (91.5% for A83 and 78.2% for AS, und=r the conditions employed here) can be simply produced without resorting to mor~ potentially artlfactual systems, e.q, isoleuoin~ starvation (Tobey and LeF, ~971), thymidlne blocking [ T o b e y e ~ , , 1967) and sodlum butyrate blocking (D'A(,na et el., |980); ( i i ) The t';o variants A83 and A8 are arrested at d~fe~rent points wis the G% phase o~ the cell cycle. A83 cells are arrested 2 h. after 8nterinq GI, whereas the A8 cells are not arrested until after 8 h. have elapsed and the cells accumulate close to the GI/S boundary (Maha e t a l . , 1975; Naha, ~979). these ceils therefore p e ~ i = the a n ~ y s ~ s of specific biochemical events occurring during the G1 ~base of the cell cycle.

In thi~ paper we have sough~ to answe~ two questions; {i) Is the Gi arrest associated with an in<:reaged synthesis of histone Ml" and if this ~s the case i~ it restricted to any particular part of e GI phase? ( i i ) Can histon8 HI synthesis be s h ~ n to occur during GI =rr~st and does the pa%tern of synthesis differ ~ithin th, G! phase and also from that seen in the n o ~ a l l y cycling cell population? Quantitative scanning micro@ensitometryhas s ~ o ~ a 69% increase i, hs H~" concentration in the G] arrested AS~ ceils (Fig. 2)~ This finding is in broad a~reement with the ~esults obtained by other workers (D'Amna et el, ISS0; Pehrson and Cole, ~98Q) though th~ magnitude of change is far less than the approximately f~ur-fold increase reported by these investigators. The fail,re to s,e any s l g n i f ~ c ~ t diffe~ene~ is ~be historic H* ~

Cell Biology International Reports, VoL 5", No. 12, December 1981

a

HIA HIB

1111

d

HMG1+2

b

C

-

"'1

HI8.......

8

""F .,B

f

+

+

Fig. 3. Microdensitometer traces of 3H-leucine labelled 5% PCA soluble proteins of the Balb/c-3T3 cell line (Figs. 3a and d) and its temperature sensitive variants A83 (Figs. 3b and e) and A8 (Figs. 3c and f) grown at 33~ (Figs. 3a, b and c) and the replicate flask incubated for 24 h. at 38~ (Figs. 3d, e and f) after SDS poiyacrylamide g e l e l e c t r o p h o r e s i s . The position of histone HI ~ is indicated by an arrow.

1112

CeUBio~gylnternationalRepo~s,

VoL ~ N o . l ~ December1981

~H-leu~ine incorporation ~att~rns in both the A8 and A83 cells indicates that this protein is synthesised at approxi~Ateiy equml rates at both the p e ~ i ~ s i v e temperature and during G1 arrest a t the non-permissive temperature. We therefore c o n c l u d e that the accumulaiion of historic HI o in th~ Gl arrested A83 cells is probabl~ due to a decrease in the rate of hlstone HI = turnover ra ther than from an increase in the xat~ of hlstone HI ~ synthesis. The absence of any significant increase in the quantity of hlstone HI o in the A8 cells can be attributed to either its shorter p e r i o d of GI arrest and/or the lack of any change in histone HI o turnover during the latter part of the G1 phase. T h e f i n d i n g that Balb/C-3T3 cells conti;lue to synth~sise hist0n~ HI during the G1 phase is in =gree~ent w i ~ other workers (Taznowka et el., 1978; Groppi a n d Coffino, 1980). It is of interest, ho~ev~r, to n o t e that w h i l e HI h i s t o n e synthesis continues in the GI arrested cell=, thsre is a shift in the relative rates of synthesis of the two subfractions HIA and HIB (Fig. 3). In the A83 and A8 cells g r o ~ at 33~ as well as in the 3T3 cells cultured at both 33 and 38~ 3H-leueine incorporation in bistone HIB is app:oxlmately equal to 58% of that seen in hs HIA. with the onset of GI arrest in the A83 cells grown 8 t 38"C, hlstone HIA SH-leucine incorporation falls eo 62~ of that seen at 33~ while in the A8 cells this value is further reduced to 29% of that seen at 33~ These changes are p a r a l l e l l e d b y a similar, though less m a r k e d decrease in sH-leuGine incorporation J.n histone HIB, falling from 72% in the A@3 ceils to 50% in the A8 cells, relative to the values obtained for the cells grown at 33~

F rom the above results it is clear that n o t o n l y does Sl histone synthesis,,as m e a s u r e d by 3H-leueine incorporation, continue throughout the G~ phase b u t that there is a shift in the r~lative rates of histone H~ a and ~IH synth~sls in favour of histone HID which becomes most pronounced in late GI. There have been numerous other reports of changes in the hist0ne Hi subfraction patter,, in response to developmental and physiological processes (see for ~xample R u d e ~ a n et el., ~974; K i s t l e r and Geroch, 1975; Hohmann and Cole, 1971); q ~ s however, the first tame to o u r knowledge that such a change has b e e n observed during G1 a r r e s t in the Cell cycle. ~t is important to n o t e that whi~e n e i t h e r the A83 nor the A8 cell lines produce a complete GI arrest at the n o n permissive t~perature the greatest rate of 3H-leuclne incorporation is seen s the A83 cell line (91.5% G1 arrest}, whereas in the more leaky AS cell line (78.2% GI arrest) a lower level of ~H-leucine incorporation is observed. ~he differences in ~H-leuoine inc~=poration into the histone Ill subfraet• a r e t~lerefore thought ~ l i k e l ~ to be due to the presence of n o n - a r r e s t e d cells b u t instead tO reflect the p a t t e r n of HI Syns occurring w i t h i n the G1 phase.

Ceil BiologylnternaNonafRepo~VoL ~ N o . 12, Decembert98;

1113

AC~O~EOCEMF.NTS The authors would like to thank Professor H.J. Evans for his support and encouragement during this work, and to ackr~owledge the financial support of the Medical Research Council and Cancer R~search Campaign. REFERENCES Briand, G., ~ l e c i k , D., Sautiere, P., Wouters, D., Vorie-Loy, 0., Biserte, S., ~ z e n , A. and C h ~ p a g n e , M. (1980) Chicken erythrocyte histone H5. IV Sequence of the carboxy-termlnal half of the molecule (96 residues) and complete sequence. FEBS Letters 112, 147-151. Bustin, M. and Cole, R.D. (1968) SpeciQs and organ specificity in very lysine~rich histones. Journal of Biological Chemistry 243, 4500-4505. D'Anna, J.A., Tobey, R.A. and Gurley, L.R. ([980) Concentrationdepemdant effects of sodium butyrate in Chinese hamstRr cells: Cell-cycle progression, innez-hls~one acetylation, histone HI deph0sphorylation, and induction of an [{I-like protein. Biochemistry 19, 2656-2671. Elgin, S.C.R. and--weintraub, H. (1975) Chromosomal proteins and chromatin structure. Annual Review of Biochemistry 44, 725-774. Groppi Jr. V.E. and C0ffino, P. (1980) GI and S phase i.a~alian cells synthesize histones at equivalent rates. Cell 2~, 195-204. Hohmann, p. and Cole, R.D. (1971) H o ~ o n a l effects on amino acid incorporation into lysine-rich h• in the mouse m ~ a x y gland. Journal of Molgeular Biology 58, 533-540. Hohm=nn, P. (19S0) Species- and cell-sp~ific expression of Hi histones in tissue culture cells. Archives of Biochemistry and Biophysics 205, 198-209. Johns, E.W. (1977) The isolation and purification of histories. In: Methods in Cell Biology XVI (stein, G., Stein, J., Kleinsmith, L.J. eds] pp 183-203. Academic Press Inc. New York, San Francisco, London. Kinkada, J.M. and Co~e, R.D. (1966) The resolution of four lysine-rich histones derived from calf thymus+ Journal of Biological Chemistry 241, 5790-5797. Kistler, W.S. and Geroeh, M.E~ (1975) An unusual pattern of lysi~e-rich historic Componcnts associated with spe~atogenesis in rat testis. Biochemical and Biophysical Research Co~unicat• 63, 378-384. L a e ~ l i , U.K. fig70) Cleavage of structural proteins during the assenf01y of the head of bacteriophage T4. Nature, London 227, 680-685. Laskay, R.A. and Mills, A.D. ([975) Quantitative film detection of ~I{ and 14c in pelyacryi~ide gels by fluorography. European Journal of Biochemistry 56, 335-341.

11 ~4

Cell Bio/ogy International Reports, VoL 5, No. 72, December 1981

Marsh, W.H. ~nd Fitzgerald, P.J. (1973) Pancreas acinar cell regeneration. XIII Histone synthesis and modification. Federation proceedings 32, 2119-2125. McGhee, J.D. and Felsenfeld, G. (19s0) Nucle0some structnr~Annual Review of Biochemistry 49, ~}15-1156. Naha, P.M., Meyer, A.L. and Hewitt, K. (1975) Mapping of the Gl phase of a m a m ~ a l i ~ cell cycle. Nature, London 258, 49-53. Naha, P.M. (1979) Complem~ntation of Gl~phase v a r i a ~ o f a m a ~ a l J a n cell cycle. Journal of Cell Science 35, 53158. Panyim, S. and Chalkley, R. (1969} A new histone found only in m~r=alian tissue s with little cell division . BioChemical and Biophysical Research C ~ u n i e a t i o n s 37, iQ42-I048. pebrson, d. and cole, R.D+ (1980) His~one al o accumulates in growth~inhibited cultured cells. Nature, London 285, 43-44. Ruderman, J.H., Baglioni, C. and Gross, p.R. (1974) Histone m ~ A and histone synthesis during embryogenesis. Nature, London 247, 36-38. Sand~rs, C. and Johns, E.W. (1974) A method for the large seals preparation of two chromatin proteins* Biochemical Society Transactions 2, 547-550. Smith, B.J. and J~hns, E.W. (~980) Historic HI ~, its locat~on in chromats Nucleic Acids Research 8, 6069-6079. SMith, B.J., Walker, J.M. and Johns, E.W. (1980) Structural homology between a ~ , a l i a n HI o subfzaction and avian erythrocyte specific histone HS. FEBS Letters 112, 42-44. ~'arnowka, M., Baqlioni, C. and Basilico, C. {1978) Synthesis of H~ histories by BHK cells in GI. Cell i-9, 163-171. TObey, R.A., Anderson, E.C. and Petersen, D.F. ([967) The effect of thymidine on the duration of GI in Chinese hamster cells. Journal of Cell. Biology 35, 53-59. Tobey, R.A. and Ley, M.D. ~ 9 7 1 ) Isoleucine-mediated regulation of genome ~eplication in various m e - - l i e n cell lines. Cancer Research 31, 46 51. yaguchi, M.,-~oy, C., Dove, M. and Seligy, v. Amino acid sequence homologies between HI and ~5 histories. Bioehemlcal and Biophyslca! Research Communications 76, 100-106.