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© 2020 by the authors., The embryonal renal cancer Wilms tumor (WT) accounts for 7% of all children’s malignancies. Its most frequent molecular defect is represented by DNA methylation abnormalities at the imprinted 11p15.5 region. Multiple imprinted methylation alterations dictated by chromosome copy-number variations have been recently demonstrated in adult cancers, raising the question of whether multiple imprinted loci were also affected in WT. To address this issue, we analyzed DNA methylation and chromosome profiles of 7 imprinted loci in 48 WT samples. The results demonstrated that methylation abnormalities of multiple imprinted loci occurred in 35% of the cases, but that they were associated with either chromosome aberrations or normal chromosome profiles. Multiple imprinted methylation changes were correlated with tumor stage and presence of metastasis, indicating that these epimutations were more frequent in highly aggressive tumors. When chromosome profiles were affected, these alterations were extended to flanking cancer driver genes. Overall, this study demonstrates the presence of multiple imprinted methylation defects in aggressive WTs and suggests that the mechanism by which they arise in embryonal and adult cancers is different., This study was funded by the Ministry of Education, Universities and Research, Research Projects of National Interest (MIUR PRIN) 2015 grant no JHLY35, Telethon-Italia grant no GGP1513, AIRC grant no. IG18671 (A.R.).

The protein MucR from Brucella abortus has been described as a transcriptional regulator of many virulence genes. It is a member of the Ros/MucR family comprising proteins that control the expression of genes important for the successful interaction of &alpha, proteobacteria with their eukaryotic hosts. Despite clear evidence of the role of MucR in repressing virulence genes, no study has been carried out so far demonstrating the direct interaction of this protein with the promoter of its target gene babR encoding a LuxR-like regulator repressing virB genes. In this study, we show for the first time the ability of MucR to bind the promoter of babR in electrophoretic mobility shift assays demonstrating a direct role of MucR in repressing this gene. Furthermore, we demonstrate that MucR can bind the virB gene promoter. Analyses by RT-qPCR showed no significant differences in the expression level of virB genes in Brucella abortus CC092 lacking MucR compared to the wild-type Brucella abortus strain, indicating that MucR binding to the virB promoter has little impact on virB gene expression in B. abortus 2308. The MucR modality to bind the two promoters analyzed supports our previous hypothesis that this is a histone-like protein never found before in Brucella.

Background: Differentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the H19 gene known as the imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMRO, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMRO methylation changes in the aetiology of BWS or SRS is unknown. Methodology/Principal Findings: We analysed the methylation status of the DMRO in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to reports, the IGF2 DMRO is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC1 methylation status and is inconsistent with the proposed silencing function of the maternal IGF2 allele. Beckwith-Wiedemann and Silver-Russell patients with IC1 methylation defects have similar methylation defects at the IGF2 DMRO, consistent with IC1 regulating methylation at IGF2 in cis. In Wilms tumour, however, methylation profiles of IC1 and IGF2 DMRO are indicative of methylation changes occuring on both parental alleles rather than in cis. Conclusions/Significance: These results support a model in which DMRO and IC1 have opposite susceptibilities to global hyper and hypomethylation during tumorigenesis independent of the parent of origin imprint. In contrast, during embryogenesis DMRO is mehtylated or demethylated according to the germline methylation imprint at the IC1, indicating different mechanisms of imprinting loss in neoplastic and non-neoplastic cells. © 2008 Murrell et al.

ZFP57 is necessary for maintaining repressive epigenetic modifications at Imprinting control regions (ICRs). In mouse embryonic stem cells (ESCs), ZFP57 binds ICRs (ICRBS) and many other loci (non-ICRBS). To address the role of ZFP57 on all its target sites, we performed high-throughput and multi-locus analyses of inbred and hybrid mouse ESC lines carrying different gene knockouts. By using an allele-specific RNA-seq approach, we demonstrate that ZFP57 loss results in derepression of the imprinted allele of multiple genes in the imprinted clusters. We also find marked epigenetic differences between ICRBS and non-ICRBS suggesting that different cis-acting regulatory functions are repressed by ZFP57 at these two classes of target loci. Overall, these data demonstrate that ZFP57 is pivotal to maintain the allele-specific epigenetic modifications of ICRs that in turn are necessary for maintaining the imprinted expression over long distances. At non-ICRBS, ZFP57 inactivation results in acquisition of epigenetic features that are characteristic of poised enhancers, suggesting that another function of ZFP57 in early embryogenesis is to repress cis-acting regulatory elements whose activity is not yet required. ZFP57 is necessary for maintaining repressive epigenetic modifications at Imprinting control regions (ICRs). In mouse embryonic stem cells (ESCs), ZFP57 binds ICRs (ICRBS) and many other loci (non-ICRBS). To address the role of ZFP57 on all its target sites, we performed high-throughput and multi-locus analyses of inbred and hybrid mouse ESC lines carrying different gene knockouts. By using an allele-specific RNA-seq approach, we demonstrate that ZFP57 loss results in derepression of the imprinted allele of multiple genes in the imprinted clusters. We also find marked epigenetic differences between ICRBS and non-ICRBS suggesting that different cis-acting regulatory functions are repressed by ZFP57 at these two classes of target loci. Overall, these data demonstrate that ZFP57 is pivotal to maintain the allele-specific epigenetic modifications of ICRs that in turn are necessary for maintaining the imprinted expression over long distances. At non-ICRBS, ZFP57 inactivation results in acquisition of epigenetic features that are characteristic of poised enhancers, suggesting that another function of ZFP57 in early embryogenesis is to repress cis-acting regulatory elements whose activity is not yet required.