δ-Crystallin gene expression and patterns of hypomethylation demonstrate two levels of regulation for the δ-crystallin genes in embryonic chick tissues

DEVELOPMENTAL

BIOLOGY

145,40-50

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&Crystallin Gene Expression and Patterns of Hypomethylation Demonstrate Two Levels of Regulation for the SCrystallin Genes in Embryonic Chick Tissues CHARLESH.SULLIVAN,~SUSANO'FARRELL,~ Departrrwn

t of Biology,

Gilm

er Hall,

AND of Virgin

Umvrrsit,y

ROBERT M. GRAINGER

ia, Ch:hrcrlottrwill~.

I&gin

ia .2z901

In this study we address two questions regarding the control of 6-crystallin gene expression in chick embryos. First WC have determined whether fi-crystallin mRNA is found outside of the developing lens, in which it is the predominant mRNA. We find that this mRNA can be detected, although at relatively low levels, in all embryonic tissues we have examined (from the definitive streak stage onward). This low level of transcription may be related to a second function for one or both of the &crystallin genes: both genes have a high degree of sequence identity to the enzyme argininosuccinate lyase. This result led us to a second set of experiments in which we reevaluated the possible role of hypomethylation in the expression of the &crgstallin genes. Previous work showed that particular HhaI and H;jnII sites in the crystallin genes undergo hypomethylation early in the process of lens differentiation when there is a burst of b-crystallin mRNA accumulation. We now find that these sites remain methylated in nonlens tissues, implying that they cannot bc required for the &crystallin gene activity found in these tissues. Other sites are constitutivelg hypomethylated, however, and may be functionally linked to this low level of gene activity. From an analysis of the kinetics of the developmentally regulated hppomethylation of H//o1 and HpaII sites we also find that complete hypomethylation of these sites is not required for activating high levels of &crystallin transcription during lens differentiation. We do find, however, that these sites approach a fully hgpomethylated state later in the lens differentiation process. Our analyses of mRNA levels and hypomethylation together lead us to propose that the K-crystallin genes are regulated by two different mechanisms, one that leads to high levels of expression in the lens and the other which is responsible for low level expression in all other tissues in the chick embryo. 8~‘1991 Academic press. I~C.

(designated 61 and 62) are present in the chicken genome (Bhat et al., 1980), most of the transcription in the lens is from the al-crystallin gene (Wawrousek et al., 1986; Parker et al, 1988). &crystallin expression does not appear to be restricted to the lens; low levels of transcripts have been detected in several nonlens tissues during development, though not in others (Agata et al., 1983; Bower et al., 1983; Jeanny et al., 1985). These results suggest that there are regional differences in the regulation of the &crystallin genes: high level expression in the lens and low level expression in some nonlens tissues. A possible explanation for the transcription of fi-crystallin outside of the lens is that this gene might also code for the urea cycle enzyme argininosuccinate lyase (ASL) based on the high degree of sequence homology between the chicken &crystallin and human ASL genes (Wistow and Piatigorsky, 1987). In ducks, one of the two &crystallin genes codes for both proteins, and duck fi-crystallin has high ASL activity (Piatigorsky et al, 1988). If one or both of the two &crystallin genes in chickens also codes for ASL, it would not be surprising if these genes were expressed in nonlens tissues. The first part of this study involved using sensitive assays for detecting 6crystallin transcripts to determine the extent of &crystallin transcription in nonlens tissues in young embryos, particularly at stages before and during lens differentiation, when this question has not been examined

INTRODUCTION

While the crystallins are the most abundant proteins in the lens, recent findings that some of these proteins are found at low levels in other tissues (Piatigorsky and Wistow, 1988) argue that the regulation of the crystallin genes is more complex than thought previously. The crystallins that are found in all vertebrates (a and the P/r superfamily) appear to be expressed only in the lens, while taxon-specific crystallins (6, t, p, and T) are not (Wistow and Piatigorsky, 1988). It has been proposed that at least some of the latter class of genes encode enzymes expressed in many tissues and that these proteins have been recruited in the lens as major constituents of the protein lattice in this tissue (Wistow and Piatigorsky, 1987). This observation raises new questions about the control of crystallin gene expression. The first question we examined was the temporal and spatial pattern of &crystallin expression in young chicken embryos. &crystallin is the predominant enzyme/crystallin expressed in developing chicken lenses (Piatigorsky, 1984). Although two a-crystallin genes

’ Current Address: Department of Biology, Grinnell College, Grinnell, IA 50112. ’ Current Address: Department of Pediatrics, Stanford University Medical Center, Stanford, CA 94305. 0012.1606/91 Copyright All rights

$3.00

I< 1991 by Academic Press. Inc. of reproduction in any form reserved.

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SULLIVAN,O'FARRELL. ANDGRAINGER

previously. We find that fi-crystallin is expressed in all tissues examined during early chicken development. This information concerning &crystallin expression was essential for addressing the other question raised here: the relationship of changes in DNA methylation and expression of the fi-crystallin genes. Previously, we have detected a reduction in crystallin gene methylation that is tightly linked to expression in the lens of the genes encoding a-crystallin (Grainger ef al., 1983; Sullivan et trb, 1989). To further elucidate the role of DNA methylation in the control of gene expression, we have examined whether these latter hypomethylation events observed in DNA from lens cells accompanied cases of &crystallin transcription observed in nonlens tissues as well. Also, the extent of hypomethylation of the al-crystallin gene was quantified at stages throughout the lens differentiation process in order to determine whether complete hypomethglation is necessary for the high levels of transcription observed in the lens. Most experiments that analyze the extent of DNA methylation around a particular gene use methyl-sensitive restriction enzymes (Bird and Southern, 1978) to compare restriction enzyme digests of DNA from a tissue where a gene is expressed to DNA from a tissue where the gene is inactive. The typical result from such studies is that nontranscribed genes are heavily methylated, while transcribed genes have reduced levels of cytosine methylation (“‘C) on both DNA strands (Adams and Burdon, 1985). In a few cases,changes in DNA methylation have been examined over time in cultured cells (Kelley et al., 1988) or following hormone treatment (Wilks et ul., 1984), but examination of hypomethylation early in the differentiation of an embryonic tissue generally has not been possible. However, because lenses can be isolated at all stages from chicken embryos, the lens system has the advantage that changes in hypomethylation can be studied through time, as lens differentiation proceeds. Thus far we have determined that the &crystallin genes are heavily methylated at 40 hr of development (stage 10 of Hamburger and Hamilton, 1951) in presumptive lens ectodermal cells, which are specified for lens formation (Karkinen-Jatiskelainen, 1978; Barabanov and Fedtsova, 1982), but have not yet begun to differentiate or synthesize high levels of & crystallin (Grainger ef uL, 1983; Sullivan et c11.,1989). By 50 hr, when 6-crystallin transcripts first accumulate in the lens (Shinohara and Piatigorsky, 1976), hypomethylation is observed within the bl-crystallin gene at one H~xxII site in intron 15 (Grainger ef al., 1983) and two HhaI sites in intron 2 (Sullivan et cxh,1989). We report here that these latter hypomethylation events are confined to the lens, but that the kinetics of hypomethylation at these sites lag behind the increase in &crystallin gene expression. Seven other HpaII sites in both &crys-

&h/sttr/lih

E.r(uTssiruI (1tit/ N!/po?wth !/lntion

41

tallin genes are constitutively hypomethylated in all tissues (Sullivan et a,Z.,1989). The different methylation patterns in lens and nonlens tissues together with the very different levels of expression in lens and nonlens tissues lead us to propose that two different mechanisms are likely to control fi-crystallin gene expression in the chick embryo. MATERIALS

AND METHODS

Tissue Collect ion, and DNA Isoln t ion Chicken eggs (Truslow Farms, Inc., Chestertown, MD) were incubated at 38°C until the appropriate stage of lens development had been reached (O’Rahilly and Meyer, 1959). Lens and nonlens tissues were isolated from embryos, quickly frozen in liquid nitrogen, and stored at -70°C until used. DNA was extracted as described previously (Sullivan and Grainger, 1987). Assay of’ Hypom.eth ylution Purified DNA samples were assayed for hypomethylation by restriction endonuclease digestion with Hr,aII (which cleaves the sequence CCGG, but not C”“CGG) or with HhaI (which cleaves the sequence GCGC but not GmeCGC). A second enzyme, BarnHI, was included in digests to compare fragments from the present study with earlier work. Specific restriction fragments from the &crystallin genes were identified by hybridizing 32P-labeled (nick translated) 6-crystallin gene probes to DNA fragments separated on agarose gels and transferred to nitrocellulose (Southern, 1975). Two 6crystallin gene probes were used (see Fig. 1): pXr 2, a cDNA from exons 7 through 17 at the 3’ end of 61 (Bhat and Piatigorsky, 1979); and a short genomic probe 5’ to 61 designated pal.3 (Borras et al., 1985). Conditions for restriction enzyme digestions and hybridizations have been described (Grainger cf al., 1983). To quantify the extent of hypomethylation in DNA, autoradiograms were scanned on a Gilford multimedia densitometer and the band intensities were quantified with a PerkinElmer LCI-100 integrator. RNA Isolation Tissue samples were thawed in 10 mMTris-HCl (pH 8.0), 1% sodium dodecyl sulfate, 1 mM EDTA, 0.1% diethyl pyrocarbonate, and extracted three times with an equal volume of phenol:chloroform:isoamyl alcohol (24:24:1) and once with an equal volume of chloroform:isoamyl alcohol (24:l). Glycogen (BoehringerMannheim) was added to a concentration of 20 pg/ml and total nucleic acids were recovered by precipitation with 2.5 vol of 95% ethanol after the addition of 0.3 M sodium acetate.

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DEVELOPMENTALBIOLOGY 1

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FIG. 1. Restriction maps of the two &crystallin genes of the chicken (compiled from exons (shaded boxes), introns (open boxes), and restriction sites for BumHI (A), HpaII study are shown.

Because the amount of RNA recovered from these samples was quite small (0.1-5 pug from 100-200 tissue fragments), total RNA generally was used for electrophoresis. The amount of RNA in individual samples was quantified by scanning densitometry of ethidium bromide stained gels using known concentrations of poly(A)- RNA as a standard. For anterior and posterior portions of 40-hr embryos, poly(A)+ RNA was prepared in order to achieve greater sensitivity in Northern blot analysis. To prepare poly(A)+ RNA, tissues were homogenized in GuSCN buffer (Jonas et al., 1985) and extracted in phenol:chloroform:isoamyl alcohol (25:24:1). RNA was precipitated once with an equal volume of isopropyl alcohol and again with 2.5 MLiCl. Poly(A)’ RNA was obtained by chromatography on oligo(dT)-cellulose (Sambrook et al., 1989). RNA yield was quantified by measuring absorbance at 260 nm.

Nickerson et CL, 1985,1986). The map shows (O), and HhaI (m). The positions of all probes

Northern Blot Analysis

RNA was separated by electrophoresis and transferred to Gene Screen membranes (New England Nuclear) as described by Church and Gilbert (1984). Membranes were placed in prehybridization buffer (50% deionized formamide, 1% SDS, 100 pg/ml calf thymus DNA, 5~ SSC [lx SSC is 0.15 h4 NaCl, 0.015 M sodium citrate], 0.05% ficoll, 0.05% polyvinylpyrillidone) at 62°C for 4 hr, then in hybridization buffer (prehybridization buffer which also contained the 32P-labeled probe described above), at 62°C for 16-20 hr. Filters were washed twice for 5 min at room temperature then five times for 30 min each at 67°C in a solution of 1% SDS and 0.1~ SSC. Filters were exposed to Kodak XOmat Film with intensifying screens at -7O”C, usually for 2-3 days. Analysis of Levels of Krystallin

Subcloning of pK’r2 and RNA Probe Synthesis

A PstI fragment from p6Cr2 was subcloned into the transcription vector pGEM-2 (Promega Biotec) to allow preparation of labeled RNA probes following procedures outlined by Sambrook et al. (1989). The resulting plasmid (pGEMGCr2a; see Fig. 1) was linearized with EcoRI to yield the appropriate template for the transcription reaction. The RNA transcript was radiolabeled by including [w~~P]CTP (800 Ci/mmol, New England Nuclear) in the reaction mixture.

locations of used in this

mRNA

The &crystallin mRNA abundance in samples was quantified by comparing densitometric scans of sample lanes on X-ray films with known amounts of a 13-day lens standard included on each gel. Calculation of the abundance of &crystallin mRNA in 13-day lens RNA was based on the following published values: approximately 1% of the total RNA is polyadenylated and 6crystallin is 75% of the mRNA at this time (Zelenka and Piatigorsky, 1974). In order to compare the relative abundance of a-crystallin mRNA in various tissues an estimate was made of

43 the contribution of &crystallin mRNA to the total poly(A)’ RNA in each sample (percentage of poly(A)+ RNA) as a convenient measure of its abundance. For 40-hr poly(A)+ RNA samples this estimate was made by dividing the estimated amount of &crystallin mRNA by the amount of poly(A)+ RNA loaded in a lane. As mentioned above, &crystallin mRNA levels were calculated by comparing the intensity of the &crystallin signal from a sample to that from a 13-day lens standard. The levels of &crystallin mRNA in all other samples (which were from total RNA) were calculated by dividing the estimated amount of crystallin mRNA in the sample by the amount of poly(A)+ RNA calculated to be in each sample. We estimate that poly(A)’ RNA in these samples is approximately 1% of total RNA (based on recovery of poly(A)+ RNA from 40-hr chick embryos [Charlebois, 19881 and from 15-day lenses [Zelenka and Piatigorsky, 19741). We are not certain that this estimate of poly(A)+ RNA is exactly correct for all stages and tissues examined, but it provides a reasonable basis for a useful absolute estimate. The accuracy of this estimate has no bearing on the relative levels of crystallin mRNA from tissue to tissue, however, since the same value was used in determining the divisor in all of these calculations. This quantitation procedure allowed us to compare the relative levels of &crystallin mRNA in different tissue samples analyzed on the same gel. Polp(A)RNA was used as a negative control on every gel to ensure that any signal seen was not due to binding to 1% rRNA, which co-migrates with &crystallin mRNA on RNA gels.

In order to estimate the number of &crystallin copies per cell in stage 4 embryos, we first calculated that an embryo contains 5 X lo5 cells, based on the total DNA content per embryo (measured by the assay of Labarca and Paigen, 1980) and the estimation of 3.7 pg of DNA per chicken nucleus (Milstone and Piatigorsky, 1977). This information was used, together with the levels of mRNA measured at this stage and the length of the fi-crystallin mRNA (1900 bases; Nickerson and Piatigorsky, 1984), to calculate the average &crystallin RNA copy number per cell. RESULTS

The observations that &crystallin and ASL may be the same protein, suggested that &crystallin might be found in all tissues in the chicken embryo. As mentioned

a

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FIG. 2. Northern blot analysis of krystallin transcripts found in: (a) 40 PR of total RN.4 from 84.hr brain; (h) 20 kg total RNA from 84-hr heart; (cj 20 PR total RNA from 84-hr limb bud; (d) 5 wg poly(A) RNA; and (e) 9 na of a standard of total RNA from a G-day lens. Thcl arrow indicates the six of K-crystallin mRNA.

earlier, low levels of &crystallin expression have been detected in some, but not all, chick tissues at 84 hr of development by Agata et al. (1983). Our first set of experiments quantified &crystallin expression in these tissues using a sensitive RNA probe transcribed from the plasmid pGEMGCr2a. &Crystallin transcripts were detected in brain and limb bud (Figs. 2a and 2~) and scanning densitometry revealed these transcripts represented approximately 0.001% of the poly(A)+ RNA in each tissue (Table 1). These results agree well those of Agata et al. (1983). In addition, transcripts were detected in the heart (Fig. 2b) at a level of approximately 0.0003%, of poly(A)’ RNA (Table 1). Our RNA probes apparently can detect lower levels of mRNA than the cDNA probe used by Agata ef ul. (1983), since &crystallin was not detected in the heart in this latter study. When 5 pg of poly(A)- RNA was loaded on a gel, no &crystallin signal was detected after a l-day exposure (Fig. 2d) or after a lo-day exposure (not shown). The RNA probe described above was then used to examine a-crystallin expression in the lens and other regions of chicken embryos during the time of lens differentiation. The burst of &crystallin transcription in the lens at 50 hr is followed quickly by the appearance of detectable protein in some lens cells by 52 hr of development (Zwaan and Ikeda, 1968). An increasing fraction of lens cells activate &crystallin synthesis over the next 32 hr, so that by 84 hr of development all lens cells stain positively with a-crystallin antibody (Zwaan and Ikeda, 1968; Brahma and van Doorenmaalen, 1971). The protein continues to accumulate in lens cells to a maximum (as judged by immunofluorescence) until 7 days of devel-

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DEVELOPMENTALBIOLOGY

TABLE1 6-CRYSTALLIN mRNA ASAPERCENTAGEOFTHEESTIMATEDPOLY(A)+ RNALEVELSINVARIOUSTISSUESOFCHICKENEMBRYOS 1%hr embryo

(stage 40.hr embryo (stage anterior (poly(A)+) posterior (poly(A)+) head ectoderm head mesoderm neural tube trunk ectoderm trunk mesoderm 52-hr lens 5%hr lens 65.hr headless bodies lens X4-hr hrain heart limb bud lens

4) 10)

0.05% 0.03% 0.03% 0.06%’ 0.02% 0.02% 0.01% 0.03% 0.85% 1.8%’ 0.01% 2.7%’ 0.001%’ 0.0003’% 0.001%’ 3.4%

~Vofe. Data shown are based on the average of two measurements from densitometric scans of autoradiograms shown in Figs. 2 and 3 and other similar experiments (data not shown). The levels of &crystallin mRNA are expressed as a percentage of the estimated poly(A)+ RNA as described under Materials and Methods. The anterior (poly(A)‘) and posterior (poly(A)‘) samples come from gels where poly(A)+ RNA was analyzed, while all other samples come from analysis of tot,al RNA. The variation seen between samples was no more than twofold, except in the case of the stage 4 embryos, where there was a threefold variation hetwren the replicates. The variation seen is not a serious concern, however, because our interpretation of these data is primarily qualitative.

V0~~~~145.1991

els of &crystallin increased with age from approximately 0.03% of poly(A)+ RNA in anterior or posterior regions of 40-hr embryos, to approximately 3.4% of poly(A)+ RNA in lenses from 84 hr embryos (Table 1). Because both the anterior and posterior regions were positive for fi-crystallin RNA, we were interested in determining whether the transcripts detected in 40-hr embryos were expressed in a tissue-specific manner (e.g. within the ectoderm) or whether expression was more widespread. Embryos were dissected into head ectoderm and trunk ectoderm (covering the somites), as well as neural tube and trunk mesoderm. All tissues contained &crystallin mRNA (Fig. 3c-3f). When the level of &crystallin expression was quantified by scanning densitometry, all tissues tested had similar levels of expression: &crystallin mRNA ranged from O.Ol-0.06% of poly(A)+ RNA (Table 1). Finally, we were interested in determining whether the &crystallin genes are ever transcribed in embryos at even earlier stages, during gastrulation, when lens induction is not likely to have begun (Saha et al., 1989). R.NA was therefore isolated from 1% to 19-hr embryos at the primitive streak stage (stage 4 of Hamburger and Hamilton, 1951). &crystallin transcripts were detected at a level of 0.05% of poly(A)+ RNA (Fig. 3g and Table 1). This level of expression represents approximately six copies of fi-crystallin mRNA per cell in these young embryos.

a

opment. After that, the signal is greatly diminished in some regions of the lens, particularly the anterior epithelium, yet overall transcription of &crystallin in the lens increases until a month after hatching (Hejtmancik et al., 1985). In order to examine the extent of a-crystallin expression, total lens RNA was isolated from embryos at 52, 58, 65, and 84 hr of development, as was poly(A)+ RNA from 40-hr embryos cut into anterior and posterior portions (the cut was made just anterior to the heart). Tissues were taken at the latter time because by 40 hr a large area of the head ectoderm, including the presumptive lens ectoderm, has a lens-forming bias. Although head ectoderm does not show any signs of overt lens differentiation at 40 hr, when this ectoderm is cultured in vifro, it will differentiate into lens-like structures containing high levels of fi-crystallin (Barabanov and Fedtsova, 1982; Grainger et nl., in preparation). As expected, lenses from 52 hr and older were positive for &crystallin (Table l), but there were also low levels of mRNA detected in tissues from 40-hr embryos (Figs. 3a and 3b). Scanning densitometry indicated that the lev-

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FIG. 3. Northern blot analysis of &crystallin transcripts found in: (a) 2 pg poly(A)’ RNA from 40-hr anterior embryos; (h) 2 wg poly(A)’ RNA from 40-hr posterior embryos: (c) 2.5 ~(a of total RNA from head ectoderm; (d) 2.8 fig of total RNA from trunk ectoderm; (e) 4.5 wg of total RNA from trunk mesoderm; (f) 3.0 pg of total RNA from neural tuhe: (g) 7 pg of total RNA from stage 4 embryos: and (h) 9 ng of total RNA from 13.day lenses, included as a standard.

SULLIVAN,

O’FARRELL,

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45

from 40-hr headless embryos is digested with HhaI and BamHI, two Bant-HI-BamHI restriction fragments of 5.8 kb (Barn’-Bam3) and 3.6 kb (Barn’-Bam3) are detected with the probe ~61.3. The appearance of two fragments is due to a polymorphism at Barn2 (Sullivan et nl., 1989). As we have shown previously (Sullivan et ah, 1989), DNA from 13-day embryonic lenses shows extensive hy.- 5.8 pomethylation when cleaved with HhnI and BamHI. - 4.3 The precursor fragments of 5.8 and 3.6 kb are greatly - 3.6 reduced, while many smaller bands appear as HhaI sites 1,3, and 4 (see Fig. 1) acquire sensitivity to HhaI due to hypomethylation (Fig. 4b). DNA from 84 hr lenses also contained hypomethylated Hh,aI sites, but not to the extent as that observed at 13 days (Fig. 4~). - 2.1 Because &crystallin transcripts have been detected in brain, heart and limb buds from 84-hr embryos, we examined the methylation pattern of the 6-crystallin genes in these nonlens tissues. When DNA was isolated from these tissues and digested with HhaI and BarnHI, there was no evidence of hypomethylation of HhaI sites (Figs. 4d-4f). As was the case with DNA from 40-hr FIG. 3. TIN.4 methylation patterns at Hhd sites in the 61.crystallin embryos, hypomethylation at these sites does not correyenc in chicken embryos. DNA samples (2 rg) were digested with late with low levels of &crystallin expression outside of HhtrI and RtrwHI and probed with nick-translated pfi1.3. Samples the lens. were from: (a) 10-hr headless embryos; (b) 1%day lens; (cl 3.5day Hypomethylation of Hpa7 in the dl-crystallin gene, lens: (d) M-day brain; (e) X5-day heart; and (f) X5-day limb bud. The positions of major restriction fragments (in kb) referred to in the test the other site examined in earlier studies of hypometharc indicated ylation during lens differentiation, was also examined in nonlens tissues. This site is the 5’ end of a transient 5.0 kb restriction fragment that first appears in DNA from lens ectoderm collected from 50-hr embryos (Grainger et al., 1983), when high levels of mRNA accumulation begin. This fragment contains an internal Previously, we have shown that Hh,aI sites 3 and 4 in HpaII site (site 8 on Fig. 1) that becomes hypomethyintron 2 (designated Hhd and Hha4 on Fig. 1; Sullivan et lated later (by 75 hr) and is detected by pfiCr2 as a 1.8-kb (xl., 1989) and HJMII site ‘7(designated Ifpa?) in intron 15 fragment with this second HiIn site as its 3’ border (Grainger et al., 1983) of the 61 gene become hypometh(Sullivan and Grainger, 1987). Therefore, the extent of ylated in DNA from differentiating lenses beginning at hypomethylation of Hpa’ was followed by monitoring 50 hr, when high levels of &crystallin mRNA accumulathe appearance of both the 5.0- and 1.8-kb fragments. The pattern of methylation of the Hptr7 from 40-hr tion commence. The extent of hypomethylation of these three sites has been examined in DNA from nonlens headless embryos and differentiating lenses is shown in Fig. 5. DNA from 40-hr headless embryos, again tissues during early development to determine whether hypomethylation is required for the low level of gene previously treated as a control (Grainger et al., 1983), contained two large BamHI-BnmHI restriction fragexpression outside of the lens. The extent of hypomethylation of Hha3 and Hha“ is ments of approximately 10.6 and 7 kb from the 3’ end of presented in Fig. 4. In our earlier studies we were un- the fil- and fi2-crystallin genes, respectively (Fig. 5a). aware of &crystallin expression in 40-hr embryos, and There are no detectable levels of the 5.0- and 1.8-kb because no detectable hypomethylation at any of these fragments. There are, however, two smaller HpaII/ and 3.1 kb sites was found (Sullivan et al., 1989), this sample was BamHI fragments of 3.3 kb (Barn3-Hpa’) treated as a control. However, because we have now de- (Barn’-Hpu”) present in all chick tissues examined betected transcripts in tissues from 40-hr embryos, this cause of constitutive hypomethylation in all tissues of these two Hp,aII sites in the 61 and 62genes, respectively sample is an example of a nonlens tissue in which hypo(Sullivan et al., 1989). Hypomethylation of these latter methylation of these sites in the 61 gene is not required two sites could be related to the low levels of transcripfor low levels of transcription (Fig. 4a). When DNA a

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FIG. 5. DNA methylation patterns at HnoII sites in the 61- and d2-crystallin genes in chicken embryos. DNA samples (2 ~9) were digested with HnaII and BamHI and probed with nick-translated p8Cr2. Samples were from: (a) 40-hr headless embryos; (b) la-day lens; (c) 3.5.day lens; (d) 3.5-day brain; (e) 3.5-day heart; and (f) 3.5-day limb bud. The positions of major restriction fragments (in kb) referred to in the text are indicated.

tion observed throughout the embryo (see Discussion). DNA samples from 13-day lenses digested with HpaII and BamHI, included for purposes of comparison, show extensive hypomethylation as judged by the disappearance of the large precursor fragments and the appearance of many shorter restriction fragments, including the 1.8-kb fragment (Fig. 5b). Even by 84 hr, DNA from some lens cells is becoming hypomethylated at many of the same sites observed from 13-day lens samples (Fig. 5~). The status of Hpa7 was examined in DNA from several nonlens tissues which express low levels of d-crystallin mRNA. DNA from brain, heart, and limb bud showed no evidence of hypomethylation at Hpa’ (Figs. 5d-5f). Therefore, hypomethylation at Hpa7, as well as at the two HhaI sites discussed above, is not associated with low-level transcription in nonlens tissues, but is confined to the lens where expression is high. Is Complete Hypomethylation of the dl-Crystallin Gene Required for High Levels of Expression in the Lens?

Because the hypomethylation detected in the lens at the HhaI sites (Hha3 and Hha4) and HpaII site (Hpu?)

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were not associated with low levels of transcription outside of the lens, we wanted to learn whether these hypomethylation events are required for expression in the lens. One way of establishing whether this hypomethylation is necessary is to determine whether complete hypomethylation of these sites parallels the burst of mRNA accumulation observed early in lens differentiation. The extent of hypomethylation of the three sites within the 61-crystallin gene was examined in lenses at 4 days when all lens cells are first positive for a-crystallin synthesis, as well as at several later stages: at 6 days when all cells are continuing to accumulate the protein; at 8,10, and 13 days when an increasing number of cells have reduced levels of protein; and at 20 days (just before hatching). The kinetics of hypomethylation of the site Hha3 are shown in Fig. 6. The extent of hypomethylation was determined by scanning densitometry of autoradiograms (not shown) to quantify the disappearance of two large precursor BamHI-BamHI restriction fragments beginning at 50 hr when Hha3 first begins to become hypomethylated. Hypomethylation of this site reduces the 5.8-kb precursor fragment to 4.3 kb in size; the 3.6-kb fragment is reduced to 2.1 kb in size (see Figs. 1 and 4). Figure 6 shows the contributions of the 5.8- and 3.6-kb fragments as a percentage of the total signal at different times of development. Even though the number of cells engaged in &crystallin synthesis reaches 100% at 84 hr, this site is not hypomethylated in DNA from all lens cells, as measured by the persistence of both precursor fragments in close to 60% of the DNA from lens cells. During the interval from Day 8 to Day 13 when some cells have ceased d-crystallin synthesis and the

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FIG. 6. Kinetics of hypomethylation of site Hha3 entiation. The disappearance of the 5% and 3.6-kb striction fragments was quantitated in HhaI/BamHI DNA during chicken development. Lanes from (data not shown) were scanned to determine what signal was still present in the sum of the 5% and

during lens differHhaI-BumHI redigests of lens an autoradiogram portion of the total 3.6-kb fragments.

SULLIVAN, O'FARRELL, ANDGRAINGER

protein is no longer detectable in anterior epithelial cells, hypomethylation is still increasing. By 13 days about 20% of the DNA from lens cells still contains uncut restriction fragments. Therefore, the extent of hypomethylation of Hha3 in the a,-crystallin gene lags behind the increase in &crystallin producing cells in the lens. The kinetics of hypomethylation at the Hpa7 site showed a similar pattern of increasing hypomethylation during lens development (data taken from autoradiograms not shown). Here the analysis was done differently because of the availability of the restriction enzyme 1Msp1,an isoschizomer of HpaII, which cuts at the same CCGG sequence regardless of the methylation status of the internal cytosine. Therefore, the pattern of restriction fragments obtained from an lMsp1 digest is identical to that obtained with HpaII if the particular restriction site is fully hypomethylated. By comparing the extent of digestion at an individual site in HpaII digests to this site in an MspI digest, the extent of hypomethylation at this site can be readily determined. The extent of hypomethylation of Hpa7 was quantified in this way by summing the contributions of the 5.0-kb restriction fragment, representing the early transient hypomethylation event, and the 1.8-kb fragment (the end product) from a HpaII/BamHI digest. The sum of these percentages was then divided by the percent of the signal present in the 1.8-kb fragment in MspI/BamHI digests of chick DNA (Sullivan and Grainger, 1987). The kinetics of hypomethylation at the Hpa7 site show an initial rapid rate of hypomethylation, which then levels off (Fig. 7). When all lens cells are positive for 6crystallin synthesis by Day 4,71% of the DNA is hypomethylated. When the intensity of 6-crystallin expression reaches a maximum at 7 days, a little over 80% of the DNA is hypomethylated. As was seen for HhaI site 3, the extent of hypomethylation lags behind the percentage of cells synthesizing the protein. However, unlike hypomethylation at the Hha3 site, which never reaches 100%) hypomethylation of Hpu7 approaches 100%’ by 13 days and this level is maintained until hatching (Fig. 7). DISCIJSSION

Control qf Crystallin Nonlens Tissues

Gene Expression in Lens and

Many studies over the past twenty years have documented one level of regulation of the &crystallin genes in chicken embryos: high levels of expression of &crystallin in the lens (Piatigorsky, 1984; Wistow and Piatigorsky, 1988). Our work contributes new information documenting a second level of regulation: there is lowlevel expression of &crystallin mRNA throughout the

6-Cqstallin

E.qn-essim cm1 H~ypometh ylatim

47

FIG. 7. Kinetics of hypomethylation of site Hpu7 during lens differentiation. The appearance of the 5.0- and 1%kb restriction fragments was quantitated in H&I/BarnHI digests of lens DNA during chicken development. Lanes from an autoradiogram (data not shown) were scanned to determine what portion of the total signal was present in both the 5.0- and 1%kb fragments. This sum was then divided by that obtained for the percentage of the 1.8.kb fragment in M,spI/BamHI digests, thereby expressing hypomethylation at site Hpa7 as a percentage of the theoretical maximum.

rest of the embryo. Several studies have detected the mRNA for &crystallin in many nonlens tissues before (Agata et c&Z., 1983; Bower et ah, 1983; Jeanny et al., 1985), but the increased sensitivity of our methods has allowed us to detect &crystallin transcripts in other tissues previously thought to be negative for expression as well as to show for the first time expression throughout the embryo at earlier developmental stages at or before the time when lens differentiation begins. Which of the two &crystallin genes is responsible for the low level of mRNA throughout the embryo has not yet been thoroughly investigated. From our data there is no way to determine the ratio of expression of the 61 and 62 genes. While transcription in the lens is primarily from the al-crystallin gene, low levels of 62 expression have been detected (Parker et al, 1988). Preliminary evidence argues that both genes are transcribed outside of the lens in chicken embryos, and in some tissues 6, may be expressed preferentially to 61 (J. Piatigorsky, personal communication). In order to learn more about the significance of these low levels of &crystallin gene expression, we thought it would be useful to quantify mRNA levels at the earliest times that &crystallin was detected. At 18 hr (stage 4), we calculated that approximately six copies of &crystallin mRNA were present per cell. Similar levels of expression were observed throughout tissues of the 40-hr embryo. By 84 hr, the levels of &crystallin mRNA from several nonlens tissues had declined by a factor of lo-

48

DEVELOPMENTALBIOLOGY

100, based on relative values of 6-crystallin abundance that were determined from Table 1. Thus while there are two general levels of &crystallin mRNA, high in the lens and low elsewhere in the embryo, there is some modulation of expression among nonlens tissues. These data suggest either that the older embryonic tissues contain less than one copy of &crystallin mRNA per cell or alternatively that expression may be heterogeneous, with a subset of cells expressing the gene at high levels. In the heart of 5-day chicken embryos, where a-crystallin transcripts have been detected by in, situ hybridization, it has been reported that there is such a heterogeneity of expression, with only 0.1% of the heart cells containing enough a-crystallin mRNA (lo3 copies per cell) to be detectable by this method (Jeanny et al., 1985). What is the function of these low levels of transcripts in nonlens tissues? Because &crystallin mRNA appears to be ubiquitous, it is possible that these transcripts encode the widely distributed enzyme ASL. One (or both) of the &crystallin genes appears to code for ASL, based on the high degree of sequence homology between the human ASL and chicken b-crystallin and the ability of cDNA probes to either gene to hybridize to common restriction fragments from chicken DNA (Piatigorsky et al., 1988). In addition, low levels of ASL activity were found in lens homogenates and purified chick &crystallin protein has some ASL activity (Wistow and Piatigorsky, 1987). It is interesting to note that very low levels of enzyme activity were found in several nonlens tissues from chicken embryos (lo3 times lower than in the lens; Piatigorsky et al., 1988). Thus, if one or both of the two &crystallin genes is also coding for ASL, then continuous, low-level transcription of the 61 and/or 62crystallin genes would be expected. Although it is certainly quite possible that the &crystallin mRNA detected in nonlens tissues is also coding for ASL, at least some of the expression, especially where levels of expression are very low, could be a result of nonfunctional “leaky” transcription as has been proposed to occur for other genes, for example, the elastase I gene in rats (Swift et al., 1984). Our expression studies have provided a new perspective on earlier hypomethylation data which has linked changes in hypomethylation to regulation of &crystallin expression. We find that the temporally regulated hypomethylation that we observed beginning at 50 hr at Hha3, Hha4 (Sullivan et al., 1989) and Hpa7 (Grainger et ah, 1983) in the al-crystallin gene is spatially regulated in the young embryo as well. When the extent of hypomethylation of these same three sites was examined in DNA from nonlens tissues of 84-hr chicken embryos, we found that they remained fully methylated, even though fi-crystallin was expressed at low levels. However, DNA in nonlens tissues from both genes was not methylated

V0~~~~145,1991

at seven other HpaII sites, identical to the pattern of hypomethylation seen previously, where these sites were identified as being constitutively hypomethylated in tissues that were examined (Sullivan et ah, 1989). In our earlier experiments the significance of this observation was unclear, but we now propose that hypomethylation at these sites reflects the ubiquitous expression of the a-crystallin genes. Genes that are expressed continuously often have such a region of constitutive hypomethylation (Stein et al., 1983). These results can be assembled into a model to link data on hypomethylation with data on expression. Low levels of expression found in all tissues are linked to the constitutive hypomethylation observed. Constitutive hypomethylation is found at HpaII sites in both &crystallin genes, consistent with the data that both the aland fi2-crystallin genes are transcribed in nonlens tissues. High levels of &crystallin mRNA in the lens are primarily due to 61-crystallin transcription (Wawrousek et al., 1986; Parker et ah, 1988). The hypomethylation events seen exclusively in the lens are at HhaI and HpaII sites found only in the al-crystallin gene (Sullivan ef al., 1989). Hypomethylation of these sites and high levels of tissue-specific d-crystallin gene expression in the lens, which both begin at 50 hr of development, therefore, appear to be linked. Thus there are two distinct patterns of &crystallin gene expression and modification in embryonic chick tissue. Although our data on expression do not allow us to predict whether different levels of &crystallin mRNA in lens and nonlens tissues are a result of control at the transcriptional or post-transcriptional levels, the different methylation patterns in lens and nonlens tissues suggest at least some of the differences in expression are likely to involve transcriptional control. Having defined these two levels of expression, it should be possible to study directly the regulatory factors involved in these two ways of controlling 6-crystallin gene expression. Th,e Relationship of Hypomethylation and Transcriptional Control of the &Crystallin Genes Our data suggest that levels of &crystallin gene expression can occur in the absence of complete hypomethylation of HpaII and HhaI sites in this gene, and identify specific restriction sites that could be examined for a role in transcriptional control. However, it is very possible that important methylation changes might occur at other cytosine residues that are not recognized by the restriction enzymes available or that methylation changes at HpaII clustered at the 5’ end of 61 (Fig. 1) would go undetected because very small restriction fragments are produced. Also, restriction enzyme analysis cannot distinguish between complete methylation

SULLIVAN, O’FARRELL, AND GRAINGER

and methylation of only one strand of DNA (hemimethylation). However, the technique of genomic sequencing (Church and Gilbert, 1984) might be used to address these possibilities. In one case, this technique has been used to show that although complete hypomethylation lags behind expression of the vitellogenin gene in liver cells treated with estradiol (Wilks et ul., 1982,1984), the appearance of hemimethylated DNA precedes the onset of transcription (Saluz et ul., 1986). Of interest, the regions undergoing hemimethylation are upstream of the gene in a region of binding of the hormone-receptor complex with DNA at the site thought to be important for gene activation (Jost et ul., 1984). Therefore, strandspecific changes in methylation may be the functionally relevant alteration in DNA methylation that regulates gene activity. This remains to be investigated further as a possible mechanism of regulation of the &crystallin gene. On the other hand it is quite possible that although hypomethylation of particular sites in the al-crystallin gene occurs during the burst of crystallin mRNA accumulation associated with lens differentiation, these events are not required for Scrystallin transcription in lens and nonlens cells. A similar separation of hypomethylation from expression has been observed for the K-immunoglobulin genes (Kelley et al., 1988). Precocious expression of K-genes can be induced in pre-B lymphocytes, but this gene activity is not accompanied by hypomethylation. However, K-expression is tightly linked to hypomethylation in B-cells and plasma cells. The kinetics of hypomethylation of the al-crystallin gene argue against a role in initiating transcription as well, but is consistent with an alternate role such as in stabilizing transcriptionally active chromatin, as suggested by Kelley ef al. (1988) for the K-genes. It should be possible to examine whether there are changes in chromatin structure, such as DNase sensitivity, that occur with the same kinetics as the changes in DNA methylation we have observed. We thank John Marshall for assistance in preparing the figures. This work was supported by grants from the American Cancer Society (VC-306), National Institutes of Health (EY-05542), National Science Foundation (PCM-82.08266), and the Thomas F. and Kate Miller Jeffress Memorial Trust (J-24) to R.M.G., who was also supported by a National Institutes of Health Research Career Development Award (HD-00289). C.H.S. was supported by National Institutes of Health Training Grant HD-07192 and a National Institutes of Health National Research Service Award (HD-06422).

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