Replication timing properties within the mouse distal chromosome 7 imprinting cluster

Genomic imprinting is characterized by allele-specific expression of genes within chromosomal domains. Here we show, using fluorescence in situ hybridization (FISH) analysis, that the large chromosomal domain of the mouse distal chromosome 7 imprinting cluster, approximately 1 Mb in length between p57Kip2 and H19 genes, replicates asynchronously between the two alleles during S-phase. At the telomeric side of this domain, we found a transition from asynchronous replication at the imprinted p57Kip2 gene to synchronous replication at the Nap2 gene. Two-color FISH suggested that the paternal allele of this whole domain replicates earlier than its maternal allele. Treatment of the cells with a histone deacetylase inhibitor abolished this allele-specific feature accompanied with accelerated replication of the later-replicating allele at a domain level. Allele-specific asynchronous replication was observed even in ES cells. These results suggest that this imprinting cluster consists of a large replication domain which is already found at the early stage in development.     

Multiple Mechanisms Regulate Imprinting of the Mouse Distal Chromosome 7 Gene Cluster

Genomic imprinting is an epigenetic process that results in the preferential silencing of one of the two parental copies of a gene. Although the precise mechanisms by which genomic imprinting occurs are unknown, the tendency of imprinted genes to exist in chromosomal clusters suggests long-range regulation through shared regulatory elements. We characterize a 800-kb region on the distal end of mouse chromosome 7 that contains a cluster of four maternally expressed genes, H19, Mash2, Kvlqt1, and p57^Kip2, as well as two paternally expressed genes, Igf2 and Ins2, and assess the expression and imprinting of Mash2, Kvlqt1, and p57^Kip2 during development in embryonic and extraembryonic tissues. Unlike Igf2 and Ins2, which depend on H19 for their imprinting, Mash2, p57^Kip2, and Kvlqt1 are unaffected by a deletion of the H19 gene region, suggesting that these more telomeric genes are not regulated by the mechanism that controls H19, Igf2, and Ins2. Mutations in human p57^Kip2 have been implicated in Beckwith-Wiedemann syndrome, a disease that has also been associated with loss of imprinting of IGF2. We find, however, that a deletion of the gene has no effect on imprinting within the cluster. Surprisingly, the three maternally expressed genes are regulated very differently by DNA methylation; p57^Kip2 is activated, Kvlqt1 is silenced, and Mash2 is unaffected in mice lacking DNA methyltransferase. We conclude that H19 is not a global regulator of imprinting on distal chromosome 7 and that the telomeric genes are imprinted by a separate mechanism(s).     

Alterations of H19 Imprinting and IGF2 Replication Timing Are Infrequent in Beckwith–Wiedemann Syndrome

Beckwith–Wiedemann syndrome (BWS) is an overgrowth disorder resulting from dysregulation of multiple imprinted genes through a variety of distinct mechanisms. A frequent alteration in BWS involves changes in the imprinting status of the coordinately regulated IGF2 and H19 genes on 11p15. Patients have been categorized according to alterations in the imprinted expression, allele-specific methylation, and regional replication timing of these genes. In this work, IGF2/H19 expression, H19 DNA methylation, and IGF2 regional replication timing were studied in nine karyotypically normal BWS fibroblasts and two BWS patients with maternally inherited 11p15 chromosomal rearrangements. Informative patients (9/9) maintained normal monoallelic H19 expression/methylation, despite biallelic IGF2 expression in 6/9. Replication timing studies revealed no changes in the pattern of asynchronous replication timing for both a patient with biallelic IGF2 expression and a patient carrying an 11p15 inversion. In contrast, a patient with a chromosome 11;22 translocation and normal H19 expression/methylation exhibited partial loss of asynchrony and a shift toward earlier replication times. These results indicate that in BWS, (1) H19 imprinting alterations are less frequent than previously estimated, (2) IGF2 imprinting and H19 imprinting are not necessarily coordinated, and (3) alterations in regional replication timing are generally not correlated with either chromosomal rearrangements or the imprinting status of IGF2 and H19.     

Parental alleles of an imprinted mouse transgene replicate synchronously

Molecular features of imprinted genes include differences in expression, methylation, and the timing of DNA replication between parental alleles. Whereas methylation differences always seem to be associated with differences in expression, differences in the timing of replication between parental homologs are not always seen at imprinted loci. These observations raise the possibility that differences in replication timing may not be an essential feature underlying genomic imprinting. In this study, we examined the timing of replication of the two alleles of the imprinted RSVIgmyc transgene in individual embryonic cells using fluorescence in situ hybridization (FISH). The cis-acting signals for RSVIgmyc imprinting are within RSVIgmyc itself. Thus, allele-specific differences in replication, if they indeed govern RSVIgmyc imprinting, should be found in RSVIgmyc sequences. We found that the parental alleles of RSVIgmyc, which exhibit differences in methylation, replicated at the same time. Synchronous replication was also seen in embryonic cells containing a modified version of RSVIgmyc that exhibited parental allele differences in both methylation and expression. These findings indicate that maintenance of expression and methylation differences between alleles does not require a difference in replication timing. The differences in replication timing of endogenous imprinted alleles detected by FISH might therefore reflect structural differences between the two alleles that could be a consequence of imprinting or, alternatively, could be unrelated to imprinting. Dev. Genet. 23:275–284, 1998. © 1998 Wiley-Liss, Inc.     

Alterations of H19 imprinting and IGF2 replication timing are infrequent in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder resulting from dysregulation of multiple imprinted genes through a variety of distinct mechanisms. A frequent alteration in BWS involves changes in the imprinting status of the coordinately regulated IGF2 and H19 genes on 11p15. Patients have been categorized according to alterations in the imprinted expression, allele-specific methylation, and regional replication timing of these genes. In this work, IGF2/H19 expression, H19 DNA methylation, and IGF2 regional replication timing were studied in nine karyotypically normal BWS fibroblasts and two BWS patients with maternally inherited 11p15 chromosomal rearrangements. Informative patients (9/9) maintained normal monoallelic H19 expression/methylation, despite biallelic IGF2 expression in 6/9. Replication timing studies revealed no changes in the pattern of asynchronous replication timing for both a patient with biallelic IGF2 expression and a patient carrying an 11p15 inversion. In contrast, a patient with a chromosome 11;22 translocation and normal H19 expression/methylation exhibited partial loss of asynchrony and a shift toward earlier replication times. These results indicate that in BWS, (1) H19 imprinting alterations are less frequent than previously estimated, (2) IGF2 imprinting and H19 imprinting are not necessarily coordinated, and (3) alterations in regional replication timing are generally not correlated with either chromosomal rearrangements or the imprinting status of IGF2 and H19.     

Identification of a putative DNA replication origin in the λ-aminobutyric acid receptor subunit β3 and α5 gene cluster on human chromosome 15q11-q13, a region associated with parental imprinting and allele-specific replication timing

The region containing the GABA_A receptor β3 and α5 subunit-encoding genes is subject to parental imprinting and is organized in different allele-specific replication timing domains. A 60-kb domain displaying a maternal early/paternal late pattern of allele-specific replication timing asynchrony is nested within a larger region displaying the opposite pattern. The proximal portion of this maternal early replicating domain is incorporated into phage clone λ84. In order to identify DNA structures which may be associated with the boundary between the replication domains, phage λ84 has been subcloned into smaller fragments and several of these have been analyzed by nucleotide sequencing. A plot of helical stability for 13 kb of contiguous sequence reveals several A +T-rich regions which display potential DNA unwinding. The plasmid subclones from phage λ84 have been analyzed for bent DNA and one of these, p82, contains bent DNA and overlaps with the region of highest potential helical instability. Of the seven plasmids tested, only p82 shows strong autonomous replication activity in an in vitro replication assay, with replication initiating within the genomic insert. These results suggest that a putative origin of DNA replication contained within p82 may play a role in establishing the allele-specific replication timing domains in the GABA_A receptor subunit gene cluster.     

Regulation of the Cyclin-Dependent Kinase Inhibitor p57Kip2 Expression by p63

The cyclin-dependent kinase (CDK) inhibitor p57^Kip2 is a negative regulator of cell proliferation, binding to a variety of cyclin-CDK complexes and inhibiting their kinase activities. The p57^Kip2 gene was recognized as a target gene for p73β, one member of the p53 family. In spite of this, the phenotypes of p73 and p57Kip2 knock out mice do not resemble each other while there is a phenotypic overlap betweeen the p57^Kip2 null mice, the p63 null mice and patients affected by p63 associated syndromes, suggesting that p57^Kip2 could be indeed a downstream target of p63. By ChIP we determined that in the HaCaT cell line the δNp63α protein is associated to three different regions of the p57Kip2 gene. δNp63 can activate both the endogenous p57^Kip2 gene and a reporter vector containing a -2191 promoter fragment of the p57^Kip2 gene. Natural p63 mutants, associated to the AEC syndrome, show a partial or complete lack of transactivation potential of the p57^Kip2 promoter, while three other natural p63 mutants, associated to the EEC, LMS and SHFM-4 syndromes, were less affected. These data suggests that p63 play an important role in the regulation of p57^Kip2 expression and that this regulation is subverted in AEC p63 mutants.     

The mouse Chromosome 7 distal imprinting domain maps to G-bands F4/F5

Mouse Chromosome (Chr) 7 distal to band F3 on the physical map is known to be subject to imprinting, maternal duplication (MatDp) of the region leading to a late embryonic lethality, while paternal duplication (PatDp) causes death in utero before 11.5 dpc. Using a new mouse reciprocal translocation T(7;11)65H to produce MatDp for distal Chr 7, we have mapped the region subject to imprinting more precisely to bands 7F4/F5 on the cytogenetic map. Fluorescence in situ hybridization (FISH) studies on mitotic and meiotic chromosomes of a T65H heterozygote show that the imprinted gene Igf2 is located in the same region. This was confirmed by the finding that embryos with MatDp of bands 7F4/F5 did not express Igf2. We suggest that other members of the imprinted domain containing Igf2, namely Mash2, H19, Ins2, and p57 ^K1P2 , are also located in 7F4/F5 and that some or all of these genes may be responsible for the two imprinting lethalities seen with MatDp and PatDp for this region. Received: 13 October 1996 / Accepted: 8 December 1996     

Relaxation of imprinting in Prader-Willi syndrome

We describe two Prader-Willi syndrome (PWS) patients who exhibit maternal uniparental disomy (UPD) of chromosome 15 and unusual patterns of gene expression and DNA replication. Both were diagnosed during infancy as having PWS; however, their growth and development were atypical compared with others with this condition. Weight was below normal in the first patient, and height and development were within normal limits in the second individual. Hyperphagia and polyphagia were not evident in either patient. Genotypes at multiple genomic loci, allele-specific methylation, gene expression, and DNA replication were analyzed at D15S9 [ZNF127], D15S63 [PW71], SNRPN, PAR5, IPW, and D15S10 in these patients. The maternal imprint (based on the absence of gene expression, synchronous replication, and methylation of both alleles) was retained at SNRPN in these patients, as is the case in others with UPD. By contrast, cells from the first individual expressed PAR5 and ZNF127, whereas the second expressed a single IPW allele. Asynchronous DNA replication was observed in both patients at all loci, except SNRPN. These findings show that a subset of imprinted genes can be transcribed in some PWS patients with maternal UPD and that asynchronous DNA replication is coordinated with this pattern of gene expression. Relaxed imprinting in these patients is consistent with their milder phenotype. Received: 19 June 1998 / Accepted: 2 October 1998     

Functional analysis of the p57 KIP2 gene mutation in Beckwith-Wiedemann syndrome

p57 ^KIP2 is a potent tight-binding inhibitor of several G₁ cyclin/cyclin-dependent kinase (Cdk) complexes, and is a negative regulator of cell proliferation. The gene encoding p57 ^KIP2 is located at 11p15.5, a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome (BWS). Previously we demonstrated that p57 ^KIP2 is imprinted and only the maternal allele is expressed in both mice and humans. We also showed mutations found in p57 ^KIP2 in patients with BWS that were transmitted from the patients’ carrier mothers, indicating that the expressed maternal allele was mutant and that the repressed paternal allele was normal. In the study reported here, we performed functional analysis of the two mutated p57 ^KIP2 genes. We showed that the nonsense mutation found in the Cdk inhibitory domain in a BWS patient rendered the protein inactive with consequent complete loss of its role as a cell cycle inhibitor and of its nuclear localization. We also showed that the mutation in the QT domain, although completely retaining its cell cycle regulatory activity, lacked nuclear localization and was thus prevented from performing its role as an active cell cycle inhibitor. Consequently, no active p57 ^KIP2 would have existed, which might have caused the disorders in BWS patients. Received: 7 November 1998 / Accepted: 19 December 1998     

Methylation analysis of the imprinted DLK1-GTL2 domain supports the random parental origin of the IGH-involving del(14q) in B-cell malignancies

Leukemias/lymphomas with IGH-involving del(14q)¹ commonly lose the DLK1-GTL2 imprinted domain that comprises several paternally and maternally expressed genes, including a cluster of microRNAs. Given that deletion of this region could lead to inactivation of a monoallelically expressed tumor suppressor gene, our study aimed at determination of the parental origin of del(14q/IGH). The designed allele-specific methylation study of the DLK1/GTL2 intergenic differentially methylated region allowed us to determine the parental origin of del(14q/IGH) in 9/20 analyzed cases. In six cases del(14q/IGH) was of the paternal origin and in three cases of the maternal origin. These findings argue against the concept that a TSG/anti-oncomir located in the imprinted region is systematically inactivated by a targeted deletion of its functional allele.     

CDK inhibitor p57Kip2 is downregulated by Akt during HER2-mediated tumorigenicity

HER2/neu oncogene is frequently deregulated in cancers, and the (PI3K)-Akt signaling is one of the major pathways in mediating HER2/neu oncogenic signal. p57^Kip2, an inhibitor of cyclin-depependent kinases, is pivotal in regulating cell cycle progression, but its upstream regulators remain unclear. Here we show that the HER2-Akt axis is linked to p57^Kip2 regulation, and that Akt is a negative regulator of p57^Kip2. Ectopic expression of Akt can decrease the expression of p57^Kip2, while Akt inhibition leads to p57^Kip2 stabilization. Mechanistic studies show that Akt interacts with p57^Kip2 and causes cytoplasmic localization of p57^Kip2. Akt phosphorylates p57 on Ser 282 or Thr310. Akt activity results in destabilization of p57 by accelerating turnover rate of p57 and enhancing p57 ubiquitination. Importantly, the negative impact of HER2/Akt on p57 stability contributes to HER2-mediated cell proliferation, transformational activity and tumorigenicity. p57 restoration can attenuate these defects caused by HER2. Significantly, Kaplan-Meier analysis of tumor samples demonstrate that in tumors where HER2 expression was observed, high expression levels of p57^Kip2 were associated with better overall survival. These data suggest that HER2/Akt is an important negative regulator of p57^Kip2, and that p57 restoration in HER2-overexpressing cells can reduce breast tumor growth. Our findings indicate the applicability of employing p57 regulation as a therapeutic intervention in HER2-overexpressing cancers.     

Allele-specific binding of ZFP57 in the epigenetic regulation of imprinted and non-imprinted monoallelic expression

BackgroundSelective maintenance of genomic epigenetic imprints during pre-implantation development is required for parental origin-specific expression of imprinted genes. The Kruppel-like zinc finger protein ZFP57 acts as a factor necessary for maintaining the DNA methylation memory at multiple imprinting control regions in early mouse embryos and embryonic stem (ES) cells. Maternal-zygotic deletion of ZFP57 in mice presents a highly penetrant phenotype with no animals surviving to birth. Additionally, several cases of human transient neonatal diabetes are associated with somatic mutations in the ZFP57 coding sequence.ResultsHere, we comprehensively map sequence-specific ZFP57 binding sites in an allele-specific manner using hybrid ES cell lines from reciprocal crosses between C57BL/6J and Cast/EiJ mice, assigning allele specificity to approximately two-thirds of all binding sites. While half of these are biallelic and include endogenous retrovirus (ERV) targets, the rest show monoallelic binding based either on parental origin or on genetic background of the allele. Parental-origin allele-specific binding is methylation-dependent and maps only to imprinting control differentially methylated regions (DMRs) established in the germline. We identify a novel imprinted gene, Fkbp6, which has a critical function in mouse male germ cell development. Genetic background-specific sequence differences also influence ZFP57 binding, as genetic variation that disrupts the consensus binding motif and its methylation is often associated with monoallelic expression of neighboring genes.ConclusionsThe work described here uncovers further roles for ZFP57-mediated regulation of genomic imprinting and identifies a novel mechanism for genetically determined monoallelic gene expression.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0672-7) contains supplementary material, which is available to authorized users.     

A novel pleckstrin homology-related gene family defined by Ipl/Tssc3, TDAG51, and Tih1: tissue-specific expression, chromosomal location, and parental imprinting

We previously described a gene, Ipl (Tssc3), that is expressed selectively from the maternal allele in placenta, yolk sac, and fetal liver and that maps within the imprinted domain of mouse distal Chromosome (Chr) 7/human Chr 11p15.5 (Hum Mol Genet 6, 2021, 1997). Ipl is similar to TDAG51, a gene that is involved in FAS/CD95 expression. Here we describe another gene, Tih1 (TDAG/Ipl homologue 1), with equivalent sequence similarity to Ipl. Structural prediction indicates that the products of these three genes share a central motif resembling a pleckstrin-homology (PH) domain, and TIH1 protein has weak sequence similarity to the PH-domain protein SEC7/CYTOHESIN. Like Ipl, Tih1 is a small gene with a single small intron. Tih1 maps to distal mouse Chr 1 and human Chr 1q31, chromosomal regions that have not shown evidence for imprinting and, in contrast to Ipl, Tih1 is expressed equally from both parental alleles. Ipl, Tih1, and TDAG51 have overlapping but distinct patterns of expression. Tih1 and TDAG51 are expressed in multiple fetal and adult tissues. In contrast, during early mouse development Ipl mRNA and protein are highly specific for two tissues involved in maternal/fetal exchange: visceral endoderm of the yolk sac and labyrinthine trophoblast of the placenta. These findings highlight the dominance of chromosomal context over gene structure in some examples of parental imprinting and extend previous evidence for placenta-specific expression of imprinted genes. The data also define a new subfamily of PH domain genes. Received: 10 June 1999 / Accepted: 26 July 1999