The need for enhancers is acquired upon formation...

The need for enhancers is acquired upon formation of a diploid nucleus during early mouse development Encarnacion Martinez-Salas, Doreen Y. Cupo/ and Melvin L. DePamphilis

Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110 USA

The activity of the polyoma virus (PyV) origin of DNA replication was used as a sensitive assay for enhancer function in one- and two-cell mouse embryos by injecting embryos with plasmid DNA containing different PyV ori configurations, allowing them to continue development in vitro, and then measuring plasmid DNA replication. Replication always required the PyV origin 'core' sequence in cis and PyV large tumor antigen (T-Ag) in trans. In developing two-cell embryos, DNA replication also required an enhancer in cis. Two copies of part of PyV enhancer 3 0 element) was sevenfold better than one copy, and enhancer 3 was better than enhancer 1 + 2 (a element). Competition between ori configurations suggested that enhancers bound specific proteins required for replication and transcription. In contrast, DNA injected into one-cell embryos did not need an enhancer for replication, and no competition for replication factors was observed between different ori configurations. In fact, ori core replicated about ninefold better in one-cell embryos than the complete origin did in developing two-cell embryos. Therefore, core contains all the cis-acting information necessary to initiate DNA replication. Because one-cell embryos that replicated injected DNA retained their pronuclei and remained one-cell embryos, enhancers are not needed in mammalian development until a diploid nucleus is formed.

[Key Words: Enhancer; mouse; embryos; pronuclei; polyoma virus]

Received April 13, 1988; revised version accepted July 18, 1988.

Enhancers are cis-acting sequences that activate RNA polymerase II transcription of linked genes independently of their orientation and distance and play a major role in determining cell specificity of gene transcription (Serfling et al. 1985). Enhancers also function as components of several eukaryotic origins of DNA replication (DePamphilis 1988). For example, in polyoma virus (PyV), the genetically defined origin of DNA replication (ori) consists of two components, a core (oh core) and an enhancer (see Fig. 1). PyV ori core determines where DNA replication begins (Hendrickson et al. 1987) and functions only in the presence of PyV large tumor antigen (T-Ag) and mouse DNA primase-DNA poly-merase-a (Murakami et al. 1986). However, oh core must be associated with an enhancer to initiate replication in permissive mouse cell cultures (Veldman et al. 1985; Hassell et al. 1986), as well as in the living animal from a preimplantation embryo to an adult (Wirak et al. 1985; Rochford et al. 1987). The ability of an enhancer to stimulate ori-core-dependent DNA replication in a particular mouse cell type correlates closely with its ability to stimulate transcription (Veldman et al. 1985; Campbell and Villarreal 1986; DePamphilis et al. 1986; Hassell et al. 1986; Mueller et al. 1988). Mutations that alter one function also alter the other (Linney and Donerly 1983; Tang et al. 1987; Satake et al. 1988), and cis-acting

P̂resent address: Department of Chemistry, Bates College, Lewiston, Maine 04240 USA.

alterations of the PyV enhancer region can allow viral DNA replication in mouse cells that are normally non-permissive (Amati 1985). Either enhancer orientation can activate PyV ori core from a distance (deVilliers et al. 1984), although a subsequent study concluded that the enhancer must be in close proximity to the AT-rich side of oh core (Hassell et al. 1986). Therefore, it appears that enhancer elements stimulate initiation of DNA replication at oh core by a mechanism that is similar, if not identical, to that used to stimulate transcription at a promoter.

The PyV enhancer region is contained within a 241-bp DNA fragment defined by Bell and Pvull restriction sites that can be subdivided into domains 1, 2, and 3, which interact to produce full enhancer capacity (Fig. 1). Enhancer domains 1 -H 2 and 3 correspond closely with the borders of two functionally redundant elements, a (or A -H D) and ^ (or B -H C), that are part of the origin of PyV DNA replication. DNA binding sites for putative enhancer-specific proteins have been identified that correspond to replication elements A, B, and C. Enhancer activity can be provided by either the a or (3 elements, depending on the cell type, or by non-PyV enhancers (deVilliers et al. 1984; Campbell and Villarreal 1986). Two copies of the minimal PyV a (A) element are sufficient to activate oh core fully in mouse fibroblasts, whereas three copies of this sequence are necessary to activate 10-30% of the PyV early promoter (Veldman et

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Maitinez-Salas et al.

al. 1985). Thus, because DNA replication is undetectable when PyV oh core is not associated with a functional enhancer, oh core activity provides an exceptionally sensitive assay for measuring enhancer activity. Therefore, we used this sensitive assay to determine at what point in mammalian development enhancers become regulatory elements.

Injection of DNA into mouse oocytes revealed that both RNA polymerase II and III promoters are recognized but that full expression of polymerase II promoters does not require association with an enhancer (Brinster et al. 1981; DePamphilis et al. 1986; Chalifour et al. 1987). However, enhancers are utilized in developing two-cell embryos. DNA injected into a two-cell embryo does not replicate unless it contains a functional oh such as the PyV oh core linked with the PyV p element (Wirak et al. 1985; Chalifour et al. 1986). Transient gene expression assays confirmed that the p element (enhancer 3) stimulated the herpes simplex virus (HSV) thymidine kinase promoter in two-cell embryos as it does in differentiated cells (DePamphilis et al. 1986). Thus, the first time that enhancer elements are recognized during mammalian development may be when transcription is activated in the nucleus of a two-cell embryo. We have tested this hypothesis by measuring the replication of plasmid DNA containing various PyV oh configurations following injection into one- and two-cell mouse embryos.

Remarkably, enhancers were not required for DNA replication (or gene expression) in one-cell embryos that retained their pronuclei and did not cleave, whereas enhancers were required in developing two-cell embryos. Thus, enhancer elements are not needed for activating origins of replication and transcription until a diploid nuclear structure is formed. Furthermore, these experiments demonstrated that the pronuclei of fertilized mouse eggs could recognize as oh the core element of PyV, a sequence that was not recognized as oh in any subsequent stage of mouse development.

Results

Developing two-cell embryos uhlize enhancers

Previous studies revealed that plasmids containing the complete PyV ori replicate when injected into two-cell mouse embryos in the presence of a functional PyV T-Ag gene (Wirak et al. 1985). Furthermore, only the a- to p-core and p-core configurations (Fig. 1) were active in these experiments, suggesting that wild-type PyV enhancer 3, but not enhancer 1-1-2, was active in preim-plantation mouse embryos. These results were confirmed and extended by showing that core cannot replicate in developing two-cell embryos but that either a- or p-core can replicate, providing that neither replication factors nor enhancer-specific binding factors are removed through competition with another DNA sequence.

PyV sequences cloned into pML-1 (Fig. 1) were injected into one of the nuclei of a two-cell embryo, and

surviving embryos were cultured for up to 48 hr before measuring the amount of plasmid DNA replication. During this time, 80% of the surviving two-cell embryos continued development to the morula and early blastocyst stages. Development of the remaining 20% was arrested at the two- and four-cell stages. Therefore, embryos were segregated according to the extent of their development (Wirak et al. 1985) before assaying plasmid DNA replication by its sensitivity to Dpnl restriction endonuclease, as described in Experimental procedures. DNA resistant to Dpnl digestion represented newly replicated DNA exclusively, whereas the total amount of plasmid DNA was determined from the sample cut only at a single site. From 30% to 80% of the plasmid DNA was lost soon after injection, presumably because some of the injected DNA leaked into the cytoplasm where it was degraded rapidly. Nevertheless, the remaining DNA was stable, regardless of whether or not it replicated. Newly replicated plasmid DNA normally was not detected until after 24 hr postinjection, and it reached a maximum level by 48 hr (Wirak et al. 1985). Therefore, we routinely collected DNA samples at 0, 24, and 48 hr postinjection. The total amount of plasmid DNA present at 24 hr was defined as the baseline for calculating the net increase in newly replicated DNA {Dpnl resistant) at 48 hr.

As observed previously by Wirak et al. (1985), the complete PyV genome cloned into pML-1 (pBla, Fig. 1) replicated when injected into the nuclei of two-cell embryos that subsequently underwent further development (Fig. 2). Analysis of the cis- and trans-acting sequences that were required for replication of the injected DNA revealed that only plasmids containing both the p and core elements of PyV ori (a-p core and p core; Fig. 1) replicated and only if coinjected with a second plasmid containing a functional PyV T-Ag gene (pBla; Fig. 2). Plasmids containing a core failed to replicate in the presence of pBla. Furthermore, core also did not replicate under these conditions (Fig. 2). These results suggested that proteins interacting with the a element are either absent or present at low levels in preimplantation embryos. Alternatively, a core may simply be less effective at competing for replication initiation factors (e.g., T-Ag initiation complex) than the complete ori (pBla).

To eliminate competition between ori in pBla (a-p core) and a core or core, the experiment was repeated using pSVE in place of pBla. pSVE contains the SV40 enhancer-promoter ori region (nucleotides 1047-5171) joined to PyV DNA from position 154 to 4632, replacing the entire PyV ori region (Fig. 1). pSVE did not replicate in two-cell embryos (Fig. 2) because SV40 T-Ag was absent (Chalifour et al. 1986). However, it did produce PyV T-Ag when used to transform mouse fibroblasts (MuUer et al. 1984). Functional T-Ag was also produced in preimplantation embryos, as p core replicated as well or better when coinjected with pSVE than with pBla (Fig. 2). Furthermore, a core replicated equally as well as p core when coinjected with pSVE (Fig. 2). Thus, the complete PyV ori (a-p core) binds replication factors more tightly than a core, preventing initiation of replication

1116 GENES & DEVELOPMENT




Enhancers in early mouse development

by a core. Despite the absence of competition for replication initiation factors, PyV oh core still failed to replicate following injection into two-cell embryos (Fig. 2). Therefore, an enhancer sequence must be linked with core in order to initiate replication in developing two-cell embryos.

Enhancer-specific factors are required for DNA replication

The ability of a core to initiate replication in the absence of competition from a complete PyV ori suggested

that either viral T-Ag or cellular enhancer-1 + 2-spe-cific binding proteins (e.g., PEAl, PEA2; Fig. 1) were limiting for initiation of PyV DNA replication in developing two-cell embryos. To test the possibility that unregulated production of T-Ag by pSVE could overcome competition between two different ori configurations, a or p core was coinjected with both pBla and pSVE. a Core failed to replicate, whereas p core did replicate (Fig. 3), revealing that production of additional T-Ag by pSVE could not overcome competition between PyV ori configurations. To test the possibility that PyV ori configurations were competing for enhancer-specific binding

Bell PvuII PvuII I

Hpall Bgll

5015 5055 5095 5135 5175 5215 5255 5295/0 40 SOi 120 160 bp U I I 1-J I I __1_JE I I I 1 I \

'•Y y y y ' ' y y y

y y y ^ y y y

y y y y y y ' ' < y y y y y y

y y y y y y ^ ' y y y y y y

EH

mRNAs T-ag binding sites

Enhancer region

Origin of ( D ) e g C g X I DNA Replication

"Enhancer protein" binding sites

pBla[ a-P-core-

P-core •

a-core-

PEAl PEA2 EEC PEBl E441 4632 4632:

5039 90

5131 90

5039 51301 5265 90

core- 5265 90

a-p[4632 149 1560

pF44114632 | ^ F 9 mutation (5233) 2962

5186 I 2962

4632 IJ5239 pFlOl

pF441AT = pF441 with nucleotides 695 to 1054 deleted. pFlOlAT = pFlOl with nucleotides 695 to 1054 deleted.

Figure 1. Polyoma virus oii region and pML-1 plasmids containing various PyV oii configurations. Two sets of genetically defined cis-acting sequences are required for PyV DNA replication (summarized in Fig. 8 of DePamphilis and Bradley 1986). In addition to core, MuUer et al. (1983) and Hassell et al. (1986) have identified a and p, and Tyndall et al. (1981) and Veldman et al. (1985) have identified A, B, C, and D in the same region. Minimal sequences are designated by solid boxes, with additional sequences required for full activity indicated by open boxes. Sequences that enhance gene expression (1, 2, and 3) have also been identified (Herbomel et al. 1984; Mueller et al. 1988). The origin of bidirectional DNA replication is defined by transition sites from discontinuous to continuous DNA synthesis (Hendrickson et al. 1987), indicated on each side of oii core by arrows. In addition, early (E) and late (L) mRNA start sites (Salzman et al. 1986), strong (A, B, and C) and weak (1, 2, and 3) T-Ag DNA-binding sites defined by DNase I protection (Cowie and Kamen 1984; Dilworth et al. 1984), and binding sites for putative enhancer-specific proteins defined by nuclease protection [PEAl and PEA2 (Piette and Yaniv 1987); EEC (Ostapchuk et al. 1986; Fujimura 1986); PEBl (Bohnlein and Gruss 1986; Piette and Yaniv 1986); F441 (Kovesdi et al. 1987)] are indicated. The numbering system is explained in DePamphilis and Bradley (1986). Plasmids consist of pML-1 sequences (thin line) containing PyV sequences (shaded bar) whose terminal nucleotide positions are indicated. Broken line designates two sequences joined together. pBla, a-p core, p core, a core, core, and a-p plasmids were constructed by MuUer et al. (1983) and described by Wirak et al. (1985). The structure of each plasmid and its pML-1 vector was confirmed by restriction site analysis. pSVE (MuUer et al. 1984), pSVori (Decker et al. 1987), and pJYM (Wirak et al. 1985) have been described. WTV3, pFlOl, pF441, and pFl 11 are described in Fujimura et al. (1981). pFlOlAT and pF441 AT were constructed by cutting pFlOl and pF441 with Avfll restriction endonuclease and recovering the large fragment by electrophoresis in a 3% NuSieve (FMC Corp.) agarose gel that was then recircularized. Analysis of restriction sites in the new plasmid confirmed the absence of 359 bp from the T-Ag gene.

GENES & DEVELOPMENT 1117




Martinez-Salas et al.

PyV T - ^ provided by r

PyV{pB1a) SV40 (pSVE)

ori- sequence - core a-core p-core I I I 1 I 1 Dpn1 - - + - + - +

core a-core p-core a-p n r

1

pBlaorpSVE-

core, a - cx)re or p- core -

Figure 2. cis- and trans-acting sequence requirements for plasmid DNA replication following injection into two-cell embryos. Plasmids carrying PyV oh sequences, designated core, a core, p core, and a-p (Fig. 1), were coinjected with either pBla or pSVE into one of the nuclei of two-cell embryos. The ratio of any two coinjected plasmids was routinely set at 1 : 1 on a weight basis. Each embryo received 0.3-0.5 pg of total DNA, and the survivors were cultured in vitro for up to 48 hr. Cells were then divided into two groups: those that were two- or four-cell embryos and those that had developed into morula. Each lane shows the results with DNA extracted from an average of 20 embryos at 48 hr postinjection and digested either with Sail (-) or with both Sail and Dpnl( -h). DNA products were fractionated by gel electrophoresis, transferred to a nylon membrane, hybridized to pML-1 [̂ ^P]DNA, and detected by autoradiography (see Experimental procedures). In addition, DNA from four embryos was collected routinely —20 min after injection, and DNA from 10 embryos was collected routinely at 24 hr postinjection to calculate changes in the amount of plasmid DNA.

proteins, a or p core was coinjected with both a - p and pSVE. Again, a core did not replicate, whereas p core did (Fig. 3), revealing that the PyV enhancer region alone was capable of binding one or more factors required for initiation of replication by a core. T-Ag was not one of these factors because the major T-Ag DNA-binding sites present in pBla are absent in a - p (Fig. 1). The fact that p core replicated under conditions that inhibited a core indicates that a-specific and p-specific binding factors

differ either in their binding affinity or in their concentration in developing two-cell embryos.

Requirement for enhancers to activate PyV ori core appears after fusion of pronuclei in one-cell embryos

The same combinations of plasmids that were injected into two-cell embryos were also injected into the male pronucleus of one-cell embryos to determine whether or

pSVE +

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pSVE +

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pSVE 4-

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pSVE 4-

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a-p-(x-core orp--Gore-

48 "I r 48 1 r 1

? r

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48 ' ' 0 48 +- - 4.'

%

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Figure 3. Competition between PyV on elements in developing two-cell embryos. Two-cell embryos were injected with one of the following combinations: pSVE + a-p 4- a core ( 1 : 1 : 1), pSVE -I- a-P -I- p core ( 1 : 1 : 1), pSVE + pBla -I- a core (0.5 : 1 : 1), and pSVE -I- pBla + p core (0.5 : 1 : 1). Surviving embryos were cultured in vitro for up to 48 hr with samples taken at 0, 24, and 48 hr postinjection and processed as described in Fig. 2. Because the agarose concentration in panel 2 was different than in the other three experiments, the position of 10 pg of pSVE marker is also shown (*). Each lane contains DNA from an average of 18 morula, except for 0 hr postinjection, where only two to four embryos were used per lane.






not enhancer elements were required immediately after fertilization. Surviving embryos were cultured for 48 hr and segregated according to their stage of development. One-cell embryos contained two pronuclei and one or two visible polar bodies, whereas unfertilized eggs contained one polar body and no pronuclei. At least 99% of uninjected one-cell embryos underwent cleavage to the two-cell stage. However, an average of 20% of the one-cell embryos injected with buffer or 26% of those injected with DNA arrested development prior to cell cleavage. No correlation was observed between the fraction of cells that failed to cleave and the replication ability, amount (0.1-2 pg per embryo), structure (linear or form I DNA) of the DNA injected, or the type of injection buffer used [standard phosphate injection buffer, 10 mM Tris-HCl (pH 8.0) plus 1 mM EDTA, or a solution of both]. An average of 83% of the embryos that failed to cleave within 48 hr also retained both pronuclei.

In those embryos that remained at the one-cell stage, all plasmids replicated efficiently as long as they contained PyV oh core and PyV T-Ag was provided by either the same or another plasmid (Fig. 4). Thus, core replicated as efficiently as any other plasmid. Only the a -p region still did not exhibit oh activity (Fig. 4). Furthermore, in contrast to developing two-cell embryos, no competition between oh configurations was observed in one-cell embryos. pBla did not inhibit replication of a core, and core alone replicated in the presence of either pBla or pSVE (Fig. 4). In fact, DNA rephcation observed with either core or a core was not decreased when increasing amounts of pBla were coinjected (data not shown). These results demonstrate that the need for enhancer elements is acquired in cleavage-stage embryos sometime after fusion of pronuclei into a diploid nucleus and prior to development into morula.

In contrast to the one-cell embryos that did not undergo cleavage, one-cell embryos that progressed to the two-cell stage failed to rephcate the injected DNA. Core and pBla were coinjected into one-cell embryos, which were then allowed to continue development for 48 hr

before being separated into two groups—one-cell embryos that remained as one-cell embryos and one-cell embryos that developed into two- and four-cell embryos. As before, one-cell embryos replicated both core and pBla, but cells that underwent cleavage failed to replicate either, although the injected DNA was stable (Fig. 5). This was true even when the injected embryos were incubated for 4 days until they developed into morula. A faint band of Dpizl-resistant pBla DNA was detected in some experiments if DNA was extracted from 25-30 embryos (Fig. 5). We never observed significant plasmid DNA replication in two- to eight-cell embryos, regardless of whether the DNA was injected into one-cell embryos that underwent cleavage or into two-cell embryos that failed to develop into morula. Thus, the maximum amount of plasmid DNA replication observed always required 48 hr of incubation postinjection and depended on the developmental state of the embryo.

One-cell embryos contain high levels of PyV ori-specific initiahon factors

As shown previously for developing two-cell embryos (Wirak et al. 1985), PyV T-Ag was required specifically for replication of the PyV ori in one-cell embryos as well. Core did not replicate when injected alone into one-cell embryos, but it did replicate when coinjected with pBla (Fig. 5). SV40 T-Ag, provided by pJYM (pML-l containing the complete SV40 genome) did not support PyV ori core replication (data not shown), although it did support pJYM replication (Fig. 5) at the same low level observed previously when it was injected into two-cell embryos (Wirak et al. 1985). Conversely, PyV T-Ag provided by pBla did not support replication of a pML-1 plasmid containing the SV40 ori region (pSVori, Fig. 5). Therefore, initiation of DNA replication in one-cell embryos required the same stringent interaction between homologous ori core and T-Ag components that is observed for PyV and SV40 in differentiated cells (DePamphilis and Bradley 1986). Several putative cellular ori sequences

PyV T-ag pravided by -

ori- sequence -Dpn1 -

PyV(pB1a)

core a-core p--core I 11 1 I [

1 r SV40 (pSVE)

core a-core p~core a-p I 1 1 1 I 1 I 1

pBlaorpSVE-

a - p -core, a - core or p-- core - 0B

Figure 4. cis- and trans-acting sequences required for plasmid DNA replication following injection into one-cell embryos. Plasmids carrying PyV ori sequences, designated core, a core, p core, and a-p (Fig. 1), were coinjected at a weight ratio of 1 : 1 with either pBla or pSVE into the male pronucleus of one-cell embryos. Each embryo received 0.3-0.5 pg of DNA, and the survivors were cultured in vitro for up to 48 hr. Cells were then divided into two groups: those that remained as one-cell embryos and those that developed into two- and four-cell embryos. Each lane shows the results with DNA extracted from three to five one-cell embryos at 48 hr postinjection and processed as in Fig. 2.






Py V T-ag -SV40 T-ag -

PyV on -SV40 ori -

Dpn1 -pBlaorpJYM-

core or pSVori -# embryos _

lane

'- +"- +". +' T̂ + 1 r

+ +

4 1-cell

26 2-cell

5 1-cel 1

4 -cell

1 r

+ + ^nP

12 1-cell

Figure 5. Requirements for PyV ori-dependent DNA replication following injection into one-cell embryos. PyV T-Ag is provided by pBla, and SV40 T-Ag is provided by pJYM. PyV oh is represented by either PyV oh core (core) or the complete PyV oh region (pBla). SV40 oh is the complete SV40 oh region cloned into pML-1 (pSVori) or the whole SV40 genome cloned into pML-1 (pJYM). DNA extracted from the number of embryos indicated was appHed to each gel lane. DNA was extracted at 48 hr postinjection from embryos at the developmental stage indicated. Treatment of the samples was identical to that in Fig. 2.

w^ere also tested for their ability to replicate after injection into one-cell or two-cell embryos, but no replication was detected (DePamphilis et al. 1988b). Therefore, the ability of preimplantation mouse embryos to replicate injected DNA appeared to be specific for viral origins of replication.

Remarkably, PyV DNA replication in the pronuclei of one-cell embryos was about ninefold more efficient than in the diploid nuclei of developing two-cell embryos. For example, one-cell embryos that remained as one-cell embryos after 48 hr in vitro had increased the total amount of plasmid DNA per embryo by 14-fold, with all of the DNA resistant to Dpnl (Fig. 6). Two-cell embryos that progressed to morula by 48 hr contained about the same amount of plasmid DNA that was present initially, but most of it was now Dpnl resistant (0.5- to 2-fold change in DNA per embryo). Similar results were obtained with each plasmid that contained a functional oh configuration, and one-cell embryos invariably averaged an increase in plasmid DNA per embryo ninefold greater than developing two-cell embryos. Because this increased replication efficiency was observed only with PyV ori-containing plasmids in the presence of PyV T-Ag-expressing plasmids, one-cell embryos appear to contain higher levels of the PyV permissive cell factor(s) than embryos at later stages of development.

To evaluate the importance of DNA structure on DNA replication, p core was cut at a single site with either Pstl (3'-protruding ends) or Seal (even ends) to produce linear molecules. Linear p core and form I pSVE were coinjected into one-cell embryos,- however, more than 90% of the linear DNA was degraded rapidly. The remaining molecules formed circular monomers and then replicated extensively; large-molecular-weight concatemers were not detected in the total DNA sample fractionated before digestion with restriction endonu-

cleases. Therefore, DNA consisting of —80% covalently closed, superhelical molecules (form I) was injected routinely.

1-Cell 2-Cell I —

hours post-mj. - 0 Dpn1 - ' - +'^

gel origin -

pSVE

oc-p- core -

Dpn1 products -

# embryos lane

- 3 30

Figure 6. Comparison of the efficiency of DNA replication following injection into one- and two-cell embryos, a-(3 core and pSVE (1:1) were coinjected into the male pronucleus of one-cell embryos and one of the nuclei of two-cell embryos. Cells were harvested at 0, 24, and 48 hr after injection and segregated according to their stage of development. The number of embryos collected at 0 and 48 hr postinjection is given. For injected one-cell embryos, DNA was extracted from one-cell embryos. For injected two-cell embryos, DNA was extracted from two-cell embryos at 0 hr postinjection and from morula at 48 hr postinjection.






Duplication of part of enhancer 3 increases transcription in two-cell but not in one-cell, embryos

We considered the possibility that mouse embryos, hke mouse undifferentiated and embryonic cell lines (Amati 1985), respond to specific enhancer elements not represented in the wild-type PyV enhancer region. For example, the single-base-pair change in PyV host-range mutant F441 allows this virus to replicate in embryonal carcinoma (EC) F9 cells, as well as in mouse fibroblasts (Fujimura et al. 1981). Therefore, we tested the abiUty of this mutant to replicate in mouse preimplantation embryos.

pF441 (Fig. 1) injected into two-cell embryos replicated to the same extent as its wild-type parent pWTV3 (Fig. 7). pWTV3 replication was equivalent to pBla. Therefore, the F441 mutation had no advantage over PyV wild-t3^e enhancer in two-cell embryos. However, a tandem duplication of a 54-bp region containing the F441 mutat ion replicated at least sevenfold better in developing two-cell embryos (pFlOl, Fig. 7) than its parent wild-type genome (pWTV3, Fig. 7). Because one copy of the F441 mutat ion was essentially the same as wild-type PyV in two-cell embryos, the result with pFlOl revealed that two copies of the p element in tandem are more efficient than one copy. This would also explain why p F l l l , containing a duplication of a 31-bp region that includes the F9 mutation, replicated only twice as well as pF441 (data not shown).

Increased replication of pFlOl appeared to result from increased levels of T-Ag production, because pFlOl increased the replication efficiency of wild-type PyV coin-jected into the same cell. Although pBla replication alone was never detected at 24 hr postinjection, pBla and pFlOl both replicated by 24 hr after they were coin-jected into two-cell embryos (Fig. 8). This stimulation of replication by pFlOl was also observed in embryos that

did not develop beyond the two- or four-cell stage (Fig. 7, '48*'; Fig. 8, '2 -h 4 celP). The efficiency of replication for both pFlOl and pBla was at least sixfold better in embryos that had developed to morula by 48 hr than in embryos that remained at the two- and four-cell stages (Fig. 8, 'morula'). This trans-sicting stimulation of pBla suggested that the increased ability of pF 101 to replicate in two-cell embryos resulted froin an increased rate of T-Ag synthesis rather than a more efficient ori.

To test this hypothesis, most of the T-Ag-coding sequence in pFlOl and pF441 was deleted in pF441AT and pFlOlAT (Fig. 1). As expected, neither pF441AT nor pFlOlAT replicated when injected alone, but each replicated two-fold when coinjected with pSVE (Fig. 8). Therefore, pFlOl ori was equivalent to wild-type PyV ori in developing two-cell embryos. To demonstrate that the primary effect of the F101 enhancer was at the level of transcription, pFlOlAT was coinjected with pBla. Neither plasmid replicated (Fig. 8), revealing that the FlOl enhancer region competes more effectively for enhancer-specific binding proteins in two-cell embryos than the wild-type PyV enhancer. Because T-Ag can only be provided in this experiment by pBla, T-Ag is not produced and neither plasmid replicates.

The FlOl enhancer had no effect in one-cell embryos. The average of 15 experiments, in which plasmids carrying the PyV ori were injected into one-cell embryos and assayed for replication 48 hr later, gave a value of 8.5 ± 1.4 (s.E.M.) for the relative increase in pBla, pWTV3, pF441, p F l l l , or pFlOl DNA (Fig. 7). This was equivalent to the high level of replication achieved by pFlOl in developing two-cell embryos (Fig. 7, 'DNA rep/ embryo'). Because pFlOlAT did not inhibit plasmid DNA replication when coinjected with pBla into one-cell embryos, enhancer-binding proteins were not required for activation of the PyV early gene promoter of pBla in the pronucleus of one-cell embryos.

2-Cell Embryos 1-Cell Embryos

pWTV3 pF101 f—:: " T ~ i r

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DNA Rep tmbryo

14 8

Figure 7. Ability of PyV enhancer mutations to activate PyV oii core. pFlOl, pF441, (Fig. 1) or their parental wild-type PyV genome (pWTV3) was injected into one-cell and two-cell embryos. Embryos were harvested at 0, 24, and 48 hr after injection (hrs p.i.), and their DNA processed as described in Fig. 2. For injected two-cell embryos, only morula were scored at 48 hr (10 embryos per lane for pWtV3 and pF441; 4 embryos per lane for pFlOl). For injected one-cell embryos, only one-cell embryos were scored (three to five embryos per lane). No DNA replication was detected in two- and four-cell embryos collected at 48 hr, except when pFlOl was injected (48*). Relative amount of DNA replication per embryo is indicated at the bottom of each lane. The efficiency of plasmid DNA replication per embryo was calculated by dividing the amount of Dpizl-resistant DNA per embryo present at 48 hr by the total amount of plasmid DNA present at 24 hr. The amount present at 24 hr was used as a baseline, as -30% of the DNA present immediately after injection was degraded by this time, presumably because it leaked into the cytoplasm.






hrs p.i. -Dpn1 -

# embryos lane

pB1a +

pF101

pB1a pB1a pSVE pSVE + + + +

pF101ATpF441AT pF101AT pF441AT

§ 2-cell s i

2+4 cell 2+4 cell

30 morula

13 morula

Figure 8. Activation of PyV oh in developing two-cell embryos by increasing levels of PyV T-Ag. A 1 : 1 weight ratio of pBla + pFlOl, pBla + pFlOlAT, or pBla + pF441AT was injected into two-cell embryos. Similarly, a 0.5 : 1 weight ratio of pSVE + pFlOlAT or pSVE -I- pF441AT was injected into two-cell embryos. At 24 and 48 hr postinjection (hr p.i.), embryos were segregated into those that developed into morula and those that were still at the two- or four-cell stages. DNA was extracted from the number of embryos indicated per lane and processed as in Fig. 2. The upper band in the blot corresponds to pBla or pSVE, and the lower band to pFlOl, pFlOlAT, or pF441AT, as indicated at the top.

Discussion

The results presented in this paper and summarized in Figure 9 reveal two surprising facts about the role of enhancers in replication and expression of genes. First, enhancers are not always needed in vivo as a component of the PyV oh) oh core alone contains all the cis-acting information required to initiate DNA replication. Second, the need for enhancers is acquired after formation of a diploid nucleus. The difference between one- and two-cell embryos with respect to enhancer requirements and efficiency at which injected genes are replicated and expressed appears to reflect a difference between pronuclei and nuclei. All of the observations that we attribute to one-cell embryos occurred exclusively in one-cell embryos that remained one-cell embryos and retained their two pronuclei. Although DNA was injected into the male pronucleus because of its greater size, the same results would likely occur in the female pronucleus. The germinal vesicle in mouse oocytes is the precursor to the female pronucleus in one-cell embryos. Expression of SV40 early genes (T-Ag and t-Ag) does not require an enhancer when injected into the oocyte germinal vesicle but does require an enhancer for expression in differentiated cells (Chalifour et al. 1987). Therefore, we suggest that the need for enhancer elements during normal mammalian development is acquired only after the pronuclei have fused. Furthermore, this acquisition occurs at a specific stage in development. Injected one-cell embryos that developed into two- or four-cell embryos were very poor at plasmid DNA replication (Fig. 5) and gene expression (Chen et al. 1987; D. Wirak, un-publ.). Similar results were observed with injected two-cell embryos that failed to develop past the four-cell stage. Significant plasmid replication was observed by the four-cell stage only with a - p * - p * core (pFlOl, Fig. 8). Therefore, even though the same amount of t ime elapsed following injection of plasmid DNA into either one- or two-cell embryos, enhancer-dependent DNA rep

lication was difficult to detect until development progressed beyond the four-cell stage.

The conclusions we have drawn based on the need for enhancers to activate PyV oh core can be generalized to include enhancer activation of promoters. Other laboratories have shown that the ability of enhancer elements to activate PyV oh core in somatic cells is correlated directly with their ability to activate a promoter (Linney and Donerly 1983; deVilliers et al. 1984; Veldman et al. 1985; Campbell and Villarreal 1986; Hassell et al. 1986; Tang et al. 1987; Satake et al. 1988). In addition, we have shown that a duplication of the p element (pFlOl) markedly stimulated T-Ag synthesis from the PyV early gene promoter when injected into two-cell embryos but had no effect on T-Ag-dependent DNA replication in one-

Experimental Conditions

colnjected plasmid—^

DNA Injected Into —•

PyV orl-conflgurations:

CC—p-core

p - core

a-p*- core a-p* -p* -core

a - core core

a-p

Origin Activity

PyV orl/PyV T-ag SV40 ori/PyV T-ag

1-Cell 2-Cell 1-Cell 2-Cell

Figure 9. Summary of cis-acting sequence requirements for replication of DNA injected into one- and two-cell mouse embryos. PyV T-Ag was provided either by pBla (contains PyV oh region), or pSVE (contains SV40 oh region). PyV oh configurations are described in Fig. 1. a-p* core is pF441T, and a-p*-p* core is pF 10IT. A bar indicates where Dpizl-resistant DNA was observed. Thick bars represent a ninefold increase in the average amount of replication relative to thin bars. (0) DNA replication was not detected. No DNA replication was observed in the absence of PyV T-Ag.






cell embryos (Figs. 7 and 8). The (3 element, but not the a element, also stimulates gene expression from the HSV thymidine kinase promoter in developing two-cell embryos and in mouse fibroblasts (DePamphilis et al. 1986). Like the PyV oh, this promoter was about 10 times more active in one-cell than in two-cell embryos, regardless of the presence or absence of the PyV enhancer region (D. Wirak, unpubl.). The metallothionein promoter has also been found to be 10 times more active in one-cell than in two-cell embryos (Chen et al. 1987). This inducible promoter still responds to metal ion stimulation in one-cell embryos (Brinster et al. 1982). In fact, full activity of a crippled HSV thymidine kinase promoter can be restored in one-cell embryos by substituting the consensus sequence of the metal-ion-responsive element for the mutated thymidine kinase promoter elements (Stuart et al. 1984).

The question arises as to the biological significance of one-cell embryos that are arrested in their development. The experiments described above show clearly that although injected one-cell embryos arrest pronuclear development, they still can replicate and transcribe DNA, at least with injected plasmids. In fact, one-cell embryos that are arrested in S phase by treatment with aphidi-colin express injected genes at least 10 times better than two-cell embryos do (Chen et al. 1987). Because gene expression does not begin until about 18 hr after injection (Chen et al. 1987), translation of injected genes coincides with the normal onset of zygotic gene expression in late two-cell embryos. Bolton et al. (1984) have shown that treatment of one-cell embryos with either aphidicolin or cytochalasin D inhibits pronuclear fusion but does not prevent synthesis of late two-cell-specific polypeptides. Therefore, we suggest that the developmental clock for gene expression continues despite the arrest of pronuclear development. Consequently, events observed in pronuclei of arrested one-cell embryos would represent the effects of pronuclear structure and organization on gene expression and DNA replication rather than artifacts of the experimental protocol.

Acquisition of enhancer function during embryonic development may begin when zygotic gene expression is activated. This happens in the late two-cell stage in mammals (Taylor and Piko 1987) and at the midblastula transition (MBT) that follows 12 cell cleavages in amphibians (Newport and Kirschner 1982). Krieg and Melton (1987) injected into fertilized Xenopus eggs a gene that is normally among the first to be expressed at the MBT and observed that the injected gene is expressed at the MBT but only if its enhancer element is present. Hiromi and Gehring (1987) observed a similar result in Drosophila. Thus, the appearance of enhancer function coincides with the onset of the developmental program in a variety of organisms.

The same enhancer element is recognized at different times during mammalian development. Previous studies have shown that mouse EC cell lines share many characteristics with primitive endoderm that arises from the inner cell mass during the late stage of blastocyst development (Hogan et al. 1983). One striking characteristic

of EC cells is their inability to utilize enhancer elements found in PyV, SV40, and retroviruses, even when DNA is microinjected (Kelly and Condamine 1982; Taketo et al. 1983). Thus, promoters that require enhancer elements for their activity in somatic cells are inactive in EC cells unless either the EC cells are induced to differentiate or the enhancers are altered genetically (Amati 1985). These requirements reflect the appearance of somatic cell-specific enhancer recognition proteins upon cell differentiation and the presence of embryonic versions of these proteins in EC cells (Fig. 1; Kovesdi et al. 1987; Kryszke et al. 1987), consistent with the notion that these viral genes are not expressed in preimplanta-tion mouse embryos (Kelly and Condamine 1982). However, a host range mutation (pF441, Fig. 1) that allows EC F9 cells to utilize PyV enhancer 3 was neither better nor worse than wild-type enhancer 3 when injected into one- or two-cell embryos (Fig. 7). Two copies of this region (pFlOl, Fig. 1) did stimulate the PyV early gene promoter to produce more T-Ag (Fig. 7), but it was no better than wild-type PyV enhancers at activating oh core (Fig. 8). The fact that coinjection of pFlOl with wild-type PyV plasmid pBla stimulated pBla replication in developing two-cell embryos (Fig. 8), but not in F9 cells (Fujimura and Linney 1982), confirmed that wild-type PyV enhancer 3 is a functional part of oh in mouse embryos but not in mouse EC cells. These results are consistent with previous reports that maximum stimulation of promoters requires several tandem copies of the minimal a element, whereas maximum stimulation of the PyV oh core requires only two copies (Veldman et al. 1985). Thus, some enhancer-specific proteins (e.g., those binding to enhancer 3) that are present soon after formation of two-cell embryos are later repressed and then reappear in differentiated tissues.

Why is an enhancer needed in a diploid nucleus but not in a pronucleus? Enhancers appear to play essentially the same role in DNA replication as they do in DNA transcription by facilitating the interaction of replication or transcription proteins with oh core or promoter components, respectively (DePamphilis 1988; DePamphilis et al. 1988b). Proteins that bind to enhancer elements can either stabilize binding of a T-Ag initiation complex to oh core or modify the chromatin structure to make oh core more accessible to the T-Ag initiation complex. The first mechanism appears to be involved in stabilizing an initiation complex formed at the plasmid R6K oh (Mukherjee et al. 1988), whereas the second mechanism appears in the observation that TATA-box-binding protein TFIID can prevent nucleo-some-mediated repression of a promoter (Workman and Roeder 1987). Proteins that bind to SV40 promoter and enhancer elements are responsible for creating nuclease-hypersensitive sites in oh core (Jongstra et al. 1984; Gerard et al. 1985), and, in the apparent absence of chromatin structure, PyV oh core can initiate replication in vitro (Prives et al. 1987). Perhaps the chromatin structure formed on plasmids injected into pronuclei is more 'open' than chromatin structure formed on plasmids injected into a diploid nucleus. Alternatively, high con-






centrations of PyV permissive cell factor [e.g., DNA pri-m a s e - D N A polymerase-a (Murakami et al. 1986)] may overcome the need for an enhancer to stabilize the T-Ag initiation complex. Experiments are in progress to identify which mechanism is operative.

Acknowledgments

Plasmids pSVE, pBla, a-p core, a core, p core, a-p, and core were kindly provided by John Hassell, and pF441, pFlOl, pFl 11, and pWTV3 were kindly provided by Elwood Linney. We are indebted to Jose Sogo for critically reading this manuscript and to Miriam Miranda for help with the embryology.

Experimental procedures

Microinjection of mouse embryos

Escherichia coh DH5 was transformed with plasmid DNA and processed as described by Timmis et al. (1978), except that the clear lysate was incubated with 0.5 mg/ml of proteinase K, extracted with phenol, and subjected to two consecutive cesium chloride-ethidium bromide equilibrium gradients. The 260:280 ratio was between 1.80 and 2.0, and from 70% to 80% of the DNA was superhelical. DNA was diluted with injection buffer (Wirak et al. 1985) to 0.05-0.5 mg DNA/ml.

Embryos were isolated from CD-I female Swiss albino mice (Charles River Breeding Lab), 7-8 weeks old, and cultured as described previously (Wirak et al. 1985; Hogan et al. 1986; De-PamphiHs et al. 1988a), except that Whitten and Biggers' medium was supplemented with 40 |XM E D T A .

Microinjection procedures have been described in detail (De-Pamphilis et al. 1988a). Embryos submerged in Whitten and Biggers' medium supplemented with 21 mM HEPES (pH 7.4) and 4.2 mM NaHCOg were injected in the nucleus with 0.1-1 pg of DNA, as calculated from DNA extracted from embryos at time zero and quantitated by hybridization, in which standard amounts of DNA were also included. Approximately 100 embryos were injected with the same injection pipette containing a single DNA sample. From 50% to 70% of the embryos survived injection and cultured for up to 48 hr.

Detection of DNA rephcation

At various times after injection, a sample of embryos was washed by transferring them to 100 |xl of 140 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.2 mg/ml bovine serum albumin (BSA) and then to a siliconized tube containing 40 \x\ of 150 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 1 mM EDTA that was placed at - 70°C. When all embryos were harvested, cells were thawed on ice, lysed in 0.5% SDS, and digested with 0.5 mg/ml of proteinase K in the presence of 5 |xg yeast tRNA for 1 hr at 50°C, followed by 1 hr at 37°C. Lysates were extracted once with phenol (1 : 1) and then precipitated twice with 75% ethanol in the presence of 2.5 M ammonium acetate.

DNA samples were resuspended in 40 jil of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgClj, 10 mM 2-mercaptoeth-anol, 100 M-g/ml of BSA, and 1 mM spermidine. Spermidine is important to allow complete cleavage at restriction sites. Half of the sample was incubated at 37°C for 2 hr with 3 units of Sail, and half with 3 units of Sail and 3 units of Dpnl. DNA digestion products were fractionated by electrophoresis in 0.6% or 0.4% agarose gels at room temperature in Tris-borate buffer (Maniatis et al. 1982), transferred from the gel to GeneScreen Plus membranes (New England Nuclear), and then hybridized to pML-1 PPJDNA at a sp. act. of 0.5 x 10^ to 1 x 10^ cpm/|xg. Prehybridizations and hybridizations were carried out at 65°C, as described by the manufacturer. Partially wet membranes were exposed to Kodak X-OMAT AR film at - 70°C with a Du-pont Cronex Lightening Plus intensifying screen for 1-3 days. The relative intensity of the bands in two different exposures of the autoradiograms was determined by densitometry with a Cliniscan densitometer.

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E Martínez-Salas, D Y Cupo and M L DePamphilis nucleus during early mouse development.The need for enhancers is acquired upon formation of a diploid

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