Keiji Tanimoto

Faithful maintenance of allele-specific methylation at imprinting control regions (ICR) is essential for genomic imprinting. Using transgenic mice (TgM), we investigated sequence requirements for paternal methylation acquisition and maternal hypomethylation maintenance during early embryogenesis. We previously showed that a 2.9-kb mouse H19 ICR acquires post-fertilization paternal allele-specific DNA methylation via a critical 118-bp region; its deletion from the endogenous H19 ICR abolishes maintenance of germ cell-established paternal methylation. In contrast, TgM with the 2.8-kb rat H19 ICR ortholog failed to acquire post-fertilization imprinted methylation. Although another paternally methylated ICR, the IG-DMR of the Dlk1–Dio3 locus, acquired paternal methylation post-fertilization in TgM, the imprinted state was lost due to maternal de novo methylation post-implantation. Here, we extended previously analyzed ICR sequences to reexamine conservation of the activity across different species and genomic loci. Extending the rat H19 ICR upstream to include candidate cis -motifs still failed to establish imprinted methylation. Conversely, TgM with an extended mouse IG-DMR sequence protected maternal IG-DMR from genome-wide de novo DNA methylation after implantation, maintaining its imprinted DNA methylation state. This finding provides the first in vivo evidence for a bipartite IG-DMR model: 5′ domain for paternal methylation acquisition and 3′ domain for maternal hypomethylation maintenance.

Genomic imprinting is a crucial epigenetic mechanism directing parent-of-origin-specific gene expression in mammals, and it is regulated by allele-specific DNA methylation at imprinting control regions (ICRs). In this canonical imprinting mechanism, maintenance factors preserve germline-derived DNA methylation at ICRs during genome-wide demethylation after fertilization. However, our studies of the Igf2/H19 locus reveal that allele-specific de novo DNA methylation after fertilization, termed ‘post-fertilization imprinted methylation,’ also contributes to imprint maintenance. In this process, epigenetic imprints other than DNA methylation are thought to guide the recognition of parental alleles by de novo methyltransferases in the next generation, acting in concert with maintenance factors to ensure stable DNA methylation at canonical imprinted loci. Recently identified non-canonical imprinting, in contrast, relies on non-DNA methylation marks that are converted into DNA methylation only after implantation. Despite differences in the timing of this conversion, post-fertilization imprinted methylation may share similarities with non-canonical imprinting. Collectively, these findings suggest that the robustness of canonical imprinting is supported by a two-tiered regulatory system.

Genomic imprinting at the mouse Igf2/H19 locus is controlled by the H19 ICR, within which paternal allele-specific DNA methylation originating in sperm is maintained throughout development in offspring. We previously found that a 2.9 kb transgenic H19 ICR fragment in mice can be methylated de novo after fertilization only when paternally inherited, despite its unmethylated state in sperm. When the 118 bp sequence responsible for this methylation in transgenic mice was deleted from the endogenous H19 ICR, the methylation level of its paternal allele was significantly reduced after fertilization, suggesting the activity involving this 118 bp sequence is required for methylation maintenance at the endogenous locus. Here, we determined protein binding to the 118 bp sequence using an in vitro binding assay and inferred the binding motif to be RCTG by using a series of mutant competitors. Furthermore, we generated H19 ICR transgenic mice with a 5-bp substitution mutation that disrupts the RCTG motifs within the 118 bp sequence, and observed loss of methylation from the paternally inherited transgene. These results indicate that imprinted methylation of the H19 ICR established de novo during the post-fertilization period involves binding of specific factors to distinct sequence motifs within the 118 bp sequence.

Background: Allele-specific methylation of the imprinting control region (ICR) is the molecular basis for the genomic imprinting phenomenon that is unique to placental mammals. We previously showed that the ICR at the mouse H19 gene locus (H19 ICR) was unexpectedly established after fertilization and not during spermatogenesis in transgenic mice (TgM), and that the same activity was essential for the maintenance of paternal methylation of the H19 ICR at the endogenous locus in pre-implantation embryos. To examine the universality of post-fertilization imprinted methylation across animal species or imprinted loci, we generated TgM with two additional sequences. Results: The rat H19 ICR, which is very similar in structure to the mouse H19 ICR, unexpectedly did not acquire imprinted methylation even after fertilization, suggesting a lack of essential sequences in the transgene fragment. In contrast, the mouse IG-DMR, the methylation of which is acquired during spermatogenesis at the endogenous locus, did not acquire methylation in the sperm of TgM, yet became highly methylated in blastocysts after fertilization, but only when the transgene was paternally inherited. Since these two sequences were evaluated at the same genomic site by employing the transgene co-placement strategy, it is likely that the phenotype reflects the intrinsic activity of these fragments rather than position-effect variegation. Conclusions: Our results suggested that post-fertilization imprinted methylation is a versatile mechanism for protecting paternal imprinted methylation from reprogramming during the pre-implantation period.

Monoallelic gene expression at the Igf2/H19 locus is controlled by paternal allele-specific DNA methylation of the imprinting control region (H19 ICR) that is established during spermatogenesis. We demonstrated that the H19 ICR fragment in transgenic mice acquires allele-specific methylation only after fertilization, which is essential for maintaining its allelic methylation during early embryogenesis. We identified a DNA element required for establishing postfertilization methylation within a 118 bp (m118) region. A previously generated knock-in mouse whose endogenous H19 ICR was substituted with the human H19 ICR (hIC1; 4.8 kb) sequence revealed that the hIC1 sequence was partially methylated in sperm, although this methylation was lost by the blastocyst stage, which we assume is due to a lack of an m118-equivalent sequence in the hIC1 transgene. To identify a cis sequence involved in postfertilization methylation within the hIC1 region, we generated three transgenic mouse lines (TgM): one carrying an 8.8 kb hIC1 sequence joined to m118 (hIC1+m118), one with the 8.8 kb hIC1 and one with the 5.8 kb hIC1 sequence joined to m118 (hIC1–3′+m118). We found that the hIC1–3′ region was resistant to de novo DNA methylation throughout development. In contrast, the 5′ portion of the hIC1 (hIC1–5′) in both hIC1+m118 and hIC1 TgM were preferentially methylated on the paternal allele only during preimplantation. As DNA methylation levels were higher in hIC1+m118, the m118 sequence could also induce imprinted methylation of the human sequence. Most importantly, the hIC1–5′ sequence appears to possess an activity equivalent to that of m118.

Background: Paternal allele-specific DNA methylation of theH19imprinting control region (ICR) regulates imprinted expression of theIgf2/H19genes. The molecular mechanism by which differential methylation of theH19ICR is established during gametogenesis and maintained after fertilization, however, is not fully understood. We previously showed that a 2.9-kbH19ICR fragment in transgenic mice was differentially methylated only after fertilization, demonstrating that two separable events, gametic and post-fertilization methylation, occur at theH19ICR. We then determined that CTCF/Sox-Oct motifs and the 478-bp sequence of theH19ICR are essential for maintaining its maternal hypomethylation status and for acquisition of paternal methylation, respectively, during the post-fertilization period.Results: Using a series of 5′-truncatedH19ICR transgenes to dissect the 478-bp sequence, we identified a 118-bp region required for post-fertilization methylation activity. Deletion of the sequence from the paternal endogenousH19ICR caused loss of methylation after fertilization, indicating that methylation activity of the sequence is required to protect endogenousH19ICR from genome-wide reprogramming. We then reconstructed a synthetic DNA fragment in which the CTCF binding sites, Sox-Oct motifs, as well as the 118-bp sequence, were inserted into lambda DNA, and used it to replace the endogenousH19ICR. The fragment was methylated during spermatogenesis; moreover, its allele-specific methylation status was faithfully maintained after fertilization, and imprinted expression of the bothIgf2andH19genes was recapitulated.Conclusions: Our results identified a 118-bp region within theH19ICR that is required for de novo DNA methylation of the paternally inheritedH19ICR during pre-implantation period. A lambda DNA-based artificial fragment that contains the 118-bp sequence, in addition to the previously identifiedciselements, could fully replace the function of theH19ICR in the mouse genome.

Long-range associations between enhancers and their target gene promoters have been shown to play critical roles in executing genome function. Recent variations of chromosome capture technology have revealed a comprehensive view of intra- and interchromosomal contacts between specific genomic sites. The locus control region of the β-globin genes (β-LCR) is a super-enhancer that is capable of activating all of the β-like globin genes within the locus in cis through physical interaction by forming DNA loops. CTCF helps to mediate loop formation between LCR-HS5 and 3’HS1 in the human β-globin locus, in this way thought to contribute to the formation of a “chromatin hub”. The β-globin locus is also in close physical proximity to other erythrocyte-specific genes located long distances away on the same chromosome. In this case, erythrocyte-specific genes gather together at a shared “transcription factory” for co-transcription. Theoretically, enhancers could also activate target gene promoters at the identical loci, yet on different chromosomes in trans, a phenomenon originally described as transvection in Drosophilla. Although close physical proximity has been reported for the β-LCR and the β-like globin genes when integrated at the mouse homologous loci in trans, their structural and functional interactions were found to be rare, possibly because of a lack of suitable regulatory elements that might facilitate such trans interactions. Therefore, we re-evaluated presumptive transvection-like enhancer-promoter communication by introducing CTCF binding sites and erythrocyte-specific transcription units into both LCR-enhancer and β-promoter alleles, each inserted into the mouse ROSA26 locus on separate chromosomes. Following cross-mating of mice to place the two mutant loci at the identical chromosomal position and into active chromation in trans, their transcriptional output was evaluated. The results demonstrate that there was no significant functional association between the LCR and the β-globin gene in trans even in this idealized experimental context.

Long-range associations between enhancers and their target gene promoters have been shown to play critical roles in executing genome function. Recent variations of chromosome capture technology have revealed a conprehensive view of intra- and inter-chromosomal contacts between specific genomic sites. The locus control region of the β-globin genes (β-LCR) is a super-enhancer that is capable of activating all of the β-like globin genes within the locus in cis through physical interaction by forming DNA loops. CTCF helps to mediate loop formation between LCR-HS5 and 3’HS1 in the human β-globin locus, in this way thought to contribute to the formation of a “chromatin hub”. The β-globin locus is also in close physical proximity to other erythrocyte-specific genes located long distances away on the same chromosome. In this case, erythrocyte-specific genes gather together at a shared “transcription factory” for co-transcription. Theoretically, enhancers could also activate target gene promoters on different chromosomes in trans , a phenomenon originally described as transvection in Drosophilla . Although close physical proximity has been reported for the β-LCR and the β-like globin genes when integrated at the mouse homologous loci in trans , their structural and functional interactions were found to be rare, possibly because a lack of suitable regulatory elements that might facilitate trans interactions. Therefore, we re-evaluated presumptive transvection-like enhancer-promoter communication by introducing CTCF binding sites and erythrocyte-specific transcription units into both LCR-enhancer and β-promoter alleles, each inserted into the mouse ROSA26 locus on separate chromosomes. Following cross-mating of mice to place the two mutant loci at the identical chromosomal position and into active chromation in trans , their transcriptional output was evaluated. The results demonstrate that there was no significant functional association between the LCR and the β-globin gene in trans even in this idealized experimental context.

Genomic imprinting is governed by allele-specific DNA methylation at imprinting control regions (ICRs), and the mechanism controlling its differential methylation establishment during gametogenesis has been a subject of intensive research interest. However, recent studies have reported that gamete methylation is not restricted at the ICRs, thus highlighting the significance of ICR methylation maintenance during the preimplantation period where genome-wide epigenetic reprogramming takes place. Using transgenic mice (TgM), we previously demonstrated that the H19 ICR possesses autonomous activity to acquire paternal-allele-specific DNA methylation after fertilization. Furthermore, this activity is indispensable for the maintenance of imprinted methylation at the endogenous H19 ICR during the preimplantation period. In addition, we showed that a specific 5′ fragment of the H19 ICR is required for its paternal methylation after fertilization, while CTCF and Sox-Oct motifs are essential for its maternal protection from undesirable methylation after implantation. To ask whether specific cis elements are sufficient to reconstitute imprinted methylation status, we employed a TgM co-placement strategy for facilitating detection of postfertilization methylation activity and precise comparison of test sequences. Bacteriophage lambda DNA becomes highly methylated regardless of its parental origin and thus can be used as a neutral sequence bearing no inclination for differential DNA methylation. We previously showed that insertion of only CTCF and Sox-Oct binding motifs from the H19 ICR into a lambda DNA (LCb) decreased its methylation level after both paternal and maternal transmission. We therefore appended a 478-bp 5′ sequence from the H19 ICR into the LCb fragment and found that it acquired paternal-allele-specific methylation, the dynamics of which was identical to that of the H19 ICR, in TgM. Crucially, transgene expression also became imprinted. Although there are potential binding sites for ZFP57 (a candidate protein thought to control the methylation imprint) in the larger H19 ICR, they are not found in the 478-bp fragment, rendering the role of ZFP57 in postfertilization H19 ICR methylation a still open question. Our results demonstrate that a differentially methylated region can be reconstituted by combining the activities of specific imprinting elements and that these elements together determine the activity of a genomically imprinted region in vivo.

The renin-angiotensin system plays an essential role in blood pressure homeostasis. Because renin activity is reflected as a blood pressure phenotype, its gene expression in the kidney is tightly regulated by a feedback mechanism; i.e., renin gene transcription is suppressed in a hypertensive state. To address the molecular mechanisms controlling hypertension-responsive mouse renin (mRen) gene regulation, we deleted either 5′ (17-kb) or 3′ (78-kb) regions of the endogenous mRen gene and placed the animals in a hypertensive environment. While the mRen gene bearing the 3′ deletion was appropriately downregulated, the one bearing the 5′ deletion lost this hypertension responsiveness. Because the 17-kb sequence exhibited enhancer activity in vivo and in vitro, we narrowed down the enhancer to a 2.3-kb core using luciferase assays in As4.1 cells. When this 2.3-kb sequence was removed from the endogenous mRen gene in the mouse, its basal expression was dramatically reduced, and the hypertension responsiveness was significantly attenuated. Furthermore, we demonstrated that the angiotensin II signal played an important role in mRen gene suppression. We propose that in a hypertensive environment, the activity of this novel enhancer is attenuated, and, as a consequence, mRen gene transcription is suppressed to maintain blood pressure.