Imprinted genes are expressed from one allele according to their parent of origin, and many are essential to mammalian embryogenesis. Here we show that the ɛ-sarcoglycan gene (Sgce) and Zac1 (Lot1) are both paternally expressed imprinted genes. They were identified in a subtractive screen for imprinted genes using a cDNA library made from novel parthenogenetic and wild-type fibroblast lines. Sgce is a component of the dystrophin-sarcoglycan complex, Zac1 is a nuclear protein inducing growth arrest and/or apoptosis, and Zac1 is a potential tumor suppressor gene. Sgce and Zac1 are expressed predominantly from their paternal alleles in all adult mouse tissues, except that Zac1 is biallelic in the liver and Sgce is weakly expressed from the maternal allele in the brain. Sgce and Zac1 are broadly expressed in embryos, with Zac1 being highly expressed in the liver primordium, the umbilical region, and the neural tube. Sgce, however, is strongly expressed in the allantoic region on day 9.5 but becomes more widely expressed throughout the embryo by day 11.5. Sgce is located at the proximal end of mouse chromosome 6 and is a candidate gene for embryonic lethality associated with uniparental maternal inheritance of this region. Zac1 maps to the proximal region of chromosome 10, identifying a new imprinted locus in the mouse, homologous with human chromosome 6q24-q25. In humans, unipaternal disomy for this region is associated with fetal growth retardation and transient neonatal diabetes mellitus. In addition, loss of expression of ZAC has been described for a number of breast and ovarian carcinomas, suggesting that ZAC is a potential tumor suppressor gene.
Organs are programmed to acquire a particular size during development, but the regulatory mechanisms that dictate when dividing progenitor cells should permanently exit the cell cycle and stop producing additional daughter cells are poorly understood. In differentiated tissues, tumor suppressor genes maintain a constant cell number and intact tissue architecture by controlling proliferation, apoptosis and cell dispersal. Here we report a similar role for two tumor suppressor genes, the Zac1 zinc finger transcription factor and that encoding the cytokine TGFβII, in the developing retina.
Using loss and gain-of-function approaches, we show that Zac1 is an essential negative regulator of retinal size. Zac1 mutants develop hypercellular retinae due to increased progenitor cell proliferation and reduced apoptosis at late developmental stages. Consequently, supernumerary rod photoreceptors and amacrine cells are generated, the latter of which form an ectopic cellular layer, while other retinal cells are present in their normal number and location. Strikingly, Zac1 functions as a direct negative regulator of a rod fate, while acting cell non-autonomously to modulate amacrine cell number. We implicate TGFβII, another tumor suppressor and cytokine, as a Zac1-dependent amacrine cell negative feedback signal. TGFβII and phospho-Smad2/3, its downstream effector, are expressed at reduced levels in Zac1 mutant retinae, and exogenous TGFβII relieves the mutant amacrine cell phenotype. Moreover, treatment of wild-type retinae with a soluble TGFβ inhibitor and TGFβ receptor II (TGFβRII) conditional mutants generate excess amacrine cells, phenocopying the Zac1 mutant phenotype.
We show here that Zac1 has an essential role in cell number control during retinal development, akin to its role in tumor surveillance in mature tissues. Furthermore, we demonstrate that Zac1 employs a novel cell non-autonomous strategy to regulate amacrine cell number, acting in cooperation with a second tumor suppressor gene, TGFβII, through a negative feedback pathway. This raises the intriguing possibility that tumorigenicity may also be associated with the loss of feedback inhibition in mature tissues.
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 p57Kip2, as well as two paternally expressed genes, Igf2 and Ins2, and assess the expression and imprinting of Mash2, Kvlqt1, and p57Kip2 during development in embryonic and extraembryonic tissues. Unlike Igf2 and Ins2, which depend on H19 for their imprinting, Mash2, p57Kip2, 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 p57Kip2 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; p57Kip2 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).
The cerebellum is composed of a diverse array of neuronal subtypes. Here we have used a candidate approach to identify Zac1, a tumor suppressor gene encoding a zinc finger transcription factor, as a new player in the transcriptional network required for the development of a specific subset of cerebellar nuclei and a population of Golgi cells in the cerebellar cortex.
We found that Zac1 has a complex expression profile in the developing cerebellum, including in two proliferating progenitor populations; the cerebellar ventricular zone and the external granular layer overlying posterior cerebellar lobules IX and X. Zac1 is also expressed in some postmitotic cerebellar neurons, including a subset of GABAergic interneurons in the medial cerebellar nuclei. Notably, GABAergic interneurons in the cerebellar nuclei are derived from the cerebellar ventricular zone, where Zac1 is also expressed, consistent with a lineage relationship between these two Zac1+ populations. Zac1 is also expressed in a small subset of cells in the posterior vermis, including some neurogranin-immunoreactive (NG+) Golgi cells, which, based on short-term birthdating, are derived from the EGL, where Zac1 is also expressed. However, Zac1+ cells and NG+ Golgi cells in the cerebellar cortex also display unique properties, as they are generated within different, albeit overlapping, time windows. Finally, consistent with the expression profile of Zac1, two conspicuous abnormalities were found in the cerebellum of Zac1 null mice: the medial cerebellar nuclei, and not the others, were significantly reduced in size; and the number of Golgi cells in cerebellar lobule IX was reduced by approximately 60% compared to wild-type littermates.
The data presented here indicate that the tumor suppressor gene Zac1 is expressed in a complex fashion in the developing cerebellum, including in two dividing progenitor populations and in specific subsets of postmitotic neurons, including Golgi cells and GABAergic neurons in the medial nuclei, which require Zac1 for their differentiation. We thus conclude that Zac1 is a critical regulator of normal cerebellar development, adding a new transcriptional regulator to the growing list of factors involved in generating neuronal diversity in the developing cerebellum.
This study used the MG-63 osteosarcoma cell line to investigate the demethylation of the phosphate and tension homolog (PTEN) gene promoter and the change in PTEN gene expression levels, which are caused by the methylation inhibitor 5-azacytidine (5-Zac), and the association between the two. Different concentrations of 5-Zac (0, 5 and 10 μmol/l) were added into the MG-63 cell culture medium and the cells were cultured for 72 h. The following techniques were performed on the cells: Western blot analysis to detect the PTEN protein; reverse transcription-polymerase chain reaction (PCR) to detect the mRNA transcription levels of the PTEN gene; flow cytometry to detect the cell apoptotic rate; and sodium bisulfate to deal with the DNA of each group. The genes of the PTEN promoter and the transcription factors specificity protein 1 (Sp1) and Myc were PCR amplified and transformed into Escherichia coli, then a number of clones were selected for sequencing and the methylation status of the amplified PTEN promoter fragment was detected. Following culture of the MG-63 cells with 5-Zac at concentrations of 0, 5 and 10 μmol/l for 72 h, the expression levels of PTEN protein in each group were gradually increased, presenting a concentration-dependent effect: Group 0 μmol/l compared with groups 5 and 10 μmol/l, P<0.05; and group 5 μmol/l compared with group 10 μmol/l, P=0.007. The mRNA expression levels of the PTEN gene significantly increased. The apoptotic rates of groups 0, 5 and 10 μmol/l were 0.69±0.42, 2.50±0.30 and 6.59±0.62%, and significant differences (P<0.01) were observed between every two groups. The bisulfate DNA sequencing results of three groups showed that, following the treatment with 5-Zac, the binding of the CG site to transcription factors was affected by demethylation. The average rate of demethylation indicated a statistical difference among the three groups. In conclusion, the methylation inhibitor 5-Zac leads to a significant increase in the expression levels of the tumor suppressor gene PTEN in the MG-63 osteosarcoma cell line in vitro. The expression levels of mRNA and the cellular apoptotic rate were also increased. The elevated activation and expression levels of the PTEN gene may be associated with the low methylation levels of the CG site that binds to the transcription factors Sp1 and Myc in the PTEN gene promoter, and they promote the combination of the transcription factors and the gene promoter.
DNA demethylation; PTEN gene; PTEN expression; 5-azacytidine; MG-63 human osteosarcoma cell line
Beckwith-Wiedemann syndrome (BWS) is a model imprinting disorder resulting from mutations or epigenetic events involving imprinted genes at chromosome 11p15.5. Thus, germline mutations in CDKN1C, uniparental disomy (UPD), and loss of imprinting of IGF2 and other imprinted genes have been implicated. Many familial BWS cases have germline CDKN1C mutations. However, most BWS cases are sporadic and UPD or putative imprinting errors predominate in this group. We have identified previously a subgroup of sporadic cases with loss of imprinting (LOI) of IGF2 and epigenetic silencing of H19 proposed to be caused by a defect in a distal 11p15.5 imprinting control element (designated BWSIC1). However, many sporadic BWS patients show biallelic IGF2 expression in the presence of normal H19 methylation and expression patterns. This and other evidence suggested the existence of a further imprinting control element (BWSIC2) at 11p15.5. Recently, we showed that a subgroup of BWS patients have loss of methylation (LOM) at a differentially methylated region (KvDMR1) within the KCNQ1 gene centromeric to the IGF2 and H19 genes. We have now analysed a large series of sporadic cases to define the frequency and phenotypic correlates of epigenetic abnormalities in BWS. LOM at KvDMR1 was detected by Southern analysis or a novel PCR based method in 35 of 69 (51%) sporadic BWS without UPD. LOM at KvDMR1 was often, but not invariably associated with LOI of IGF2. KvDMR1 LOM was not detected in BWS patients with putative BWSIC1 defects and cases with KvDMR1 LOM (that is, putative BWSIC2 defects) invariably had a normal H19 methylation pattern. The incidence of exomphalos in putative BWSIC2 defect patients was not significantly different from that in patients with germline CDKN1C mutations (20/29 and 13/15 respectively), but was significantly greater than that in patients with putative BWSIC1 defects (0/5, p=0.007) and UPD (0/22, p<0.0001). These findings are consistent with the hypothesis that LOM of KvDMR1 (BWSIC2 defect) results in epigenetic silencing of CDKN1C and variable LOI of IGF2. BWS patients with embryonal tumours have UPD or a BWSIC1 defect but not LOM of KvDMR1. This study has further shown how (1) variations in phenotypic expression of BWS may be linked to specific molecular subgroups and (2) molecular analysis of BWS can provide insights into mechanisms of imprinting regulation.
Keywords: Beckwith-Wiedemann syndrome; epigenotype-phenotype correlations; imprinting
Imprinted genes play a critical role in brain development and mental health, although the underlying molecular and cellular mechanisms remain incompletely understood. The family of basic helix-loop-helix (bHLH) proteins directs the proliferation, differentiation, and specification of distinct neuronal progenitor populations. Here, we identified the bHLH factor gene Tcf4 as a direct target gene of Zac1/Plagl1, a maternally imprinted transcriptional regulator, during early neurogenesis. Zac1 and Tcf4 expression levels concomitantly increased during neuronal progenitor differentiation; moreover, Zac1 interacts with two cis-regulatory elements in the Tcf4 gene locus, and these elements together confer synergistic activation of the Tcf4 gene. Tcf4 upregulation enhances the expression of the cyclin-dependent kinase inhibitor gene p57Kip2, a paternally imprinted Tcf4 target gene, and increases the number of cells in G1 phase. Overall, we show that Zac1 controls cell cycle arrest function in neuronal progenitors through induction of p57Kip2 via Tcf4 and provide evidence for cooperation between imprinted genes and a bHLH factor in early neurodevelopment.
Beckwith-Wiedemann syndrome (BWS) is a human imprinting disorder with a variable phenotype. The major features are anterior abdominal wall defects including exomphalos (omphalocele), pre- and postnatal overgrowth, and macroglossia. Additional less frequent complications include specific developmental defects and a predisposition to embryonal tumours. BWS is genetically heterogeneous and epigenetic changes in the IGF2/H19 genes resulting in overexpression of IGF2 have been implicated in many cases. Recently germline mutations in the cyclin dependent kinase inhibitor gene CDKN1C (p57KIP2) have been reported in a variable minority of BWS patients. We have investigated a large series of familial and sporadic BWS patients for evidence of CDKN1C mutations by direct gene sequencing. A total of 70 patients with classical BWS were investigated; 54 were sporadic with no evidence of UPD and 16 were familial from seven kindreds. Novel germline CDKN1C mutations were identified in five probands, 3/7 (43%) familial cases and 2/54 (4%) sporadic cases. There was no association between germline CDKN1C mutations and IGF2 or H19 epigenotype abnormalities. The clinical phenotype of 13 BWS patients with germline CDKN1C mutations was compared to that of BWS patients with other defined types of molecular pathology. This showed a significantly higher frequency of exomphalos in the CDKN1C mutation cases (11/13) than in patients with an imprinting centre defect (associated with biallelic IGF2 expression and H19 silencing) (0/5, p<0.005) or patients with uniparental disomy (0/9, p<0.005). However, there was no association between germline CDKN1C mutations and risk of embryonal tumours. No CDKN1C mutations were identified in six non-BWS patients with overgrowth and Wilms tumour. These findings (1) show that germline CDKN1C mutations are a frequent cause of familial but not sporadic BWS, (2) suggest that CDKN1C mutations probably cause BWS independently of changes in IGF2/H19 imprinting, (3) provide evidence that aspects of the BWS phenotype may be correlated with the involvement of specific imprinted genes, and (4) link genotype-phenotype relationships in BWS and the results of murine experimental models of BWS.
Keywords: Beckwith-Wiedemann syndrome; CDKN1C (p57KIP2) mutation
Paternal repression of the imprinted H19 gene is mediated by a differentially methylated domain (DMD) that is essential to imprinting of both H19 and the linked and oppositely imprinted Igf2 gene. The mechanisms by which paternal-specific methylation of the DMD survive the period of genome-wide demethylation in the early embryo and are subsequently used to govern imprinted expression are not known. Methyl-CpG binding (MBD) proteins are likely candidates to explain how these DMDs are recognized to silence the locus, because they preferentially bind methylated DNA and recruit repression complexes with histone deacetylase activity. MBD RNA and protein are found in preimplantation embryos, and chromatin immunoprecipitation shows that MBD3 is bound to the H19 DMD. To test a role for MBDs in imprinting, two independent RNAi-based strategies were used to deplete MBD3 in early mouse embryos, with the same results. In RNAi-treated blastocysts, paternal H19 expression was activated, supporting the hypothesis that MBD3, which is also a member of the Mi-2/NuRD complex, is required to repress the paternal H19 allele. RNAi-treated blastocysts also have reduced levels of the Mi-2/NuRD complex protein MTA-2, which suggests a role for the Mi-2/NuRD repressive complex in paternal-specific silencing at the H19 locus. Furthermore, DNA methylation was reduced at the H19 DMD when MBD3 protein was depleted. In contrast, expression and DNA methylation were not disrupted in preimplantation embryos for other imprinted genes. These results demonstrate new roles for MBD3 in maintaining imprinting control region DNA methylation and silencing the paternal H19 allele. Finally, MBD3-depleted preimplantation embryos have reduced cell numbers, suggesting a role for MBD3 in cell division.
Genomic imprinting is a specialized system of gene regulation whereby only one copy of a gene is used, either the maternal or the paternal copy. Misregulation of imprinting in humans results in developmental disorders such as Beckwith-Wiedemann Syndrome, and is implicated in many cancers. Study of imprinted genes in mice can lead to a greater understanding of these diseases as well as insight into gene regulation. Many imprinted genes are associated with methylation on the silenced allele. The imprinted gene H19 is maternally expressed and paternally methylated in a region upstream of the promoter known as the differentially methylated domain. This region is required for proper imprinted expression of H19 and its upstream imprinted neighbor Igf2. Our studies have explored the requirement for methyl-CpG binding protein 3 (MBD3) in silencing of the paternal allele. MBD3 is known to be part of a repressive complex that resides at silenced genes. In our experiments, we have shown that MBD3 is required for imprinting of H19, and is also required for the maintenance of methylation on the paternal allele. Finally, the MBD3 protein can be found at the differentially methylated domain. The identification of a protein required for silencing of the paternal allele of H19 is an important step in understanding regulation of this gene.
Context: Beckwith–Wiedemann syndrome (BWS) arises by several genetic and epigenetic mechanisms affecting the balance of imprinted gene expression in chromosome 11p15.5. The most frequent alteration associated with BWS is the absence of methylation at the maternal allele of KvDMR1, an intronic CpG island within the KCNQ1 gene. Targeted deletion of KvDMR1 suggests that this locus is an imprinting control region (ICR) that regulates multiple genes in 11p15.5. Cell culture based enhancer blocking assays indicate that KvDMR1 may function as a methylation modulated chromatin insulator and/or silencer.
Objective: To determine the potential consequence of loss of methylation (LOM) at KvDMR1 in the development of BWS.
Methods: The steady state levels of CDKN1C gene expression in fibroblast cells from normal individuals, and from persons with BWS who have LOM at KvDMR1, was determined by both real time quantitative polymerase chain reaction (qPCR) and ribonuclease protection assay (RPA). Methylation of the CDKN1C promoter region was assessed by Southern hybridisation using a methylation sensitive restriction endonuclease.
Results: Both qPCR and RPA clearly demonstrated a marked decrease (86–93%) in the expression level of the CDKN1C gene in cells derived from patients with BWS, who had LOM at KvDMR1. Southern analysis indicated that downregulation of CDKN1C in these patients was not associated with hypermethylation at the presumptive CDKN1C promoter.
Conclusions: An epimutation at KvDMR1, the absence of maternal methylation, causes the aberrant silencing of CDKN1C, some 180 kb away on the maternal chromosome. Similar to mutations at this locus, this silencing may give rise to BWS.
Beckwith-Wiedemann syndrome (BWS) is a loss-of-imprinting pediatric overgrowth syndrome. The primary features of BWS include macrosomia, macroglossia, and abdominal wall defects. Secondary features that are frequently observed in BWS patients are hypoglycemia, nevus flammeus, polyhydramnios, visceromegaly, hemihyperplasia, cardiac malformations, and difficulty breathing. BWS is speculated to occur primarily as the result of the misregulation of imprinted genes associated with two clusters on chromosome 11p15.5, namely the KvDMR1 and H19/IGF2. A similar overgrowth phenotype is observed in bovine and ovine as a result of embryo culture. In ruminants this syndrome is known as large offspring syndrome (LOS). The phenotypes associated with LOS are increased birth weight, visceromegaly, skeletal defects, hypoglycemia, polyhydramnios, and breathing difficulties. Even though phenotypic similarities exist between the two syndromes, whether the two syndromes are epigenetically similar is unknown. In this study we use control Bos taurus indicus X Bos taurus taurus F1 hybrid bovine concepti to characterize baseline imprinted gene expression and DNA methylation status of imprinted domains known to be misregulated in BWS. This work is intended to be the first step in a series of experiments aimed at determining if LOS will serve as an appropriate animal model to study BWS.
The use of F1 B. t. indicus x B. t. taurus tissues provided us with a tool to unequivocally determine imprinted status of the regions of interest in our study. We found that imprinting is conserved between the bovine and human in imprinted genes known to be associated with BWS. KCNQ1OT1 and PLAGL1 were paternally-expressed while CDKN1C and H19 were maternally-expressed in B. t. indicus x B. t. taurus F1 concepti. We also show that in bovids, differential methylation exists at the KvDMR1 and H19/IGF2 ICRs.
Based on these findings we conclude that the imprinted gene expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 ICRs are conserved between human and bovine. Future work will determine if LOS is associated with misregulation at these imprinted loci, similarly to what has been observed for BWS.
KvDMR1; H19/IGF2 ICR; KCNQ1OT1; CDKN1C; PLAGL1; Beckwith-Wiedemann syndrome; Methylation; Genomic imprinting; Epigenetics; Bovine
Zac encodes a zinc finger protein that promotes apoptosis and cell cycle arrest and is maternally imprinted. Here, we show that Zac contains transactivation and repressor activities and that these transcriptional activities are differentially controlled by DNA binding. Zac transactivation mapped to two distinct domains. One of these contained multiple repeats of the peptide PLE, which behaved as an autonomous activation unit. More importantly, we identified two related high-affinity DNA-binding sites which were differentially bound by seven Zac C2H2 zinc fingers. Zac bound as a monomer through zinc fingers 6 and 7 to the palindromic DNA element to confer transactivation. In contrast, binding as a monomer to one half-site of the repeat element turned Zac into a repressor. Conversely, Zac dimerization at properly spaced direct and reverse repeat elements enabled transactivation, which strictly correlated with DNA-dependent and -independent contacts of key residues within the recognition helix of zinc finger 7. The later ones support specific functional connections between Zac DNA binding and transcriptional-regulatory surfaces. Both classes of DNA elements were identified in a new Zac target gene and confirmed that the zinc fingers communicate with the transactivation function. Together, our data demonstrate a role for Zac as a transcription factor in addition to its role as coactivator for nuclear receptors and p53.
Beckwith-Wiedemann syndrome (BWS) is a fetal overgrowth and human imprinting disorder resulting from the deregulation of a number of genes, including IGF2 and CDKN1C, in the imprinted gene cluster on chromosome 11p15.5. Most cases are sporadic and result from epimutations at either of the two 11p15.5 imprinting centres (IC1 and IC2). However, rare familial cases may be associated with germline 11p15.5 deletions causing abnormal imprinting in cis. We report a family with BWS and an IC2 epimutation in which affected siblings had inherited different parental 11p15.5 alleles excluding an in cis mechanism. Using a positional-candidate gene approach, we found that the mother was homozygous for a frameshift mutation in exon 6 of NLRP2. While germline mutations in NLRP7 have previously been associated with familial hydatidiform mole, this is the first description of NLRP2 mutation in human disease and the first report of a trans mechanism for disordered imprinting in BWS. These observations are consistent with the hypothesis that NLRP2 has a previously unrecognised role in establishing or maintaining genomic imprinting in humans.
A small set of genes (imprinted genes) are expressed in a “parent-of-origin” manner, a phenomenon known as genomic imprinting. Research in human disorders associated with aberrant genomic imprinting provided insights into the molecular mechanisms of genomic imprinting and the role of imprinted genes in normal growth and development. Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth syndrome associated with developmental abnormalities and a predisposition to embryonic tumours. BWS results from alterations in expression or function of imprinted genes in the imprinted gene cluster at chromosome 11p15. Although BWS may be caused by a variety of molecular mechanisms, to date, all the genetic and epigenetic defects associated with BWS have been limited to 11p15.5. We report a family with two children affected with BWS and an epigenetic defect at 11p15.5 in which the primary genetic defect mapped outside the imprinted gene cluster. Using autozygosity mapping, we found an extended homozygous region on chromosome 19q13.4 (containing NLRP2 and NLRP7 genes) in the mother. Homozygous inactivating mutations in NLRP7 in women have been associated previously with abnormal imprinting and recurrent hydatidiform moles. We identified a homozygous frameshift mutation in NLRP2 in the mother of the two children with BWS implicating NLRP2 in the establishment and/or maintenance of genomic imprinting/methylation.
Although many of the questions raised by the discovery of imprinting have been answered, we have not yet accounted for tissue- or stage-specific imprinting. The Kcnq1 imprinted domain exhibits complex tissue-specific expression patterns co-existing with a domain-wide cis-acting control element. Transcription of the paternally expressed antisense non-coding RNA Kcnq1ot1 silences some neighboring genes in the embryo, while others are unaffected. Kcnq1 is imprinted in early cardiac development but becomes biallelic after midgestation. To explore this phenomenon and the role of Kcnq1ot1, we used allele-specific assays and chromosome conformational studies in wild-type mice and mice with a premature termination mutation for Kcnq1ot1. We show that Kcnq1 imprinting in early heart is established and maintained independently of Kcnq1ot1 expression, thus excluding a role for Kcnq1ot1 in repressing Kcnq1, even while silencing other genes in the domain. The exact timing of the mono- to biallelic transition is strain-dependent, with the CAST/EiJ allele becoming activated earlier and acquiring higher levels than the C57BL/6J allele. Unexpectedly, Kcnq1ot1 itself also switches to biallelic expression specifically in the heart, suggesting that tissue-specific loss of imprinting may be common during embryogenesis. The maternal Kcnq1ot1 transcript is shorter than the paternal ncRNA, and its activation depends on an alternative transcriptional start site that bypasses the maternally methylated promoter. Production of Kcnq1ot1 on the maternal chromosome does not silence Cdkn1c. We find that in later developmental stages, however, Kcnq1ot1 has a role in modulating Kcnq1 levels, since its absence leads to overexpression of Kcnq1, an event accompanied by an aberrant three-dimensional structure of the chromatin. Thus, our studies reveal regulatory mechanisms within the Kcnq1 imprinted domain that operate exclusively in the heart on Kcnq1, a gene crucial for heart development and function. We also uncover a novel mechanism by which an antisense non-coding RNA affects transcription through regulating chromatin flexibility and access to enhancers.
Imprinted genes express only one copy and the active allele depends on the parental origin. These genes are usually organized in groups and are subject to regulation over long distances. Misregulation in these domains can lead to developmental diseases and cancer. The paternally expressed Kcnq1ot1 non-coding RNA emerges from an intron of the Kcnq1 gene in antisense direction and silences several neighboring genes. The Kcnq1 gene is initially imprinted but becomes biallelic during development of the heart. To understand the mechanisms underlying this behavior, we performed expression and conformational studies of the Kcnq1 domain during this transition and found that the early stage imprinting of Kcnq1 is not dependent on transcription of Kcnq1ot1, thus revealing the existence of a distinct mechanism of silencing of the paternal Kcnq1 allele. Surprisingly, Kcnq1ot1 loses its imprinted expression specifically in the heart in the same time frame as Kcnq1. Absence of Kcnq1ot1 does lead to overexpression of Kcnq1 in later cardiac development, an event characterized by increased chromatin flexibility allowing the Kcnq1 promoter to establish ectopic regulatory contacts. Thus, we have uncovered novel regulatory mechanisms that will help our understanding of tissue specificity in imprinted domains and how they can go awry in disease.
The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27KIP1, an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27KIP1 expression and/or localization are often observed in tumor cells, further confirming its central role in regulating the cell cycle. Recently, germline mutations in CDKN1B have been associated with the inherited multiple endocrine neoplasia syndrome type 4, an autosomal dominant syndrome characterized by varying combinations of tumors affecting at least two endocrine organs. In this study we identified a 4-bp deletion in a highly conserved regulatory upstream ORF (uORF) in the 5′UTR of the CDKN1B gene in a patient with a pituitary adenoma and a well-differentiated pancreatic neoplasm. This deletion causes the shift of the uORF termination codon with the consequent lengthening of the uORF–encoded peptide and the drastic shortening of the intercistronic space. Our data on the immunohistochemical analysis of the patient's pancreatic lesion, functional studies based on dual-luciferase assays, site-directed mutagenesis, and on polysome profiling show a negative influence of this deletion on the translation reinitiation at the CDKN1B starting site, with a consequent reduction in p27KIP1 expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27KIP1 activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27KIP1 activity can also be modulated by an uORF and mutations affecting uORF could change p27KIP1 expression. This study adds the CDKN1B gene to the short list of genes for which mutations that either create, delete, or severely modify their regulatory uORFs have been associated with human diseases.
Gene expression can be modulated at different steps on the way from DNA to protein including control of transcription, translation, and post-translational modifications. An abnormality in the regulation of mRNA and protein expression is a hallmark of many human diseases, including cancer. In some eukaryotic genes translation can be influenced by small DNA sequences termed upstream open reading frames (uORFs). These elements located upstream to the gene start codon may either negatively influence the ability of the translational machinery to reinitiate translation of the main protein or, much less frequently, stimulate protein translation by enabling the ribosomes to bypass cis-acting inhibitory elements. CDKN1B, which encodes the cell cycle inhibitor p27KIP1, includes an uORF in its 5′UTR sequence. p27KIP1 expression is often reduced in cancer, and germline mutations have been identified in CDKN1B in patients affected with a syndrome (MEN4) characterized by varying combinations of tumors in endocrine glands. Here we show that a small deletion in the uORF upstream to CDKN1B reduces translation reinitiation efficiency, leading to underexpression of p27KIP1 and coinciding with tumorigenesis. This study describes a novel mechanism by which p27KIP1 could be underexpressed in human tumors. In addition, our data provide a new insight to the unique pathogenic potential of uORFs in human diseases.
Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth syndrome associated with a characteristic pattern of visceromegaly and predisposition to childhood tumours. BWS is a genetically heterogeneous disorder; most cases are sporadic but approximately 15% are familial and a small number of BWS patients have cytogenetic abnormalities involving chromosome 11p15. Genomic imprinting effects have been implicated in familial and non-familial BWS. We have investigated the molecular pathology of 106 sporadic BWS cases; 17% (14/83) of informative cases had uniparental disomy (UPD) for chromosome 11p15.5. In each case UPD appeared to result from a postzygotic event resulting in mosaicism for segmental paternal isodisomy. The critical region for isodisomy was refined to a 25 cM interval between D11S861 and D11S2071 which contained the IGF2, H19, and p57(KIP2) genes. In three cases isodisomy for 11q markers was detected but this did not extend further than 11q13-q21 suggesting that complete chromosome 11 disomy may not produce a BWS phenotype. The allele specific methylation status of the H19 gene was investigated in 80 sporadic BWS cases. All 13 cases with UPD tested displayed hypermethylation consistent with an excess of paternal H19 alleles. In addition, five of 63 (8%) cases with normal biparental inheritance had H19 hypermethylation consistent with an "imprinting centre" mutation (ICM) or "imprinting error" (IE) lesion. The phenotype of patients with putative ICM/IE mutations was variable and overlapped with that of non-UPD sporadic BWS cases with normal H19 methylation. However, exomphalos was significantly (p < 0.05) more common in the latter group. These findings may indicate differential effects on the expression of imprinted genes in chromosome 11p15 according to the precise molecular pathology. Analysis of H19 methylation is useful for the diagnosis of both UPD or altered imprinting in BWS and shows that a variety of molecular mechanisms may cause relaxation of IGF2 imprinting in BWS.
Heterogeneous molecular defects affecting the 11p15.5 imprinted gene cluster are associated with the opposite growth disorders Beckwith-Wiedemann Syndrome (BWS) and Silver Russell syndrome (SRS). Maternal deletions of the centromeric domain usually result in BWS, but paternal deletions have been so far associated with normal phenotype. Here we describe a case of recurrent severe Intra-Uterine Growth Restriction (IUGR) with paternal transmission of an 11p15.5 60 kb deletion.
Methods and results
Chromosome microarray (CMA), PCR and DNA sequencing analyses showed that two fetuses conceived by a normal couple inherited from their father a 60 kb deletion encompassing the Imprinting Control Region of the 11p15.5 centromeric domain. The two fetuses died in utero with severe growth restriction. PCR amplification of parental DNAs indicated that the father carried the mutation in the mosaic state. DNA methylation and gene expression analyses showed that the deletion led to an imprinting alteration restricted to the centromeric domain and resulting in silencing of KCNQ1OT1 and activation of CDKN1C and PHLDA2.
Our data demonstrate that the phenotype associated with 11p15.5 deletions is strongly influenced by the size of the region involved and indicate imprinting defects leading to CDKN1C and PHLDA2 activation as cause of severe IUGR.
Epigenetics; Imprinting; Chromosomal; Molecular genetics
p57Kip2, a cyclin-dependent kinase inhibitor, is considered to be a candidate tumor suppressor gene that has been implicated in Beckwith-Wiedemann syndrome and sporadic cancers. In addition, decreased expression of p57Kip2 protein has been frequently observed in pancreatic, lung, breast, bladder, gastrointestinal tract and prostate cancers. However, p57Kip2 gene mutations are rare in these cancers suggesting that other unknown mechanisms might be at play in reducing its expression. The aim of this study was to investigate the molecular mechanism of down-regulation of p57Kip2 in prostate cancer.
We observed a significant negative correlation between the expression of p57Kip2 and microRNA-21 (miR-21) in prostate cancer samples and after androgen deprivation with castration in the CWR22 human prostate cancer xenograft model. We report that miR-21 targeted the coding region and decreased p57Kip2 mRNA and protein levels in prostate cancer cells. Conversely, inhibition of endogenous miR-21 by an anti-miR-21 inhibitor strongly induced p57Kip2 expression. Furthermore, we found that knockdown of p57Kip2 reversed the effects of the anti-miR-21 inhibitor on cell migration and anchorage-independent cell growth.
Our results indicate that miR-21 is able to downregulate p57Kip2 expression by targeting the coding region of the gene and is also able to attenuate p57Kip2 mediated functional responses. This is the first report demonstrating that p57Kip2 is a novel target of miR-21 in prostate cancer and revealing a novel oncogenic function of this microRNA.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-212) contains supplementary material, which is available to authorized users.
p57Kip2; microRNA-21 and prostate cancer
A cluster of imprinted genes at chromosome 11p15.5 is associated with the growth disorders, Silver–Russell syndrome (SRS) and Beckwith–Wiedemann syndrome (BWS). The cluster is divided into two domains with independent imprinting control regions (ICRs). We describe two maternal 11p15.5 microduplications with contrasting phenotypes. The first is an inverted and in cis duplication of the entire 11p15.5 cluster associated with the maintenance of genomic imprinting and with the SRS phenotype. The second is a 160 kb duplication also inverted and in cis, but resulting in the imprinting alteration of the centromeric domain. It includes the centromeric ICR (ICR2) and the most 5′ 20 kb of the non-coding KCNQ1OT1 gene. Its maternal transmission is associated with ICR2 hypomethylation and the BWS phenotype. By excluding epigenetic mosaicism, cell clones analysis indicated that the two closely located ICR2 sequences resulting from the 160 kb duplication carried discordant DNA methylation on the maternal chromosome and supported the hypothesis that the ICR2 sequence is not sufficient for establishing imprinted methylation and some other property, possibly orientation-dependent, is needed. Furthermore, the 1.2 Mb duplication demonstrated that all features are present for correct imprinting at ICR2 when this is duplicated and inverted within the entire cluster. In the individuals maternally inheriting the 160 kb duplication, ICR2 hypomethylation led to the expression of a truncated KCNQ1OT1 transcript and to down-regulation of CDKN1C. We demonstrated by chromatin RNA immunopurification that the KCNQ1OT1 RNA interacts with chromatin through its most 5′ 20 kb sequence, providing a mechanism likely mediating the silencing activity of this long non-coding RNA.
Genomic imprinting is an epigenetic phenomenon restricting gene expression in a manner dependent on parent of origin. Imprinted gene products are critical regulators of growth and development, and imprinting disorders are associated with both genetic and epigenetic mutations, including disruption of DNA methylation within the imprinting control regions (ICRs) of these genes. It was recently reported that some patients with imprinting disorders have a more generalised imprinting defect, with hypomethylation at a range of maternally methylated ICRs. We report a cohort of 149 patients with a clinical diagnosis of Beckwith–Wiedemann syndrome (BWS), including 81 with maternal hypomethylation of the KCNQ1OT1 ICR. Methylation analysis of 11 ICRs in these patients showed that hypomethylation affecting multiple imprinted loci was restricted to 17 patients with hypomethylation of the KCNQ1OT1 ICR, and involved only maternally methylated loci. Both partial and complete hypomethylation was demonstrated in these cases, suggesting a possible postzygotic origin of a mosaic imprinting error. Some ICRs, including the PLAGL1 and GNAS/NESPAS ICRs implicated in the aetiology of transient neonatal diabetes and pseudohypoparathyroidism type 1b, respectively, were more frequently affected than others. Although we did not find any evidence for mutation of the candidate gene DNMT3L, these results support the hypotheses that trans-acting factors affect the somatic maintenance of imprinting at multiple maternally methylated loci and that the clinical presentation of these complex cases may reflect the loci and tissues affected with the epigenetic abnormalities.
Beckwith-Wiedemann syndrome (BWS); KCNQ1OT1; methylation; imprinting; imprinting disorder; hypomethylation of imprinted loci (HIL)
Derivation of induced pluripotent stem (iPS) cells is mainly an epigenetic reprogramming process. It is still quite controversial how genomic imprinting is reprogrammed in iPS cells. Thus, we derived multiple iPS clones from genetically identical mouse somatic cells. We found that parentally inherited imprint was variably lost among these iPS clones. Concurrent with the loss of DNA methylation imprint at the corresponding Snrpn and Peg3 imprinted regions, parental origin-specific expression of the Snrpn and Zim1 imprinted genes was also lost in these iPS clones. This loss of parental genomic imprinting in iPS cells was likely caused by the reprogramming process during iPS cell derivation because extended culture of iPS cells did not lead to significant increase in the loss of genomic imprinting. Intriguingly, one to several paternal chromosomes appeared to have acquired de novo methylation at the Snrpn and Zac1 imprinted regions in a high percentage of iPS clones. These results might have some implications for future therapeutic applications of iPS cells. Since DNA methylation imprint can be completely erased in some iPS clones at multiple imprinted regions, iPS cell reprogramming may also be employed to dissect the underlying mechanisms of erasure, reacquisition and maintenance of genomic imprinting in mammals.
Transcriptional activation by nuclear hormone receptors is mediated by the 160-kDa family of nuclear receptor coactivators. These coactivators associate with DNA-bound nuclear receptors and transmit activating signals to the transcription machinery through two activation domains. In screening for mammalian proteins that bind the C-terminal activation domain of the nuclear receptor coactivator GRIP1, we identified a new variant of mouse Zac1 which we call mZac1b. Zac1 was previously discovered as a putative transcriptional activator involved in regulation of apoptosis and the cell cycle. In yeast two-hybrid assays and in vitro, mZac1b bound to GRIP1, to CREB-binding protein (CBP) and p300 (which are coactivators for nuclear receptors and other transcriptional activators), and to nuclear receptors themselves in a hormone-independent manner. In transient-transfection assays mZac1b exhibited a transcriptional activation activity when fused with the Gal4 DNA binding domain, and it enhanced transcriptional activation by the Gal4 DNA binding domain fused to GRIP1 or CBP fragments. More importantly, mZac1b was a powerful coactivator for the hormone-dependent activity of nuclear receptors, including androgen, estrogen, glucocorticoid, and thyroid hormone receptors. However, with some reporter genes and in some cell lines mZac1b acted as a repressor rather than a coactivator of nuclear receptor activity. Thus, mZac1b can interact with nuclear receptors and their coactivators and play both positive and negative roles in regulating nuclear receptor function.
Background: Mutations in the imprinted gene CDKN1C account for approximately 10% of Beckwith–Wiedemann syndrome (BWS) cases. Fibroblasts from BWS patients with loss of methylation (LOM) at the imprinting control region (ICR) KvDMR1 have reduced CDKN1C expression. Another group of BWS patients with downregulated CDKN1C expression but with normal methylation at KvDMR1 has been identified.
Objective: To investigate the mechanism of CDKN1C silencing in BWS in these two classes of patients.
Methods: The CDKN1C promoter region was analysed for changes in DNA methylation using bisulphite sequencing, and for alterations in chromatin structure using the chromatin immunoprecipitation (ChIP) assay.
Results: There was only spurious CpG methylation of the CDKN1C promoter in fibroblast DNA from both normal individuals and patients with BWS, irrespective of the methylation status of KvDMR1. There was no detectable change in chromatin structure at the CDKN1C promoter in patients with LOM at KvDMR1. BWS patients with downregulated CDKN1C and normal methylation at KvDMR1 had depletion of dimethylated H3-K4 and enrichment of dimethylated H3-K9 and HP1γ at the CDKN1C promoter, suggesting that in these cases gene silencing is associated with repressive chromatin changes.
Conclusions: CDKN1C may be downregulated by multiple mechanisms including some that do not involve promoter methylation. In BWS patients with normal methylation at KvDMR1 and reduced expression of CDKN1C, repressive chromatin may play a role, but the absence of methylation and repressive chromatin structure at the CDKN1C promoter in BWS patients with LOM at KvDMR1 argues for a direct role of this epimutation in silencing CDKN1C.
Beckwith‐Wiedemann syndrome (BWS) is an overgrowth disorder with increased risk of paediatric tumours. The aetiology involves epigenetic and genetic alterations affecting the 11p15 region, methylation of the differentially methylated DMR2 region being the most common defect, while less frequent aetiologies include mosaic paternal 11p uniparental disomy (11patUPD), maternally inherited mutations of the CDKN1C gene, and hypermethylation of DMR1. A few patients have cytogenetic abnormalities involving 11p15.5.
Screening of 70 trios of BWS probands for 11p mosaic paternal UPD and for cryptic cytogenetic rearrangements using microsatellite segregation analysis identified a profile compatible with paternal 11p15 duplication in two patients.
Fluorescence in situ hybridisation analysis revealed in one case the unbalanced translocation der(21)t(11;21)(p15.4;q22.3) originated from missegregation of a cryptic paternal balanced translocation. The second patient, trisomic for D11S1318, carried a small de novo dup(11)(p15.5p15.5), resulting from unequal recombination at paternal meiosis I. The duplicated region involves only IC1 and spares IC2/LIT1, as shown by fluorescent in situ hybridisation (FISH) mapping of the proximal duplication breakpoint within the amino‐terminal part of KvLQT1.
An additional patient with Wolf‐Hirschorn syndrome was shown by FISH studies to carry a der(4)t(4;11)(p16.3;p15.4), contributed by a balanced translocation father. Interestingly, refined breakpoint mapping on 11p and the critical regions on the partner 21q and 4p chromosomal regions suggested that both translocations affecting 11p15.4 are mediated by segmental duplications. These findings of chromosomal rearrangements affecting 11p15.5–15.4 provide a tool to further dissect the genomics of the BWS region and the pathogenesis of this imprinting disorder.
Beckwith‐Wiedemann syndrome; chromosomal rearrangements; imprinting 11p15 region; segmental duplicons
Silver–Russell syndrome (SRS) is a heterogeneous disorder characterised by severe intrauterine and postnatal growth retardation, limb and body asymmetry, a typical facial appearance and less common dysmorphisms. Recently, epimutations and maternal duplications affecting the short arm of chromosome 11 have been shown to have a crucial role in the aetiology of the disease. Disturbances in the same genomic region cause the overgrowth disorder Beckwith–Wiedemann syndrome (BWS). In BWS, mutations in the telomeric as well as in the centromeric imprinting centres (ICR1 and ICR2) in 11p15 can be observed. In SRS, methylation defects in the imprinted region in 11p15 were considered to be restricted to the telomeric ICR1. They can be detected in about 30% of patients. This article reports on the first patient with SRS with a cryptic duplication restricted to the centromeric ICR2 domain in 11p15. The maternally inherited duplication in this patient included a region of 0.76–1 Mbp and affected the genes regulated by the ICR2, among them CDKN1C and LIT1. This study provides evidence for a role for this imprinting centre in the aetiology of SRS and shows that SRS presents a picture genetically opposite to that of BWS.