Developmental changes in the expression of high mobility group ...

Communication Developmental Changesin the Expression of High Mobility Group Chromosomal Proteins* (Received for publication, September 6, 1990)

Massimo P.CrippaS, Joanne M. Nickols, and Michael BustinSll From the Laboratoriesof $Molecular Carcinogenesis, National Cancer Institute and $MolecularBiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

The high mobility group (HMG) chromosomal proteins may modulatethe structure of distinct regions in chromatin, thereby affecting processes such as development and differentiation. Here we report that the levels of the HMG chromosomal proteins and their mRNAs change significantly during erythropoiesis. Erythroid cells from 5-day chicken embryos contain 2.5-10 times more HMG mRNAs than cells from 14day embryos, whereas circulating cells from adult animals are devoid of HMG and most other mRNAs. Nuclear run-off experiments and Northern analysis of RNA fromvarious developmental stages and from Percoll-fractionated cells indicate that the genes aretranscribed in early cells of either the primitive or definitive erythroid lineage. The rate of synthesis of the various HMGs changes during erythropoiesis; in erythroid cells from 7-day embryos the ratio of HMG-14b or HMG-17 to HMG-14a is, respectively, 8 and 10 times lower than in 9-day erythroids. HMG-l4a, the major chicken HMG-14 species, is synthesized mainly in primitive cells, while HMG-14b is preferentially synthesized in definitive cells. Thus, the change from primitive to definitive erythroid lineage during embryogenesis is accompanied bya change in the expression of HMG chromosomal proteins. Conceivably, these changes may affect the structure of certain regions in chromatin; however, it is not presently clear whether the switch in HMG protein gene expression is a consequence or a prerequisite for proper differentiation.

THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 266, No. 5, Issue of February 15, pp. 2712-2714,1991 Printed in U.S.A.

During chickenembryogenesis the circulating erythrocytes have a defined developmentalprogram, involving twodistinct cell lineages. The primitive lineage, the cells of which synthesize the embryonic @‘ and @”, predominates from the startof erythropoiesis to day 5. After this time, the definitive erythroid lineage, the cells of which synthesize the later globins (pH and PA),gradually becomes the major population throughout the restof embryogenesis. The redcells are readily available at every stage of maturity and remain nucleated throughout life, thereby allowing the analysisof the structure, as well as the expression of genes of interest. The putative involvement of HMGproteinsinchromatinstructureand gene expression lead usto investigate the expression of the proteins during erythropoiesis. In this study we report that the relative signiflevels of the HMG proteins and their mRNAs change icantly duringerythropoiesis. MATERIAL ANDMETHODS

Celk-Fertilized eggs and normal adult chicken erythroids were M D ) . Erythrocytes purchasedfromTruslowFarm(Chestertown, from embryos were obtained by nicking an amniotic blood vessel. Reticulocytes were obtained by wing bleeding of adult chickensmade anemic by injections of I-acetyl-3-phenylhydrazine. R N A Analysis-Total RNA was extracted from erythrocytes of different age embryos and from reticulocytes of adult anemicchicken by the guanidinium/cesium chloride method, as described (IO). For Northern analysis, the RNA was fractionated on 1%(w/v) agaroseformaldehyde gel, transferred to Genescreen Plus membranes and hybridized with the probes described in the legend to Figs. 1 and 2, according to the method of Church and Gilbert (11).Primer extension and nuclear run-offswere done as described (10). The probe used for primer extension experiments isdescribed in the text. Protein Extraction and Gel Electrophoresis-HMG proteins were extracted with 5% (w/v) perchloric acid (1)and fractionated on 15% polyacrylamide-sodium dodecyl sulfate gels, as described (19). Labeled proteins were obtained by suspending cells in lysine-depleted minimal essential medium containing dialyzed fetal bovine serum for 20 h with [14C]lysine (0.1 pCi/ml). The proteins were extracted, fractionated, and stained inpolyacrylamide gels. The gels were dried and exposed to Amersham Hyperfilm 0-max for 30 days a t -70 ” C . RESULTS AND DISCUSSION

Northern analysesof total RNA extractedfrom circulating erythroids of embryos, and anemic and normal chickens indicate that the steady-statelevels of HMG mRNAs decrease during embryogenesis (Fig.1).The HMGmessages are absent The HMG protein class is among the most abundant and from reticulocytes, with the exception of HMG-14b mRNA, ubiquitous nonhistones found in thenuclei of higher eucary- the level of which is greatly reduced. The mRNA extracted otes (1, 2). HMG-1 and -2 are single strand DNA-binding from 5-day erythroids containssignificantly more HMG mesproteins (3) which recognize specific DNA structures rather sage than that extracted from 7-day or older embryos. Comthan sequences(4, 5). HMG-14and -17 are nucleosomal pared with 5-day red cells, 14-day redcells contain 10% HMGbindingproteins (6), which may modulatethechromatin 14a mRNA and 40% mRNA for either HMG-l4b, -17, or -1, structure of transcriptionally activegenes (7, 8). Chicken indicating that the HMG-14a message decreases more rapidly erythrocytes containtwo forms of HMG-14. The main HMG- than that of the other HMGs. As an internal control to the 14 component, HMG-l4a, has a higher molecularweight than experimentalsystem, we haveused the well characterized the minor component, named HMG-l4b, which is the true expression of the @*-globingene. The data in Fig. 1 indicate homologue of the mammalian HMG-14(9). that, during the same period, the mRNA level of @*-globin increases significantly. * The costs of publication of this article were defrayed in part by The decrease of HMGmRNA levels suggests that the the payment of page charges. This article must therefore be hereby messages are synthesizeda t early stagesof differentiation. To marked “aduertisement” in accordance with 18 U.S.C. Section 1734 define better the developmental stage a t which these HMG solely to indicate this fact. nuclear run-off experiments ll To whom correspondence should be addressed: Bldg. 37, Rm. mRNAs are actually transcribed, were done. In both5- and 9-day embryos the levels of HMG3D12, NCI, NIH, Bethesda,M D 20892.

HMG Proteins in Developing Chicken Erythrocytes

2713 (A)

DAYS

(B) Age of embryos Idays) :. 5 7 9 1 2 1 4 dc"

Age of embryos ldaysl 5 7 9 1 4

(C)

(6)

9 __-

5

7

STAIN LABEL

STAIN LABEL

STAIN LABEL

" "

" -

HMG-14a-

- H5 - HMG-1 "4G-14a -HMG-14b -HMG-l7

" -

- pA-globin

-

HMG-17 - w

FIG.4. Synthesis of HMG proteins during erythropoiesis. Cells from ( A ) 5-day. ( H ) 7-day, and ( C )9-day chicken embryos were studied. Lanes A-C, respectively, contained 10-20 pg of total proteins. The proteins areidentified on the right. TABLE I

1

pA-globin-

- 23s - 18s

FIG. 1. Northern analysis of the steady-statelevels of mRNA for HMGs in chicken erythrocytes during development. SO Fg of total RNA were fractionat.ed on 1% (w/v) agaroseformaldehyde gels and analyzed using the following probes: HMG14a,an oligonucleotide correspondingto residues 101-120 of the cDNA (13);HMG-14hcDNA(14);HMG-17 cDNA (15);HMG-1 cDNA (16). After autoradiographic exposure, the blots were stripped and rehybridizedwitha probe for the BA-globin gene (17).The multiple hands for HMC-1 mRNA correspond tomessages of different sizes, due to multiple polyadenylation signals in the gene (18).A, HMG-l4a, HMG-l4h, and HMG-17; R,HMG-1; C,ethidium bromide stain of the gel used in A. The position of the ribosomal RNA is indicated.

Relative ratios of newly synthesized (laheled)HMG proteins in erythrocytes from chicken embryos during the shift from primitiw to definitive cell lineqe The bands visualized by autoradiography were quantitated with a Beckman DU8 spectrophotometer. The proteins were isolated and analyzed as described under "Materials and Methods." H M C ration Day 14h/14a

17/14a

17/14h

0.16 1.25

0.045 0.13 0.47

0.84 0.38

S 7 9

of the HMG mRNA detected in circulating erythrocytes is actually transcribed at an earlierstage of maturation. T o examine further thispossibility, we analyzed the earlier blast cell stages of the definitive lineage, separate from both the (A) (B) primitive and definitive mature erythrocytes. Thisis accomplished by fractionating erythrocytesfrom 6-day embryos on Age of embryos Ida@ S _ L 7 4 5 6 7 Percoll density gradients (12) to yield two defined bands of T Total B T Total B cells, each of which is collected for RNA extraction. Thecell lineage comprising each band is determinedby primer extension analysisof the typeof globin mRNA thecells synthesize, yuy *d -HMG14b c, p, or A. The sequence spanningnucleotides +98 to +118 of which is common to all chicken @-globinmRNAs, @ ~ w ~ ~ H M G lPA-globin, 7 is used as the primer. Since this common primer sequence is FIG. 2. HMG mRNA is enriched in erythroid precursor located a t different distances from the respective CAP sites cells. A, primer extension (10) using a probe which detects all the (+1) of each @-globintranscript, each transcript's extension globin transcripts (d. M. Nickol, unpublisheddata;seetext). /3"- product of differentlength is unmistakably identified and Globin representsembryonictranscripts; BA-globin representsthe quantified. This analysis andmicroscopic examination of the adult transcript. Erythrocytes from 6-day embryos were fractionated cells in the bands indicate that the top band (Fig. 2 A , lune on Percoll gradients (12). The RNA was prepared from the unfractionatedcells (Total) and from the top (T) or hottom ( R )hand. Note 67') of the Percoll gradients is a composite containing a small t.hat the bottom band isdevoid of BA-glohin.R,Northern analysis of proportion of definitive6-dayerythroblastsandprimitive HMG mRNA. Note that the top hand of day 6 (67') is highly enriched erythrocytes, whereas the bottom band (Fig. 2 A , lune 6B) in HMG mRNAs as compared with the bottom band (6R). contains pure erythrocytesof the primitivelineage. Northern analysis of the same fractions(Fig. 2R) indicates that the top DAYS band is highly enriched in HMG mRNAs as compared with either the total or the bottom band. The results support our 3 5 7 9 1 2 1 4 E R conclusion that HMG gene transcriptionis an early event in " the differentiation of both the primitive and definitive cell lineages. The messages detected in circulating cells reflect the relative stability of the HMG mRNAs and thepercentage of various erythroid cells in the circulation. However, the data FIG. 3. Steady-state HMG protein levels in chicken erythdo not exclude the possibility that thedecrease in the mRNA rocytes during development. Proteins were extracted with 5% levels reflects degradation of maternal messages, which were (v/v) perchloric acid (1), fractionated on 15% polyacrylamide-sodium dodecyl sulfate gels (19)andstainedwith CoomassieBlue. The stored in precursor cells. Analysis of the steady-state levels of HMGproteins at numbers a t the top of the lanes indicate the age of the embryos (days post-fertilization) from which the samples were taken. E, erythro- various stagesof erythropoiesis reveals that theprotein profile cytes; R, reticulocytes. also changes during development (Fig. 3). In 3- and 5-day cells the major protein is HMG-14a and HMG-17 is barely 14b and HMG-17 mRNA synthesis arebarely detectable and visible, whereas HMG-14b cannot be detected. The ratio of HMG-14b or -17 to HMG-14a increases up to 9 days and significantly lower than those of globin mRNA which were following developmental used as controls (not shown). The resultssuggest that most remainsconstantthroughoutthe "y

"

2714

HMG Proteins in Developing Chicken Erythrocytes

stages. Densitometricanalysesindicatethattheratio of stages of erythrocyte maturation is correlated with a high HMG-17 to HMG-14a in erythroid cells from 7-day embryos activity of gene transcription. Furthermore, the shift in HMGis 4-fold lower than in erythrocytesfrom adult chickens. The 14 synthesis, which corresponds to the replacement of the changes are due mainly to a decreasein the amounts of HMG- primitive erythroid lineage by the definitive lineage in the 14a. population of circulating cells, indicates the possibility that The synthesis of HMG proteins during erythropoiesis is the two different forms of HMG-14 may play a role in the analyzed to see if it corresponds with the steady-state HMG selection of the cell-specific genes transcribed. It is not presprotein levels and can be correlated to the switch from the ently clear whether the switch in HMG gene expression is a primitive to the definitive erythroid lineage of circulating consequence or a prerequisite for proper differentiation. erythrocytes. Erythroid cells are incubated with ['4C]lysine, REFERENCES and the HMG proteins are fractionated by electrophoresis and visualized by Coomassie Blue staining and autoradiogra- 1. Johns, E. W. (1982) The HMG Chromosomal Proteins, Academic Press, New York phy (Fig. 4). In 5-dayembryos the most prominent band 2. Bustin, M., Lehn,D., and Landsman,D. (1990) Biochim. Biophys. correspondsto HMG-14a. The HMG-17 signal is weak, Acta 1049, 231-243 whereas HMG-14b is not visible on the autoradiogramseven 3. Isackson, P. J., Fishback, J. L., Bidney, L. D., and Reeck, G. when the gel is overloaded. In 7-day embryos, the relative (1979) J. Biol. Chern. 254, 5569-5574 synthesis of HMG-14a decreases significantly and the incor- 4. Hamada, H., and Bustin, M. (1985) Biochemistry 24,1428-1433 5. Bianchi, M. E., Beltrame, M., and Paonessa, G. (1989) Science poration of ['4C]lysine into HMG-14b isdetectable. At 9 days 243,1056-1059 the synthesis of HMG-14a is lower than that of HMG-14b. 6. Schroter, H., and Bode, J. (1982) Eur. J. Biochem. 127,429-436 The changes in HMG synthesis, are most obvious when the 7. Albanese, I., and Weintraub, H. (1980) Nucleic Acids Res. 8, relative ratios between the proteins are calculated (Table I). 2787-2805 Between day 5 and 9 the relative ratio of HMG-17 to HMG- 8. Weisbrod, S., Groudine, M., and Weintraub, H. (1980) Cell 19, 289-301 14a increases 10-fold. Between day 7 and 9 the relative ratio 9. Srikantha, T., Landsman, D., and Bustin,M. (1989) J. Mol. Biol. of HMG-14b to HMG-14a increases %fold, and the ratio of 211,49-61 HMG-17 to HMG-14b decreases approximately 2-fold. Al10. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, though the synthesis of HMG-14a is significantly reduced, J. G., Smith, J. A,, and Strhul, K. (1990) Current Protocols in this protein quantitatively remains themajor member of the Molecular Biology, pp. 4.10.1-4.10.9, John Wiley & Sons, Inc., HMG-14/-17 family, most probably due to the low turnover New York rate of HMG proteins. The change in the synthesis of the two 11. Church, G., and Gilbert, W. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1991-1995 HMG-14 proteinscoincides with the population change from 12. Lois, R., and Martinson, H. G. (1989) Biochemistry 28, 2281primitive to definitive cell lineage in the embryonic circula2287 tion. 13. Dodgson, J. B., Browne, D. L., and Black, A. J. (1988) Gene The results indicate that the steady-state levels of HMG (Amst.) 6, 287-295 mRNAs decrease during chicken erythrocytedevelopment in 14. Srikantha, T.,Landsman, D., and Bustin, M. (1988) J. Biol. Chem. 263, 13500-13503 a differentially timed way. As the circulating redcells mature from erythroblasts t o erythrocytes andas the definitive line- 15. Landsman, D., and Bustin, M. (1987) Nucleic Acids Res. 15,6750 age replacesthe earlier primitive lineage there isa major shift 16. Paonessa, G., Frank, R., and Cortese, R. (1987) Nucleic Acids Res. 15,9077 in HMG-14 synthesis, from-14a to -14b, and alsomore subtle 17. Crippa, M. P., Nickol, J. M., and Bustin, M. (1990) J. Mol. Biol., changes in the synthesis of the other HMG proteins. The in press shift in HMG synthesis is similar to observed that forhistones 18. Lee, K.-L. D., Pentecost, B. T., D'Anna, J. A., Tobey, R.A., Gurley, L. R., and Dixon, G. H. (1987) Nucleic Acids Res. 15, H1 and H5(20). Reduction in HMG mRNAlevels also occurs 5051-5068 during myogenesis in rat(21) suggestingthat down-regulation 19. Laemmli, U. K. (1970) Nature 227,680-685 of HMG mRNAmay be associated with tissue differentiation.20. Neelin, J. M., Callahan, P. X., Lamb, D. C., and Murray, K. The proteins of the HMG-14/-17 family have been associ(1964) Can. J. Biochern. 42, 1743-1752 ated with transcriptionally active chromatin (7, 8). Our data 21. Pash, M. J., Bhorjee, J. C., Patterson, B. M., and Bustin, M. (1990) J. Biol. Chem. 265, 4197-4199 suggest that the synthesis of HMG proteins during the early