Control Mechanisms Regulating Gene Expression ... - Science Direct

DEVELOPMENTAL

BIOLOGY

81,

255-265 (1981)

Control Mechanisms Regulating Gene Expression during Normal Differentiation of Myeloid Leukemic Cells: Differentiation Defective Mutants Blocked in mRNA Production and mRNA Translation BARBARA HOFFMAN-LIEBERMANN, DAN LIEBERMANN, AND LEO SACHS Department of Genetics, Weizmann Institute of Science, Rehovot, 76100,Israel Received April 10, 1980; accepted in revised form July 8, 1980 Regulation of gene expression during myeloid cell differentiation has been analyzed using clones of myeloid leukemic cells that differ in their competence to be induced to differentiate by the normal macrophage- and granulocyte-inducing protein MGI. Changes in the relative rate of synthesis for specific proteins were compared to changes in the relative amounts of corresponding translatable poly(A)+ mRNAs, assayed in the reticulocyte cell-free translation system, using two-dimensional gel electrophoresis. Of the 217 proteins which changed during MGI-induced differentiation of normally differentiating MGI+D+ leukemic cells, 136 could be identified as products of cell-free translation. Eighty-four percent of the 70 decreases in synthesis, most of which occurred early during differentiation, were not accompanied by a parallel decrease in the amount of translatable mRNA, but were accompanied by a parallel shift of the corresponding mRNAs from the polysomal to the monosomal and free mRNA fractions. These results indicate that most of the early decreases in the synthesis of proteins were translationally regulated. In contrast, 81% of the proteins which increased in synthesis and 71% of the proteins that were induced de nova were regulated at the level of mRNA production. Experiments with differentiation defective mutants have shown that they were blocked both at the level of mRNA production and mRNA translation. The data with these mutants have suggested that there were different subsets of translationally regulated proteins which were separately regulated. The translational blocks for several proteins’ in these mutant clones have also made it possible to identify additional translational sites of regulation for protein changes that were controlled at the level of mRNA production during normal differentiation. The results indicate that translational regulation may predominantly have a different function in cell differentiation than regulation by mRNA production, and that differentiation-defective mutants can be blocked at either level.

INTRODUCTION

An in vitro experimental system has been established to study the control mechanisms that regulate differentiation of normal and leukemic myeloblasts to mature macrophages or granulocytes (Sachs, 1974, 1978, 1980; Hoffman-Liebermann and Sachs, 1978; Liebermann and Sachs, 1978; Lotem and Sachs, 1979). We have shown that during differentiation of fully differentiable MGI+D+ mouse myeloid leukemic cells, induced by the normal macrophage- and granulocyte-inducing protein MGI, there was a programmed sequential change in the rate of synthesis of 217 of the 450 proteins detected using two-dimensional polyacrylamide gel electrophoresis (Liebermann et al., 1980). The g-day developmental program was initiated with a decrease in the synthesis of many proteins within the first hour, whereas the synthesis of new proteins only occurred later, mostly between the second and fourth day. Both the MGI+D+ leukemic and normal myeloblasts showed a similar sequence of protein changes during differentiation. The normal developmental program was thus maintained in the MGI+D+ leukemic cells. Sixty-six protein changes

were induced by MGI in partially differentiatable MGI+D- clones, whereas only 12 or 16 protein changes were induced in MGIIDclones which had not been induced by MGI for any previously known differentiation-associated property (Sachs, 1978; Liebermann et cd., 1980). The changing pattern of proteins synthesized during myeloid differentiation reflect changes in gene expression. An understanding of the regulation of the synthesis of these changing proteins would help to elucidate the mechanisms involved in the control of the flow of information from genes to proteins during differentiation. Changes in the amount of a particular mRNA can be measured by changes in the amount of its product synthesized in a cell-free translation system, thus revealing changes in the expression of individual genes. This has an advantage over the hybridization studies carried out to examine RNA sequence complexities in a variety of developmental systems (Affara et al., 1979; Paterson and Bishop, 1977; Affara and Daubas, 1979), in that only RNA which is message is being studied and that specific gene products can be examined. 255

0012-1606/81/020255-11$02.00/O Copyright All rights

0 1981 by Academic Press, Inc. of reproduction in any form reserved.

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In the present studies, changes in the translation products from the reticulocyte cell-free system (Pelham and Jackson, 1976), primed by poly(A)+ mRNA isolated from cells during different stages of myeloid differentiation, were compared to changes in the proteins synthesized by intact cells pulse labeled with [35S]methionine at the same developmental stage. The protein patterns were analyzed by two-dimensional gel electrophoresis. By this analysis, it is possible to ascertain when the mRNA coding for a specific protein is present and to correlate it with the synthesis of the corresponding protein in intact cells (Alton and Lodish, 1977a). The results indicate that during myeloid cell differentiation many protein changes are translationally regulated, in addition to other changes regulated by mRNA production. Using differentiation-defective mutants, we have been able to identify blocks at the level of mRNA production and mRNA translation. In addition, evidence is presented that translationally regulated proteins can be subdivided into separately regulated classes, and that there are additional sites of regulation, at the level of translation, for some protein changes which are also regulated at the level of mRNA production during normal differentiation. MATERIALS

AND METHODS

Cells and cell culture. MGI+D+ clone 11 and MGI+Dclone 5 were derived (Fibach et al., 1973) from a spontaneous myeloid leukemia in an SL mouse. MGI-Dclone 6 was derived from an X-irradiation-induced myeloid leukemia in an SJL/J mouse (Lotem and Sachs, 1976). The cells were established in culture and cloned as previously described (Fibach et al., 1973). Cells were cultured in Eagle’s medium with a four fold concentration of amino acids and vitamins (H-21; Grand Island Biological, N. Y.) and 10% (v/v) inactivated (56’C for 30 min) fetal calf serum. The source of MGI was conditioned medium from B-day-old rat embryo primary cultures containing 10% fetal calf serum. The rat embryos were obtained from 13-day pregnant rats 3 hr after intravenous injection with 100 pg of Salmonella typhimurium lipopolysaccharide (Difco). This conditioned medium was used at a final concentration of 5%. Cells were seeded at varying densities to give a final density of 1.5-2 X lo6 cells/ml when harvested. Pulse labeling cells with [35SJmethionine and preparation of cell extracts. Cells at a density of 1.5-2 X 106/ ml were washed twice in methionine-free Eagle’s medium, resuspended in methionine-free medium containing 10% fetal calf serum with or without MGI, and incubated for 30 min, followed by another 30 min with 250 &i/ml [35S]methionine (1090 Ci/mmole; Amersham). Cells were harvested and washed twice with 0.25 M sucrose, 5 mM MgClz, 0.01 M Tris-HCl (pH 8.0); son-

VOLUME 81, 1981

icated in 5 mM MgClz, 5 mM CaClz, 0.1 M Tris (pH 7.5), 1 mM phenylmethylsulfonylfluoride; and incubated at 37°C for 30 min with DNase (50 bg/ml) and RNase (50 pug/ml). Solid urea was added to 9 M and an equal volume of lysis buffer; 9.5 M urea, 2% (w/v) NP-40, 2% ampholines (pH range 3.5 to lo), 5% @mercaptoethanol (O’Farrell, 1975), was added. RNA extraction. Total cellular RNA, used for cellfree translation, was extracted by a modification (Zeelon and Gerson, 1973) of the method described by Kirby (1968). Cells were harvested, washed twice with PBS, and the pellet was frozen at -70°C. The pellet was disrupted in 25 vol of phenol/m-cresol/water (40:5.5:52, w/ w) containing 0.04% (w/w) 8 hydroxyquinoline, 2% (w/ v) paraaminosalicylic acid (Na salt), and 0.5% (w/v) NaCl. The aqueous phase was made 3% (w/v) with NaCl and extracted two times with l/2 vol of phenol/m-cresol/water (80:11:9, w/w) containing 0.08% (w/w) 8 hydroxyquinoline. Following precipitation with 2 vol of ethanol/m-cresol (9:1, v/v), the precipitate was centrifuged and extracted three times with cold 3 M sodium acetate (pH 6.0), suspended in water, and precipitated with 2 vol of ethanol. For polysomal and postpolysomal RNA, cells were harvested, washed twice with PBS, and lysed with 10 vol of 10 mM NaCl, 3 mM NaClz, 10 mM Tris (pH 7.5), 0.25 M sucrose, 0.5% (v/v) NP-40. After standing on ice for 10 min, the nuclei were pelleted at 10009, washed once with the cell lysing solution containing 0.5% deoxycholate, and both supernatants were pooled. Diethylpyrocarbonate (0.5%, v/v) was present in all solutions. Mitochondria were pelleted at 20,OOOg.Fourteen milliliters of the postmitochondrial supernatant were layered over 11 ml of 48% sucrose (w/w, 10 mM NaCl, 10 mM Tris (pH 7.5), 3 mM MgClz). The polysomes were pelleted in the Ti 60 rotor at 47,000 rpm for 4.5 hr at 2°C. The pellet (polysomes) and the interphase with the upper layer (postpolysomal fraction) were extracted by the modified (Zeelon and Gerson, 1973) method of Kirby (1968). A#inity pur@ication of poly(A) containing RNA. Poly(A) containing RNA was purified by affinity chromatography on a l-ml oligo(dT)-cellulose column (Collaborative Research) (Aviv and Leder, 1972) substituting NaCl for KCl. Cell-free potein synthesis in rabbit reticulocyte lysates. Rabbit reticulocyte lysates were prepared according to the procedure of Pelham and Jackson (1976) and were made mRNA-dependent as described by Pelham and Jackson (1976). Reactions, in a final volume of 25 ~1 were: 60% (v/v) lysate, 3 PM hemin, 0.6 mM CaCL 1.2 mM EGTA, 0.5 mM Mg (OAc)z, 90 mM KOAc, 15 mM creatine phosphate, 100 pg/ml creatine kinase, and contained 20 &i of [?S]methionine (1090 Ci/mmole;

HOFFMAN-LIEBERMANN,LIEBERMANN,ANDSACHS

Amersham) and 0.25 pg of poly(A)+ mRNA (from either total cells or polysomal and postpolysomal fractions). Reactions were incubated at 37°C for 1 hr and assayed for hot TCA-precipitable counts. Aliquots on Whatman 3MM filters were kept first for 10 min in 10 vol of 10% cold TCA, then for 10 min in boiling 5% TCA, washed twice with 5% TCA, once with ethanol, once with ethanol:ether (l:l, v/v), once with ether, dried, and counted. Samples were prepared in the same way as cell extracts for two-dimensional polyacrylamide gel electrophoresis. The pattern of protein spots on two-dimensional gels from the cell free reaction in the absence of exogenous RNA showed only a few spots which were very weak, requiring much longer exposure times than those we used for samples with exogenous RNA. In addition, translation of specific mRNAs was linear at the concentration of RNA used. Using equal volumes of reaction mixture, the intensity of any given spot was proportional to the amount of RNA added to the reaction.

Two-dimensional

gel electrophoresis and analysis.

Two-dimensional polyacrylamide gel electrophoresis was carried out according to the method of O’Farrell (1975) with the following modifications. Only ampholines in the pH 3.5-10 range was used. The second dimension was a sodium dodecyl sulfate polyacrylamide slab gel with a 3% acrylamide stacking gel and a 7-17% acrylamide gradient in the resolving gel (Laemmli, 1970; Maizel, 1971). Gels were stained with Coomassie brilliant blue, destained, and either dried onto highstrength paper and exposed to X-ray film (Kodak SB5), or prepared for fluorography by impregnation with PPO according to the procedure of Bonner and Laskey (1974) and then, after drying, exposed to preflashed X-ray film (Laskey and Mills, 1975) at -70°C. A constant amount of radioactivity was applied to all gels (250,000 cpm) and the fluorograms were exposed for 96 hr. For each clone, three cell extracts were prepared, RNA was extracted and translated at least twice, and at least two gels were run for each sample. The criteria for identity of a spot from one gel to the next was the ability to superimpose the spot in question and surrounding spots on the two gels. Details of the analysis of two-dimensional gel profiles and the level of confidence of the analysis have been described in detail (Liebermann et al., 1980).

Sucrose density centrifugation to obtain polyribosome projiles. Postmitochondrial supernatants, prepared as described for polysomal RNA extractions, were layered onto a linear 18-ml gradient of 15-45% (w/w) sucrose made up in 10 mM NaCl, 10 mM Tris (pH 7.5), 3 mM MgCIB, and were centrifuged for 4 hr at 26,000 rpm at 1°C in a Beckman SW 27 rotor. The absorbance at 260 nm was monitored by pumping the gradients through an Isco recording spectrophotometer.

Control Mechanisms in Leukemic Cell DZfferentiation

257

[3Hjpoly(U) hybridization. RNA was hybridized to at least a lo-fold excess of [3H]polyuridylic acid (New England Nuclear) in 2 X SSC for 10 min at 45°C according to Bishop et al. (1974). After the addition of 20 pg/ml RNase A, digestion proceeded for 30 min at room temperature in 2 x SSC. At the end of the digestion period, 20 hg/ml carrier bovine serum albumin and 10 vol cold 10% TCA were added. After 30 min the precipitates were collected by filtration onto Millipore filters, washed extensively with cold 5% TCA, dried, and counted in a toluene-based scintillation fluid in a Packard Tri-Carb scintillation counter. Measurement of the rate of incorporation of [35S]methionine into TCA-insoluble material. Incorporation of [35S]methionine (1090 Ci/mmole, Amersham) into TCA-precipitable material was determined after 30 min. Cells, resuspended in PBS, were precipitated with cold 10% TCA, collected by filtration onto Whatman GF/C filters, washed extensively with cold 5% TCA, dried, and assayed for radioactivity. An equal number of cells were compared for incorporation.

Radioactivity measurements by scintillation counting of protein spots excised from gels. Regions from dried gels corresponding to spots on autoradiograms were excised, hydrated, and incubated with 0.5 ml of 90% NCS solublizer (Amersham) for 3 hr at 55°C. Radioactivity was determined by scintillation counting in a toluene based fluor. Similar sized cuts from blank areas of the gel were used to determine the background. This procedure was most accurate for major protein species from isolated regions of the gels. Total radioactivity present in each slab gel was determined by cutting the gel into 0.5 X 0.5-cm pieces and measuring the radioactivity eluted from each piece. RESULTS

One hundred and thirty-six of the 217 proteins which changed during MGI-induced differentiation of MGI+D+ clone 11 could be identified as products of cell-free translation in the reticulocyte lysate, programmed by myeloid leukemic cell poly(A)+ mRNA from different stages of MGI-induced differentiation, and identified by two-dimensional gel electrophoresis. Not all cellular proteins can be detected as products of cell-free translation, since some mRNAs may not be translated or may be only partially translated and there are polypeptides which may be translated, but not processed or modified as in intact cells. This also accounts for some of the polypeptides found in the translation products which are not detected in intact cells (Alton and Lodish, 1977a). In addition, the relative rates of synthesis of some proteins may not be the same in intact cells and in the reticulocyte lysate. The translation of specific mRNAs was found to be linear at the concentration of

DEVELOPMENTALBIOLOGYV0~~~~81,1983

258

mRNA

PROTEIN

D

t S !3

E

HOFFMAN-LIEBERMANN,

MGI+D+ (A)Rate

of synttws~s of proteins

CLONE (BlAmount

LIEBERMANN,

AND SACHS

Co&-o1 Mechanisms

259

in Leukemic Cell DZfferentiation

ii of translotablc

mRNAs

(A) Protein

(B)mRNA

J

-

iH3H

iD

9H TIME

AFTER

2D ADDITION

4D

6D OF MGI

FIG. 3. Distribution of the initiation of different types of changes in the (A) rate of synthesis of proteins and (B) amount of translatable mRNA during MGI-induced differentiation of MGI+D’ clone 11. (W) Decrease; (o) increase (H) de no~o. Changes in the amount of particular mRNAs were ascertained by changes in the amount of their corresponding proteins translated in the reticulocyte cell-free system.

FIG. 2. Schematic summary of (A) rate of synthesis of proteins and (B) amount of translatable mRNAs for the proteins which changed during MGI-induced differentiation of MGI+D+ clone 11. Only proteins which were translated in the reticulocyte cell-free system were analyzed. The numbers on the sides of the diagram correspond to the spots labeled in Fig. 1. The numbers on the outer sides are the same and alternate with the numbers in the middle. The numbers are not continuous. This summary is based on analysis of Gels (A) of proteins synthesized in cells and (B) of proteins sythesized in the reticulocyte cell-free system programmed by poly(A)+ mRNA extracted from cells 1, 3, and 9 hr, and 1, 2, 4, and 6 days after treatment with MGI. (---) Indicates times of high or maximum protein synthesis or amount of mRNA; (--) indicates intermediate levels; and (. . .) indicates low levels.

RNA used in the cell-free translation reaction. Programming the reticulocyte cell-free system with RNAs from myeloid cells at different developmental stages

can, therefore, be used to assay for changes in the relative amounts of specific mRNAs during differentiation. This study is limited only to the polypeptides whose synthesis changes in intact cells during differentiation and which could be identified by comigration with polypeptides synthesized in our cell-free assay. For both cell extracts and cell-free translation products, spots on the two-dimensional gels were recorded when they first showed a significant change in intensity from one time point to another, relative to neighboring spots which remained constant. Proteins from cell extracts with decreasing relative rates of synthesis will be called decreases, those with increasing relative rates of synthesis, increases, and new proteins will be referred to as de novo proteins.

FIG. 1. Two-dimensional gel electrophoresis of proteins from total cell extracts of MGI+D* clone 11 pulse labeled with [%]methionine for 30 min (A) untreated and incubated for (B) 3 hr, and (C) 6 days with MGI; and from reticulocyte cell-free extracts programmed with poly(A)+ mRNA isolated from MGI’D+ clone 11 (D) untreated and incubated for (E) 3 hr and (F) 6 days with MGI. For all gels, only differentiationlysates were considered. In gel (A) all proteins initially present which associated proteins whose mRNAs were translated in reticulocyte changed during differentiation are numbered. P16, P61, P97, and P254, described in detail in Fig. 4, are encircled. For gel (B) the corresponding spot is numbered if a change in synthesis was detected by 3 hr. In gel (C) all the proteins which changed, including the position of spots which disappeared, are numbered. Proteins whose mRNA was not translated in uninduced cells and P12 and P35, described in detail in Fig. 4, are encircled. In gel (D) spots corresponding to all proteins which changed during differentiation are numbered, even if the corresponding to all the proteins which changed are numbered, even if the corresponding mRNA never changed. Spots corresponding to mRNAs that were not translated in uninduced cells as well as P16, P61, P97, and P254, are encircled. For gel (E), the spot is numbered if a change in its mRNA was detected by 3 hr. The two arrows point to P16 and P61 whose mRNA did not change by 3 hr. In gel (F) spots corresponding to all the proteins which changed are numbered, even if the corresponding mRNA never changed. P12 and P35 are encircled.

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rates of synthesis of these proteins in intact cells, their respective mRNAs were shifted out of the polysome fraction (compare Figs. 4 and 5). Data from the incorporation of [35S]methionine into TCA-insoluble material and the polysome profiles suggest that the rate of protein synthesis began to decrease Seventy proteins which decreased during the course 2 clays after MGI treatment of MGI+D’ clone 11 and of MGI-induced differentiation of MGI+D+ clone 11 continued to decrease thereafter, so that by 6 days it were synthesized in our in vitro assay, and for 84% of was 21% of control (Figs. 6A and B). In MGI-treated these (59 proteins) the amount of mRNA was not al- MGI+D- clone 5, the incorporation of [35S]methionine tered when the protein decrease was first observed into TCA-insoluble material only decreased to 85% of (Figs. 1, 2, and 3, Table 1). At early times during dif- the control level by 6 days, whereas no detectable ferentiation, an even higher percentage of decreasing change was observed in MGI-D- clone 6 (Fig. 6). The proteins, 97% up to 9 hr, was translationally regulated results suggest that the decrease in the rate of synthesis (Figs. 1, 2, and 3, Table 1). Thus, translation of these of many proteins at an early stage of differentiation mRNAs was specifically restricted in the early stages was not the result of a decrease in total protein synof differentiation. For some decreasing proteins the thesis, and that the decrease in the rate of total protein mRNA decreased at later times (e.g., P7, P16, P61, P145, synthesis at later stages of differentiation was correFigs. 1, 2, and 4) and for others the mRNA remained lated with the extent of MGI-induced differentiation. In contrast to the translational regulation of most constant for at least 6 days (e.g., P4, P26, Figs. 1 and decreases, 81% of the proteins which increased and 77% 2). However, there were also protein decreases which of de novo induced proteins which were synthesized in were regulated at the level of mRNA production (e.g., our in vitro assay, were regulated by a parallel change P92, P93, and P94) and such decreases usually occurred in the amount of mRNA during differentiation (Table at later times during differentiation (Figs. 1 and 2, Ta1, Figs. 1,2, and 3). P97 and P254 (Figs. 1,2, and 4) and ble 1). The decreasing proteins which were regulated P12 and P35 (Fig. 4) are examples of proteins whose at the level of translation did not all decrease simultaneously, indicating that all these decreases were not mRNAs increased or were induced de novo in parallel with the protein. However, there were also some proregulated as one set. To substantiate that the inhibition of synthesis of teins which increased without a concomitant change in specific proteins during early differentiation is clue to their corresponding mRNAs during differentiation and regulated (e.g., translational control, the cell-free translation products which were, therfore, translationally from polysome-associated and postpolysomal (mono- P126 and P143; Figs. 1 and 2). There were nine mRNA species whose corresponding somes and smaller) mRNAs during the differentiation were analyzed. The results with P16 and P61 indicate, proteins were not found in the untreated cell extracts, that concomitant with the observed decrease in the but which appeared to be induced de nova after MGI

Regulation of Protein Changes during MGI-Induced D$f.ferentiation of MGI’D’ Clone 11: Most Early Decreases Are Translational19 Regulated Whereas Most de Novo Proteins Are Regulated at the Level of mRNA Production

TABLE 1 TIME DISTRIBUTIONOFTHE INITIATION OF DIFFERENTTYPESOF PROTEINCHANGESAND THEIR MODE OF REGULATIONDURINGDIFFERENTIATIONOFMGI+D+ CLONE11 Time distribution

Number of changes” 1 hr Type of change Decrease Increase De novo Total

3 hr

of changes after addition of MGI

9 hr

1 day

2 days

4 days

6 days

TC

mRNAd

T

mRNA

T

mRNA

T

mRNA

T

mRNA

T

mRNA

T

mRNA

T

mRNA

59 (84) 5 (19) 9 (23) 73 (54)

11 (16) 22(81) 30 (77) 63 (46)

32 1 0

1 3 0

4 2 2

0 5 1

3 1 0

0 5 4

9 0 0

2 3 0

10 1 3

7 5 11

1 0 4

1 1 9

0 0 0

0 0 5

Note. MGI+D+ clone 11 was incubated with MGI for 6 days. Total proteins from cells pulse labeled with [%l]methionine were compared with products from cell-free translation primed by poly(A)+ mRNA extracted from the cells at different times after the addition of MGI. Only the proteins which changed in intact cells and could be identified as products of translation in the reticulocyte cell-free system were analyzed. a For each type of change the percentage is given in brackets. cT, translational regulation. d mRNA, regulated by the amount of mRNA.

HOFFMAN-LIEBERMANN,

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treatment (P129, P131, P133, P134, P146, P147, P149, P159, P241; Figs. 1,2, and 4). Two proteins were detected 3 hr after addition of MGI, three after 2 days, and four after 4 days (Fig. 2). The association of these mRNAs with the polysomal or postpolysomal fractions during differentiation was determined for P131 and 134 (compare Figs. 4 and 5). The mRNAs for P131 and P134 were found in the postpolysomal fraction in untreated clone 11. However, when these proteins appeared in intact cells during differentiation, there was a parallel shift of their respective mRNAs into the polysome fraction. These data suggest that during differentiation of myeloid leukemic cells, these de novo protein changes were regulated at the level of translation of preexisting mRNAs 1

i

60

-e r

60

--I

P35

60 i I P134

60

-4 w TIME

AFTER

ADDITION

lD2D

261

Cell Differentiation

Q

g

60

E

40

+-

?1..1-

ID2D

40

TIME AFTER

ID 20

ADDITION

40

6D

OF MGI

FIG. 5. Subcellular distribution of specific mRNAs during differentiation of MGI’D+ clone 11. Radioactivity was measured for specific spots corresponding to the cell-free translation products primed by (A) polysome-associated and (A) postpolysomal poly(A)+ mRNAs during differentiation, correcting for different amounts of total radioactivity in each gel. For each time point the distribution of specific nRNAs in the polysome and postpolysomal fraction was calculated, using the above data and the relative poly(A)+ mRNA content from each cell fraction as calculated by [3H]poly(U) hybridization.

Blocks in the Production and Translation of Dgferent mRNAs in differentiation-Defective Mutants

--

-+-At-w

in Leukemic

P254

k

I

Control Mechanisms

4D

6D

OF MGI

FIG. 4. Comparison of the change in (0) the rate of protein synthesis and (0) the amount of translatable mRNA for specific proteins which decrease (P16, P61), increase (P97, P254), or were induced de 1zono (P12, P35, P131, P134). Regions of the gels corresponding to specific proteins were excised and radioactivity was measured and corrected for different amounts of total radioactivity present in each slab gel. For each protein, the corrected values are expressed as percentage of its maximum synthesis in either cell extracts (rate of protein synthesis) or reticulocyte lysates (amount of mRNA) during the time course of differentiation.

Analysis of MGI-treated differentiation-defective mutants allows further dissection of the regulatory control mechanisms during myeloid differentiation and characterization of the blocks responsible for the differentiation-defective phenotype. All the protein changes which occurred after MGI treatment of MGI+Dclone 5 and MGIID- clone 6, appeared to be regulated similarly to the parallel protein changes in MGI-treated MGI+D+ clone 11, with the exception of P249 (Figs. 2 and 7). For the majority of the proteins which did not increase or which were not induced in the defective clones, and which were regulated at the level of mRNA production during normal differentiation, the corresponding mRNAs were also either not increased or not induced, respectively. This indicates that the blocks for the synthesis of these proteins were at the level of mRNA production. These proteins are not included in the schematic summary in Fig. ‘7. We have previously shown (Liebermann et al., 1980) that the untreated differentiation-defective mutants, compared to untreated MGI+D+ clone 11, constitutively expressed the differentiated state for subsets of protein changes. The mRNAs coding for 17 out of the 27 proteins involved in MGI+D- clone 5, and for 22 out of the 43 proteins involved in MGIIDclone 6, could be assayed in the reticulocyte cell-free system. In all except eight cases, the constitutive expression of these protein

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mRNAs were already being translated in the untreated mutant clones, comprising 5 of the de nova constitutively expressed proteins in these clones (P131, P147, P159 in clone 5; P147 and P149 in clone 6; Fig. 7), some were translated only after MGI treatment (P129, P146, P149, P241) in clone 5; P159 in clone S), and some were never translated, even after MGI treatment (P134 in clone 5; P129, P131, P134, P146, P241 in clone 6). In clone 6, although the mRNA coding for P134 was a major species, no synthesis of this protein was ever detected in either untreated or MGI-treated cells (Fig. 7). Only in normal granulocyte cell extracts was P134 such a major component. That this was the same protein, was verified by comigration on two-dimensional gels of a mixture of translation products coded for by clone 6 mRNA and cell extracts from granulocytes. Analysis of the cell-free translation products of mRNA from polysomal and postpolysomal fractions before and after MGI treatment of MGIID- clone 6 showed that P134 mRNA was never associated with the polysomal fraction. The data thus indicate that in the mutant clones, 2.51

SDlpys

2.0

1

MGI’D(Ahts

of oynthcslr

CLONE 5

of profems

,MGI

+MGI

I .5 I .o

..-..,p i,, I ', ;;> ".,.I,\_,p, ..,_..'...\ .... ..-Jw 0 5 IO 15 20

0.5

ml

FIG. 6. (A) Rate of incorporation of [%]methionine into proteins and (B) polyribosomal profiles following MGI treatment of: (-) MGI+D+ clone 11; (- - -) MGI+Dclone 5; and (. . .) MGI-Dclone 6.

MGI-D- CLONE 6 1S)Amount of translotabla +hlGI

changes was regulated at the level of mRNA production. For three of these eight exceptions (P47, P116, and P145 in MGI-D- clone 6), the proteins, but not the mRNAs were missing as in the differentiated state of MGI+D+ clone 11. Constitutive expression of these three protein changes in this MGIID- clone was thus regulated at the level of translation (Fig. 7). The five other exceptions will be discussed below. Out of the 59 decreasing proteins which were translationally regulated during differentiation of MGI+D+ clone 11,6 decreased in clone 5 and 1 decreased in clone 6 after MGI treatment (Fig. 7). This suggests that multiple factors or mechanisms, rather than only one, were responsible for the translationally regulated decreases during MGI-induced differentiation. Eight of the nine mRNAs species which were present in untreated MGI+D+ clone 11 and were translated only after addition of MGI, were also present in untreated MGI+D- clone 5 and MGI-D- clone 6. Some of these

mRNAs

MGI I’-

r.t,,

I

i’-3

4/iik-+J”-wM TIME AFTER ADDITION OF MGI FIG. 7. Schematic summary of (A) rate of synthesis of proteins and (B) amount of translatable mRNAs for MGI+D- clone 5 and MGI-Dclone 6. As in Fig. 2, (0) indicates that the protein or mRNA was missing in untreated mutant cells and was detected in untreated MGI+D’ clone 11; (0) indicates the time the protein appeared in clone 11, for proteins never detected in mutant clones although their mRNAs were present, either initially or de TWUOinduced. Constitutive expression of differentiated state in clones 5 and 6: P3,12, 51, 58, 75, 101,117,131,136,147,154,159,164,186,254,271,276 in MGI+D- clone 5; P3, 4, 12, 16, 36, 47, 53, 59, 60, 61, 68, 75, 78, 116, 130, 145, 147, 149, 154, 220, 257, 271, in MGI-D- clone 6.

HOFFMAN-LIEBERMANN,

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a block at the translational level prevented the induction of translation of some of these mRNAs by MGI. Untreated MGI-D- clone 6 also contained two other mRNAs (coding for P51, P249), which were not translated before or after MGI treatment (Fig. 7) and untreated MGI+D- clone 5 also contained mRNA for P249 but only synthesized the protein after MGI treatment. It appears, therefore, that these two proteins, whose mRNAs were induced de novo in MGI+D+ clone 11 and were, therefore, regulated at the level of mRNA during normal differentiation, were also regulated at a later point(s) in the flow of information from gene to mRNA to protein. The mutants which were blocked at these later points have thus shown these additional sites of regulation. A block at the translational level in the mutant clones was also found for another group of proteins. In MGI+D- clone 5, four mRNA species were induced de novo (mRNA coding for P50, P62, P152, P272) and in MGI-D- clone 6, five mRNA species were induced de novo (mRNA coding for P62, P102, P152, P175, P272) with no concomitant de novo synthesis of their corresponding proteins (Fig. 7). All these mRNAs were also de novo induced in MGI-treated MGI+D+ clone 11, the mRNA appearing at the same time as in the differentiation-defective clones; however, in the fully differentiating clone 11 the corresponding protein was also induced (Figs. 1 and 2). Analysis of the subcellular localization of the mRNAs for P152 and P272 has shown that they were associated with the postpolysomal fraction in MGI-treated clones 5 and 6, but were found primarily associated with polysomes in MGI-treated clone 11. In these cases the mutant clones were induced by MGI to produce, at the appropriate time, specific differentiation-associated mRNAs that were translationally blocked. DISCUSSION

A comparison of the cell-free translation products from the reticulocyte cell-free system, primed by mRNA from different stages of myeloid differentiation, to the proteins synthesized in intact cells, has made it possible to distinguish between two general classes of regulation of differentiation-associated protein changes. The first, translational regulation, is when there was a change in the relative rate of synthesis of a specific protein with no change in its mRNA (Table 2). The second, regulation in the production of translatable mRNA, is when the amount of mRNA changed parallel to the change in its corresponding protein; this can be due to transcription and/or post-transcriptional processing of the RNA. A third class, post-translational regulation, could not be detected by our methodology. One hundred and thirty-six of the 217 proteins which changed during differentiation could be identified as

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Cell Differentiation

TABLE 2 SUMMARY OF TRANSLATIONAL CONTROLS REGULATING CHANGES IN THE SYNTHESIS OF PROTEINS DURING MGI-INDUCED MYELOID CELL DIFFERENTIATION Protein change 1. Decrease

Type of control Inhibition of translation

of mRNA MGI

mRNA -

less or no protein

2. Increase

Increased translation of constant amount of mRNA MGI 1 mRNA more protein

3. De novo

A. Induction of translation of preexisting mRNA MGI I mRNA -protein B. Induction of translaticm of MGI-induced mRNA MGI MGI 1 1 + mRNA -protein

products of cell-free translation. The three types of protein changes we describe, decreases, increases, and de novo induced are represented in these 136 proteins in a similar proportion as in the total of 217 protein changes. The major role played by translational regulation in the early decreases compared to the change in mRNA for most increasing (81%) and de novo induced (77%) proteins, suggests that translational regulation may predominantly have a different function in differentiation than regulation by mRNA production. The presumably more rapid translational regulation may thus be a more effective way to produce the many early decreases than regulation by mRNA production. Translational Regulation of Protein Changes during Dvferentiation In MGI-induced MGI+D+ clone 11, 84% of the proteins which decreased, and were synthesized in vitro, were regulated at the level of translation and most of these decreases occurred early after adding MGI. After the addition of MGI, there was a selective inhibition of translation of many mRNA species, although the mRNAs persisted in the cells for extended periods of time. The mRNAs coding for specific decreasing proteins were no longer polysome associated, so that these proteins were no longer being synthesized. During the early stages of differentiation of the slime mold Dietyostelium discoideum there was cessation of synthesis of five specific polypeptides, the mRNAs for these proteins remaining in the cells for much longer times (Alton and Lodish, 1977a,b). In addition, there was a precipitous drop in the overall rate of polypeptide chain initiation within the first 5 min of development. It has --.been suggested that changes in the initiation machinery

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which cause a reduction in the overall rate of chain initiation can also cause a preferential inhibition of translation of specific mRNAs (Alton and Lodish, 1977b; Lodish, et al., 1978). In contrast, there was no change in total protein synthesis in differentiating myeloid cells until 2 days after the addition of MGI, whereas the decrease in synthesis of many proteins occurred much earlier. This suggests that during myeloid differentiation the change in the translation machinery, which discriminated against the translation of specific mRNAs did not involve a decrease in the translation of all mRNAs. MGI treatment of the differentiation-defective clones resulted in a decrease in the translation of only a subset of the mRNAs inhibited during differentiation on MGI+D+ cells. This indicates that all the decreases are not regulated as one set, and that there are subsets of mRNAs that can be separately subject to specific inhibition of translation. The translational inhibition of each mRNA or subset of mRNAs may be the result of loss of activity of an element in the translational machinery required specifically by these mRNAs, or a decrease in a factor required in high amounts for these mRNAs and only in small quantities for other mRNAs. Alternatively, specific inhibitors of translation may be responsible. Selective inhibition of translation of mRNAs also occurs after infection with certain viruses or treatment with interferon (Revel and Groner, 1978). The use of homologous cell-free systems from untreated and MGI-treated MGI+D+ clone 11 and the differentiation-defective clones, primed by untreated clone 11 mRNA, should make it possible to determine if the translational inhibition of specific mRNAs in differentiating intact cells can be obtained in vitro. This would then allow further dissection of the translational elements involved in the decrease in translation of specific mRNAs. Some increases in the synthesis of specific proteins were translationally regulated in the differentiating myeloid cells; the mRNA remained constant although the level of protein increased. This may be due to changes in the translational machinery during differentiation which optimize the translation of these mRNA species. In addition, nine mRNA species in untreated MGI+D+ clone 11 myeloblasts were translated only after the addition of MGI. It was shown with two of these proteins that only when the protein appeared in the cells was its corresponding mRNA associated with the polysome fraction, indicating that in untreated cells these proteins were not being synthesized. Both the sequential induction of the different proteins coded for by these mRNAs during differentiation, as well as the differential expression of these mRNAs in the differentiation-defective clones, suggest multiple mechanisms or factors for controlling the translation of these

VOLUME81,1981

mRNAs. Sea urchin eggs (Weinberg, 1977) and Atiemia salina cysts (Filipowitz et al., 1975) also contain untranslated (masked) mRNAs which are stored until the correct signals allow them to be translated. Gene Expression at the Level of mRNA Production In addition to translational regulation during myeloid differentiation, there was also a change in the population of translatable mRNAs. Forty-six percent of the proteins which changed during MGI-induced differentiation of MGI+D+ clone 11 and which were synthesized in the cell-free system, were regulated by the amount mRNA, either at the level of transcription or post-transcriptional processing. Regulation of gene expression at the level of mRNA production also takes place during differentiation of Dictyostelium discoideum (Alton and Lodish, 1977a), myogenesis (Paterson and Bishop, 1977; Buckingham, 1977), erythropoiesis (Affara and Daubas, 1979), estrogen control of the vitellogenin gene (Skipper and Hamilton, 1977), and thyroid and glucocorticoid hormone control of growth hormone production (Martial et aZ.,1977; Tushinkski et aZ., 1977). Regulation at the level of both mRNA production and translation has been reported for hepatic CY~,, globulin synthesis (Kurtz et al., 1978). The differentiation-defective MGI+D- and MGI-Dclones constitutively expressed certain protein changes compared to the uninduced MGI+D+ cells (Lieberman et al., 1980; Sachs, 1980). For most of these protein changes, there was also constitutive expression of the differentiated state for the corresponding mRNAs, so that constitutive expression of these protein changes was regulated at the level of mRNA production. There were, however, also some cases in which the constitutive expression of protein changes was regulated at the level of translation. It will be of interest to determine the mode of regulation of another subset of protein changes that was induced by MGI in normal myeloblasts and was constitutively expressed in all the leukemic clones (Liebermann et al., 1980; Sachs, 1980). Identification of Additional Sites of Regulation bg Using wferentiation Defective Mutants The differentiation-defective mutants have allowed further dissection of the regulation of protein changes which were controlled by the amount of mRNA during normal differentiation. Some mRNAs which were induced de novo with their corresponding proteins in MGI+D+ clone 11, were present a p&ri in untreated MGI-D- clone 6, or were induced de novo in MGItreated MGI+D- clone 5 and MGI-D- clone 6, with no detectable synthesis of the corresponding protein. The differentiation-defective clones were thus blocked for these proteins at the level of translation, demonstrating

HOFFMAN-LIEBERMANN,LIEBERMANN,AND SACHS

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additional sites of regulation which would otherwise not have been detected. One possibility is that the mutant clones may be missing specific factor(s) required for the translation of these mRNAs, which were either present in the untreated MGI+D+ clone or were induced in this clone after MGI treatment. The fully differentiable and differentiation-defective clones of myeloid leukemic cells have thus made it possible to identify and dissect the sites of regulation that control myeloid cell differentiation. The availability of these different clones provides a particularly favorable system to gain further insight into the finely tuned events controlling gene expression during differentiation.

KURTZ, D. T., CHAN, K. M., and FEILGELSON,P. (1978). Translational control of hepatic (Y+ globulin synthesis by growth hormone. Cell 15,743-750. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LASKEY, R. A., and MILLS, A. D. (1975). Quantititative film detection of ‘H and % in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341. LIEBERMANN,D., and SACHS,L. (1978). Co-regulation of type C RNA virus production and cell differentiation in myeloid leukemic cells.

B.H. is a recipient of a Mildred Werner League Cancer Research Fellowship of the Israel Cancer Research Fund. We thank Varda Negreanu for excellent technical assistance. This research was supported by a grant from the Hermann and Lilly &hilling Foundation.

LODISH, H. F., BERGMANN,J. E., and ALTON, T. H. (1978). Regulation of messenger RNA translation. In “Brookhaven Symposia in Biology” (C. W. Anderson, ed.), Vol. 29, pp. 309-331. Brookhaven National Lab, New York. LOTEM, J., and SACHS,L. (1976). Control of Fc and C3 receptors on myeloid leukemic cells. J. Immunol. 117, 580-586. LOTEM,J. and SACHS,L. (1979). Regulation of normal differentiation in mouse and human myeloid leukemic cells by phorbol esters and the mechanism of tumor promotion. Proc. Nat. Acad. Sci. USA 76, 5158-5162. MAIZEL, J. V., JR. (1971). Polyacrylamide gel electrophoresis of viral proteins. In “Methods of Virology,” Vol. 5, pp. 1’79-246.Academic Press, New York. MARTIAL, J. A., BAXTER,J. D., GOODMAN,H. M., and SEEBURG,P. H. (1977). Regulation of growth hormone messenger RNA by thyroid and glucocorticoid hormones. Proc. Nat. Acad. Sci. USA 74,1816-1820. O’FARRELL, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250,4007-4021. PATERSON,B. M., and BISHOP, J. 0. (1977). Changes in the mRNA population of chick myeloblasts during myogenesis in vitro. Cell 12, 751-765. PELHAM, H. R. B., and JACKSON,R. J. (1976). An efficient mRNAdependent translation system from reticulocyte lysates. EM. J.

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