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Induced Differentiation of Murine Erythroleukemia Cells: Cellular and Molecular Mechanisms
INDUCED DIFFERENTIATION OF MURINE ERYTHROLEUKEMIA CELLS: CELLULAR AND MOLECULAR MECHANISMS Richard A. Rifkind, Michael Sheffery, and Paul A. Marks DeWitt Wallace Research Laboratory and the Sloan-Kettering Division, Graduate School of Medical Sciences, Memorial Sloan-Kettering Cancer Center. New York. New York
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Terminal Cell Division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Characteristics of Inducer-Mediated Differentiation . . . . . . . . . . . . . . . . . . . . . . B. Commitment to Terminal Cell Division C. Commitment to Terminal Cell Division D. Commitment Involves a Change for Which Cells Retain a “Memory” . . . . . . E. Protein p53 and the Onset of Terminal Cell Division. . . . . . . . . . . . . . . . . . . . . 111. Gene Expression, DNA St during Induced Differentiation A. Murine Globin Gene 1 B. Transcriptional Regulation of Globin Genes during Development and Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Posttranscriptional Regulation of Globin Gene Expression . . . . . . . . . . . . . . . .
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IV. Differentiation and the Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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I. Introduction Erythropoiesis constitutes one of the best characterized developmental lineages in higher organisms; the progressive developmental stages have been identified and aspects of the control mechanisms regulating development, including the effects of hemopoietic growth factors, are increasingly well defined (Marks and Rifkind, 1978). Terminal cell differentiation in this lineage has been described in terms of morphological and biochemical changes, as well as events at the molecular level, including the generation of differentiation-specific mRNAs (globin mRNAs) and other differentiationspecific products (Marks and Rifkind, 1978; Terada et al., 1972; Ramirez et al., 1975; Sassa, 1980). Molecular probes for the definition of specific gene expression in this lineage are readily available. The murine erythroleukemia cell (MELC), a virus-transformed precursor approximating, in terms of its developmental potential, the colony forming 149 ADVANCES IN CAh’CER RESEARCH. VOL 42
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unit for erythropoiesis (CFUe) stage of the erythroid lineage, provides yet additional advantages for the study of developmentally significant regulatory mechanisms during erythroid cell differentiation (Marks and Rifkind, 1978). These transformed cells, blocked in their differentiation at a particular moment in the developmental history of the erythroid cell lineage, can be cloned and retain their normal developmental potentiality. This provides a unique opportunity for defining the cellular and molecular phenotype of a cell arrested at a particular developmental stage in its lineage. Indeed, as will be developed in this article, a number of features of chromatin and DNA structure, in the globin gene domains, have been identified in MELC which appear to characterize this developmental stage of the erythroid cell lineage. MELC can be induced, by exposure to any of a variety of chemical agents, to initiate their developmental program in a fashion which, by all tests so far applied, appears similar to the normal developmental history of nontransformed erythroid precursor cells. These cells provide an opportunity to examine, at a cellular and molecular level, events which accompany, and which may regulate, the initiation of terminal cell differentiation, including the expression of differentiation-specific genes and the initiation of terminal cell divisions. Among these, as will be developed below, is a complex set of changes of chromatin structure in the globin gene domains, which appear to be significant in the induced expression of the genes during differentiation. Additional advantages of the MELC system include the availability of different inducing agents, capable of initiating different patterns of expression of the characteristics of induced differentiation (Marks and Rifkind, 1978; Tanaka et al., 1975; Reuben et al., 1976; Nude1 et al., 1977a,b), and the availability of' variant cell lines selected for their resistance to various inducing agents and which are providing powerful tools for exploring relationships among the molecular changes which accompany induced differentiation (Ohta et al., 1976). Many aspects of the biological events which occur during induced MELC differentiation have been explored in a number of laboratories. These include, for example, factors determining morphogenetic changes (Volloch and Housman, 1982), changes in cell membrane properties, including ion fluxes (Mager and Bernstein, 1978a,b; Smith et al., 1982), cyclic nucleotide metabolism (Gazitt et al., 1978b), iron transport and heme synthesis (Sassa, 1980), and others. In this article we shall concentrate on three features of induced MELC differentiation dealing with the regulation of expression of developmentally specific genes and the relationships between differentiation and the cell cycle.
1. Studies concerning the mechanisms regulating the initiation of termi-
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nal cell division (commitment), which is a characteristic feature of the development of both normal and transformed erythroid cells. 2. The nature of mechanisms regulating the expression of differentiationspecific genes during induced differentiation; these studies, in large part, address the control of globin gene expression at the transcriptional and posttranscriptional levels, both of which may be modulated during induced differentiation. 3. Studies on the relationships between the cell division cycle, DNA synthesis, globin gene replication, and induced cell differentiation.
II. Terminal Cell Division The nature of the factors regulating cell growth in eukaryotic cells remains one of the central unresolved issues in biology today. One requirement for such a study is a homogeneous cell population which can be readily manipulated with respect to the transition from unlimited cell growth to the terminal cell divisions characteristic of many differentiating cell lineages. MELC meet this criterion and provide one attractive model for study of this problem.
A. CHARACTERISTICS OF INDUCER-MEDIATED DIFFERENTIATION MELC can be maintained in suspension culture for an essentially unlimited number of passages under appropriate conditions (Marks and Rifkind, 1978) and can be induced by a variety of agents (Table I), including TABLE 1 INDUCERS OF MELC E R Y T I ~ R ODIFFERENTIATION^ ID Polar compounds Fatty acids DNA intercalators Modified bases Phosphodiesterase inhibitors Ion-flux agents Physical agents Posttranscription-acting agent
Dirnethyl sulfoxide (MeZSO) Hexamethylene bisacetamide (HM BA) Butyric acid Actinornycin Azacytidine Methylisoxanthine Ouabain UV, X ray Hemin
a This is an abbreviated tabulation of agents which have been demonstrated to induce MELC to express characteristics of erythroid differentiation. A more complete list has been presented elsewhere (Marks and Rifkind, 1978; see also Mager and Bernstein, 1978b; Creusot et al., 1982; Ross and Sautner, 1976; Reuben et al., 1980).
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Me,SO, HMBA, and butyric acid, to express characteristics of erythroid cell differentiation including the onset of terminal cell division, as well as the expression of differentiation-specific genes. B. COMMITMENT TO TERMINAL CELLDIVISION One requirement for studying regulation of the onset of terminal cell division is an assay, at the single cell level, for the loss of capacity for cell proliferation. Such an assay has been developed for MELC (Gusella et al., 1976; Fibach et al., 1977). MELC are exposed to inducer in suspension culture, aliquots are removed after appropriate intervals and grown as a single cell suspension in semisolid medium in the absence of inducer. The growth and developmental potential of each cell is scored by examining the progeny colonies after several days. Large colonies which do not stain for hemoglobin (benzidine-negative colonies) are the progeny of uncommitted MELC. Small colonies (less than 32 cells) of benzidine-positive cells are the progeny of cells which had become committed to their program of terminal cell division (and their differentiation as erythroid cells). A small proportion of cells give rise to “mixed” colonies consisting of both benzidine-reactive and benzidine-negative cells. Analogous patterns of commitment have been observed in studies of other cell lines capable of induced terminal cell differentiation, such as the rat myoblast (Nadal-Ginard, 1978), human keratinocyte (Rheinwald, 1979), and mouse lymphocyte (Milner, 1977). Commitment, defined as the ability to express this transition from a stage of unlimited proliferation to the stage of terminal division, is dependent on the nature, as well as on the duration of exposure to and concentration of the inducer (Reuben et al., 1980). Commitment can be detected as early as 12 hr in culture with certain inducers; almost 100% of the population can be committed to terminal cell division by 48 hr in culture with HMBA, probably the most effective inducer identified (Reuben et al., 1980). C. COMMITMENT TO TERMINAL CELL DIVISION INVOLVES A MULTISTEPPROCESS Several lines of evidence suggest that the commitment of MELC is a multistep process. Inducer-mediated commitment to terminal cell division exhibits different kinetics with different inducers (Reuben et al., 1980; Nude1 et al., 1977b; and Marks et al., 1979). Variants of MELC have been developed which show altered patterns of response to inducers with respect to terminal cell division, including variants which fail to exhibit inducermediated commitment although they express other characteristics of ery-
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throid cell differentiation (Harrison, 1977; Marks et al., 1983; Eisen et al., 1978; Pragnell et al., 1980). Studies with a number of inhibitors of induced differentiation provide additional evidence. Levenson et al. (1979), examining the response of differentiating MELC to the nucleotide analog, cordycepin, which inhibits accumulation of polyadenylated mRNA, found evidence that in Me,SOmediated commitment there is a step which is sensitive to inhibition by this agent. The rate limiting step for commitment appears, however, to be insensitive to inhibition by cordycepin but sensitive to inhibition by cycloheximide, an inhibitor of protein synthesis (Levenson and Housman, 1979). Based upon work from our laboratory, analyzing patterns of induction in normal and variant MELC cell lines, it has been suggested that there is a precommitment (“initiation”) phase during which certain inducer-mediated metabolic changes occur, including, perhaps, alterations in membrane permeability, cell volume, and CAMP concentration (Marks and Rifkind, 1978; Eisen et al., 1978; Gazitt et al., 1978a,b; Mager and Bernstein, 1978a,b; Smith et al., 1982). This is followed by a period during which changes occur which appear to involve the accumulation of a factor or factors which may be responsible for the commitment to terminal cell division and, perhaps, for the expression of genes characteristic of terminal differentiation (Chen et al., 1982). There then follow those changes which characterize expression of the terminal erythroid cell phenotype (Marks and Rifkind, 1978; Harrison, 1977; Marks et al., 1982), including accumulation of newly synthesized globin mRNA (Ross et al., 1972), the sequential induction of heme-synthesizing enzymes (Sassa, 1980), the progressive loss of the capacity for cell division (Gusella et al., 1976; Fibach et al., 1977), and other changes (Boyer et al., 1972; Ostertag et al., 1972; Eisen et al., 1977). A CHANCE FOR WHICHCELLS D. COMMITMENT INVOLVES RETAIN A “MEMORY”
Tumor promoters, such as 12-0-tetradecanoylphorbol-13-acetate(TPA), are potent inhibitors of inducer-mediated differentiation of MELC (for review see Marks et al., 1982) as well as other cell lineages (Diamond et al., 1977; Ishii et al., 1978). When MELC are exposed to both HMBA and TPA, the phorbol ester suppresses the onset of terminal cell division as well as the accumulation of newly synthesized a-and (3-globin mRNA and hemoglobin (Marks et al., 1982). If MELC are transferred from medium containing both inducer (HMBA) and inhibitor (TPA) into medium without either agent, such cells retain, for a period of time, a “memory” of their prior exposure to the inducer (Fibach et al., 1979). Although the nature of this memory, at a molecular level, remains to be defined, the memory for prior exposure to
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inducer can persist for more than one cell cycle, and may reflect the accumulation of that factor (or factors) noted above. The phenomenon of memory for HMBA-mediated commitment has also been demonstrated using another potent inhibitor of induced differentiation, the glucocorticoid, dexamethasone (Santoro et al., 1978; Tsiftsoglou et al., 1979; Osborne et a l . , 1982). Dexamethasone suppresses expression of inducer-mediated MELC terminal cell division, as well as inhibiting the accumulation of cytoplasmic a- and P-globin mRNAs, globins, and hemoglobins (Chen et al., 1982; Lo et a l . , 1978; Scher et al., 1978; Mierendorfand Mueller, 1981). We have demonstrated that dexamethasone inhibits commitment to terminal cell division at a stage in the multistep process of inducer-mediated MELC commitment which is not rate limiting for this process (Chen et a l . , 1982). It has been shown, as well, that dexamethasone inhibits the accumulation of nuclear globin mRNA sequences, suggesting that the steroid acts to block the inducer-mediated increase in globin gene expression (Marks et al., 1982). However, Shaul et al. (1981)have suggested that TPA inhibits the transport of globin mRNA to the cytoplasm during culture of MELC with Me,SO, implicating a step involved in the transport or stability of mRNA. It remains to be determined whether inhibition of inducer-mediated MELC commitment by TPA and dexamethasone reflects an action of these agents at a transcriptional or a posttranscriptional level and how the inhibitors’ effects on globin gene expression are related to their effects in inhibiting commitment to terminal cell division.
E. PROTEINp53 A N D
THE
ONSETOF TERMINAL CELLDIVISION
Little is known of the molecular or biochemical mechanisms which are specifically implicated in the signal for the actual onset of termination of cell division, as noted above. HMBA-mediated MELC commitment to terminal cell division is coordinated with the expression of other markers of erythroid differentiation, among which we have focused our attention on the globin genes. Inducers which initiate commitment to terminal cell division also initiate a complex series of changes in the structure of globin gene chromatin and increase the rate of transcription at the al-and Pmaj-globin gene loci (Sheffery et al., 1982, 1983a; Profous-Juchelka et al., 1983). However, the accumulation of globin mRNA is not itself sufficient to initiate commitment to terminal cell division. Hemin, for example, increases the rate of a-and Pglobin mRNA and hemoglobin accumulation (Ross and Sautner, 1976), but does not induce commitment to terminal cell division (Gusella et al., 1980; Profous-Juchelka et a l . , 1983). On the other hand, imidazole, an inhibitor of heme accumulation, blocks Me2SO-induced accumulation of hemoglobin,
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but does not block commitment of terminal cell division (Gusella et al., 1982). The first manifestation of termination of cell division in MELC appears to be a transient prolongation of the G, phase of the cell cycle which can be detected in the first cell cycle which follows one complete S phase in inducer (Terada et al., 1977). Eventually, after 4-5 cell divisions, induced MELC are permanently arrested in G,. Evidence has accumulated suggesting that synthesis of a labile protein in early G , may control the entry of cells into S phase (Rossow et al., 1979). A nuclear protein (p53), initially recognized by its elevated levels in transformed cells (DeLeo et al., 1979)and by its capacity to bind to SV40 T antigen (Lane and Crawford, 1979; Linzer and Levine, 1979), has been identified as possibly important in determining normal cell cycle progression from G, to S (Milner and Milner, 1981; Mercer et al., 1982). It has also been suggested that p53 protein levels may be regulated in relation to a cell’s status in the differentiation of a cell lineage (Oren et al., 1982; Chandrasekarau et al., 1982). We have recently shown, by microscopic immunofluorescence, flow microfluorimetry, and immunoprecipitation after [35S]methionine incorporation, that during induced differentiation of MELC there is a decrease in p53 synthesis and in the cell content of this protein (Shen et al., 1983). Although the role of p53 protein in MELC or other cells has not been established with certainty, the principal relationship established by these studies is between down-regulation of p53 synthesis and the loss of cellular proliferative capacity. Hemin, which can induce globin gene expression, but does not induce commitment to terminal cell division, does not induce a fall in p53 synthesis or content, while a commitment-resistant cell variant (Rl), which can express certain characteristics of the MELC developmental program (Marks et al., 1983), also fails to decrease its p53 content in response to inducer (Shen et al., 1983). Taken together, these observations suggest that down-regulation of p53 protein is a part of the coordinate program of events which occur during induced MELC differentiation, and may prove to be significant to the onset of termial cell division. Ill. Gene Expression, DNA Structure; and Chromatin Configuration during Induced Differentiation
The murine erythroleukemia cell is a model cell system well suited to the study of mechanisms which control the coordinated expression of sets of unlinked differentiation-specific genes during development. The globin genes provide a paradigm for such a study; induction results in a 10- to 20fold increase in the rate of accumulation of globin mRNAs, regulated to a large degree by increased transcription (Hofer et al., 1982), although post-
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transcriptional levels of control have also been implicated (Profous-Juchelka et al., 1983). Two types of hemoglobin, Hblnaj (containing a-and P1naj-globinpolypeptides) and Hblnirl (a-and P’”’”-polypeptides) can be found in MELC. In uninduced MELC there is a low but detectable level of Hbmill,reflecting a low level of expression of the a-and plllill-globingenes (Nudel et al., 1977b). Polar inducers, such as M e 2 S 0 and HMBA, initiate accumulation of more HblnaJ than HbInin; the fatty acid inducers (such as butyric acid) induce roughly equal amounts of both proteins, while hemin induces, predominately, accumulation of Hblllill(Nudel et d.,1977b; Curtis et d.,1980).
A. MURINEGLOBINGENEDOMAINS Detailed physical maps of the murine a-and P-globin gene domains have been extensively characterized (Konkel et al., 1978, 1979; Nishioka and Leder, 1979). A 60 kb region of DNA containing the p-globin genes and a 40 kb region containing a-globin genes have been cloned into h-bacteriophage vectors (Leder et al., 1980). Within the 60 kb p domain, located on chromosome 7, seven loci have been identified which correspond to p-globin genes or pseudogenes. Identified loci include (in the 5’ to 3’ order of transcriptional polarity) the pln“J-and P1nill-globingenes. The 40 kb region contains (in the 5’ to 3’ direction) a-embryonic globin genes and the al-and a2-adult globin genes. These are not linked to the (3 domain, and are found on chromosome 11 (Leder et al., 1981). The complete nucleotide sequence of the al-globin gene has been determined (Nishioka and Leder, 1979). Of the several globin “pseudogenes” (which do not appear to be expressed), the best characterized are the a3-and a,-globin genes. These genes are not physically linked to the embryonic, a 1 and a2 genes, and have been located on chromosomes 15 and 17, respectively (Leder et al., 1981). B. TRANSCRIPTIONAL REGULATIONOF GLOBIN GENESD U RI N G DEVELOPMENT A N D DIFFERENTIATION Much evidence suggests that globin gene expression during development and differentiation is regulated at the level of gene transcription (Groudine et al., 1981; Groudine and Weintraub, 1981; Weintraub et al., 1981; Landes and Martinson, 1982; Landes et al., 1982; Villeponteau et al., 1982). Transcriptional regulation of globin gene expression in the mouse has been best characterized in MELC. Using the nuclear RNA chain elongation assay, Hofer et u1. (1982)have demonstrated a 10- to 20-fold increase in plnaj-globin gene transcription during induced differentiation. Transcription initiates
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predominately near the cap site, occurs predominately off the coding strand, and extends approximately 1.5 kb beyond the poly(A) addition site before terminating. Likewise transcription of the al-globin gene increases some 10to 20-fold during induction (Sheffery et al., 1984), initiating at or near the cap site and terminating, apparently, in a region 50 to 250 bp 3' to the putative polyadenylation site. An increase i n a,-globin gene transcription can be detected within 2 to 3 cell cycles (about 36 hr) following exposure to the inducer, HMBA; the rate of transcription continues to increase for at least 48 hr. C. POSTTRANSCRIPTIONAL REGULATION OF GLOBIN G E N EEXPRESSION Whereas HMBA, Me,SO, and other agents induce the program of MELC differentiation in a coordinate fashion, including globin gene expression and coininitment to terminal cell division, it has been demonstrated (see above) that exposure to hemin leads to accumulation of globin mRNA (and hemoglobins) but not to loss of cellular proliferative capacity. It has recently been shown that the increase in globin mRNA content detected in hemin-treated MELC is not accompanied by an increase in globin gene transcription, as measured by the technique of nuclear RNA chain elongation (ProfousJuchelka et ul., 1983). Hemin appears to act by a posttranscriptional effect upon a low but constitutive level of globin gene transcription in uninduced MELC. This effect of heinin may be mediated by changes in the processing, transport, or stability of such transcripts. The significance of this heminmediated, posttranscriptional control mechanism has yet to be determined. It should be noted, however, that induction by HMBA or other agents is characterized by an early and brisk activation of the heme synthetic enzyme system and the accumulation of cellular heme (Ebert and Ikawa, 1974; Sassa, 1976; Fibach et al., 1979).The rate of globin inRNA accumulation in induced MELC may, then, be the product of both transcriptional and posttranscriptional mechanisms of control.
D. THE ROLE o~ CHROMATIN STRUCTURE I N GLOHIN G E N EREGULATION It is likely that changes in chromatin structure, mediated in part at least by chroinosoinal proteins, play an important role in regulating the transcription of specific genes (Elgin, 1981; Weisbrod, 1982). A number of biochemical features distinguish active chromosomal regions from bulk chromatin at both the DNA and protein level. Several of these have been examined in detail with respect to the expression of globin genes during induced MELC differ-
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entiation. These include the pattern of DNA methylation, the sensitivity of chromatin to digestion by DNase I, and the appearance of sites or regions that are hypersensitive to digestion by a number of endonucleases including DNase I and S1 nuclease. Certain patterns of chromatin and DNA structure, found in uninduced MELC, do not change upon induction of terminal cell differentiation; these include the pattern of DNA methylation about both the a1and PlllaJ genes and the overall sensitivity of chromatin containing these genes, to DNase I digestion (Sheffery et al., 1982). In addition, a number of DNase I hypersensitive regions can be detected in uninduced MELC, in the region of both the Plnaj and a1 genes (Sheffery et al., 1982, 1983). These include a site located within the second intron (IVS-2) of the plnaj gene, and sites 5’ to the a,-globin gene cap site. Collectively these molecular features appear to be characteristic of a transformed erythroid cell precursor arrested in its development at approximately the CFUe stage of erythroid cell differentiation. This molecular phenotype is stably propagated in the continuously replicating, uninduced MELC; and from a functional point of view appears to characterize an erythroid precursor with at best a low, constitutive level of globin gene transcription. Each of these features of DNA and chromatin structure has been examined in detail during induced MELC differentiation.
I. DNA Methylation The pattern of DNA methylation has been cited as a heritable molecular characteristic which, at least in many instances, distinguishes expressed and unexpressed genes. We have examined the pattern of DNA methylation in the region of the plnaj- and a,-globin genes during HMBA-mediated differentiation (Sheffery et al., 1982). Cytosine methylation in the nucleotide sequence, CCGG, was assayed by the use of the methyl-sensitive isoschizomer-pair of restriction enzymes, MspI and HpaII, and other restriction enzymes. There are relatively few potentially methylated sites in the MELC globin gene domains which can be assayed by these restriction enzymes. Of the sites assayed near the plnaj-globin gene, one site is fully methylated, one partially methylated, and one is unmethylated in uninduced MELC. Most cites, but not all, assayed near the a-globin genes are unmethylated in uninduced cells. No detectable change in the pattern of methylation around either gene was observed during HMBA-mediated differentiation. Globin genes, both a1and pmaJ,in nonerythroid mouse tissues display distinctly more methylation than they do in MELC (S. Einheber and M. Sheffery, unpublished observations). It would appear likely that, within the limits of resolution of this assay, the pattern of globin gene methylation in the erythroid cell lineage is established and stably propagated at a developmental stage in erythropoiesis prior to the stage represented by the MELC.
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2. DNase 1 Sensitivity Studies of the accessibility to DNase I of MELC chromatin, in the globin gene domains, has suggested that the chromatin of uninduced cells is already in a configuration compatible with transcriptional activity (Miller et al., 1978). It has been demonstrated (Sheffery et al., 1982)that the pmaJ-and alglobin gene-associated chromatin regions are distinctly more sensitive to digestion by DNase I than is the Iga (immunoglobulin) gene (which is not expressed by cells in the erythroid lineage), in both uninduced and induced MELC. As in the case of the methylation pattern, it appears that the globin gene domains have been selectively modified during the developmental history of the erythroid lineage, establishing the globin-related chromatin in a potentially active configuration. These changes, as in the case of the methylation pattern, are stably propagated in uninduced MELC.
3. Nuclease Hypersensitivity Sites We have obtained evidence for alterations in chromatin structure which are specifically associated with inducer-mediated activation of globin gene transcription (Sheffery et al., 1982, 1983). During HMBA-induced differentiation, sites displaying a 6- to 10-fold increase in DNase I sensitivity appear in chromatin regions near the 5‘ end of the a 1and pmajgenes. The DNase I hypersensitive site which appears near the pmaJ-globin gene maps to an approximately 200 base pair region in the 5’ flanking region of that gene. That hypersensitivity site which becomes detected near the al-globin gene likewise is located in the region 5’ to the a1cap site. The changes in chromatin structure which are revealed by nuclease probes during induced differentiation are more complex, however, than simply the reconfiguration of sites at the 5’ end of the globin genes. As noted already, it has been demonstrated that there is a DNase I hypersensitive site, located within the second IVS of the pmaJ gene, which can be detected in uninduced MELC. The nuclease hypersensitivity at this site disappears during HMBA-mediated differentiation, replaced by the new hypersensitivity site which lies 5’ to the pmaJcap site. Although this complex change in chromatin configuration normally takes place in a coordinate fashion, just prior to the initiation of globin gene transcription, it has been shown (Sheffery et a l . , 1983) that in an inducer-resistant variant these two changes in chromatin configuration can be dissociated. In the R 1 variant of MELC, which is resistant to the inducing effect of HMBA on globin gene expression and commitment (Marks et al., 1983), HMBA mediates the disappearance of that hypersensitivity site located in IVS-8 but fails to generate the new site, located 5’ to the PmaJcap site, and fails to initiate transcription at the pmaJ gene.
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At the a, domain there is also a complex pattern of chromatin reconfiguration that occurs during induced differentiation (Sheffery et al., 1984). The uninduced MELC, overlapping DNase I and S1 nuclease-sensitive sites are detected 5' of the a-globin gene cap site. During induction the nuclease sensitivity of these sites increases and new, nonoverlapping DNase I and S1 sites develop, one approximately 300 base pairs 5' of the a,-globin cap site, and the other mapping to a region virtually coincident with the cap site itself. None of these changes in nuclease sensitivity occurs in the HMBAresistant MELC variant (Rl), suggesting that there may be significant differences between the chromatin-associated events that take place in the aand (3-globin gene domains during induced differentiation. IV. Differentiation and the Cell Cycle
Inducer-related events associated with the cell cycle, with DNA synthesis, and, in particular, with replication of the globin genes appear to be important in the induction of MELC differentiation and accelerated transcription of the globin genes (Gambari et al., 1979; Brown and Schildkraut, 1979; Epner et al., 1981; Levy et al., 1975). Studies in this laboratory have shown that inducing agents must be present during at least one cell cycle to initiate differentiation (Levy et al., 1975)and that late GI-early S may be the period of particular significance for subsequent globin gene expression (Gambari et al., 1978, 1979). Since DNA replication has been implicated in the transition from an inactive to an active chromosome structure in several systems (Groudine and Weintraub, 1981; Holtzer et al., 1972; Dienstman and Holtzer, 1975), it has been speculated that the changes in globin gene chromatin configuration which are associated with enhanced globin gene expression in MELC may be introduced during replication of the genes, and that the presence of the inducer at a restricted time in the cell division cycle may be critical to the reconfiguration of these gene domains. In this context, it has been found that genes whose expression is stimulated during MELC differentiation (the a- and (3-globin genes) are replicated during early S (Epner et al., 1981; Furst et al., 1981). Each of these aspects of induced differentiation is discussed below.
A. THE CELLCYCLEA N D INDUCED DIFFERENTIATION Evidence suggesting a relationship between the cell cycle (possibly DNA synthesis), and the onset of erythroid cell differentiation, particularly the onset of globin gene expression, in normal and transformed erythroid cells, has come from a number of studies. It was demonstrated a number of years ago that one effect of erythropoietin on normal erythropoietin-sensitive tar-
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get cells (probably the CFUe) is an acceleration of DNA synthesis which precedes the accumulation of globin messenger RNA (Djaldetti et a l . , 1972; Rainirez et al., 1975). Studies using inhibitors of DNA synthesis have added to this evidence implicating DNA synthesis and the cell cycle in the initiation of globin gene expression during normal erythropoiesis (Marks et al., 1977). Additional evidence has come from studies on MELC differentiation. For example, when cultures are initiated at high cell densities, the time required for cell doubling is prolonged and the extent of differentiation is decreased; under these conditions, the higher the rate of cell proliferation, the greater the proportion of cells induced to differentiate (Rifiind and Marks, 1982). More direct evidence for a role of the cell cycle in induced MELC differentiation comes from studies with synchronized cell populations, employing several independent methods for the induction of synchronized cell growth. Using 2 mM thymidine to inhibit progression of the cells past the G,/S interface, Levy et al. (1975) demonstrated that induction requires exposure to inducing agents as the cells move through at least one cell cycle. This conclusion has been reached, as well, using synchronization by nutrient deprivation (McClintock and Papaconstantinou, 1974) and by the use of a temperature-sensitive cell cycle mutant (Harrison, 1977). Geller et al. (1978) showed that commitment to initiate terminal cell division and other features of the differentiated phenotype is accomplished most quickly by cultures of MELC synchronized in the G, or G, phase of the cell cycle, suggesting that early S phase is important for induction. More recently, using thymidine, thymidine plus hydroxyurea, or centrifugal elutriation for the synchronization of MELC in G, or at the G,/S interface, it was demonstrated that exposure to inducer during one cell cycle resulted in the initiation of accumulation of globin messenger RNA during the subsequent cell cycle, beginning during G,. The critical time in the cell cycle for subsequent initiation of globin messenger RNA accumulation is G, or early S (Gambari et al., 1978, 1979). Finally, using the phorbol ester, an inhibitor of induced differentiation, it was demonstrated (Gambari et al., 1980) that the TPAsensitive period for inhibition of globin synthesis is confined to that cell cycle in the presence of inducer which precedes the onset of globin mRNA accumulation. Evidence suggesting that it is the onset of globin gene transcription, and not merely the accumulation of globin messenger RNA, that is sensitive to a cell cycle-related effect of inducers has been obtained from studies examining the kinetics of globin mRNA accumulation during induction with various inducing agents. When M ELC are induced to differentiate by polar-planar compounds such as HMBA and Me,SO, the first accumulation of globin mRNA is detected approximately 12 hr after the initiation of culture with
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inducer (the cell division cycle is approximately 12 hr). On the other hand, when MELC are induced to accumulate globin mRNA by exposure to hemin, mRNA accumulation can be detected in less than 6 hr and under certain circumstances in as little as 2 hr, a time too short for a significant cell cycle requirement (Nude1 et al., 1977b). As already described (see above) the initiation of globin mRNA accumulation by hemin is accomplished by a posttranscriptional effect upon a constitutive low rate of globin gene transcription and is accompanied by no detectable increase in transcription at the globin gene loci. Taken together these observations suggest that the cell cycle dependence of inducer effect is related to the transcription-modulating effects of these agents; an agent which modifies globin gene expression through a posttranscriptional mechanism, such as hemin, functions in a manner which is, apparently, independent of the cell division cycle.
B. GLOBINGENEREPLICATION Taken together, studies using both inducers of differentiation and inhibitors of differentiation have suggested that there is a requirement for the cell cycle for induced differentiation and the critical period may lie in late G, or early S phase. Based upon speculation that reconfiguration of globin geneassociated chromatin during replication in the presence of chemical inducers of differentiation may be critical for induced gene transcription, studies were undertaken to define the time in the cell cycle during which both a-and pglobin genes undergo DNA replication (Epner et al., 1981). For these studies, centrifugal elutriation (based upon cell volume) was employed for cell synchronization in order to avoid artifacts introduced by the use of inhibitors of DNA synthesis. Newly replicated DNA sequences were prepared from synchronized cells cultured for short periods with 5-bromodeoxyuridine, and the BUdR-containing, newly replicated DNA isolated by CsCl gradient centrifugation and analyzed by hybridization with cloned probes for the aand p-globin gene sequences. Both a- and p-globin gene sequences, in MELC, are replicated early in S phase, whereas ribosomal RNA gene sequences are replicated throughout the cell cycle. These observations are consistent with the speculation that gene replication may be an important event, implicated in the reconfiguration of globin genes required for the activation of transcription. V. Summary
Study of inducer-mediated differentiation of murine erythroleukemia cells provides insights into the cellular and molecular mechanisms implicated in cell differentiation. The loss of proliferative capacity is revealed to be a
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complex multistep process during which the cells progress through a series of stages, including a precoinmitment “initiation” stage, a stage suggestive of the accumulation of commitment-related factors, and, finally, a stage of expression of the characteristics of the differentiated state. Cell cycle arrest in G, phase of the cell cycle may, in part at least, be related to downregulation of protein p53 synthesis. Expression of induced differentiation is accompanied by an acceleration of transcription at the globin loci, and possibly by posttranscriptional modulation of globin mRNA accumulation, as well. Cells at the stage of erythroid cell development represented by the transformed, differentiation-arrested MELC, have acquired a unique DNA structure and chromatin configuration around the globin genes which distinguish them from other, nonerythroid cells; additional complex changes in chromatin configuration accompany, and probably precede, inducer-mediated acceleration of globin gene transcription during terminal differentiation. Passage through G, and early S phase of the cell cycle, in the presence of inducer, is critical for subsequent globin gene expression and may be important in establishing the chromatin reconfiguration required for gene expression.
ACKNOWLEDGMENTS These studies were supported, in part, by grants from the National Cancer Institute (PO1 CA-31768 and CA-08748) and the Bristol-Myers Cancer Research Program.
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I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Terminal Cell Division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Characteristics of Inducer-Mediated Differentiation . . . . . . . . . . . . . . . . . . . . . . B. Commitment to Terminal Cell Division C. Commitment to Terminal Cell Division D. Commitment Involves a Change for Which Cells Retain a “Memory” . . . . . . E. Protein p53 and the Onset of Terminal Cell Division. . . . . . . . . . . . . . . . . . . . . 111. Gene Expression, DNA St during Induced Differentiation A. Murine Globin Gene 1 B. Transcriptional Regulation of Globin Genes during Development and Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Posttranscriptional Regulation of Globin Gene Expression . . . . . . . . . . . . . . . .
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I. Introduction Erythropoiesis constitutes one of the best characterized developmental lineages in higher organisms; the progressive developmental stages have been identified and aspects of the control mechanisms regulating development, including the effects of hemopoietic growth factors, are increasingly well defined (Marks and Rifkind, 1978). Terminal cell differentiation in this lineage has been described in terms of morphological and biochemical changes, as well as events at the molecular level, including the generation of differentiation-specific mRNAs (globin mRNAs) and other differentiationspecific products (Marks and Rifkind, 1978; Terada et al., 1972; Ramirez et al., 1975; Sassa, 1980). Molecular probes for the definition of specific gene expression in this lineage are readily available. The murine erythroleukemia cell (MELC), a virus-transformed precursor approximating, in terms of its developmental potential, the colony forming 149 ADVANCES IN CAh’CER RESEARCH. VOL 42
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unit for erythropoiesis (CFUe) stage of the erythroid lineage, provides yet additional advantages for the study of developmentally significant regulatory mechanisms during erythroid cell differentiation (Marks and Rifkind, 1978). These transformed cells, blocked in their differentiation at a particular moment in the developmental history of the erythroid cell lineage, can be cloned and retain their normal developmental potentiality. This provides a unique opportunity for defining the cellular and molecular phenotype of a cell arrested at a particular developmental stage in its lineage. Indeed, as will be developed in this article, a number of features of chromatin and DNA structure, in the globin gene domains, have been identified in MELC which appear to characterize this developmental stage of the erythroid cell lineage. MELC can be induced, by exposure to any of a variety of chemical agents, to initiate their developmental program in a fashion which, by all tests so far applied, appears similar to the normal developmental history of nontransformed erythroid precursor cells. These cells provide an opportunity to examine, at a cellular and molecular level, events which accompany, and which may regulate, the initiation of terminal cell differentiation, including the expression of differentiation-specific genes and the initiation of terminal cell divisions. Among these, as will be developed below, is a complex set of changes of chromatin structure in the globin gene domains, which appear to be significant in the induced expression of the genes during differentiation. Additional advantages of the MELC system include the availability of different inducing agents, capable of initiating different patterns of expression of the characteristics of induced differentiation (Marks and Rifkind, 1978; Tanaka et al., 1975; Reuben et al., 1976; Nude1 et al., 1977a,b), and the availability of' variant cell lines selected for their resistance to various inducing agents and which are providing powerful tools for exploring relationships among the molecular changes which accompany induced differentiation (Ohta et al., 1976). Many aspects of the biological events which occur during induced MELC differentiation have been explored in a number of laboratories. These include, for example, factors determining morphogenetic changes (Volloch and Housman, 1982), changes in cell membrane properties, including ion fluxes (Mager and Bernstein, 1978a,b; Smith et al., 1982), cyclic nucleotide metabolism (Gazitt et al., 1978b), iron transport and heme synthesis (Sassa, 1980), and others. In this article we shall concentrate on three features of induced MELC differentiation dealing with the regulation of expression of developmentally specific genes and the relationships between differentiation and the cell cycle.
1. Studies concerning the mechanisms regulating the initiation of termi-
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nal cell division (commitment), which is a characteristic feature of the development of both normal and transformed erythroid cells. 2. The nature of mechanisms regulating the expression of differentiationspecific genes during induced differentiation; these studies, in large part, address the control of globin gene expression at the transcriptional and posttranscriptional levels, both of which may be modulated during induced differentiation. 3. Studies on the relationships between the cell division cycle, DNA synthesis, globin gene replication, and induced cell differentiation.
II. Terminal Cell Division The nature of the factors regulating cell growth in eukaryotic cells remains one of the central unresolved issues in biology today. One requirement for such a study is a homogeneous cell population which can be readily manipulated with respect to the transition from unlimited cell growth to the terminal cell divisions characteristic of many differentiating cell lineages. MELC meet this criterion and provide one attractive model for study of this problem.
A. CHARACTERISTICS OF INDUCER-MEDIATED DIFFERENTIATION MELC can be maintained in suspension culture for an essentially unlimited number of passages under appropriate conditions (Marks and Rifkind, 1978) and can be induced by a variety of agents (Table I), including TABLE 1 INDUCERS OF MELC E R Y T I ~ R ODIFFERENTIATION^ ID Polar compounds Fatty acids DNA intercalators Modified bases Phosphodiesterase inhibitors Ion-flux agents Physical agents Posttranscription-acting agent
Dirnethyl sulfoxide (MeZSO) Hexamethylene bisacetamide (HM BA) Butyric acid Actinornycin Azacytidine Methylisoxanthine Ouabain UV, X ray Hemin
a This is an abbreviated tabulation of agents which have been demonstrated to induce MELC to express characteristics of erythroid differentiation. A more complete list has been presented elsewhere (Marks and Rifkind, 1978; see also Mager and Bernstein, 1978b; Creusot et al., 1982; Ross and Sautner, 1976; Reuben et al., 1980).
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Me,SO, HMBA, and butyric acid, to express characteristics of erythroid cell differentiation including the onset of terminal cell division, as well as the expression of differentiation-specific genes. B. COMMITMENT TO TERMINAL CELLDIVISION One requirement for studying regulation of the onset of terminal cell division is an assay, at the single cell level, for the loss of capacity for cell proliferation. Such an assay has been developed for MELC (Gusella et al., 1976; Fibach et al., 1977). MELC are exposed to inducer in suspension culture, aliquots are removed after appropriate intervals and grown as a single cell suspension in semisolid medium in the absence of inducer. The growth and developmental potential of each cell is scored by examining the progeny colonies after several days. Large colonies which do not stain for hemoglobin (benzidine-negative colonies) are the progeny of uncommitted MELC. Small colonies (less than 32 cells) of benzidine-positive cells are the progeny of cells which had become committed to their program of terminal cell division (and their differentiation as erythroid cells). A small proportion of cells give rise to “mixed” colonies consisting of both benzidine-reactive and benzidine-negative cells. Analogous patterns of commitment have been observed in studies of other cell lines capable of induced terminal cell differentiation, such as the rat myoblast (Nadal-Ginard, 1978), human keratinocyte (Rheinwald, 1979), and mouse lymphocyte (Milner, 1977). Commitment, defined as the ability to express this transition from a stage of unlimited proliferation to the stage of terminal division, is dependent on the nature, as well as on the duration of exposure to and concentration of the inducer (Reuben et al., 1980). Commitment can be detected as early as 12 hr in culture with certain inducers; almost 100% of the population can be committed to terminal cell division by 48 hr in culture with HMBA, probably the most effective inducer identified (Reuben et al., 1980). C. COMMITMENT TO TERMINAL CELL DIVISION INVOLVES A MULTISTEPPROCESS Several lines of evidence suggest that the commitment of MELC is a multistep process. Inducer-mediated commitment to terminal cell division exhibits different kinetics with different inducers (Reuben et al., 1980; Nude1 et al., 1977b; and Marks et al., 1979). Variants of MELC have been developed which show altered patterns of response to inducers with respect to terminal cell division, including variants which fail to exhibit inducermediated commitment although they express other characteristics of ery-
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throid cell differentiation (Harrison, 1977; Marks et al., 1983; Eisen et al., 1978; Pragnell et al., 1980). Studies with a number of inhibitors of induced differentiation provide additional evidence. Levenson et al. (1979), examining the response of differentiating MELC to the nucleotide analog, cordycepin, which inhibits accumulation of polyadenylated mRNA, found evidence that in Me,SOmediated commitment there is a step which is sensitive to inhibition by this agent. The rate limiting step for commitment appears, however, to be insensitive to inhibition by cordycepin but sensitive to inhibition by cycloheximide, an inhibitor of protein synthesis (Levenson and Housman, 1979). Based upon work from our laboratory, analyzing patterns of induction in normal and variant MELC cell lines, it has been suggested that there is a precommitment (“initiation”) phase during which certain inducer-mediated metabolic changes occur, including, perhaps, alterations in membrane permeability, cell volume, and CAMP concentration (Marks and Rifkind, 1978; Eisen et al., 1978; Gazitt et al., 1978a,b; Mager and Bernstein, 1978a,b; Smith et al., 1982). This is followed by a period during which changes occur which appear to involve the accumulation of a factor or factors which may be responsible for the commitment to terminal cell division and, perhaps, for the expression of genes characteristic of terminal differentiation (Chen et al., 1982). There then follow those changes which characterize expression of the terminal erythroid cell phenotype (Marks and Rifkind, 1978; Harrison, 1977; Marks et al., 1982), including accumulation of newly synthesized globin mRNA (Ross et al., 1972), the sequential induction of heme-synthesizing enzymes (Sassa, 1980), the progressive loss of the capacity for cell division (Gusella et al., 1976; Fibach et al., 1977), and other changes (Boyer et al., 1972; Ostertag et al., 1972; Eisen et al., 1977). A CHANCE FOR WHICHCELLS D. COMMITMENT INVOLVES RETAIN A “MEMORY”
Tumor promoters, such as 12-0-tetradecanoylphorbol-13-acetate(TPA), are potent inhibitors of inducer-mediated differentiation of MELC (for review see Marks et al., 1982) as well as other cell lineages (Diamond et al., 1977; Ishii et al., 1978). When MELC are exposed to both HMBA and TPA, the phorbol ester suppresses the onset of terminal cell division as well as the accumulation of newly synthesized a-and (3-globin mRNA and hemoglobin (Marks et al., 1982). If MELC are transferred from medium containing both inducer (HMBA) and inhibitor (TPA) into medium without either agent, such cells retain, for a period of time, a “memory” of their prior exposure to the inducer (Fibach et al., 1979). Although the nature of this memory, at a molecular level, remains to be defined, the memory for prior exposure to
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inducer can persist for more than one cell cycle, and may reflect the accumulation of that factor (or factors) noted above. The phenomenon of memory for HMBA-mediated commitment has also been demonstrated using another potent inhibitor of induced differentiation, the glucocorticoid, dexamethasone (Santoro et al., 1978; Tsiftsoglou et al., 1979; Osborne et a l . , 1982). Dexamethasone suppresses expression of inducer-mediated MELC terminal cell division, as well as inhibiting the accumulation of cytoplasmic a- and P-globin mRNAs, globins, and hemoglobins (Chen et al., 1982; Lo et a l . , 1978; Scher et al., 1978; Mierendorfand Mueller, 1981). We have demonstrated that dexamethasone inhibits commitment to terminal cell division at a stage in the multistep process of inducer-mediated MELC commitment which is not rate limiting for this process (Chen et a l . , 1982). It has been shown, as well, that dexamethasone inhibits the accumulation of nuclear globin mRNA sequences, suggesting that the steroid acts to block the inducer-mediated increase in globin gene expression (Marks et al., 1982). However, Shaul et al. (1981)have suggested that TPA inhibits the transport of globin mRNA to the cytoplasm during culture of MELC with Me,SO, implicating a step involved in the transport or stability of mRNA. It remains to be determined whether inhibition of inducer-mediated MELC commitment by TPA and dexamethasone reflects an action of these agents at a transcriptional or a posttranscriptional level and how the inhibitors’ effects on globin gene expression are related to their effects in inhibiting commitment to terminal cell division.
E. PROTEINp53 A N D
THE
ONSETOF TERMINAL CELLDIVISION
Little is known of the molecular or biochemical mechanisms which are specifically implicated in the signal for the actual onset of termination of cell division, as noted above. HMBA-mediated MELC commitment to terminal cell division is coordinated with the expression of other markers of erythroid differentiation, among which we have focused our attention on the globin genes. Inducers which initiate commitment to terminal cell division also initiate a complex series of changes in the structure of globin gene chromatin and increase the rate of transcription at the al-and Pmaj-globin gene loci (Sheffery et al., 1982, 1983a; Profous-Juchelka et al., 1983). However, the accumulation of globin mRNA is not itself sufficient to initiate commitment to terminal cell division. Hemin, for example, increases the rate of a-and Pglobin mRNA and hemoglobin accumulation (Ross and Sautner, 1976), but does not induce commitment to terminal cell division (Gusella et al., 1980; Profous-Juchelka et a l . , 1983). On the other hand, imidazole, an inhibitor of heme accumulation, blocks Me2SO-induced accumulation of hemoglobin,
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but does not block commitment of terminal cell division (Gusella et al., 1982). The first manifestation of termination of cell division in MELC appears to be a transient prolongation of the G, phase of the cell cycle which can be detected in the first cell cycle which follows one complete S phase in inducer (Terada et al., 1977). Eventually, after 4-5 cell divisions, induced MELC are permanently arrested in G,. Evidence has accumulated suggesting that synthesis of a labile protein in early G , may control the entry of cells into S phase (Rossow et al., 1979). A nuclear protein (p53), initially recognized by its elevated levels in transformed cells (DeLeo et al., 1979)and by its capacity to bind to SV40 T antigen (Lane and Crawford, 1979; Linzer and Levine, 1979), has been identified as possibly important in determining normal cell cycle progression from G, to S (Milner and Milner, 1981; Mercer et al., 1982). It has also been suggested that p53 protein levels may be regulated in relation to a cell’s status in the differentiation of a cell lineage (Oren et al., 1982; Chandrasekarau et al., 1982). We have recently shown, by microscopic immunofluorescence, flow microfluorimetry, and immunoprecipitation after [35S]methionine incorporation, that during induced differentiation of MELC there is a decrease in p53 synthesis and in the cell content of this protein (Shen et al., 1983). Although the role of p53 protein in MELC or other cells has not been established with certainty, the principal relationship established by these studies is between down-regulation of p53 synthesis and the loss of cellular proliferative capacity. Hemin, which can induce globin gene expression, but does not induce commitment to terminal cell division, does not induce a fall in p53 synthesis or content, while a commitment-resistant cell variant (Rl), which can express certain characteristics of the MELC developmental program (Marks et al., 1983), also fails to decrease its p53 content in response to inducer (Shen et al., 1983). Taken together, these observations suggest that down-regulation of p53 protein is a part of the coordinate program of events which occur during induced MELC differentiation, and may prove to be significant to the onset of termial cell division. Ill. Gene Expression, DNA Structure; and Chromatin Configuration during Induced Differentiation
The murine erythroleukemia cell is a model cell system well suited to the study of mechanisms which control the coordinated expression of sets of unlinked differentiation-specific genes during development. The globin genes provide a paradigm for such a study; induction results in a 10- to 20fold increase in the rate of accumulation of globin mRNAs, regulated to a large degree by increased transcription (Hofer et al., 1982), although post-
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transcriptional levels of control have also been implicated (Profous-Juchelka et al., 1983). Two types of hemoglobin, Hblnaj (containing a-and P1naj-globinpolypeptides) and Hblnirl (a-and P’”’”-polypeptides) can be found in MELC. In uninduced MELC there is a low but detectable level of Hbmill,reflecting a low level of expression of the a-and plllill-globingenes (Nudel et al., 1977b). Polar inducers, such as M e 2 S 0 and HMBA, initiate accumulation of more HblnaJ than HbInin; the fatty acid inducers (such as butyric acid) induce roughly equal amounts of both proteins, while hemin induces, predominately, accumulation of Hblllill(Nudel et d.,1977b; Curtis et d.,1980).
A. MURINEGLOBINGENEDOMAINS Detailed physical maps of the murine a-and P-globin gene domains have been extensively characterized (Konkel et al., 1978, 1979; Nishioka and Leder, 1979). A 60 kb region of DNA containing the p-globin genes and a 40 kb region containing a-globin genes have been cloned into h-bacteriophage vectors (Leder et al., 1980). Within the 60 kb p domain, located on chromosome 7, seven loci have been identified which correspond to p-globin genes or pseudogenes. Identified loci include (in the 5’ to 3’ order of transcriptional polarity) the pln“J-and P1nill-globingenes. The 40 kb region contains (in the 5’ to 3’ direction) a-embryonic globin genes and the al-and a2-adult globin genes. These are not linked to the (3 domain, and are found on chromosome 11 (Leder et al., 1981). The complete nucleotide sequence of the al-globin gene has been determined (Nishioka and Leder, 1979). Of the several globin “pseudogenes” (which do not appear to be expressed), the best characterized are the a3-and a,-globin genes. These genes are not physically linked to the embryonic, a 1 and a2 genes, and have been located on chromosomes 15 and 17, respectively (Leder et al., 1981). B. TRANSCRIPTIONAL REGULATIONOF GLOBIN GENESD U RI N G DEVELOPMENT A N D DIFFERENTIATION Much evidence suggests that globin gene expression during development and differentiation is regulated at the level of gene transcription (Groudine et al., 1981; Groudine and Weintraub, 1981; Weintraub et al., 1981; Landes and Martinson, 1982; Landes et al., 1982; Villeponteau et al., 1982). Transcriptional regulation of globin gene expression in the mouse has been best characterized in MELC. Using the nuclear RNA chain elongation assay, Hofer et u1. (1982)have demonstrated a 10- to 20-fold increase in plnaj-globin gene transcription during induced differentiation. Transcription initiates
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predominately near the cap site, occurs predominately off the coding strand, and extends approximately 1.5 kb beyond the poly(A) addition site before terminating. Likewise transcription of the al-globin gene increases some 10to 20-fold during induction (Sheffery et al., 1984), initiating at or near the cap site and terminating, apparently, in a region 50 to 250 bp 3' to the putative polyadenylation site. An increase i n a,-globin gene transcription can be detected within 2 to 3 cell cycles (about 36 hr) following exposure to the inducer, HMBA; the rate of transcription continues to increase for at least 48 hr. C. POSTTRANSCRIPTIONAL REGULATION OF GLOBIN G E N EEXPRESSION Whereas HMBA, Me,SO, and other agents induce the program of MELC differentiation in a coordinate fashion, including globin gene expression and coininitment to terminal cell division, it has been demonstrated (see above) that exposure to hemin leads to accumulation of globin mRNA (and hemoglobins) but not to loss of cellular proliferative capacity. It has recently been shown that the increase in globin mRNA content detected in hemin-treated MELC is not accompanied by an increase in globin gene transcription, as measured by the technique of nuclear RNA chain elongation (ProfousJuchelka et ul., 1983). Hemin appears to act by a posttranscriptional effect upon a low but constitutive level of globin gene transcription in uninduced MELC. This effect of heinin may be mediated by changes in the processing, transport, or stability of such transcripts. The significance of this heminmediated, posttranscriptional control mechanism has yet to be determined. It should be noted, however, that induction by HMBA or other agents is characterized by an early and brisk activation of the heme synthetic enzyme system and the accumulation of cellular heme (Ebert and Ikawa, 1974; Sassa, 1976; Fibach et al., 1979).The rate of globin inRNA accumulation in induced MELC may, then, be the product of both transcriptional and posttranscriptional mechanisms of control.
D. THE ROLE o~ CHROMATIN STRUCTURE I N GLOHIN G E N EREGULATION It is likely that changes in chromatin structure, mediated in part at least by chroinosoinal proteins, play an important role in regulating the transcription of specific genes (Elgin, 1981; Weisbrod, 1982). A number of biochemical features distinguish active chromosomal regions from bulk chromatin at both the DNA and protein level. Several of these have been examined in detail with respect to the expression of globin genes during induced MELC differ-
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entiation. These include the pattern of DNA methylation, the sensitivity of chromatin to digestion by DNase I, and the appearance of sites or regions that are hypersensitive to digestion by a number of endonucleases including DNase I and S1 nuclease. Certain patterns of chromatin and DNA structure, found in uninduced MELC, do not change upon induction of terminal cell differentiation; these include the pattern of DNA methylation about both the a1and PlllaJ genes and the overall sensitivity of chromatin containing these genes, to DNase I digestion (Sheffery et al., 1982). In addition, a number of DNase I hypersensitive regions can be detected in uninduced MELC, in the region of both the Plnaj and a1 genes (Sheffery et al., 1982, 1983). These include a site located within the second intron (IVS-2) of the plnaj gene, and sites 5’ to the a,-globin gene cap site. Collectively these molecular features appear to be characteristic of a transformed erythroid cell precursor arrested in its development at approximately the CFUe stage of erythroid cell differentiation. This molecular phenotype is stably propagated in the continuously replicating, uninduced MELC; and from a functional point of view appears to characterize an erythroid precursor with at best a low, constitutive level of globin gene transcription. Each of these features of DNA and chromatin structure has been examined in detail during induced MELC differentiation.
I. DNA Methylation The pattern of DNA methylation has been cited as a heritable molecular characteristic which, at least in many instances, distinguishes expressed and unexpressed genes. We have examined the pattern of DNA methylation in the region of the plnaj- and a,-globin genes during HMBA-mediated differentiation (Sheffery et al., 1982). Cytosine methylation in the nucleotide sequence, CCGG, was assayed by the use of the methyl-sensitive isoschizomer-pair of restriction enzymes, MspI and HpaII, and other restriction enzymes. There are relatively few potentially methylated sites in the MELC globin gene domains which can be assayed by these restriction enzymes. Of the sites assayed near the plnaj-globin gene, one site is fully methylated, one partially methylated, and one is unmethylated in uninduced MELC. Most cites, but not all, assayed near the a-globin genes are unmethylated in uninduced cells. No detectable change in the pattern of methylation around either gene was observed during HMBA-mediated differentiation. Globin genes, both a1and pmaJ,in nonerythroid mouse tissues display distinctly more methylation than they do in MELC (S. Einheber and M. Sheffery, unpublished observations). It would appear likely that, within the limits of resolution of this assay, the pattern of globin gene methylation in the erythroid cell lineage is established and stably propagated at a developmental stage in erythropoiesis prior to the stage represented by the MELC.
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2. DNase 1 Sensitivity Studies of the accessibility to DNase I of MELC chromatin, in the globin gene domains, has suggested that the chromatin of uninduced cells is already in a configuration compatible with transcriptional activity (Miller et al., 1978). It has been demonstrated (Sheffery et al., 1982)that the pmaJ-and alglobin gene-associated chromatin regions are distinctly more sensitive to digestion by DNase I than is the Iga (immunoglobulin) gene (which is not expressed by cells in the erythroid lineage), in both uninduced and induced MELC. As in the case of the methylation pattern, it appears that the globin gene domains have been selectively modified during the developmental history of the erythroid lineage, establishing the globin-related chromatin in a potentially active configuration. These changes, as in the case of the methylation pattern, are stably propagated in uninduced MELC.
3. Nuclease Hypersensitivity Sites We have obtained evidence for alterations in chromatin structure which are specifically associated with inducer-mediated activation of globin gene transcription (Sheffery et al., 1982, 1983). During HMBA-induced differentiation, sites displaying a 6- to 10-fold increase in DNase I sensitivity appear in chromatin regions near the 5‘ end of the a 1and pmajgenes. The DNase I hypersensitive site which appears near the pmaJ-globin gene maps to an approximately 200 base pair region in the 5’ flanking region of that gene. That hypersensitivity site which becomes detected near the al-globin gene likewise is located in the region 5’ to the a1cap site. The changes in chromatin structure which are revealed by nuclease probes during induced differentiation are more complex, however, than simply the reconfiguration of sites at the 5’ end of the globin genes. As noted already, it has been demonstrated that there is a DNase I hypersensitive site, located within the second IVS of the pmaJ gene, which can be detected in uninduced MELC. The nuclease hypersensitivity at this site disappears during HMBA-mediated differentiation, replaced by the new hypersensitivity site which lies 5’ to the pmaJcap site. Although this complex change in chromatin configuration normally takes place in a coordinate fashion, just prior to the initiation of globin gene transcription, it has been shown (Sheffery et a l . , 1983) that in an inducer-resistant variant these two changes in chromatin configuration can be dissociated. In the R 1 variant of MELC, which is resistant to the inducing effect of HMBA on globin gene expression and commitment (Marks et al., 1983), HMBA mediates the disappearance of that hypersensitivity site located in IVS-8 but fails to generate the new site, located 5’ to the PmaJcap site, and fails to initiate transcription at the pmaJ gene.
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At the a, domain there is also a complex pattern of chromatin reconfiguration that occurs during induced differentiation (Sheffery et al., 1984). The uninduced MELC, overlapping DNase I and S1 nuclease-sensitive sites are detected 5' of the a-globin gene cap site. During induction the nuclease sensitivity of these sites increases and new, nonoverlapping DNase I and S1 sites develop, one approximately 300 base pairs 5' of the a,-globin cap site, and the other mapping to a region virtually coincident with the cap site itself. None of these changes in nuclease sensitivity occurs in the HMBAresistant MELC variant (Rl), suggesting that there may be significant differences between the chromatin-associated events that take place in the aand (3-globin gene domains during induced differentiation. IV. Differentiation and the Cell Cycle
Inducer-related events associated with the cell cycle, with DNA synthesis, and, in particular, with replication of the globin genes appear to be important in the induction of MELC differentiation and accelerated transcription of the globin genes (Gambari et al., 1979; Brown and Schildkraut, 1979; Epner et al., 1981; Levy et al., 1975). Studies in this laboratory have shown that inducing agents must be present during at least one cell cycle to initiate differentiation (Levy et al., 1975)and that late GI-early S may be the period of particular significance for subsequent globin gene expression (Gambari et al., 1978, 1979). Since DNA replication has been implicated in the transition from an inactive to an active chromosome structure in several systems (Groudine and Weintraub, 1981; Holtzer et al., 1972; Dienstman and Holtzer, 1975), it has been speculated that the changes in globin gene chromatin configuration which are associated with enhanced globin gene expression in MELC may be introduced during replication of the genes, and that the presence of the inducer at a restricted time in the cell division cycle may be critical to the reconfiguration of these gene domains. In this context, it has been found that genes whose expression is stimulated during MELC differentiation (the a- and (3-globin genes) are replicated during early S (Epner et al., 1981; Furst et al., 1981). Each of these aspects of induced differentiation is discussed below.
A. THE CELLCYCLEA N D INDUCED DIFFERENTIATION Evidence suggesting a relationship between the cell cycle (possibly DNA synthesis), and the onset of erythroid cell differentiation, particularly the onset of globin gene expression, in normal and transformed erythroid cells, has come from a number of studies. It was demonstrated a number of years ago that one effect of erythropoietin on normal erythropoietin-sensitive tar-
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get cells (probably the CFUe) is an acceleration of DNA synthesis which precedes the accumulation of globin messenger RNA (Djaldetti et a l . , 1972; Rainirez et al., 1975). Studies using inhibitors of DNA synthesis have added to this evidence implicating DNA synthesis and the cell cycle in the initiation of globin gene expression during normal erythropoiesis (Marks et al., 1977). Additional evidence has come from studies on MELC differentiation. For example, when cultures are initiated at high cell densities, the time required for cell doubling is prolonged and the extent of differentiation is decreased; under these conditions, the higher the rate of cell proliferation, the greater the proportion of cells induced to differentiate (Rifiind and Marks, 1982). More direct evidence for a role of the cell cycle in induced MELC differentiation comes from studies with synchronized cell populations, employing several independent methods for the induction of synchronized cell growth. Using 2 mM thymidine to inhibit progression of the cells past the G,/S interface, Levy et al. (1975) demonstrated that induction requires exposure to inducing agents as the cells move through at least one cell cycle. This conclusion has been reached, as well, using synchronization by nutrient deprivation (McClintock and Papaconstantinou, 1974) and by the use of a temperature-sensitive cell cycle mutant (Harrison, 1977). Geller et al. (1978) showed that commitment to initiate terminal cell division and other features of the differentiated phenotype is accomplished most quickly by cultures of MELC synchronized in the G, or G, phase of the cell cycle, suggesting that early S phase is important for induction. More recently, using thymidine, thymidine plus hydroxyurea, or centrifugal elutriation for the synchronization of MELC in G, or at the G,/S interface, it was demonstrated that exposure to inducer during one cell cycle resulted in the initiation of accumulation of globin messenger RNA during the subsequent cell cycle, beginning during G,. The critical time in the cell cycle for subsequent initiation of globin messenger RNA accumulation is G, or early S (Gambari et al., 1978, 1979). Finally, using the phorbol ester, an inhibitor of induced differentiation, it was demonstrated (Gambari et al., 1980) that the TPAsensitive period for inhibition of globin synthesis is confined to that cell cycle in the presence of inducer which precedes the onset of globin mRNA accumulation. Evidence suggesting that it is the onset of globin gene transcription, and not merely the accumulation of globin messenger RNA, that is sensitive to a cell cycle-related effect of inducers has been obtained from studies examining the kinetics of globin mRNA accumulation during induction with various inducing agents. When M ELC are induced to differentiate by polar-planar compounds such as HMBA and Me,SO, the first accumulation of globin mRNA is detected approximately 12 hr after the initiation of culture with
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inducer (the cell division cycle is approximately 12 hr). On the other hand, when MELC are induced to accumulate globin mRNA by exposure to hemin, mRNA accumulation can be detected in less than 6 hr and under certain circumstances in as little as 2 hr, a time too short for a significant cell cycle requirement (Nude1 et al., 1977b). As already described (see above) the initiation of globin mRNA accumulation by hemin is accomplished by a posttranscriptional effect upon a constitutive low rate of globin gene transcription and is accompanied by no detectable increase in transcription at the globin gene loci. Taken together these observations suggest that the cell cycle dependence of inducer effect is related to the transcription-modulating effects of these agents; an agent which modifies globin gene expression through a posttranscriptional mechanism, such as hemin, functions in a manner which is, apparently, independent of the cell division cycle.
B. GLOBINGENEREPLICATION Taken together, studies using both inducers of differentiation and inhibitors of differentiation have suggested that there is a requirement for the cell cycle for induced differentiation and the critical period may lie in late G, or early S phase. Based upon speculation that reconfiguration of globin geneassociated chromatin during replication in the presence of chemical inducers of differentiation may be critical for induced gene transcription, studies were undertaken to define the time in the cell cycle during which both a-and pglobin genes undergo DNA replication (Epner et al., 1981). For these studies, centrifugal elutriation (based upon cell volume) was employed for cell synchronization in order to avoid artifacts introduced by the use of inhibitors of DNA synthesis. Newly replicated DNA sequences were prepared from synchronized cells cultured for short periods with 5-bromodeoxyuridine, and the BUdR-containing, newly replicated DNA isolated by CsCl gradient centrifugation and analyzed by hybridization with cloned probes for the aand p-globin gene sequences. Both a- and p-globin gene sequences, in MELC, are replicated early in S phase, whereas ribosomal RNA gene sequences are replicated throughout the cell cycle. These observations are consistent with the speculation that gene replication may be an important event, implicated in the reconfiguration of globin genes required for the activation of transcription. V. Summary
Study of inducer-mediated differentiation of murine erythroleukemia cells provides insights into the cellular and molecular mechanisms implicated in cell differentiation. The loss of proliferative capacity is revealed to be a
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complex multistep process during which the cells progress through a series of stages, including a precoinmitment “initiation” stage, a stage suggestive of the accumulation of commitment-related factors, and, finally, a stage of expression of the characteristics of the differentiated state. Cell cycle arrest in G, phase of the cell cycle may, in part at least, be related to downregulation of protein p53 synthesis. Expression of induced differentiation is accompanied by an acceleration of transcription at the globin loci, and possibly by posttranscriptional modulation of globin mRNA accumulation, as well. Cells at the stage of erythroid cell development represented by the transformed, differentiation-arrested MELC, have acquired a unique DNA structure and chromatin configuration around the globin genes which distinguish them from other, nonerythroid cells; additional complex changes in chromatin configuration accompany, and probably precede, inducer-mediated acceleration of globin gene transcription during terminal differentiation. Passage through G, and early S phase of the cell cycle, in the presence of inducer, is critical for subsequent globin gene expression and may be important in establishing the chromatin reconfiguration required for gene expression.
ACKNOWLEDGMENTS These studies were supported, in part, by grants from the National Cancer Institute (PO1 CA-31768 and CA-08748) and the Bristol-Myers Cancer Research Program.
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