Mosaicism of tyrosinase-locus transcription and chromatin structure in dark vs. light melanocyte clones of homozygous chinchilla-mottled mice

DEVELOPMENTAL GENETICS 12:393-402 (1991)

Mosaicism of Tyrosinase-Locus Transcription and Chromatin Structure in Dark vs. Light Melanocyte Clones of Homozygous chinchilla-mottled Mice SUSAN PORTER, LIONEL LARUE, AND BEATRICE MINTZ Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia

ABSTRACT The chinchilla-mottled (c'") mutation at the mouse tyrosinase-encoding locus leads to a transversely striped pattern of dark- and light-grey coat colors in homozygotes. The same basic pattern occurs in various other genotypes and has previously been found to represent the clonal developmental history of melanocytes. In a homozygote such as c'"/c'", cis-acting mechanisms would be expected to account for the color differences. To search for these mechanisms, the genomic structure of the mutation was examined and compared with the wild-type, and its function was compared in cultured melanocyte clones of the respective colors. Evidence from restriction mapping indicated that the coding region of the mutant gene resembles that of the fully and uniformly pigmented wild-type. However, the upstream sequences are rearranged in the mutation. The rearrangement be- gins 5 kb 5' of the transcription initiation site and is estimated to encompass at least 30 kb of distal upstream sequence. At least two stable functional states of the i" gene were detectable: Light-cell clones have low levels of tyrosinase-specific transcription, reduced DNAase I sensitivity of tyrosinase chromatin, and no detectable hypersensitive sites near the gene; dark-cell clones have higher (but subnormal) levels of transcription, greater sensitivity of chromatin to DNAase I, and a hypersensitive site in the promoter region. The changed re- lation between the structural gene and its upstream region may separate it from cis-acting control elements, resulting in reduced and variable ability to achieve the appropriate chromatin configuration near the time of melanocyte determination; differences in expression among clonal initiator cells are then mitotically perpetuated. o 1992 ~ i i e y - t i s s , inc.

Key words: Clonal variation, gene expression, DNAase I hypersensitive sites, matrix-associated regions

INTRODUCTION There are by now numerous studies describing mo-

lecular mechanisms that enable a given mammalian

gene to generate different products in different kinds of cells. What has gone virtually unexplored in mammals, outside of the hematopoietic system, is how a gene may contribute different variants or amounts of its product in subpopulations of the same kind of cell. When this is followed by mitotic inheritance, phenotypically different clones, termed phenoclones [Mintz, 1970,19711, are formed. Yet the phenomenon is surely widespread, and exists a t many places in the genome: we have merely to look at the coat colors of mice for examples. Multicol- ored patterns of coat melanocytes are in fact common and are encoded by single-gene mutations a t many known color loci [Silvers, 19791. These natural mosaics are of special interest as the clonal heterogeneity which they exemplify could confer selective advantages of adaptability and diversification in development and evolution [Mintz, 1971, 19741. While mosaicism may occur in all cell types, its presence in the coat is most easily detected by multicoloration. Only the X-linked patterns, seen in heterozygotes, have a known basis, in dosage compensation by single-allele activity per cell [Cattanach, 19741. The most challenging occurrences of patterns are those due to specific autosomal homozygous genes, none of which has hitherto been investigated at the molecular level.

The chinchilla-mottled (cm/cm) mouse [Silvers, 19791 presents a uniquely favorable set ofadvantages for molecular analysis of clonal variation in gene expression. The allelic wild-type gene has been isolated; the developmental coat pattern has been studied; and the melanocytes creating the pattern can be grown in culture. The cm mutation occurs a t the pivotal locus in melanogenesis, encoding the enzyme tyrosinase (EC 1.14.18.1), which catalyzes at least the first two steps in melanin synthesis from tyrosine. Tyrosinase is the only well- characterized product of any of the large number of color

Received for publication October 4, 1991; accepted October 14, 1991.

Address reprint requests to Beatrice Mintz, Institute for Cancer Re- search, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadel- phia, PA 19111.

0 1992 WILEY-LISS, INC.

394 PORTER ET AL.

MATERIALS AND METHODS Mice

Fig. 1. The chinchilla-mottled (c"/c") mouse. Transverse stripes of relatively darker and lighter pigmentation are visible in the coat on head and body.

loci in the mouse. It is expressed in melanocytes and localized in melanosomes, on which melanin is depos- ited. The pigment cells in the skin of mice reside mainly in the base of the hair follicles and secrete melanosomes into the hairs as they emerge. The tyrosinase locus, classically termed the albino- or c-locus, is on chromo- some 7; its structure has been determined [Ruppert et d., 19881 and the full-length cDNA encoding the active enzyme has been obtained [Ruppert et al., 1988; Terao etal., 19891. At least 12 alternatively spliced transcripts of the gene exist [Porter and Mintz, 19911.

The coat pattern of the cmkm mouse (Fig. 1) consists of wide transverse stripes of roughly two colors: relatively dark grey and light grey, in approximately equal frequency. The colors may be locally mismatched on left and right sides and become lighter ventrally; hence they represent clones of melanocytes originating inde- pendently from the neural crest on each side of the dorsal midline and migrating dorsoventrally. The same basic pattern has been identified in many other natural genotypes of mice, including X-linked heterozygotes, and it resembles the patterns of allophenic mice experimentally produced from aggregated early em- bryo cells of different color genotypes [Mintz, 19671. In the latter, the clonality of the stripes has been confirmed by multiple strain-specific markers [Mintz and Silvers, 19701.

We describe here the structure of the cm gene in comparison with the wild-type, and the epigenetic differences in transcription and chromatin structure of C" in darkly and lightly pigmented melanocytes that are mitotically stable in culture. The mutation is a rearrangement in the upstream sequences of the gene and appears to separate the structural gene from cis-acting control elements. The defective and variable expression of the mutant gene may be due to a randomness in ability to specify the appropriate chromatin configuration a t or near the time of melanocyte determination.

The chinchilla-mottled (c") mutation arose at the MRC Radiobiology Unit, Harwell, England, in the progeny of a neutron-irradiated hybrid male from a cross between the C3H and 101 inbred strains, mated to a tester-stock female. A more lightly mottled animal with a new c-locus mutation, extreme dilution mottled (cem), arose in the ern stock. Homozygous Pic" and ceml cem breeders were obtained from Harwell. In addition to these mutants, control material was obtained chiefly from the wild-type C3H and 101 strains and also from C57BLi6.

Cell Culture Melanocyte cultures from the skin of newborn male

c"/c" mice were established as described for wild-type melanocytes [Eisinger and Marko, 1982; Tamura et al., 19871. The cells were subcultured 3-6 weeks after CY- plantation and were split a total of 5-7 times over the first 6 months. The culture medium, unless otherwise stated, comprised Ham's F10 supplemented with 48 nM 12-0-tetradecanoyl phorbol-13-acetate (TPA), 20% fetal calf serum, 50 pg proteidml of human placental extract, 1 mM glutamine, 100 unitsiml penicillin, 100 pgiml streptomycin, and 0.1 mM db CAMP. p-MSH was added, when stated, a t a concentration of 100 nM. Cell stocks were frozen in liquid nitrogen as described [Larue and Mintz, 19901. Cloning was carried out by dilution of a cell suspension to produce 2-3 colonies per 100-mm petri dish. A single colony per dish was isolated after 3 weeks, using cloning rings, and was ex- panded. This operation was repeated a total of three times.

RNA Analysis RNA was isolated from the melanocytes by guani-

dium isothiocyanate extraction as described [Maniatis et al., 19821. Northern blotting was done on Nytran (Schleicher and Schuell) as specified by the manufac- turer.

Analysis of DNAase I Sensitivity Near-confluent cultures were harvested by scraping

and washed twice in PBS. Cell pellets were resuspended in RSB (10 mM Tris-C1 pH 7.4, 10 mM NaC1, 3 mM MgC1,) plus 0.5% NP-40, at a concentration of approximately lo7 cellsiml, and dounce-homogenized. Nuclei were pelleted by centrifugation a t 2,000 rpm for 10 minutes and resuspended a t 2 x 107/ml in cold RSB; 0.5-ml samples were incubated with increasing quan- tities of DNAase I (Boehringer Mannheim) for 10 minutes a t 37°C. The reaction was stopped by the addition of an equal volume of 1% SDS, 0.6 M NaC1, 20 mM Tris-C1 pH 7.4,lO mM EDTA, 400 pgiml proteinase K, and incubated overnight a t room temperature, or for 2

MOSAIC GENE EXPRESSION IN CHZNCHZLLA-MOTTLED MICE

hours a t 37°C. The DNA was extracted with phenol and phenolichloroform and precipitated.

For Southern analysis, 6-12 pg of DNA was digested with restriction enzyme, separated by agarose gel electrophoresis, and transferred to Nytran as recommended (Schleicher and Schuell). Blots were hybridized with RNA probes, also as recommended.

In Vitro Nuclear Matrix Binding Assay Matrices from mouse spleen were prepared and as-

sayed for DNA binding in vitro essentially as described [Cockerill and Garrard, 19861. Binding assays were performed in 100 p1 of 50 mM NaC1, 10 mM Tris-HC1 pH 7.5,2 mM EDTA, 0.25 mglml BSA with 5 ng (total) end-labelled DNA fragments and matrices from approximately lo6 nuclei. Sonicated E . coli DNA at concentrations of 100 pgiml to 500 pgiml were used as competitor. Binding was carried out at room temperature for 2 hours and the matrices were diluted and washed twice with assay buffer without DNA. Matrices with bound DNA were solubilized overnight at 37°C in the presence of 0.5% SDS and 0.4 mgiml proteinase K. After phenol extraction, the DNA was separated on 0.8% agarose gel, which was fixed and dried for auto- radiography.

RESULTS Structural Analysis of the cm Gene

The mutant gene of the cmlcm mouse was analyzed for alterations in restriction pattern relative to that of the wild-type tyrosinase gene, C, to determine if a rearrangement or deletion, particularly in the upstream region, was associated with the mutant phenotype. As the cm mutation arose in a stock derived from the C3H and 101 inbred strains, these two strains were used to describe the wild-type. The maps generated are dia- grammed in Figure 2. Restriction sites common to the C and crn genes coincide within the coding region and the sequences immediately upstream and downstream. The maps diverge (as indicated by cross-hatching) at a point 5 kb 5' of the transcription initiation site, which lies within a LINE (Ll) element (a long interspersed repetitive element) [see review by Burton et al., 19861. The internal 2 kb of the element was determined by hybridization; its predicted 5' end (for which a maxi- mum size is indicated) and 3' end are bordered by dotted lines. The rearrangement is clearly not a deletion, as the farther upstream sequences are present and un- rearranged for a t least 20 kb, from a point 2.5 kb to 5 kb 5' of the first exon. The relationship or distance between the near upstream and more distal upstream sequences is unknown.

A partial restriction map of the exon-containing c"' genomic DNA revealed no irregularities. In addition, cDNAs synthesized by the polymerase chain reaction (PCR) from cmlcm skin (data not shown) did not differ detectably in size or restriction pattern from those de-

I I I , kb 0 10 20 30

r- c (W SP s PBA S S p X H H B P S p X k XPBH E H X k B A E B

V \I l / V V I I V I I w/ I/ I I

cm

Fig. 2. Structural analysis of the cm mutation. Partial restri maps were constructed from c'"/c" and from C/C (C3H strain) type (WT) genomic DNA. The transcription initiation site is indil by an arrow above the first exon (stippled). Approximately 25 upstream sequence is included. The cm region that diverges fror wild-type in restriction pattern is cross-hatched. Restriction tested and found in both wild-type and cm/cm DNA are shown ii clear regions bounded by solid lines. Areas for which restriction were not determined are represented with dashed lines. The cm b point occurs in a LINE element (open arrow) oriented as found i wild-type; the mid-part (solid lines) was confirmed by hybridiz and the predicted ends (dotted) are indicated. Probes used in the matin analysis were derived from chznchzlla (cCh/cch) genomic phage (a gift from S. Ruppert) and are designated by solid bar beled a, b, c) under the wild-type map. Restriction sites are: A, B, BglII; Bc, BclI; E, EcoRI, H, HindII; P, PstI; S, SacI; Sp, Spl XbaI.

rived from wild-type C57BLl6 skin [Porter and Mi 19911. Therefore, the coding region appears unaffe in the cm mutation.

The altered restriction map of cm is thus consis with two possibilities: an inversion, either of a genc fragment encompassing the tyrosinase coding reg or of a t least 30 kb of upstream sequences; or an in tion or translocation of similar size.

Culture of cmkm and Wild-Type C/C Melanocj To address the molecular basis of clonal variatio

expression of the c"' gene, pigment cells of the I

tively dark and light phenotypes would have to be arately examined and compared with the wild-t This is not feasible in vivo, partly because melanoc constitute a very small fraction of the cells in the s Melanocytes were therefore cultured from the dei of dorsal (fur-bearing) skin of young cmlcm mice which the respective colored stripes could be di: guished. Two lines of CiC (C57BLi6) melanocytes, previously described [Larue and Mintz, 19901, 1 used as wild-type controls grown in the standard dium, in which they become darkly melanized.

cm/c"' light-cell clones were easily obtained; sc clones were isolated and gave rise to cell lines in w the light phenotype was maintained for as long a months in the standard medium. Putatively dark I

396 PORTER ET AL.

Fig. 3. Typical melanocytes from a dark-cell line (upper pair) and a light-cell line (lower pair) in phase contrast micrographs (A, C) and transmission electron micrographs (B, D). In the dark cells, some unpigmented premelanosomes and various stages of pigmented mel-

did not become notably melanized unless the medium was transiently supplemented with 100 nM melanocyte-stimulating hormone (P-MSH) or the concentration of dibutyryl cAMP (db CAMP) was raised from 200 nM to 400 nM. Under these conditions, dark cells be- gan to appear after two passages and seven dark clonal cell lines were derived from an MSH-treated culture; all subsequently remained stably pigmented for up to 5 months in the standard medium after only the initial exposure to the inducer. MSH is known to increase melanogenesis through various pathways, including stimulation of tyrosinase specific activity [ Wong and Pawelek, 19731 which is mediated in part by increased tyrosinase transcription [Hoganson et al., 19891. The hormone reportedly acts by increasing cAMP synthesis [Johnson and Pastan, 19721 or agents that raise intra- cellular levels of CAMP. The cmlcm dark cells thus have a higher threshold than wild-type for induction of tyrosinase transcription by trans-acting factors; nevertheless, after brief exposure, they are still “marked” as dark in some way established early in their developmental lineage. At least two cell lines of each cmkm color phenotype, as well as wild-type, were used for molecular analyses. No significant differences within any of the groups were found.

Cells of the light and dark cmlcm lines remained clearly distinguishable from each other, and from wild- type. Typical cmlcm examples are seen with light and transmission electron microscopy in Figure 3. The ap- proximate numbers and distribution of melanosomes in the cells are similar but the dark cells have a larger proportion of more fully pigmented melanosomes, rel-

anosomes are present; in the light cells, unpigmented premelanosomes predominate and some lightly pigmented melanosomes are seen. A, C, x 330. B, D, ~22 ,000 .

ative to unpigmented premelanosomes, than do the light cells. While the dark melanocytes are not as dark as the wild-type, they more closely resemble them than do the light cells; the latter are more similar to, but slightly more pigmented than, albino melanocytes (data not shown). The population doubling times of the light-cell lines were slightly less than those of the dark-cell lines (e.g., 75 hours and 90 hours, respec- tively, for one light and one dark clone). No cell lines formed foci and none were able to grow in the absence of TPA. In general, the growth characteristics of the cells did not differ significantly from those of other un- transformed melanocyte lines established here from a variety of mouse color genotypes and strains.

Expression of the cm Gene To determine if the different color phenotypes due to

cm might result from differences in the amount of tyrosinase RNA, total RNA from the cultured melanocytes was analyzed by Northern blot. The results, shown in Figure 4, clearly indicate that the level of tyrosinase message in the cmlcm phenotypically light clones is significantly less than in the phenotypically dark clones; tyrosinase expression in the dark clones is in turn less than in the wild-type. These differences follow the rank order of pigmentation in the cultured cells: 14 pg melaninilight cmlcm cell, 160 pgidark cmkm cell, and, as previously reported [Larue and Mintz, 19901, 229 pgiwild-type cell. They also reflect the relative differences in the original coat colors and in total tyrosinase expression in skin from the mice (data not shown).

MOSAIC GENE EXPRESSION IN CHINCHILLA-MOTTLED MICE 397

1 1 1

28s -

18s -

p-actin - Fig. 4. Northern blot analysis of wild-type and c"/c" tyrosinase

RNA. Total RNA (10 pg) from wild-type (WT) cultured melanocytes, and from one clone of light and one of dark c"/c" melanocytes, were hybridized on a Northern blot with a tyrosinase cDNA anti-sense RNA probe. The tyrosinase RNA level decreases from wild-type to dark c"/c" and is barely detectable in light c"/c". Hybridization to a p-actin probe was used as a loading control.

DNAase I Hypersensitivity of the Proximal Promoter Region

At least two transcriptional states of the c"' gene apparently coexist in the animal, in pigment cells of identical genotype and environment. Because the defect occurs in cis, it was of interest to learn whether differences in the chromatin structure associated with the mutation could account for functional variability of the gene. We therefore investigated whether the exis- tence or pattern of DNAase I hypersensitive sites in the promoter region differed between genotypes (CIC and cm/cm) and between phenotypes (light and dark c"1 c"' melanocytes). Nuclei were isolated from the melanocyte cell cultures and incubated with increasing quan- tities of DNAase I. The DNA was purified, digested with EcoRI, and processed for Southern analysis. The blots were probed with a sequence abutting the 5' end of the genomic EcoRI restriction fragment encompassing the first exon (probe b of Fig. 2). The results (Fig. 5) reveal a hypersensitive site within 100 bp of the major transcription initiation site [Ruppert et al., 19881 in the wild-type; the site is notably diminished or absent in the light cmlcm cell clones but present approximately normally in the dark cmlcm cell clones. Other, much weaker, hypersensitive sites were sometimes visible farther upstream (within 2 kb) of the primary site and in the first intron, but a comparison between cell clones was not feasible.

To ensure that absence of the major hypersensitive

P/P PIP WT light dark -n-

DNAasel - - - 4.8 -

2.4 - 4

Fig. 5. DNAase I hypersensitivity of wild-type and em tyrosinase promoters. Nuclei were prepared from light and dark c"/c" melanocyte clones, and from wild-type melanocytes, and digested with increasing concentrations (arrows) of DNAase I at 37°C for 10 minutes. DNA was extracted and digested to completion with EcoRI. After electrophoresis and transfer to Nytran, the samples were hybridized with an RNA probe (probe b in Fig. 2) recognizing the 5' 370 bp of the genomic EcoRI fragment encompassing the first exon (the 4.8 kb main band). The sub-band which maps to within 100 bp of the transcription initiation site (arrowhead) is present in the wild-type and dark c"/c" melanocyte clones but reduced or absent in the light c"/c" melanocyte clone.

site in the light clones was specific for their cmlcm phenotype, DNAase I hypersensitivity was examined in the ribosomal protein S16 gene. As seen in Figure 6 , two relevant sub-bands mapping in the upstream por- tion of the S16 gene appear at similar intensities and positions in the light as well as the dark cmlcm cells.

Upstream DNAase I Hypersensitivity As the c"' proximal promoter and coding region is

separated from the DNA sequence that normally re- sides >5 kb upstream of the gene, it was important to analyze this region of the wild-type for evidence of regulatory or structural function. DNAase I hypersensitivity of the region was therefore examined. As seen in Figure 7, there is in fact a strong hypersensitive site in the wild-type DNA a t approximately 15 kb upstream from the transcription start site that is absent in the equivalent region (that is, within 25 kb of the upstream sequence) of c"' (Fig. 7A). While the DNA sequence which generated this hypersensitive site is not expected to be present in the cm restriction fragment analyzed, the question was whether a hypersensitive site may have arisen here fortuitously or by long-range interaction with tyrosinase sequences.

Of further interest was whether the specific sequence corresponding to the - 15 kb hypersensitive site is still hypersensitive in the cmlP cells. A Southern blot (Fig. 7B) of PstI-cut DNA, hybridized with probe a of Figure 2, reveals that this sequence (up to 500 bp in length) is in fact hypersensitive in both light- and dark-cell cmlcm

398 PORTER ET AL,.

light dark light dark A

DNAase I - DNAasel - 0- 0-

9 4 - 6 6 -

2.0 -

1.4 - 4 4

23 - 19 - 17 - 15 - 12 - 10 -

6.6-

Fig. 6. DNAase I hypersensitivity of a control gene the ribosomal protein S16 gene Nuclei were prepared and digested with DNAase I as in Figure 5 DNA was restricted with Hind11 and hybridized with a probe from the second intron of the ribosomal protein S16 gene The indicated hypersensitive sites (arrowheads) are present a t equivalent intensities in all cell lines and are located upstream of the first exon __lll

Cm/@ Cm/P WT light dark B

chromatin a t a site equivalent to the wild-type. The DNAaSel -n n ever, weaker than that in the dark cells. hypersensitivity in the light-cell chromatin is, how- 23 -

9 4 - 6 6 -

4.4 - General Sensitivity to DNAase I at the Tyrosinase Locus

7

Are the DNAase I hypersensitivity differences between the light- and dark-cell em proximal promoter chromatin reflected in the overall sensitivity of the locus to DNAase I? In other words, is a more compact chromatin structure responsible for non-permissive- ness or inaccessibility of the locus to trans-acting factors? Figure 8 illustrates the relative sensitivities of the tyrosinase-locus chromatin in the wild-type, and in dark and light P I C m melanocytes, relative to their in- trinsic ribosomal protein (L32), immunoglobulin (Cp), or actin genes. Incremental DNAase I digestions were performed on nuclei, and DNA was processed for South- ern analysis. In the cm/cm light cells, an 8-kb HindIII restriction fragment encompassing exon 2 was analyzed in comparison with the 3.2-kb HindIII L32 fragment and the 2.2-kb HindIII C p fragment. In a separate DNAase I series, a 4-kb BgZII fragment including exon 1 (and excluding non-transcribed sequences) was compared to a 3.7-kb p-actin fragment. In each case, the tyrosinase sequence in cmkm light cells was less sensitive to DNAase I than fully "open" chromatin, but was not as resistant as that of a fully repressed locus. To avoid hypersensitive regions in the dark-cell and wild-

2.3 - 2.0 -

Fig. 7. DNAase I hypersensitivity of wild-type and cm sequences upstream of the tyrosinase locus. Nuclei were prepared and digested with DNAase I, and the DNA was extracted and analyzed by South- ern blot. A DNA sequences with 19 kb of the tyrosinase structural gene in the C57BW6 wild-type strain, or within approximately 25 kb in c"/c", were analyzed for hypersensitivity. The DNAs were digested with ApaI and hybridized with an RNA probe from the 3' 300 bp (within exon 1) of the genomic ApaI restriction fragment (probe c in Fig. 2). The arrowhead indicates the sub-band a t approximately 15 kb upstream of the transcription start site which is absent in both dark- and light-cell c"/c" chromatin. B: DNA separated (in c") from the coding region was analyzed for hypersensitivity. Probe a of Figure 2 was used on DNA cut with PstI. The indicated hypersensitive-region fragment (bracketed) is the same as demonstrated for the wild-type in A. (Two lanes in the dark-cell analysis in B were out of sequence in the original blot and are correctly repositioned here.)

In Vitro Matrix Binding Assay To investigate the functional significance of the - 15

kb hypersensitive site, particularly the possible relationship of its relocation to the phenotype of em, DNA

type chromatin, a smaller (EcoRIIBglII) restriction fragment encompassing the tyrosinase gene exon 1 was analyzed in these cells. The DNAase I sensitivity of this sequence in both the dark and wild-type cells was in- distinguishable from that of L32. Thus, a correlation exists between the overall chromatin configuration and the presence or absence of a hypersensitive site in the proximal promoter region.

sequences surrounding the site were assayed for nuclear matrix attachment ability. Matrix-associated regions (MARS) have been implicated in the isolation of integrated genes from the effects of neighboring sequences [Stief et al., 1989; Bonifer et al., 19901, although they are unlikely to be the only elements with this ability [reviewed by Wilson et al., 19901. If the em phenotype represents a position effect of the newly jux-

tyr

L32

CP


@/c? light c?@ dark WT

8

3.2

2.2

2.2

3.2

2.2

2.2

3.2

2.2

actin 3.7

Fig. 8. General sensitivities to DNAase I a t the tyrosinase locus. Nuclei were digested with DNAase I and the DNA was prepared and cut with the appropriate restriction enzyme. Southern blots were hybridized with RNA probes derived from the L32 ribosomal protein locus (intron 3), the Cp immunoglobulin locus (exons 1 and 21, and tyrosinase-locus exons 1 (light- and dark-cells) or 2 (light-cells) of

c"/c'". A p-actin cDNA probe was used in a separate series as a control for DNAase I sensitivity of the light-cell c"/c" tyrosinase exon 1 sequence. The sizes of the bands are indicated at the right of each strip. Both tyrosinase-specific fragments in the light c"/c" melanocytes show decreased sensitivity to DNAase I relative to expressed controls, even though they are of higher molecular weights.

taposed DNA, then in the event that such an element exists a t the tyrosinase locus, i t would be expected to map within the rearranged DNA. We looked specifi- cally in the vicinity of the - 15 kb hypersensitive site, as MARS frequently overlap and reside close to regulatory elements [Gasser and Laemmli, 1986; Cockerill and Garrard, 1986; Jarman and Higgs, 1988; Phi-Van and Stratling, 1988; Farache et al., 19901 which may exhibit DNAase I hypersensitivity.

The in vitro association assay [Cockerill and Gar- rard, 19861 which was used relies on the specific binding of radiolabeled MAR-containing DNA fragments to isolated nuclear matrices in the presence of unlabeled prokaryotic competitor DNA. Figure 9 demonstrates that, of the 5 kb surrounding the -15 kb hypersensitive sites, three fragments encompassing approximately 3.6 kb exhibit MAR activity. Vector and tyrosinase coding sequences, and the 3' terminal fragment of this series, showed no significant binding.

Tyrosinase Expression in cemlcem Mice Extreme dilution mottled (cem), an allelic mutation

that arose in the cmlcm stock, results in a reduction in coat and eye pigmentation relative to cm without alter- ing the overall coat color pattern. Pigmented tissue from cemlcem mice was analyzed for tyrosinase expres-

vec - - 3.0

c -

CDNA - d -

a -

b - - 0.65

E Y ? C t F H

a b C d

H lkb

to learn if further reguiatory modu1ation Fig. 9. Localization ofan in vitro MAR near the -15 kb hypersen- here. The defect

is posttranscriptional, as there is no visible reduction of tyrosinase message in Cem/Cem relative to Cm/Cm eyes (data not shown). The level of tyrosinase RNA in the skin, which has than the eye, was too low in the Cm/Cm and Cem/Cem mice to be accurately quantitated.

suggest, however, that the sitive region. Nuclear matrices from mouse spleen were incubated with radiolabeled fragments (input) encompassing approximately - 13 to - 18 kb of the tyrosinase locus, as well as a tyrosinase cDNA Ityr2 in Terao et al., 19891, in the presence of 300 pgiml E. coli competitor DNA. Matrix-bound DNA was purified and electrophoretically resolved. Indicated fragments a 4 are identified on the map below, where the hypersensitive site is represented by an arrow. The vector sequence (pBluescript I1 SK + ) is abbreviated vec.

fewer pigmented

400 PORTER ET AL.

DISCUSSION

The rearrangement found in the c"' mutation sepa- rates the tyrosinase structural gene along with 5 kb of 5' flanking sequence from distal upstream sequences. The size of the rearrangement is unknown; however, a minimum of 30 kb (determined by restriction analysis) is estimated to be involved. The breakpoint of the rearrangement occurs within a member of the L1 LINE element family, which has been implicated in other rearrangements in the mouse genome [Shyman and Weaver, 1985; Philbrick et al., 19901. The restriction pattern obtained here could arise by recombination between the element and a rearranged or further trun- cated element, such as comprise the majority of the L1 sequences [see Burton et al., 19861.

DNA regions spanning active genes that are readily degraded by DNAase I and other nucleases, or that have specific hypersensitive sites, are taken to exemplify a more open configuration and to be associated with transcriptional activity [Weintraub et al., 1981; Emerson and Felsenfeld, 19841. The chromatin structure of the proximal upstream sequences and tyrosinase coding region of the c"' gene in dark cells is indis- tinguishable from that of the wild-type in terms of DNAase I sensitivity and pattern of hypersensitivity. TranscriptZion of t,he mutant gene is nevertheless less than in the wild-type. This may indicate that an enhancer or farther-upstream control element important to full expression of the gene has been separated, by the rearrangement, from the c"' coding sequences. Alter- natively, transcription may be negatively influenced by the rearranged DNA adjacent to the tyrosinase promoter, by a mechanism not involving chromatin alteration. The chromatin of the dark-cell ~"'lc"' may also differ from the wild-type in ways not examined here.

Chromatin configuration differs markedly, however, in the two cmlcm phenotypic classes. In the light cells i t displays an intermediate sensitivity to DNAase I, whereas in the dark cells it appears as sensitive to the enzyme as chromatin of fully active genes. In addition, a hypersensitive site near the dark-cell c"' transcription initiation site is absent or reduced in the light-cell case. A hypersensitive region a t -15 kb in the wild-type is present in both phenotypes of cmlcm cells (in the rearranged position), but is weaker in the light crnlcm cells than in the dark cells. Thus, both hypersensitive sites in the light cells are less accessible to normal melanocyte trans-acting factors than are equivalent regions in the wild-type. The correlation between the relative hypersensitivity of the two sites within each cmlcm phenotype is consistent with a communication between the two DNA segments although physical linkage has not been established. A diagrammatic summary of the hypersensitivity and transcriptional alterations in cmlcm cells is shown in Figure 10.

The differences between cmlcm light- and dark-cell clones are reminiscent of the chromatin alterations of

Fig. 10. Schematic representation of the cm defect. The upper seg- ment of the diagram represents the upstream region and first exon (solid box) of the wild-type tyrosinase gene. The lower segments represent the cm allele in phenotypically dark and light melanocytes. The relationship between both sides of the breakpoint at -5 kb in ern is unknown. Vertical arrows indicate DNAase I hypersensitive sites, with the thickness of the arrows representing degrees of hypersensitivity. Relative levels of tyrosinase transcription initiating a t the first exon are represented by horizontal arrows.

a- and P-globin genes upon transcriptional induction in erythroleukemia cells [Hofer et al., 1982; Sheffery et al., 19841 and also to some extent of those in normal erythrocyte development [Weintraub et al., 19811. Whether the chromatin configuration of the light-cell tyrosinase gene represents a developmental precursor to the fully open configuration in vivo is unknown.

The properties of the tyrosinase-locus chromatin in these cells may be contrasted with the chromatin changes thought to be responsible for position effect variegation in Drosophila. There, the introduction of a gene into a heterochromatic region appears to result in the spread of heterochromatin into the gene in some clones but not in others [reviewed by Tartof et al., 19891. This ordoff regulation is reflected in the phenotypes of the clones, which are generally, not always [Frisardi and MacIntyre, 19841, extremes, i.e., wild- type or null.

A strong DNAase I hypersensitive site is situated approximately 15 kb upstream from the wild-type tyrosinase transcription initiation site. In the c"' mutation, this upstream sequence is separated from the coding region and no hypersensitive site is formed in the newly relocated DNA. The apparent lack of consistency in the determination of chromatin structure may be related to the separation of the gene from sequences that control this aspect of gene expression and/or that isolate the gene from influences from neighboring DNA (position effects).

In connection with this hypothesis, the upstream hypersensitive region was analyzed for matrix binding activity. MARS are generally 0.6 to 1 kb in length, are characteristically A + T-rich, and often have conserved sequence motifs, including topoisomerase I1 recogni- tion sites [reviewed by Gasser and Laemmli, 19871. Re-


cent evidence has implicated functional correlations between MARs and the control of chromosomal do- mains; this is consistent with the observed position- independent expression of integrated genes containing such elements [Bonifer et al., 1990; Stief et al., 19891. The region surrounding or adjacent to the -15 kb hypersensitive site in fact exhibited matrix binding activity, as defined by the ability of cloned DNA to reas- sociate with nuclear matrices in vitro; the assay has identified the same sites as those determined in vivo [Cockerill and Garrard, 1986; Phi-Van and Stratling, 19881. Since MARs frequently “cohabit” with regulatory elements [Gasser and Laemmli, 19871, these results provide further evidence for a regulatory role of this region in defining the active state of the domain. Sequences farther upstream or downstream of the locus have not yet been analyzed for hypersensitivity or regulatory activity. The sequences immediately upstream (2.5 kb) from the tyrosinase coding region lack this function, as shown by the susceptibility of this region to chromosomal position effects in transgenic mice with coat-color changes [Bradl et al., 19911.

Apart from the possibility that the cm gene may be separated from an element insulating the wild-type gene from position effects, the mechanisms responsible for alternatives in the determination of chromatin structure are unknown. The newly relocated DNA in all likelihood plays a role in the phenotypic instability of the allele. A separation of the murine alloantigen gene Ly-6C from distal upstream sequences (also mediated by a LINE element) results in expression of the gene in only a small proportion of bone marrow cells, and not a t all in other normally expressing cell types [Philbrick et al., 19901. Although clonality has not been investigated in this case, there is clearly a similar aspect of randomness in the establishment of expression, associated in both cases with an interruption by foreign sequences in the upstream flanking region.

The neighboring DNA could influence tyrosinase expression in a number of ways, including the spread of a higher-order chromatin structure or an alteration or randomness in the timing of DNA replication. The association of replication origins [Farache et al., 1990; Razin et al., 19861 or terminators [Dijkwel and Hamlin, 19881 with MARs may in this situation be significant. DNA secondary structure or methylation could also influence chromatin formation. Although a CpG site in the first exon is unmethylated in both the dark and the light cmlcm cell clones (data not shown), it is unknown whether the upstream, rearranged, sequences are dif- ferentially methylated. A high degree of methylation has been regarded as incompatible with formation of active chromatin [Keshet et al., 19861, and may exert a dominant effect over a gene-sized domain.

There are many examples, particularly in Drosoph- ila and mice, of natural patterns, or of patterns resulting from gene transfer, that represent clonal pheno- tvDic variation established etigeneticallv among cells

of the same type. Those that have been detected rely on a limited array of favorable markers, such as pigmentary differences or histochemically visualizable dispar- ities, and are probably only a small sample of the phenotypic variants occurring among cells of a given type. It has been proposed that, in mammals, such phenoclones are a commonplace and that cellular heterogeneity may provide for greater flexibility in development, response to environmental change, disease, and aging. Genetic mechanisms favoring establishment of increased heterogeneity may thus have had some evo- lutionary advantage [Mintz, 1970, 19711.

Cases of mosaic expression of genes in mammals are increasingly coming to light. Recent examples include several genes in the developing rat intestinal epithelium [Rubin et al., 19891 and variation in expression of foreign genes in transgenic mice, e.g., an integrated fatty acid-binding protein-human growth hormone fusion gene expressed in the intestine [Sweetser et al., 19881, and production of a heritable coat-color clonal pattern in mice with a tyrosinase-SV40 early region fusion gene [Bradl et al., 19911 or a metallothionein- tyrosinase fusion gene [Mintz and Bradl, 19911.

Deciphering the cis-acting mechanisms of a gene such as cm should greatly increase our understanding, not only of heterogeneity of gene expression, but, more broadly, of the basis for developmental determination and higher-order gene control.

ACKNOWLEDGMENTS We thank John Burch of this Institute for valuable

discussions and suggestions. This work was supported by U.S. Public Health Service grants HD-01646 and CA-42560 to B.M., by CA-06927 and RR-05539 to the Fox Chase Cancer Center, and by an appropriation to the Center from the Commonwealth of Pennsylvania.

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Mosaicism of tyrosinase-locus transcription and chromatin structure in dark vs. light melanocyte clones of homozygous chinchilla-mottled mice