Molecular Biology Reports 16: 33-38, 1992. 9 1992 Kluwer Academic Publishers. Printed in Belgium.

33

Histone H1 ~ mRNA and protein accumulate early during retinoic acid induced differentiation of synchronized embryonal carcinoma cells F.J. van Hemert, L.J.C. Jonk & O.H.J. Destr6e*

Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands (* requests for offrints) Received 22 August 1991; accepted in revised form 26 September 1991

Key words: differentiation, embryonal carcinoma cells, replacement histone variant, teratocarcinoma cells

Abstract

The very lysine-rich replacement histone variant H 1~ is found to be present in different murine (C1003, PC 13, P19) and human (Tera-2) embryonal carcinoma cell lines. The proportion of H 1~ increases upon induction of differentiation of the different cell lines by various treatments. In undifferentiated PC 13 EC cells H1 ~ m R N A is present at a low level. During retinoic acid induced differentiation of mitotically synchronized PC13 EC cells, accumulation of H1 ~ mRNA starts in the first cell cycle. The H1 ~ protein level starts to increase in the second synchronous cycle preceding changes in the cycle parameters that become apparent in the third cycle. The results provide further support for an important role of H 1~ in the control of cellular differentiation in early mammalian development.

Abbreviations: E C - embryonal carcinoma, R A - retinoic acid, D A P T - 4'-6-diamino-2-phenylindole, SDS - sodium dodecylsulphate, D M S O -dimethyl sulfoxide, T C A - trichloro acetic acid

Introduction

Embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, are used as a model system to gain insight into certain aspects of early mammalian development since they have many properties in common with cells of the inner cell mass of the blastocyst. Differentiation of EC cells is marked by dramatic changes in phenotype and in proliferation rate [ 1]. These changes are characteristics for the type of EC cell as well as for the inducing agent and depend on alterations in the transcription rate of specific genes. Among the genes with a modified level of expression after

induction of differentiation are those coding for polypeptide growth factors and their receptors, transcription factor, intra- and extracellular enzymes and structural proteins [ 1]. Some of these genes are activated rapidly after the induction of differentiation like Hox 1.6 (Era1) [2] and the retinoic acid receptor fl [3]. The induction of other genes is much slower and may depend on the expression of the early genes. Still other genes become transcriptionally repressed during EC cell differentiation [4]. The reprogramming of transcriptional activity during cellular differentiation is accompanied by changes in the structure of the nuclear chromatin.

34 The organization of chromatin structure at the supranucleosomal level is subject to the local arrangement of the histone H1 subtypes [5]. The replacement histone variant H1 ~ accumulates in many cells and tissues, when the growth rate decreases [6]. The degree of H1 ~ induction varies with cell type and treatment [7, 8]. The avian homologue of H 1o, H5 has been overexpressed in rat sarcoma cells. The resultant inhibition of DNA replication and arrest of cells in G1 suggest that H5 participates in the control of DNA replication and cell proliferation [9]. Evidence has also been provided indicating that H1 ~ plays a role in the regulation of gene expression. Highly inducible genes may be segregated into chromatin regions, which are depleted in H1 ~ [10]. In tissues which are dependent on hormones for their function and maintenance, H1 ~ expression depends on the presence of the maintenance hormone [ 11 ]. An early increase in H1 ~ m R N A has been demonstrated during differentiation of F9 EC cells [ 12]. In the present investigation we have analyzed the expression of the H 1~ gene at the protein and RNA level in different EC cell lines of murine and human origin. We find both H1 ~ protein and m R N A to be present in the undifferentiated EC cells. Differentiation induced by various induction mechanisms leads to accumulation of H1 ~ protein. The PC13 EC cell system has been demonstrated to be an excellent system to study cyclerelated events during the process of differentiation [13]. Therefore, this system was used to investigate the relation between H1 ~ mRNA as well as protein induction and the appearance of cell cycle lengthening.

Materials and methods

Cell culture Embryonal carcinoma cells were maintained and induced to differentiate as described [ 13-16]. The clonal variants Epi-7, Mes-1 and End-2 derived from P19 EC cells have been characterized previously [ 13, 17]. PC13cells were synchronized by mitotic selection and cultured in the presence of

1 0 - 6 M RA [13]. For cell cycle analysis, cells suspended in growth medium with or without serum were diluted with an equal volume of 10 mM TrisHC1 pH 7.5, 40 mM MgC12, 20 #g/ml DAPI (4'-6-diamino-2-phenylindole), 0.1 ~o Triton X-100 and analyzed immediately using a Partec SPU 32 PAS II Fluorescence Activated Cell Sorter. A fibroblast-like cell line, Fib9, has been cloned from C1003 cells after a 2 day period of serum deprivation.

RNA purification and blot hybridization Total RNA from cells and tissues was purified according to Chirgwin et al. [18], Total RNA (25 #g per lane) was fractionated on 1.2~o agarose gels containing 6.6~o formaldehyde and transferred to nitrocellulose filters [19]. The amount of total RNA applied to each slot was determined by visual inspection of the EtBr fluorescence of the 28S and 18S ribosomal RNAs in the test gels. Murine H1 ~ cDNA was kindly provided by Dr. H. Eisen (Seattle), cloned Xenopus histone H3 DNA was a gift of Dr. J.G. Schilthuis. Probes were labeled by random priming according to the instructions supplied (Boehringer). Hybridization and autoradiography were performed according to standard procedures [19].

Histone extraction and immunoblotting Very lysine-rich histones were extracted from cells and tissues with 5 ~o TCA as described previously [20]. After electrophoresis on 12~o polyacrylamide gels in the presence of sodium dodecylsulphate (SDS-PAGE), the proteins were transferred to nitrocellulose membranes (Schleicher & Schuell) by means of electroblotting at pH 9.29.5. The blots were processed for immunostaining with diaminobenzidine as a substrate for horse radish peroxidase conjugated goat-anti-mouse IgG (Nordic). The characterization of monoclonal antibodies against the very lysine-rich histone variants has been described before [20].

35 Results H1 ~ protein in murine and human embryonal carcinoma cells

The presence of H 1~ protein in the murine EC cell lines C1003, PC13 and P19 and the human line Tera-2 is illustrated in Fig. 1. H 1~ is present in the various EC cell lines at different levels. Induction of differentiation of C1003 both by serum deprivation (Fig. 1, lane 3) and addition of RA of the serum-containing growth medium (Fig. 1, lane 4) leads to a level of H 1~ protein comparable to that in the cloned derivative Fib9 (Fig. 1, lane 5). A cross-reaction with the more abundant variants H 1b'd'e can be observed and this is attributable to the large amount of total H 1 protein applied in order to detect the relatively low abundant H1 ~ variant [20]. Histone H1 ~ is present in PC13 EC cells at a low level (Fig. 1, lane 6). Its amount is found to be increased after differentiation induced by RA (Fig. 1, lane 7). An increase in the amount of H 1~ is also observed after induction of differentiation of P19 EC cells both by RA in monolayer culture (Fig. 1, lane 9) and by D M S O in cultured aggregates (Fig. 1, lane 10). Human Tera-2 cells, treated with RA, also show an increased proportion of H1 ~ (Fig. 1, cf lanes 11 and 12).

H1 ~ protein and mRNA accumulation in synchronously proliferating PC13 EC cells

PC13 EC cells were synchronized by mitotic selection and immediately induced to differentiate with RA. The flow-cytometric determination of the cell cycle progression showed that cell divisions take place at about 12 and 22 hours after mitotic selection both in EC cells and after RA addition. When the second cycle and the first part of the third cycle (Fig. 2) were compared it became evident that in a fraction of the cell population the cycling time increased as indicated by the peak persisting at the G1 position during the third cycle. The accumulation of histone H1 ~ protein during RA induced differentiation of synchronously proliferating PC13 cells is illustrated in Fig. 3. The amount of H1 ~ is very low in dividing PC13 EC cells (Fig. 3, lane 1), and increases steadily during the second and third cycles (12-30 hours) after mitotic selection and RA addition. Steady state levels of H1 ~ m R N A during RAinduced differentiation synchronized PC13 cells have been determined by blot hybridization as illustrated in Fig. 4. H 1~ cDNA hybridizes with two m R N A species (about 2.1 and 0.9 kb in size) from mouse cells and tissues, although the gene for H 1~ is known to be single-copied in the mouse

Fig. 1. H 1~ in different murine and human EC cells and their differentiated derivatives. Very lysine-rich histones were isolated from EC cells or their differentiated derivatives. Equal amounts of very lysine-rich protein were applied to each slot and staining of the blot was done simultaneously with anti-H 1~ and anti-H ~ as control. After electrophoresis through 12% SDS-gels, the proteins (1/tg per lane) were transferred to nitrocellulose membranes by electroblotting. The upper band represents the histone variants H1 b, H1 d and H e staining aspecifically, while the histone variants HI a and H1 c comprise the middle band, stained with the anti-Hie-specific monoclonal antibody. 1. Control: liver 2. C1003 EC 3. C1003 minus serum, 5 days 4. C1003 + RA 5. Fib 9 cells 6. PC 13 EC 7. PC 13 + RA 8. P19 EC 9. P19 + RA 10. P19 with DMSO (1%), 5 days 11. Tera-2 EC 12. Tera-2 + RA. + RA: indicates cells cultured in the presence of 10- 6 M RA for 5 days.

36

-RA

+RA

22h

24h

6)

"5

26h

r

z

=>

n'-

28h

30h

Channel Number

Fig. 2. Flow-cytometric analysis of synchronized PC 13 cells cultured in the absence ( - RA) or presence ( + RA) ofretinoic acid, Distribution of cells during progression through the third cycle after mitotic selection. At the times indicated (2230 hours) cells were harvested, stained with DAPI and analyzed by Fluorescence Activated Cell Sorting.

genome. RNA products of similar size have been observed in F9 cells after treatment with RA [ 1]. In the synchronously differentiating PC 13 cells, the H1 ~ m R N A concentration increases during the first cell cycle and remains high during the second and third cycles. Discussion

Our results demonstrate that histone H1 ~ is present in different EC cells lines both from mu-

rine and human origin. The proportion of H1 ~ increases during differentiation of the EC cells into different derivatives, induced by various treatments. These results confirm and extend data on H1 ~ accumulation in the nullipotent F9 EC cell line [ 12]. We find no obvious correlation between the proportion of H 10 and the differentiation pathway of the different EC cell lines e.g. PC13 showing the lowest content of H 1~ is considered to be less pluripotent compared to P19 [15, 161. The accumulation of H 1~ during differentiation of the different EC cell lines is independent of the inducing stimulus. P19 EC cells induced with RA differentiate in neurectodermal derivatives, while after treatment with D M S O they acquire a mesodermal phenotype [15, 16]. A lengthening of the cell cycle is a general feature of EC cell differentiation [12, 13, 21]. In synchronized PC13 EC cells, H 1~ m R N A accumulation starts during the first cell cycle after addition of retinoic acid (RA), followed by an increase in H 1~ protein content during the second cycle and lengthening of the generation time in the third cycle. Sun et al. [9] have demonstrated that the avian H5/histone, which is structurally related to H 1~ plays an active role in the control of cell proliferation in transfected rat sarcoma cells. However, it remains to be determined of the H 1~ accumulation affects the expression of other genes and, in doing so, controls proliferation during EC cell differentiation. H 1~ expression is induced by steroid and peptide hormones in tissues depending on the maintenance hormone involved [11]. The rapid increase in H1 ~ m R N A concentration during EC cell differentiation as described here and by others [ 12] may be due to transcriptional activation and/or posttranscriptional regulation. A rapid induction of H1 ~ mRNA, within one hour, has been observed in differentiating murine erythroleukaemia (MEL) cells after treatment with HMBA [22]. From studies on DMSOtreated, transfected MEL cells, Cheng et al. [22] conclude that the H 1~ gene is negatively regulated by c-myc. These authors suggest that an inverse relationship between c-myc and H1 ~ expression

37

Fig. 3. Accumulation of histone H1 ~ protein during RA-induced differentiation of synchronized PC 13 cells. Very lysine-rich histones (1/lg) were subjected to polyacrylamide gel (12%) electrophoresis and transferred to nitrocellulose filter. An extract from adult mouse liver has been applied to the outer slots (liv/lys). The blot was stained simultaneously with monoclonal antibodies against the histone variants H1 ~ and H1 c. As before, the upper band represents the histone variants H1 b, H1 d and H1 e staining aspecifically, while the histone variants H 1a and H 1r comprise the middle band, stained with the anti-H 1c specific monoclonal antibody. The number above each lane indicates the time of culturing in the presence of retinoic acid after mitotic selection.

may be a more general phenomenon. Studies with different EC cell lines suggest that early and transient changes in the expression of members of the myc family may also be instrumental in an enhanced accumulation of H 1~ transcripts during early EC cell differentiation [4, 23]. As myc protein(s) are helix-loop-helix proteins that bind to DNA directly and indirectly and may function as transcriptional regulators [24], the H1 ~ gene might be a target for negative regulation by these

proteins. Further analysis of the regulation of H 1o expression during EC cell differentiation should contribute to a better understanding of the role of H 1~ in the control of differentiation in early mammalian development.

Acknowledgements The authors thank Dr. C.L. Mummery, Dr. P.T. van der Saag, Dr. S.W. de Laat, Dr. A.J. Dur-

Fig. 4. Accumulation of H1 ~ mRNA during RA-induced differentiation of synchronized PC13 cells. Total RNA was extracted from synchronized PC13 cells grown in the presence of RA for the times indicated (0-30 hours). The blot was hybridized with the HI ~ cDNA clone, washed and reprobed with histone H3 DNA. Positions of the 28S and 18S ribosoal RNAs are marked as well as the appearance of cell divisions in the culture (M).

38 ston, Mr. A. Bardoel and Mr. F. Vervoordeldonk for their contributions. We thank Dr. H. Eisen (Seattle, USA) for providing H1 ~ cDNA. This research was supported by the foundation for Medical and Health Research (Medigon) of the Netherlands Organization for the Advancement of Pure Research (NWO). References 1. Adamson ED & Hogan BLM (1984) Differentiation 27: 152-157 2. LaRosa GJ & Gudas LJ (1988) Mol. Cell. Biol. 8: 39063917 3. Zelent A, Mendelsohn C, Kastner P, Krust A, Garnier JM, Ruffenach F, Leroy P & Chambon P (1991) EMBO J 10:71-81 4. Finklestein R & Weinberg RA (1988) Oncogene Res. 3: 287-292 5. Pederson DS, Thoma F & Simpson RT (1986) Ann. Rev. Cell Biol. 2:117-147 6. Lennox RW & Cohen LH (1983) J. Biol. Chem. 258: 262-268 7. Hall JM & Cole JD (1985) J. Biol Chem. 261:5168-5174 8. Chabanas A, Khoury E, Goeltz P, Froussard P, Gjerset R, Dod B, Eisen H & Lawrence JJ (1985) J. Mol. Biol. 183:141-151 9. Sun J-M, Wiaderkiewicz R & Ruiz-Carillo A (1989) Science 245:68-71

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