Regulation of Tissue-Specific Gene Expression in Microcell Hybrids

METHODS: A Companion to Methods in Enzymology 9, 30–37 (1996) Article No. 0005

Regulation of Tissue-Specific Gene Expression in Microcell Hybrids Mathew J. Thayer Department of Molecular and Medical Genetics, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, Oregon 97201

Microcell-mediated chromosome transfer has been used to study genetic loci that can either activate or repress tissuespecific gene expression. We have used microcell hybrids to study activation of the muscle phenotype, as well as to identify a genetic locus responsible for inhibiting expression of the myogenic determination gene MyoD in primary fibroblasts. We have shown that microcell transfer of human fibroblast chromosome 11, which contains the MyoD locus, into the embryonic fibroblast cell line 10T1/2 results in activation of human MyoD and, consequently, activation of the entire muscle program of differentiation. In addition, chromosome segregation analysis indicates that the continued presence of human chromosome 11 is not required for maintenance of the myogenic phenotype. In contrast, whole-cell hybrids between 10T1/2 cells and primary nonmuscle cells fail to activate the muscle phenotype, suggesting the presence of MyoD inhibitory loci. Microcell hybrids retaining human fibroblast chromosome 4 fail to activate MyoD expression. Analysis of chromosome fragment-containing hybrids localizes the repressing activity to a small region on 4p. q 1996 Academic Press, Inc.

Patterns of tissue-specific gene expression can be altered experimentally by crossing mammalian cells of different types. Long before molecular cloning techniques were available, somatic cell hybridization experiments were utilized to study cellular differentiation. These methods continue to be important tools for the study of determination and differentiation of mammalian cells. In general, hybrid cells formed by fusing dissimilar cell types fail to express the tissue-specific products of either parent, a phenomenon known as extinction (reviewed in 1). Extinction of differentiated traits was well documented by Weiss and collaborators, who have studied expression of liver-specific traits in hybrids formed by fusing hepatoma cells with a variety of other cell types (2–5). That extinction of particular hepatic traits can be mediated by discrete, trans-dominant, genetic loci was first demonstrated by Killary

and Fournier (1984). Extinction is not an irreversible change: hybrid segregants that have lost extinguisher loci reexpress previously extinguished traits (6, 7). In addition, activation of previously silent tissue-specific products encoded by heterologous parental genomes has been observed (6, 8–11). Historically, the phenomenon of gene activation in somatic cell hybrids was observed in chromosomally unbalanced hybrids or in hybrids that had segregated some but not all of the chromosomes of one parent. In either case, extinction of tissue-specific functions does not occur, and heterologous gene activation results. Therefore, activation can occur when there is an absence of extinction. This article is a brief review of the methods and cellular phenotypes observed in microcell hybrids between primary skin fibroblasts and 10T1/2 cells.

ACTIVATION OF TISSUE-SPECIFIC GENE EXPRESSION Microcell hybrids have been used to study commitment and differentiation of muscle cells (11). This system takes advantage of a remarkable property of immortalized mouse embryo fibroblast cell lines. Taylor and Jones (1979) showed that incorporation of 5-azaCytidine (azaC) into the DNA of the mouse embryonic fibroblast cell line C3H10T1/2 (10T1/2) can generate colonies capable of forming muscle, fat, and cartilage. Demethylation and subsequent expression of specific loci involved in determining each cell type are thought to be involved in the conversion process (12). The frequency of myogenic conversion (as high as 50%) after treatment with azaC is consistent with the activation of a small number of genes, possibly even a single gene (13). Genomic DNA transfection experiments indicate that myoblast DNA, but not 10T1/2 DNA, converts 10T1/2 cells into stably determined myoblasts at frequencies consistent with transfer of a single genetic locus (14, 15). The conversion of 10T1/2 cells to muscle

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1046-2023/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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by transfection of myoblast DNA but not 10T1/2 DNA supports the hypothesis that a myogenic determination gene that is inactive in 10T1/2 cells becomes structurally modified during conversion of 10T1/2 cells to the myoblast lineage following treatment with azaC. The genomic transfection experiments also suggest that 10T1/2 cells do not express trans-acting factors that repress the transferred myogenic determination gene. The conversion of 10T1/2 cells to muscle with azaC, as well as with transfected genomic DNA isolated from myoblasts, indicates that a muscle regulatory locus is negatively regulated by a cis mechanism (possibly by methylation) in these cells and that 10T1/2 cells do not contain trans-negative regulators for muscle committment. To determine directly whether MyoD expression is repressed in cis or in trans in primary nonmuscle tissue, we constructed 10T1/2 microcell hybrids retaining primary human fibroblast chromosome 11 (which contains the human MyoD locus) (16). We assumed that if human MyoD expression is repressed in cis in primary fibroblasts, then human MyoD would remain inactive when human chromosome 11 is transferred into 10T1/2 cells. In contrast, if expression of human fibroblast MyoD is repressed in trans, and if 10T1/2 cells fail to express a trans-acting inhibitor of MyoD expression, then human MyoD would be expressed when human chromosome 11 is transferred into 10T1/2 cells. Microcell Fusion There are many biochemical markers whose expression can be selectively fixed in cultured cells; however, specific auxotrophic mutants are generally required. Therefore, the most commonly used approach for fixing different donor chromosomes in microcell hybrids makes use of cloned dominant selectable markers. These markers are used to ‘‘tag’’ donor chromosomes by random insertion of recombinant retroviral vectors or by insertion of transfected DNA. This strategy allows one to transfer previously nonselectable chromosomes from virtually any cell line into another (17). Previous studies on the insertion of recombinant retroviral vectors into specific human chromosomes had generated a mouse Swiss 3T6 (HGPRT0) cell line (HDm-18) containing human fibroblast chromosome 11, into which the retroviral vector ZIPneoSV(X)1 was integrated (17). Microcell hybrids retaining human chromosome 11 were generated by fusing HDm-18 microcells with 10T1/2 recipient cells using concanavalin A coated bullets as described in Killary and Lott (17A). Briefly, HDm-18 cells were exposed to 0.06 mg/ml colcemid for 48 h and enucleated by centrifugation at 29,000g for 30 min. Microcells were collected and filtered through an 8-mm filter and fused to 10T1/2 recipient cells by exposure to 50% PEG for 60 s on 25-cm flasks. Following selection in G418 and HAT containing

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medium, seven individual clones [designated 10T1/ 2(11n)1 through 7] were isolated using cloning rings. Each clone was expanded into 150-cm plates and the cells extracted for RNA and DNA. The presence of human MyoD was confirmed in all 10T1/2(11n) clones by Southern blot hybridization (Fig. 1). Determination of Celluar Phenotype One important criterion for determining cellular phenotype is a reliable assay. The muscle phenotype is a particularly easy one to score, since muscle cells grown in culture will fuse to form multinucleated myotubes. We have used this morphological assay combined with immunohistochemistry to score cells for the muscle phenotype. To determine whether the microcell hybrids generated above were myogenic, each clone was plated at clonal density on 150-cm plates and grown to approximately 104 cells per colony. The cultures were shifted to differentiation inducing media (DMEM plus 2% horse serum) and scored for myosin heavy chain expression using alkaline phosphatase-linked immunostaining. All seven 10T1/2(11n) clones fused to form myosin heavy chain-positive multinucleate myotubes when cultured under differentiation-inducing conditions. Thus, transfer of human fibroblast chromosome 11 into 10T1/2 cells is sufficient to activate the muscle program. In order to assess activation of a particular gene product in interspecific hybrids, a species-specific assay must be used. To determine whether activation of the human MyoD locus had occurred in the 10T1/2(11n)

FIG. 1. Retention of human MyoD genomic sequences in 10T1/ 2(11n) series microcell hybrids. Parental and hybrid cell DNAs (5 mg) were digested to completion with HindIII, electrophoresed on agarose gels, and transferred to a nylon membrane. The blot was hybridized with labeled mouse MyoD cDNA probe. Lane designations: HSF, human skin fibroblast; 10T1/2, 10T1/2; 1–7, 10T1/2(11n)1 through 7. Human-specific restriction fragments are indicated.

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series clones, expression of human MyoD RNA was assayed by RNase protection. The probe was a uniformly 32 P-labeled T3 RNA transcript corresponding to the 3* end of a human MyoD cDNA (18). This probe detects an RNA species of approximately 195 nucleotides in the human rhabdomyosarcoma cell line RD (Fig. 2, lane R). As expected, this probe fails to detect MyoD transcripts in primary human skin fibroblast RNA (Fig. 2, HSF); the probe also fails to detect mouse MyoD transcripts expressed in the myoblast cell line F3; thus it detects only human MyoD RNA. The probe detected RNA species coincident with human MyoD transcripts in all seven 10T1/2(11n) hybrids (Fig. 2, clones 1–7). Thus, the human fibroblast MyoD gene was activated in myogenic 10T1/2 microcell hybrids retaining human fibroblast chromosome 11. Another method for assaying species-specific gene products is to utilize species-specific probes on Northern blot hybridizations. Because expression of MyoD is subject to positive autoregulation in 10T1/2 cells (19), the 10T1/2(11n) hybrids were tested for activation of the mouse 10T1/2 MyoD gene. Figure 3 shows mouse MyoD RNA expression in 10T1/2(11n)-5 by Northern blot hybridization. The probe corresponded to the 3* end of the mouse MyoD cDNA. This probe does not detect human MyoD RNA (data not shown). Consistent with the observation that MyoD is subject to autoacti-

FIG. 2. Expression of human MyoD mRNA in the 10T1/2 microcell hybrids. RNase protection assay on 5 mg of cytoplasmic RNA extracted from cells in growth medium (G) or from cells in differentiation medium (D) for 4 days. The uniformly labeled T3 RNA probe corresponds to the 3* end of the Myf-3 cDNA. This probe detects a fragment of approximately 195 nucleotides from human MyoD mRNA, but does not detect mouse MyoD mRNA. Lane designations: HSF, human skin fibroblasts; 10T1/2, 10T1/2; 1–7, 10T1/2(11n)-1 through 7; R, human rhabdomyosarcoma cell line RD; F3, aza-myoblasts; M, marker.

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vation, mouse MyoD mRNA was easily detected in this clone.

SEGREGATION ANALYSIS The loss of chromosomes of one of the parental cells from somatic cell hybrids is termed chromosome segregation. Chromosome segregation has been used to observe reexpression of previously extinguished traits, as well as to observe activation of previously silent gene products (reviewed in 20). To determine whether activation of the mouse MyoD gene, mediated by transfer of human chromosome 11, results in stable expression of mouse MyoD, we generated hybrid segregants that had lost human chromosome 11. 10T1/2(11n)-5 was propagated under nonselective conditions for 2 weeks to permit segregation of human chromosome 11 and plated at clonal density, and subclones were isolated. To determine which subclones had segregated chromosome 11, each subclone was plated onto two 60-mm dishes, one containing G418 and the other without G418. Four subclones, designated 10T1/2(11n)-5k, 5l, 5n, and 5p, displayed no growth in G418-containing media and therefore displayed the phenotype (G418s) expected if chromosome 11 were lost. To determine whether these subclones had lost the human MyoD gene, Southern blot analyses were performed that

FIG. 3. Expression of mouse MyoD in 10T1/2 microcell hybrids and segregants. Northern analysis (5 mg of total RNA) of mouse MyoD and myosin light chain 1/3 (MLC) expression in growing (G) or differentiated (D) cultures of F3 aza-myoblasts, 10T1/2, 10T1/2(11n)-5, and the G418s segregant 5n. The probe corresponded to the 3*-untranslated region of the mouse MyoD cDNA. The bottom panel shows an ethidium bromide stain of the samples used for the Northern analysis.

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showed that all four G418s segregants had lost the human-specific MyoD restriction fragments (data not shown). Expression of mouse (10T1/2) MyoD RNA was determined in the G418s subclone 10T1/2(11n)-5k using the mouse-specific probe by Northern blot hybridization. Expression of mouse MyoD RNA was detected in segregant 5k (Fig. 3). Furthermore, the continued expression of MyoD RNA correlated with the continued expression of the muscle phenotype: subclone 5k continued to express myosin light chain 1/3 (Fig. 3). A second round of subcloning showed that the myogenic segregants 5k and 5n gave rise to only myogenic subclones and retained the muscle phenotype when cultured continuously for more than 60 population doublings. Thus, once activated in trans by human chromosome 11, mouse MyoD and the muscle phenotype are stably expressed.

REPRESSION OF TISSUE-SPECIFIC GENE EXPRESSION Activation of human MyoD in the 10T1/2 microcell hybrids is consistent with the possibility that factors present in 10T1/2 cells are capable of activating a previously silent MyoD gene. Alternately, activation of human MyoD expression might be due to the absence of trans repression in 10T1/2 cells. To test these possibilities further, whole-cell hybrids, formed by fusing 10T1/ 2 cells with primary human fibroblasts, were prepared.

FIG. 4. Retention of human and mouse MyoD genomic sequences in the 10HSF series hybrids. Southern analysis of DNAs (5 mg) extracted from 10T1/2, human skin fibroblasts (HSF), and 10HSF-a through g (a–g). The blot was probed with labeled mouse MyoD cDNA. Human-specific restriction fragments are indicated.

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For this analysis, a hygromycin-resistant transfectant of 10T1/2 cells was fused with G418-resistant primary human skin fibroblasts. Both cell types were plated to a combined density of approximately 70% confluence in 25-cm2 flasks and fused by exposure to 50% PEG for 60 s. Following selection in hygromycin and G418, seven hybrids, designated the 10HSF series clones, were isolated and expanded. Six of the 10HSF hybrid clones were screened for retention of human chromosomes by staining fixed metaphase spreads with alkaline Giemsa, which differentiates human and rodent chromosomes (21). All six clones exhibited complex karyotypes with six or more human chromosomes retained at high frequency, plus a full complement of 10T1/2 chromosomes. In addition, all seven 10HSF clones retained both mouse and human MyoD restriction fragments, as assayed by Southern blot hybridization (Fig. 4). However, in contrast to the 10T1/2(11n) hybrids, which contain only human chromosome 11, none of the 10HSF series clones expressed significant amounts of human MyoD RNA (Fig. 5), and all were suppressed in their myogenic potential. To identify the human fibroblast chromosome(s) involved in inhibiting MyoD expression in the 10T1/2 1 HSF hybrids, karyotypic analyses were performed. The human chromosomes retained in each clone were identified by screening genomic Southern blots with probes corresponding to genes or random unique sequences known to reside on particular human chromosomes (Table 1). In addition to the whole-cell hybrid clones, the 10HSm microcell hybrids, generated by transfer-

FIG. 5. Expression of human MyoD mRNA in the 10HSF series whole-cell hybrids. Human-specific RNase protection assay on 5 mg cytoplasmic RNA extracted from cells in growth medium (G) or in differentiation medium (D) for 4 days. Lane designations: HSF, human skin fibroblasts; 10T1/2, 10T1/2; a–g, 10HSF-a through g; R, human rhabdomyosarcoma cell line RD; F3, 10T1/2 derived aza-myoblasts, tRNA, and M, marker.

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ring random chromosomes to 10T1/2 cells, were also analyzed (Table 1). All of these hybrids displayed a reduced level of myogenesis compared to that in the chromosome 11 monochromosomal hybrids. Chromosome 8 was present in all eight lines that retained human chromosome 11. Interestingly, chromosome 4 was also highly represented, being present in seven of eight chromosome 11 containing nonmyogenic lines; however, alkaline Giemsa staining shows that the one discordant line, 10HSF-d, does retain several unidentified chromosomal fragments that may come from chromosome 4. Thus, these data suggested that either human fibroblast chromosome 4 or 8 was involved in inhibiting MyoD expression.

microcell donors in fusions with 10T1/2 cells. These microcell fusions generated the 10T1/2(4n) and 10T1/ 2(8n) series microcell hybrids, containing human fibroblast chromosomes 4 and 8, respectively. Because microcell fusion often generates hybrids that contain chromosome fragments (22), we expected that only a fraction of the hybrids in either series would retain the putative inhibitory locus. To test the effects of the introduced chromosomes on MyoD expression, the 10T1/2(4n) and 10T1/2(8n) hybrids were tested for the ability to be converted to muscle following treatment with azaC. Since the endogenous MyoD gene is known to be activated in 10T1/ 2 cells treated with azaC (19, 23), and this activation results in conversion of these cells to muscle, a failure of the hybrids to convert to muscle after azaC treatment would indicate the presence of an inhibitory locus. Cultures of each hybrid clone were exposed to azaC for 24 hr, plated at clonal density, and grown to approximately 104 cells per colony. Conversion to the muscle phenotype was assayed by myosin heavy chain immunostaining. Four of five clones from the 10T1/2(4n) series hybrids could not be converted to muscle at a frequency similar to control 10T1/2 cells (Fig. 6). The relatively low (1–5%) but detectable myogenic conversion of some of the chromosome 4 hybrids, such as 10T1/2(4n)-3, -6, -8, and -10, is due to loss of chromo-

Repression of MyoD by Fibroblast Chromosome 4 To further dissect the role of chromosomes 4 and 8 in inhibition of MyoD expression, each chromosome was transferred individually into 10T1/2 cells. Previous studies, regarding insertion of transfected DNA into specific human chromosomes, had generated cell lines containing neo insertions into human chromosomes 4 and 8 (A.M. Killary, unpublished observations). These cell lines, HA(4)-A (mouse A9 cells containing a pSV2neo insertion in human fibroblast chromosome 4) or HA(8)-A (mouse A9 cells containing a pSV2neo insertion in human fibroblast chromosome 8), were used as

TABLE 1 Karyotypic Analysis of Human Fibroblast 1 10T1/2 Hybrids and Microcell Hybrids Human chromosomes retained Clone

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

X

% MHC /

Microcell hybrids 10HSm-2 10HSm-3 10HSm-4 10HSm-6 10HSm-7 10HSm-8 10HSm-9 10HSm-10 10HSm-11 10HSm-13 10HSm-14 10HSm-15 10HSm-16 10HSm-18

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 / 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

/ 0 0 / / / 0 0 0 0 / / 0 0

0 0 0 / 0 0 / 0 0 0 0 0 0 0

0 0 0 / 0 0 0 0 0 0 / / 0 0

0 0 0 / / / 0 / 0 0 0 0 0 0

/ 0 0 / 0 0 0 0 0 0 / / 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 / 0 0 0 / 0 0 0

/ 0 0 0 0 0 0 0 0 0 / 0 0 0

0 0 0 / 0 0 0 / 0 / 0 / / 0

0 0 0 / 0 0 / 0 0 0 / / 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 / 0 / 0 0 / / 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 / 0 0 / 0 0 0 / / 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 / 0 0 0 0 0 0 0

0 0 0 / 0 0 / 0 0 / / 0 0 0

0 0 0 0 / 0 / 0 0 0 0 0 / 0

0 0 0 / 0 0 0 0 0 0 0 0 0 0

0 / / / / 0 / 0 / / / / / /

30 0 0 0 0 0 0 0 0 0 4 0 0 0

Hybrid clones 10HSF-b 10HSF-c 10HSF-d 10HSF-e 10HSF-f 10HSF-g

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

/ / 0 / / /

/ / / 0 0 /

0 / / / / 0

/ / / / 0 0

/ / / / / /

0 0 0 0 0 0

/ / 0 0 0 0

/ / / / / /

/ / / / 0 0

0 0 0 0 0 0

0 0 0 0 0 0

/ / 0 / 0 /

0 0 / 0 0 /

0 / 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 / 0 / 0 0

0 0 / / 0 /

/ / 0 / / /

0 / 0 / / 0

0 0 0 0 0 2

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some 4 sequences in a subpopulation of cells present in each primary hybrid clone (not shown). In contrast, all of the 10T1/2(8n) clones converted to the muscle phenotype following treatment with azaC at a frequency similar to control 10T1/2 cells. These results suggest that chromosome 4, but not 8, is involved in inhibiting the activation of MyoD and the myogenic phenotype. Expression of MyoD mRNA was affected in 10T1/ 2(4n) hybrids that had been treated with azaC. Figure 7 shows Northern blot hybridizations of RNA isolated from cultures of 10T1/2 and two 10T1/2(4n) clones treated with azaC. Consistent with the myosin heavy chain immunostaining (Fig. 6), expression of MyoD mRNA was detected in 10T1/2 and 10T1/2(4n)-1 cells treated with azaC, but not in 10T1/2(4n)-8. In addition, treatment of 10T1/2 and 10T1/2(4n)-1 cells with azaC resulted in expression of myosin light chain 1/3 (MLC) mRNA. Furthermore, consistent with a nonmuscle phenotype and a failure to activate expression of MyoD following azaC treatment, 10T1/2(4n)-8 cells also failed to express high levels of MLC mRNA. The low level of MLC gene expression seen in these cells is likely due to a minority population of segregant cells, as seen in Fig. 6. These results suggest that a locus present on human chromosome 4 inhibited activation of the mouse MyoD gene in 10T1/2 cells following azaC treatment. Incorporation of azaC into the DNA of various cell lines induces expression of a number of genes. Activation of endogenous retroviral sequences, including ev1 in chicken cells (24) and type C and intracisternal

FIG. 6. Activation of the muscle phenotype in the chromosome 4 microcell hybrids. Parental and hybrid cell cultures were treated with azaC for 24 hr, plated at clonal density, grown for 14 days, cultured for an additional 4 days in differentiation medium (DMEM plus 2% horse serum), fixed, and immunostained for myosin heavy chain by the alkaline phosphatase method. Colonies were scored as positive if they contained darkly staining multinucleated myotubes. The values represent the average of two different experiments with ú100 colonies scored for each cell line.

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type A-particle (IAP) in 10T1/2 cells (23, 25), by exposure to azaC is well documented. In addition, activation of IAP RNA in 10T1/2 cells treated with azaC is known to be independent of MyoD expression (23). Therefore, expression of IAP RNA represents a control for activation of gene expression induced by azaC that does not involve MyoD. Figure 7 (IAP panel) shows that all three cell lines, 10T1/2, 10T1/2(4n)-1, and 10T1/2(4n)-8, activate expression of IAP RNA to similar levels following treatment with azaC. These results suggest that all three cell lines incorporate azaC with similar efficiencies and that the inhibitory effects of chromosome 4 on expression of MyoD are specific.

REGIONAL LOCALIZATION OF LOCI IDENTIFIED IN MICROCELL HYBRIDS Chromosome fragmentation can occur in microcell hybrids, and these fragment-containing hybrids can be used to generate physical maps of the fragmented chromosomes (22). To determine whether the MyoD inhibi-

FIG. 7. Human fibroblast chromosome 4 inhibits muscle gene expression in azaC- treated 10T1/2 cells. Northern analysis on 5 mg of cytoplasmic RNA extracted from cells treated or not treated with azaC for 24 hr either once (11) or twice (21). RNAs were extracted from cells in differentiation medium (DMEM plus 2% horse serum) for 4 days. Northern blots were probed with cDNAs corresponding to MyoD, myosin light chain 1/3 (MLC), intracisternal type A particle (IAP), or a-tubulin (Tubulin).

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tory activity could be mapped to a specific chromosomal region, we screened the 10T1/2(4n) series hybrids for retention of specific DNA sequences, representing genes or unique sequences known to reside on human chromosome 4. Figure 8 shows a schematic representation of human chromosome 4 with approximate map positions of the markers tested. Three of the hybrids that retained the inhibitory locus, 10T1/2(4n)-3, -6, and -10, retained all markers tested and were uninformative for regional localization. However, 10T1/2(4n)1 and 10T1/2(4n)-8 retained fragments of chromosome 4. 10T1/2(4n)-1, which does not retain the inhibitory locus, retained all markers tested except the three most distal short arm markers (D4S412, MSX1 and D4S432). In contrast, 10T1/2(4n)-8, which does retain the inhibitory locus, retained only four markers, D4S397 and the three most distal short arm markers. Therefore, 10T1/2(4n)-1 and 10T1/2(4n)-8 retain a nearly nonoverlapping set of markers, having only D4S397 in common. Since 10T1/2(4n)-8 retains the MyoD inhibitory locus and 10T1/2(4n)-1 does not, the inhibitory locus must reside in the region of nonoverlap between these two hybrids. This indicates that the MyoD inhibitory locus resides on the short arm of chromosome 4 in the region of D4S412, MSX1, and D4S432.

GENERAL CONCLUSIONS AND PROSPECTS Intertypic hybrid cells provide a unique system in which entire programs of differentiation can be altered

FIG. 8. Retention of chromosome 4 markers in the 10T1/2(4n) series microcell hybrids. Schematic diagram of human chromosome 4 showing the approximate map positions of the marker loci tested. Retention of specific chromosome 4 markers in five different 10T1/ 2(4n) series hybrids is indicated. 10T1/2(4n)-3, 10T1/2(4n)-6, and 10T1/2(4n)-10 retained all markers tested. 10T1/2(4n)-1 retained all markers tested except for the three most distal short arm markers, D4S412, MSX1, and D4S432. 10T12(4n)-8 retained only four markers, D4S412, MSX1, D4s432, and D4S397.

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experimentally. Microcell hybrids with defined genotypes can be used to identify discrete genetic loci that affect tissue-specific gene expression in trans (26). One approach to the mechanisms of these phenotypic alterations involves identification of regulatory sequences, present in target genes, that are required for the observed phenotype. One can then correlate the relationsip between these sequences and other elements involved in tissue-specific gene expression. In addition, an obvious goal of these experiments is to identify the genes responsible for the phenotypic alterations. One method for characterizing genes identified by purely genetic approaches is to analyze candidate genes that map to the same chromosomal position. The chromosomal location of the human MSX1 gene, in the region of nonoverlap between the hybrids 10T1/2(4n)-1 and 10T1/2(4n)-8, together with the observation that forced expression of Msx1 in muscle cells results in a decrease in steady-state levels of MyoD mRNA (27), make MSX1 a candidate for the chromosome 4 inhibitory locus. A second method for identifying genes involved in hybrid cell phenotypes involves the use of fragment-containing hybrids and subtractive hybridization (26). This method takes advantage of the ability to define the genetic interval of interest in microcell hybrids retaining fragments of donor chromosomes. The task of identifying sequences within this interval is greatly simplified by cloning only expressed cDNAs from the region of nonoverlap. Thus, cDNAs from a fragmentcontaining hybrid are enriched for human sequences by hybridizing with RNA from a sister clone containing a smaller chromosomal fragment. The resulting enriched cDNAs can be either directly cloned or used as probe to screen existing libraries, as described in Cerosaletti and Fournier (28). Since the locus of interest will be transcribed from the region of nonoverlap between two fragment-containing hybrids, its cDNA should be enriched using this procedure. This straightforward approach provides a relatively simple method for cloning genes from within a defined chromosome segment. The ability of microcell hybrids to identify genetic loci involved in tissue-specific gene expression, combined with new molecular tools and approaches, provides a powerful approach to the identification and characterization of mammalian cell differentiation at the molecular level.

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TISSUE-SPECIFIC GENE EXPRESSION IN MICROCELL HYBRIDS 4. Weiss, M. C., Sparkes, R. S. and Bertolotti, R. (1975) Somat. Cell Genet. 1, 27–40. 5. Fougere, C., and Weiss, M. C. (1978) Cell 15, 843–854. 6. Killary, A. M. and Fournier, R. E. (1984) Cell 38, 523–534. 7. Weiss, M. C. and Chaplain, M. (1971). Proc. Natl. Acad. Sci. USA 68, 3026–3030. 8. Brown, J. E., and Weiss, M. C. (1975) Cell 6, 481–494. 9. Darlington, G. J., Rankin, J. K. and Schlanger, G. (1982) Somat. Cell Genet. 8, 403–412. 10. Pearson, S. J., Tetri, P., George, D. L. and Francke, U. (1983) Somat. Cell Genet. 9, 567–592. 11. Thayer, M. J. and Weintraub, H. (1990) Cell 63, 23–32. 12. Jones, P. A., and Taylor, S. M. (1980) Cell 20, 85–93. 13. Konieczny, S. F., and Emerson, C. P., Jr. (1984) Cell 38, 791– 800. 14. Lassar, A. B., Paterson, B. M., and Weintraub, H. (1986) Cell 47, 649–656. 15. Pinney, D. F., Pearson-White, S. H., Konieczny, S. F., Latham, K. E., and Emerson, C. P., Jr. (1988) Cell 53, 781–793. 16. Tapscott, S. J., Davis, R. L., Thayer, M. J., Cheng, P. F., Weintraub, H., and Lassar, A. B. (1988) Science 242, 405–411. 17. Lugo, T. G., Handelin, B., Killary, A. M., Housman, D. E., and Fournier, R. E. (1987) Mol. Cell. Biol. 7, 2814–2820.

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AP: Methods