Development 121, 3425-3437 (1995) Printed in Great Britain © The Company of Biologists Limited 1995
3425
Progressive maturation of chromatin structure regulates HSP70.1 gene expression in the preimplantation mouse embryo Eric M. Thompson*, Edith Legouy, Elisabeth Christians and Jean-Paul Renard Unité de Biologie du Développement, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas, France *Author for correspondence (e-mail:
[email protected])
SUMMARY In the widely studied model organisms, Drosophila and Xenopus, early embryogenesis involves an extended series of nuclear divisions prior to activation of the zygotic genome. The mammalian embryo differs in that the early cleavage phase is already characterized by regulated cell cycles with specific zygotic gene expression. In the mouse, where major activation of the zygotic genome occurs at the 2-cell stage, the HSP70.1 gene is among the earliest genes to be expressed. We investigated the developmentally regulated expression of this gene during the preimplantation period, using a luciferase transgene, with or without flanking scaffold attachment regions (SARs). Cleavage
stage-specific modifications in expression profiles were examined in terms of histone H4 acetylation status, topoisomerase II activity, and the localisation of HMG-I/Y, a nuclear protein with known affinity for the AT-tracts of SARs. We demonstrate that HSP70.1-associated transcription factors are not limiting, and that instead, there is a progressive maturation of chromatin structure that is directly involved in HSP70.1 regulation during early mouse development.
INTRODUCTION
decondensation in the short early cleavage cycles (Ner and Travers, 1994). During Xenopus embryogenesis, H1 is replaced by the maternal variant, histone B4, until the midblastula transition (MBT), again coincident with the ZGA. In the latter organism, H1 has been shown to be involved in the switch from oocyte to somatic 5S rRNA gene expression, through specific repression of the oocyte 5S rRNA gene even in the presence of an abundance of the transcriptional activator TFIIIA (Bouvet et al., 1994). Levels of histone H1 may play a role in regulating gene expression in early mouse embryos, as immunofluorescence studies have shown that H1 is not present until S-phase of the 4-cell stage (Clarke et al., 1992). An essential difference in early mammalian development, compared to that of Drosophila or Xenopus, is the absence of an extended series of nuclear divisions prior to activation of the zygotic genome. This is particularly true of the mouse, where activity of a cAMP-dependent protein kinase (Latham et al., 1992) has been implicated in establishing a transcriptionally permissive state as early as the 1-cell stage. During this minor activation of the genome (Schultz, 1993), transcriptional activity has been detected from the male pronucleus (Ram and Schultz, 1993; Christians et al., 1995). This then leads to the major ZGA at the 2-cell stage (Flach et al., 1982). Enhancers do not seem to have an important role in the regulation of episomal gene expression during the 1-cell stage, in S-phase arrested, aphidicolin-treated embryos, but become necessary to prevent repression of episomal expression from weak promoters at the 2-cell stage (Majumder et al., 1993). These results have led to the
The packaging of DNA into chromatin has important consequences for the regulation of gene activity in eukaryotic organisms. The fundamental unit of chromatin organization is the histone octamer, in which 146 base pairs of DNA are wound around a central (H3/H4)2 tetramer, flanked by two (H2A/H2B) dimers. Positioning of nucleosomes with respect to the DNA helix can have repressive effects on gene expression by blocking transcription factor access (Bogenhagen et al., 1982; Knezetic and Luse, 1986; Workman et al., 1991), or stimulatory effects through juxtaposition of activating regulatory elements (Thomas and Elgin, 1988; McPherson et al., 1993; Schild et al., 1993). Incorporation of DNA into higher order chromatin structures is mediated by binding of histone H1 to the linker DNA between nucleosomes. Histone H1 has been considered as a repressor of transcription (Shimamura et al., 1989; Laybourn and Kadonga, 1991) but recent studies suggest caution in the generalisation of such a role (Sandaltzopolous et al., 1994; Bouvet et al., 1994). In early embryonic development, switching of H1 subtypes appears to be a common theme, while alteration of core histone subtypes, as observed in the sea urchin, is much less widespread (Poccia, 1986). In Drosophila, H1 is not detected until the 9th or 10th cleavage cycle, coincident with the major activation of zygotic transcription (ZGA). Up to this point, H1 appears to be replaced by the high mobility group protein HMG-D, a protein which may generate a less compact chromatin structure, and facilitate rapid condensation and
Key words: chromatin, histone H4 acetylation, HMG-I(Y), scaffold attachment, regions, transgenesis
3426 E. M. Thompson and others proposition that chromatin structure is important in the selective regulation of early zygotic gene expression in the mouse, perhaps via modulation of acetylation levels of histones in the nucleosomal core (Wiekowski et al., 1993). Further analysis of these hypotheses is limited by the fact that gene regulation data have been generated by microinjection of various DNA constructs into early embryos. The consequences of this approach are that it is impossible to control variability in the number of DNA copies introduced, large numbers of copies are injected, subsequent embryonic development is compromised by the injection process, and it is not trivial to relate the chromatin structure of supercoiled episomal plasmid templates to that of native embryonic chromatin. A further indication that chromatin structure and nuclear architecture are involved in regulating the program of gene expression during ontogeny is the observation that a number of developmentally regulated genes are bordered by scaffold attachment regions (SARs). These include Adh, ftz, and Sgs-4 in Drosophila (Gasser and Laemmli, 1986), and the β-globin gene locus in humans (Jarman and Higgs, 1988). Originally identified as AT-rich DNA sequences which bind to preparations of the nuclear matrix and frequently map to or near domain boundaries of gene loci (Gasser et al., 1989), SARs do not appear to be as consistent in insulating transgenes from position effects as Drosophila specialized chromatin structures (scs) (Kellum and Schedl, 1992) nor to act as dominant activators of tissue-specific gene expression as do locus control regions (LCRs) (Dillon and Grosveld, 1993). More recently it has been proposed that they may be involved in mediating chromatin accessibility, through synergistic action with enhancers (Forrester et al., 1994), or via their interaction with histone H1 (closed) or proteins such as the high mobility group protein, HMG-I/Y, capable of displacing H1 from the ATtracts (open) (Zhao et al., 1993). We have investigated the effect of SAR sequences on gene expression in the early mouse embryo, a period when significant changes in chromatin structure, and particularly histone H1 concentration occur. To overcome the limitations of transient expression assays, we generated transgenic mice in which the promoter of the heat shock gene HSP70.1 (Hunt and Calderwood, 1990) directed expression of a luciferase reporter cDNA (de Wet et al., 1987). HSP70.1 is part of the multigenic, inducible, hsp70 family known to be expressed constitutively in the 2-cell mouse embryo and to be heat inducible at the blastocyst stage (Bensaude et al., 1983). A second series of transgenic lines was then produced with the above construct flanked by 5′ and 3′ SARs from the human β-interferon locus (Bode and Maass, 1988). Significant stage-specific differences were observed in the preimplantation expression profiles of the two different transgenic constructs. Modification of expression profiles in the presence of trichostatin A (TSA), which specifically inhibits histone deacetylases (Yoshida et al., 1990) and leads to hyperacetylation of core histones, was then examined and compared with the nuclear immunofluorescence staining pattern of acetylated histone H4 during early development. At embryonic stages where SAR+ lines showed increased activity, studies carried out with the topoisomerase II inhibitor, VM-26, suggested that recruitment of topoisomerase II to SAR sequences was not the mechanism responsible for the observed effect. Finally, preimplantation HMG-I/Y mRNA levels were studied by RT-PCR and its cellular localisation was analysed
by immunofluorescence, to determine whether there was any correlation between the presence of HMG-I/Y and the transgene expression profiles. Taken together, the data characterize a progressive maturation of chromatin structure in the zygotic nucleus which plays an important role in regulating gene expression during the early cleavage stages of mouse development.
MATERIALS AND METHODS Transgenic mice and transient expression experiments The transgenic mouse lines used in this study have been previously described (Thompson et al., 1994). Four lines, F2, F27, F29 and F31, with the murine HSP70.1 promoter directing firefly luciferase reporter expression, were compared with 5 lines, SF2, SF3, SF4, SF6 and SF9, harbouring the same promoter-reporter construct flanked by 5′ and 3′ SAR elements obtained from the human β-interferon locus. The ‘F’ lines contained 2, 3, 1 and 1 transgene copies and the ‘SF’ lines, 4, 3, 6, 4 and 2 transgene copies, respectively. For transient expression experiments, plasmid DNA was prepared by cesium chloride density gradient ultracentrifugation. Supercoiled plasmid DNA was microinjected as previously described (Hogan et al., 1986) at a concentration of 50 ng/µl using Narishige micromanipulators (Nikon), and an Eppendorf microinjector. Analysis of transgene expression in preimplantation embryos Mating of transgenic male mice to superovulated F1 hybrid C57BL6×CBA females (Iffa Credo), recovery of 1-cell embryos, in vitro culture conditions, and luciferase assays were as described by Thompson et al. (1994). Constitutive expression of the transgene was determined at the 2-cell, 4-cell, 8-cell and blastocyst stages. Dark current photometer background was 150±20 relative light units (RLU) and therefore, 170 RLU were subtracted from all measured values. Timing of the anlaysis was with respect to hours post human chorionic gonadotropin (HCG) injection: 2-cell, 41-42 hours; 4-cell 63-64 hours; 8-cell 73-74 hours; and blastocyst 111-112 hours post HCG. For induced expression at the blastocyst stage, embryos at 111-112 hours post HCG were heat-shocked at 43°C for 30 minutes and allowed to recover at 37°C for 5-6 hours before lysis. Treatment of transgenic embryos with drugs and inhibitors was carried out as shown in Fig. 1. Embryos were cultured from the 1-cell stage in drops of M16 medium (Hogan et al., 1986), and at the points indicated T1-T4 in the figure, were split into two groups. The test group was transferred to an equilibrated drop of M16 medium, containing a specified concentration of a given compound. The control group was transferred into a fresh equilibrated drop of M16 medium. Transfers took place towards the end of a given cleavage stage, embryos divided through to the next cell cycle, and were then harvested for analysis at the points indicated A1-A4 in Fig. 1. Stock solutions of 600 µM trichostatin A (TSA) (a gift from M. Yoshida, University of Tokyo) in 100% dimethylsulfoxide (DMSO), and 1 mM teniposide VM-26 (a gift from Sandoz laboratories) in 100% DMSO were prepared and diluted to working concentrations in M16 medium just prior to use. An exception to the above protocol was treatment of embryos with VM-26, which was only studied in SAR+ lines, at the 2-cell stage and in heat-shocked blastocysts. When this inhibitor was introduced before cell cleavage, embryos were unable to pass through to the next cell cycle. Therefore, 2-cell embryos were picked off following cleavage from the 1-cell stage and 10 µM VM-26 was introduced. Assay of luciferase activity was performed at 41-42 hours post HCG. At the blastocyst stage, embryos at 111 hours post HCG were incubated for 1 hour in 10 µM VM-26 prior to heat shock, recovery,
Chromatin maturation in mouse embryos 3427 FERTILIZATION (Transgenic ) HCG
Fig. 1. Schedule of treatment and analysis of transgenic preimplantation embryos. The program of cell cleavage is shown Onset of embryonic gene expression at the top with the onset of zygotic gene expression indicated A1 A2 A3 A4 immediately below. Timing of events is with respect to hours post HCG injection. The point of 0H 24H 48H 72H 96H 120H fertilization is roughly indicated but T1 T2 T3 T4 was not precisely controlled. On the lower scale, the times at which H post HCG incubation of embryos began in the presence of drugs and inhibitors is indicated by T1 to T4. At the corresponding points A1 to A4, embryos were harvested and analysed for transgene expression, or alternatively, fixed and studied by immunofluorescence. 1-cell
2-cell
4-cell
8-cell
16-cell
and assay as described above. VM-26 was maintained in the culture medium at 10 µM during heat shock and subsequent recovery. Statistical analyses of expression data were performed with Statview II software (Abacus Concepts, Inc.) RT-PCR analysis of preimplantation embryos Pools of 50 embryos of a given cleavage stage, were resuspended in 7.5 µl lysis buffer containing 1% NP40, 1.3× MMLV-RT buffer (GibcoBRL) and then heated at 100°C for 1 minute. Synthesis of cDNA was initiated by addition of 2.5 µl of a mixture containing 100 ng oligo-dT, 200 mM of each dNTP, 10 units of RNAse inhibitor (Boehringer), and 200 units of MMLV reverse transcriptase (GibcoBRL). Following a 1 hour incubation at 37°C, mixtures were heated for 5 minutes at 95°C. PCR amplifications of the cDNA for heat shock factor 2 (HSF2) mRNA (equivalent to 25 embryos), and HMG-I/Y mRNA (equivalent to 30 embryos), were carried out in 50 µl of 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X100, 200 mM of each dNTP, 1 unit of TAQ DNA polymerase (Bioprobe), and 25 pmoles of each primer. After an initial denaturation step at 94°C for 10 minutes, samples were subjected to 35 cycles of amplification (94°C 30 seconds, 58°C 30 seconds, 72°C 30 seconds). This was in the linear range of amplification product versus cycle number. Aliquots of 20% of each sample were run on 1.8% agarose gels and transferred to nylon membranes (hybond-N+, Amersham) for probe hybridization. Autoradiographic exposure times at −80°C were 3 hours for HSF2 and overnight for HMG-I/Y. The HMG-I/Y primers were derived from the published cDNA sequence (Johnson et al., 1988): sense primer, nucleotides 154 to 172; anti-sense primer, nucleotides 546-564. HSF2 primers were from the published cDNA sequence (Sarge et al., 1991): sense primer, nucleotides 1353-1373; anti-sense primer, nucleotides 1874-1894. The HSF2 probe was the mouse cDNA (gift from R. Morimoto, Northwestern University). The HMG-I/Y probe was cloned by RTPCR from mouse fibroblast total RNA using the primers described above. Primary culture of foetal fibroblasts Foetuses obtained by mating transgenic male mice to non-superovulated F1 hybrid C57BL6×CBA females were recovered 12-13 days post coitum and immediately killed by decapitation. Livers were removed and bodies were chopped into fine pieces with a scalpel. Subsequent preparation of fibroblasts was as described for primary culture of ear fibroblasts (Thompson et al., 1994), except that the final concentration of trypsin (Gibco) was 0.25% and collagenase was not used. Isolated fibroblasts were used in experiments after one or two passages except when aged preparations were desired. In the latter case, cells were passed four times, and then plated at low density and
Morula
Blastocyst
cultured for several days, with medium changes, until cell division had ceased. When foetal fibroblasts were treated with TSA, the incubation was for a period of 11-12 hours. Immunofluorescence confocal microscopy Prior to fixation, the zona pellucida was removed by treatment with 5 µg/ml pronase (Boehringer) in M2 medium (Hogan et al., 1986). Subsequent treatment of embryos and foetal fibroblasts was then identical, except that fibroblasts were transferred between baths attached to a coverslip, whereas embyros were unattached and transferred by mouth pipette. Two fixation protocols were used. In the first, cells were fixed in 2.5% paraformaldehyde (Sigma)/0.02% Triton X100/phosphate-buffered saline (PBS) at 37°C for 15 minutes. In the second, cells were fixed in ethanol/H2O/acetic acid, 95:4:1, for 1 hour at 4°C and then rehydrated in a series of 10-minute baths: 85% ethanol, 70% ethanol, 50% ethanol, and PBS/1% bovine serum albumin (BSA)/0.02% sodium azide. Further preparation was the same for both fixation protocols. Fixed cells were blocked in PBS/10% fetal calf serum or 10% sheep serum/0.2% Triton X-100. First antibodies and preimmune sera were diluted in PBS/2% fetal calf or sheep serum/0.2% Triton X-100 and incubations were performed overnight at 4°C. Cells were rinsed at 37°C in PBS/2% fetal calf or sheep serum/0.2% Triton X-100 for 30 seconds, 10 minutes, and 30 seconds and then incubated with fluorescein isothiocyanate- (FITC) conjugated sheep anti-rabbit IgG (1:400) (Sigma Immunochemicals) for 1 hour at 37°C. Cells were rinsed as above, counterstained in 10 µg/ml propidium iodide (Sigma) for 15 minutes at 37°C and mounted in Moviol 4-88 (Hoechst). Observations were made using a Zeiss confocal laser scanning microscope LSM-310 with a Zeiss plan neofluor 100× (NA 1.3) oil immersion objective. Images were analysed using NIH Image 1.54 software (National Institute of Health, USA). A minimum of 50 embryos were examined in 2 to 3 replicates for each cleavage stage and for each experimental treatment. Two antibodies against acetylated forms of histone H4 were used. The first recognized all acetylated histone H4 isoforms (Lin et al., 1989) (a gift from D. Allis, Syracuse University), and was used at a 1:1000 dilution. The second antibody, specific for histone H4 acetlyated at lysine 5 (Turner and Fellows, 1989) (a gift from B. Turner, University of Birmingham), detects only the most highly acetylated isoforms in mammalian somatic cells and was used at a 1:700 dilution. The anti-HMG-I/Y antibody (a gift from R. Reeves, Washington State University) was used at a 1:100 dilution. Preimmune sera were provided with the first anti-acetylated histone H4 antibody and the anti-HMG-I/Y antibody. For the second anti-lysine 5 acetylated histone H4 antibody, the control consisted of overnight incubation in the absence of first antibody, followed by incubation with the FITCconjugated second antibody.
3428 E. M. Thompson and others RESULTS
Alteration of transgene expression in response to stage-specific hyperacetylation of core histones When present in culture medium, TSA, a specific inhibitor of histone deacetylases, causes hyperacetylation of core histones, a condition usually associated with transcriptionally active
343
2-cell
10
216 268 392 508
5 109 352 251 308
0 10 8
235
4-cell
6
Luciferase Activity / Embryo (RLU x 10 -2)
4 2 0 1.5
247 128 146
*
145 183
195 155
116
8-cell 1.0 0.5 126 89 144
0 1.0 0.8
181
112
182 103
*
Blastocyst 239
0.6 241
0.4
253 295
0.2
199
144
95 126
*
0 200
71
Blastocyst 150
184
(Induced)
100
196 181 154
50
SAR-
SF9 SF3 SF2 SF6 SF4
60 243 60
F27
58
0
F29 F31 F2
SAR sequences modified the preimplantation expression profile of the HSP70.1-luciferase transgene The HSP70.1 gene is one of the few genes for which a preimplantation profile of expression has been characterized in early mouse development. RT-PCR analysis revealed that HSP70.1 is constitutively transcribed in the 2-cell embryo and is induced by heat shock at the blastocyst stage (Christians et al., 1995). Zygotic transcription of HSP70.1 begins during G2 of the 1cell stage and increases during G1 of the 2-cell stage. The arrival of S-phase during the second cell cycle signals a reduction of HSP70.1 transcription to basal levels. Constitutive basal levels of endogene expression were also observed in 4- and 8-cell embryos and in non-heat shocked blastocysts as well as in a variety of differentiated tissues and cells in culture (Thompson et al., 1994). The firefly luciferase transgene provides a sensitive single embryo assay with which to monitor HSP70.1 expression. Using purified firefly luciferase (Sigma) for calibration, 1 relative light unit (RLU) was produced by approximately 1.5 fg of luciferase. The lability of the luciferase is useful in following the kinetics of preimplantation expression. When purified luciferase was microinjected into early zygotes, to attain levels of expression similar or superior to those generated by transgenic embryos, the half-life of the luciferase was less than 2 hours (Christians et al., 1995). Initial analysis of transgenic embryos at the 2-cell and heatshocked blastocyst stages showed a statistically significant, copy number-dependent stimulation of transgene expression in SAR+ lines relative to SAR− lines (Thompson et al., 1994). Further analysis of constitutive expression at the 4-cell, 8-cell and blastocyst stages is presented in Fig. 2. At the 4-cell stage there was a drop in transgene expression as observed for the HSP70.1 endogene. In SAR− lines, the basal constitutive level was attained with the exception of the line F29, which continued to express at a slightly elevated level. All SAR+ lines exhibited absolute expression above the basal level, this being particularly true of the line SF4 in which expression was about half of that observed during the early 2-cell stage. However, at the 4-cell stage, correlation of expression with copy number was lost in SAR+ lines and on a per copy expression basis, only the line SF4 showed a clear difference when compared to SAR− lines. At the 8-cell stage, all lines had attained the basal transcriptional level. The SAR+ line, SF4, retained a slightly elevated absolute expression level but showed no difference on a per copy basis. At the blastocyst stage, low constitutive levels similar to those at the 8-cell stage were observed, though if the substantial increase in cell number, and therefore transgene copy number, is taken into account, basal levels per transgene copy were much lower. When blastocysts were heat-shocked the stimulatory effect of SAR sequences was again observed as was a positive correlation with copy number in SAR+ lines.
15
SAR+
Transgenic Mouse Line Fig. 2. Transgene expression in preimplantation mouse embryos. SAR− lines are grouped on the left and SAR+ lines on the right. Both groups are arranged in order of increasing copy number. Luciferase activity is given on the ordinate as relative light units (RLU) per embryo. Constitutive expression was measured at the 2-, 4-, 8-cell and blastocyst stages. Heat shock induced expression was also determined at the blastocyst stage. Open bars represent absolute transgene expression and shaded bars the per copy expression. The s.e.m. and the number of embryos analysed in each transgenic line are indicated above the bars. During breeding to homozygosity, it was discovered that the site of transgene insertion in the SAR− line, F27, was homozygous lethal (between day 14 and 18 post coitum). Therefore, after initial analysis of constitutive expression in 2-cell embryos and heat induced expression at the blastocyst stage, study of this line was discontinued. This is indicated by asterisks.
1000
SAR100 10 1
1000
SAR+
100 10 1 2-cell
4-cell 8-cell Blastocyst
Developmental Stage Fig. 3. The effect of TSA on constitutive transgene expression at different developmental stages. Mean luciferase activity in the presence of TSA, divided by that obtained in the absence of TSA, is plotted on a logarithmic scale for SAR− lines (upper panel) and SAR+ lines (lower panel). F29 s, F31 h, F2 n, SF9 r, SF3 d, SF2 ., SF6 j, SF4 m.
chromatin (Hebbes et al., 1988; Jeppesen and Turner, 1993). The strategy used to examine the stage-specific effect of histone hyperacetylation is outlined in Fig. 1. Embryos were exposed to TSA towards the end of a given cell cycle and expression was analysed during the following cell cycle. The results are summarized in Fig. 3 and the statistical significance is assessed in Table 1. First, we compared the effects of TSA at 0 nM, 30 nM, 75 nM, and 150 nM concentrations. Variability in response was observed among different lines, but for most a near maximal expression signal was obtained at 75 nM. Table 1. Effect of TSA on preimplantation transgene expression 2-cell Line SF9 SF3 SF2 SF6 SF4 SAR+§ F29 F31 F2 SAR−§
4-cell
n†
TvsC‡
n†
TvsC‡
258 81 198 67 110
0.62*** 1.22 2.13*** 1.55 1.72** 1.45 0.56*** 0.85 1.04 0.82
73 87 141 63 90
29.8*** 40.9*** 35.6*** 23.7*** 14.3*** 28.9 31.6*** 38.8*** 2.2 24.2
129 53 104
201 114 110
8-cell n† TvsC‡
Blastocyst n†
99 116*** 88 40 65* 182 110 258*** 174 43 343*** 79 90 19*** 80 160 90 21.5*** 134 50 3.4*** 83 110 5.0* 85 10
TvsC‡ 23.3*** 5.7*** 28.1*** 28.5*** 57.2*** 28.6 1.6*** 4.9*** 3.7** 3.4
†The total number of embryos used in 2 to 4 experimental replicates ‡The mean expression in TSA-treated embryos divided by the expression in control embryos. Significant differences (t-test) are indicated; *P
