Two novel reporter genes, LagZ and LagoZ, were derived by directed mutagenesis of the LacZ gene which has a CpG content of 9.24% (291 CpG for 3,076 bp, Figure 1a,b), which corresponds in density to CpG-rich regions of the vertebrate genome. LagZ (Figure 1c) and LagoZ (Figure 1d) have CpG densities of 1.6% (50% above CpG-poor regions in vertebrates) and 0.06% (2 CpG), respectively. Through use of alternate codons, each modified codon encodes the same amino acid as LacZ 24. A few non-conservative mutations have appeared during the mutagenesis in LagoZ 24. As we restricted the modification to CpG sequences, the AT content of the three DNA molecules remained almost the same (44.2%, 50.7% and 51.8%, respectively) and the differences in codon usage were minimal. We chose this approach, rather than a general re-encoding of LacZ with preferred mammalian codons, to test unambiguously the differences in transgene behavior resulting from changes in CpG content alone. These genes were attached to the same control elements, which included a 2.3 kb fragment of the human promoter of the α-subunit of the elongation factor 1 of translation (EF1α) 25. This promoter possesses the characteristics for widely expressed genes in particular and it is included in a CpG island with a CpG content of 6.2% (Figure 1). The reporter gene was placed in exon 2 at 160 bp from the end of the CpG-rich island. We chose widely expressed genes as it is particularly with ubiquitous promoters that transgenes have failed to reproduce correct expression patterns, despite many attempts 181921. The expression potential of these three transgenes was analyzed in vivo through the creation of transgenic mice. The testing of these transgenes in animals (as opposed to cultured cells) permitted analysis of transgene expression at multiple periods of development and in multiple cell types: here, during gametogenesis and at the blastocyst stage, when the genome is hypomethylated, and after embryonic day 9.5 (E9.5), when the genome of somatic cells is hypermethylated 2627.

In all four LacZ (Figure 2i,j,m), four LagZ (Figure 2k,n) and four LagoZ (Figure 2l,o) transgenic lines, the male germ cells expressed the transgenes. Expression includes type A spermatogonia (SpA), the small chains in Figure 2m-o. Therefore, the alteration of CpG density in the LagZ and LagoZ sequences had no gross consequence on expression in these cells. The quantitative expression of the three transgenes microinjected in 1-cell stage embryo was also indistinguishable (5.6, 5.7 and 7.1 × 10^-5 U β-gal, respectively, for LacZ, LagZ and LagoZ). Together, these observations suggest comparable RNA stability and minimal effects of codon usage on gene expression between the three molecules.

In contrast, dramatic differences were observed in the embryo between the three transgenes (see Figure 2a-d for representative examples, and Table 1). A systematic, high expression of the LagoZ transgene was always observed in embryonic and extra-embryonic tissues whatever the line (n = 4) (Figure 2d,h), whereas no expression of the LacZ transgene was observed (n = 4) (Figure 2b,f) and only a variegated expression was observed in one LagZ line (n = 4) (EFIαLagZ2, Figure 2c,g). Following differentiation of cell types, for example at P7, these differences in transgene expression were maintained (Table 1). Clearly, epigenetic controls are imposed early on the LacZ and LagZ transgenes, and cell differentiation does not erase these controls.

To test when this epigenetic control is imposed on the LacZ and LagZ transgenes, β-galactosidase (β-gal) expression was searched for in blastocysts. Both LacZ and LagZ transgenes are strongly expressed in the ICM (inner cell mass) and trophectoderm (Figure 2q,r) in the four EF1αLac and four EF1αLag transgenic lines. Therefore, the epigenetic control is imposed on the genome after the blastocyst stage but before E9.5. Altogether, these results show that the high density of CpGs in the transcribed region of LacZ is the cause of total repression of its expression in somatic cells. A low density of CpGs (LagZ gene), although higher than the corresponding sequences in EF1α gene, still provoked a total repression or, at best, a variegated expression in tissues included in the widespread expression pattern of the EF1α promoter. An absence of CpG sequences in the transcribed part of the gene (LagoZ) resulted in a complete release from this repression.

Some epigenetic controls are especially effective on repeated sequences in the genome 28. However, the study of four YacEF1αLacZ transgenic mice (Figure 1a) in which the transgene is at single copy indicated that, even in this condition, the CpG content of the transcribed region caused total repression. The expression pattern is indistinguishable from the expression pattern of EF1αLacZ: no expression of the LacZ transgene in embryonic and extra-embryonic tissues and no expression at P7 (Table 1, n = 5). As for EF1αLacZ, the male germ cells and the cells at the blastocyst stage (Figure 2p) expressed the transgene.

This repression by CpG sequences is likely to be due to their methylation, but there are several possible hypotheses to explain the role of methylation. For instance, the methylation of the reporter gene alone can by itself provoke a change in chromatin structure of the adjacent promoter leading to its silencing, an idea compatible with the fact that the promoter included in a CpG island must escape methylation. Alternatively, direct methylation of the promoter can be necessary to repress the transgenes. To test these hypotheses, the methylation patterns of EF1αLacZ and EF1αLagZ, following digestion of DNA by MspI or HpaII, were analyzed in the liver, skin and brain as these tissues exhibit a high β-gal⁺ activity in the EF1αLagoZ line (Table 1). We present the methylation patterns of the lines harboring the lowest copy number. Indeed, the presence of multiple copies in the other lines makes it impossible to correlate expression and methylation. Whatever the tissue or the line analyzed, all HpaII sites (13 for LacZ, four for LagZ) were found to be methylated in 90% to 100% of the LacZ and LagZ fragments of β-gal^- YacEF1αLacZ1, EF1αLacZ2 or EF1αLagZ1 lines (Figure 3a, lanes 2, 5 and 8, and data not shown, n = 5). Therefore, it is clear that the CpG-rich LacZ sequences are not recognized by the cells as CpG-rich islands as they are not protected from de novo methylation. Although these observations are compatible with the idea of an indirect repression of the reporter gene by methylated CpG, examination of the DNA of the β-gal⁺ EF1αLagZ2 mice indicated that, at least in this case, the repressive effect could not be attributed solely to methylation of the reporter sequences. Indeed, in these β-gal⁺ mice, the LagZ gene was fully (100%) methylated (Figure 3a, lane 11).

If the methylation of the CpGs of the reporter gene is not sufficient to repress the transgene, is the methylation of the promoter sequences contained in a CpG-rich island involved? To address this issue we examined the two HpaII DNA fragments specific to this region: the 166 bp fragment and the 167 bp fragment, and the combined 333 (166 + 167) bp EF1α fragments, the latter indicating partial methylation (Figure 4c). Surprisingly, we observed complete methylation of these sequences in β-gal^- YacEF1αLacZ1 tissues (Figure 4a, lane 1, and data not shown, n = 3, the absence of both the 170 bp and the 333-bp-long fragments) and low levels of methylation in β-gal^- EF1αLacZ2 and EF1αLagZ1 tissues (Figure 4a, lanes 3 and 5, n = 2). Therefore, methylation of the EF1α promoter sequences could explain the β-gal^- phenotype. Other observations reinforced this possibility: in the variegated β-gal⁺ EF1αLagZ2 mice, EF1α promoter sequences are only partially methylated, in contrast with β-gal^- EF1αLagZ1 (Figure 4a, lanes 7 and 5: the 170-bp-long band); in EF1αLagoZ1 mice, EF1α promoter sequences are not methylated at all (Figure 4a, lane 9: the 170-bp-long fragment).

The examination of other HpaII sites in the CpG island demonstrated additional differences between the EF1αLagoZ1 line and the LagZ and LacZ lines. The 626-bp-long fragment in Figure 4a is indicative of the methylation of sites in the first exon and in the first intron of EF1α (Figure 4c; the 5'HpaII fragments in EF1αLacZ2, EF1αLagZ1, EF1αLagZ2 and EF1αLagoZ1 transgenic lines are longer than 626 bp). This fragment is fully detected in the EF1αLagoZ1 line (Figure 4a, lane 9) but only partially present or absent in LagZ and LacZ lines (Figure 4a, lanes 1, 3, 5 and 7, and data not shown, n = 6). Therefore, although the CpG island remains fully protected from methylation when combined with a LagoZ transgene (depleted of CpGs) it is only partially protected (at least when at low copy number, as in EF1αLagoZ1) or not protected at all when it is combined with CpG-containing sequences.

These results show firstly that β-gal^- phenotypes correlate with the methylation of EF1α promoter sequences (n = 5), and β-gal⁺ phenotypes with an absence of methylation of these sequences (n = 2); and secondly that the methylation of the EF1α CpG-rich sequences is not observed when the reporter gene can not be methylated (EF1αLagoZ1 line), indicating that, in this case, the CpG-rich island is protected from methylation. Thirdly, the results show that partial or complete methylation of this CpG island occurs when it is combined with CpG-rich sequences (LacZ or LagZ, n = 5) suggesting that, in this case, the CpG-rich island is not completely protected from methylation.

The expression patterns of YacEF1αLacZ, EF1αLacZ and EF1αLagZ suggest that inappropriate methylation of EF1α occurs during the period of de novo methylation of the genome after implantation of the embryo but before E9.5. Indeed, in all three cases the blastocysts (E4.5) of these lines strongly express the transgene (n = 13, Figure 2p-r) but the embryos at E9.5 and subsequent stages do not (Figure 2a-d, n = 12 out of 13). Two other observations support this conclusion. Firstly, the absence of methylation of the EF1α CpG island in YacEF1αLacZ1, EF1αLacZ2 and EF1αLagZ2 DNA in the male germ line (Figure 4b, lanes 1, 3 and 5: the presence of the 170 and 626-bp-long fragments, and data not shown, n = 5) and also, as expected, in EF1αLagoZ1 (Figure 4b, lane 7). Secondly, the lower methylation level of the LacZ reporter sequences in this tissue when compared to somatic tissues (Figure 3b, lanes 2 and 5).

These observations also confirm that, as in the somatic tissues of EF1αLagZ2, a methylated LagZ reporter gene can correspond to a β-gal⁺ tissue (Figure 3b, lane 8). This situation also applies to LacZ reporter genes, as a significant fraction of the male germ cells harbor completely methylated LacZ reporters in EF1αLacZ2 (Figure 3b, lane 5, indicated by the arrowhead). Clearly, as in somatic tissues in the male germ line, the mere methylation of LagZ or LacZ is not sufficient to repress EF1α.

The CpG content of the LacZ plasmid was first lowered from 9.24% to 1.65% to generate the PytknlsLagZ plasmid and PytknlsLagZ was used as a template to lower the CpG content to 0.06% to generate PytknlsLagoZ. In Pytk plasmids, a bacterial promoter allowed an expression screening of the mutagenized genes in LacZ-defective bacteria (Δ-Lac). To construct LagZ and LagoZ, a polymerase chain reaction (PCR) technique was used in which the mutagenic oligonucleotide primers spanned the whole sequences but contained fewer CpG dinucleotides. These mutagenic primers were designed to preserve the integrity of the amino-acid sequence of the β-galatosidase reporter protein. After each run of PCR the DNA was purified, circularized and transfected in Δ-Lac bacteria to select the clones presenting β-galactosidase activity. The DNA sequences of LagZ and LagoZ were verified by sequencing 24.

The three versions of LacZ were associated to the promoter region of the human EF1α gene to generate pEFnlsLacZΔenh, pEFnlsLagZΔenh and pEFnlsLagoZΔenh plasmids. The LacZ reporter was also inserted by homologous recombination in yeast at the same location into a 150 kb YAC spanning the human EFIα locus (Figure 1a). Construction of plasmids and YAC will be described elsewhere. The expression of the different transgenes was controlled by transient expression experiments that gave the same activity 24 as measured using MUG quantification 29.

Each EF1α plasmid construct was first digested by the NotI restriction enzyme and then partially digested using the XhoI restriction enzyme to remove vector DNA sequences. The 7.9 kb XhoI/NotI inserts were purified and microinjected (200 copies/pl) into the male pronucleus one-cell embryo obtained from (C57BL/6JxDBA2) F1 superovulated females mated with males of the same strain. Five independent YacEF1αLacZ, four independent EF1αLacZ, four independent EF1αLagZ and four independent EIFαLagoZ mouse lines were analyzed in this study. All embryos and organs were X-gal stained as described 30.

A TaqMan probe and primer set designed to amplify the LacZ, LagZ and LagoZ transgenes and an internal control (Rapsyn gene) were used to determine transgene copy number using DNA extracted from liver.

Tissues were collected from postnatal (P5) hemizygous mice (liver, skin and brain) and from adult males (testis) using standard protocol. Twenty micrograms of DNA were digested with appropriate enzymes to liberate the fragments to be analyzed - either HpaII (methylation-sensitive) or MspI (methylation-insensitive). The DNA was analyzed by Southern hybridization as previously described 31, probes for methylation are indicated in Figures 3 and 4.