Recapitulation of gametic DNA methylation and its post-fertilization maintenance with reassembled DNA elements at the mouse Igf2/H19 locus

Abstract

Background: Paternal allele-specific DNA methylation of the H19 imprinting control region (ICR) regulates imprinted expression of the Igf2/H19 genes. The molecular mechanism by which differential methylation of the H19 ICR is established during gametogenesis and maintained after fertilization, however, is not fully understood. We previously showed that a 2.9-kb H19 ICR fragment in transgenic mice was differentially methylated only after fertilization, demonstrating that two separable events, gametic and post-fertilization methylation, occur at the H19 ICR. We then determined that CTCF/Sox-Oct motifs and the 478-bp sequence of the H19 ICR 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′-truncated H19 ICR 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 endogenous H19 ICR caused loss of methylation after fertilization, indicating that methylation activity of the sequence is required to protect endogenous H19 ICR 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 endogenous H19 ICR. The fragment was methylated during spermatogenesis; moreover, its allele-specific methylation status was faithfully maintained after fertilization, and imprinted expression of the both Igf2 and H19 genes was recapitulated. Conclusions:Our results identified a 118-bp region within the H19 ICR that is required for de novo DNA methylation of the paternally inherited H19 ICR during pre-implantation period. A lambda DNA-based artificial fragment that contains the 118-bp sequence, in addition to the previously identified cis elements, could fully replace the function of the H19 ICR in the mouse genome.

The transgenic IG-DMR sequence of the mouse Dlk1-Dio3 domain acquired imprinted DNA methylation during the post-fertilization period

“…As mentioned earlier, we identified the 118-bp sequence that is essential for post-fertilization imprinted methylation of the mouse H19 ICR during the preimplantation period [17,18,27]. We further demonstrated that this sequence, together with the CTCF and Sox/Oct binding sequences, could confer imprinted methylation activity to the non-imprinted lambda DNA sequence both in transgenic [27] and endogenous genomic contexts [28].…”

“…Since mIG-DMR analyzed at the same genomic location exhibited post-fertilization imprinted methylation, it is unlikely that the results in the rat sequence are related to the insertion site of the transgene. In addition, our previous reconstruction experiments showed differential methylation of the LCb118 fragment, in which the CTCF, Sox/Oct, and Zfp57 motifs were embedded in lambda DNA in the same context as the mouse H19 ICR and then combined with the mouse 118-bp sequence [28]. Because binding sequences for these factors are highly conserved in number and arrangement between mouse and rat H19 ICRs (Additional file 1: Fig.…”

Maternal DNMT3A-dependent de novo methylation of the paternal genome inhibits gene expression in the early embryo

“…Indeed, following polyspermic fertilization, where methylation of the maternal genome remains relatively constant, up to five paternal genomes are demethylated in the zygote 4 . Although zygotic de novo DNAme of the maternal genome has been reported for the TKZ751 transgene and Igf2 59 , ETn retroelements 60 and H3K9me2-enriched regions 61 , to our knowledge, this is the first report to identify specific regions of the paternal genome subject to de novo DNAme by the 2C stage beyond the H19 gDMR 46 , 62 , 63 .…”

Tcte1knockout influence on energy chain transportation, apoptosis and spermatogenesis – implications for male infertility

“…Two-fold rise for Igf2 gene (strongly linked to spermatogenesis, sex determination, testis development, infertility and prostate cancer; [Matsuzaki et al , 2020; Cannarella et al , 2020; Neirijnck et al , 2019; Damaschke et al , 2017]) has been also noticed. Igf2 and Klk proteins are enclosed in a common network consisted of proteins directly linked to fertility (Figure 6; Supplementary file 11; Supplementary file 12).…”

“…Our pathway analysis revealed a network including two genes with changed expression level in testis: Igf2 (2-fold increased) and Klk1b22 (8-fold decreased), supported by an interesting set of proteins linked to apoptosis and cellular processing (Figure 5; Figure 6; Supplementary file 11; Supplementary file 12). We can suggest that observed Igf2 overexpression in our study can be one of the associated factors leading to decreased sperm count and testis parameters in evaluated KO mice model because of its known relation to spermatogenesis and testis development [Matsuzaki et al , 2020; Cannarella et al , 2020; Neirijnck et al , 2019; Damaschke et al , 2017]. Documented reductions can be supported by the observed constrained expression of Klk1b22 – kallikrein mostly known from its neuronal activity; however, it cannot be excluded that we have revealed additional role of this protein, similar to the other members of the kallikrein family – role in semen liquefaction and degradation of extracellular matrix proteins in the interstitial area surrounding Leydig cells of the adult mouse testes ( Klk1b27 , Klk1b21 , Klk1b24 ) [Michaelis et al 2017; Marques et al , 2016, Du Plessis et al , 2013; Fink et al , 2007].…”