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

BACKGROUND: Selective 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. RESULTS: Here, 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. CONCLUSIONS: The work described here uncovers further roles for ZFP57-mediated regulation of genomic imprinting and identifies a novel mechanism for genetically determined monoallelic gene expression.The authors acknowledge support from the Wellcome Trust, BBSRC, and EU FP7 Initial Training Networks INGENIUM (Marie-Curie Action 290123) and EpiHealthNet (Marie Curie Action 317146).This is the final version of the article. It first appeared from BioMed Central via http://dx.doi.org/10.1186/s13059-015-0672-

VEZF1 elements mediate protection from DNA methylation

There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm β-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin stat

Trim28 Haploinsufficiency Triggers Bi-stable Epigenetic Obesity.

This is the final version of the article. It first appeared from Cell Press via http://dx.doi.org/10.1016/j.cell.2015.12.025More than one-half billion people are obese, and despite progress in genetic research, much of the heritability of obesity remains enigmatic. Here, we identify a Trim28-dependent network capable of triggering obesity in a non-Mendelian, "on/off" manner. Trim28(+/D9) mutant mice exhibit a bi-modal body-weight distribution, with isogenic animals randomly emerging as either normal or obese and few intermediates. We find that the obese-"on" state is characterized by reduced expression of an imprinted gene network including Nnat, Peg3, Cdkn1c, and Plagl1 and that independent targeting of these alleles recapitulates the stochastic bi-stable disease phenotype. Adipose tissue transcriptome analyses in children indicate that humans too cluster into distinct sub-populations, stratifying according to Trim28 expression, transcriptome organization, and obesity-associated imprinted gene dysregulation. These data provide evidence of discrete polyphenism in mouse and man and thus carry important implications for complex trait genetics, evolution, and medicine.This work was supported by funding from the Max-Planck Society, ERC (ERC-StG-281641), DFG (SFB992 “MedEp”; SFB 1052 “ObesityMechanisms”), EU_FP7 (NoE ”Epigenesys”; “Beta-JUDO” n° 279153), BMBF (DEEP), MRC (Metabolic Disease Unit - APC, SOR, GSHY, MRC_MC_UU_12012/1), Wellcome Trust (SOR, 095515/Z/11/Z) and the German Research Council (DFG) for the Clinical Research Center "Obesity Mechanisms" CRC1052/1 C05 and the Federal Ministry of Education and Research, Germany, FKZ, 01EO1001 (Integrated Research and Treatment Center (IFB) Adiposity Diseases

Mapping the mouse Allelome reveals tissue specific regulation of allelic expression

To determine the dynamics of allelic-specific expression during mouse development, we analyzed RNA-seq data from 23 F1 tissues from different developmental stages, including 19 female tissues allowing X chromosome inactivation (XCI) escapers to also be detected. We demonstrate that allelic expression arising from genetic or epigenetic differences is highly tissue-specific. We find that tissue-specific strain-biased gene expression may be regulated by tissue-specific enhancers or by post-transcriptional differences in stability between the alleles. We also find that escape from X-inactivation is tissue-specific, with leg muscle showing an unexpectedly high rate of XCI escapers. By surveying a range of tissues during development, and performing extensive validation, we are able to provide a high confidence list of mouse imprinted genes including 18 novel genes. This shows that cluster size varies dynamically during development and can be substantially larger than previously thought, with the Igf2r cluster extending over 10 Mb in placenta

A lexicon of DNA modifications: their roles in embryo development and the germline

5-methylcytosine (5mC) on CpG dinucleotides has been viewed as the major epigenetic modification in eukaryotes for a long time. Apart from 5mC, additional DNA modifications have been discovered in eukaryotic genomes. Many of these modifications are thought to be solely associated with DNA damage. However, growing evidence indicates that some base modifications, namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxylcytosine (5caC), and N6-methadenine (6mA), may be of biological relevance, particularly during early stages of embryo development. Although abundance of these DNA modifications in eukaryotic genomes can be low, there are suggestions that they cooperate with other epigenetic markers to affect DNA-protein interactions, gene expression, defense of genome stability and epigenetic inheritance. Little is still known about their distribution in different tissues and their functions during key stages of the animal lifecycle. This review discusses current knowledge and future perspectives of these novel DNA modifications in the mammalian genome with a focus on their dynamic distribution during early embryonic development and their potential function in epigenetic inheritance through the germ line

Heterochromatin and the molecular mechanisms of 'parent-of-origin' effects in animals.

Twenty five years ago it was proposed that conserved components of constitutive heterochromatin assemble heterochromatinlike complexes in euchromatin and this could provide a general mechanism for regulating heritable (cell-to-cell) changes in gene expressibility. As a special case, differences in the assembly of heterochromatin-like complexes on homologous chromosomes might also regulate the parent-of-origin-dependent gene expression observed in placental mammals. Here, the progress made in the intervening period with emphasis on the role of heterochromatin and heterochromatin-like complexes in parent-of-origin effects in animals is reviewed

Jdp2 downregulates Trp53 transcription to promote leukaemogenesis in the context of Trp53 heterozygosity

We performed a genetic screen in mice to identify candidate genes that are associated with leukaemogenesis in the context of Trp53 heterozygosity. To do this we generated Trp53 heterozygous mice carrying the T2/Onc transposon and SB11 transposase alleles to allow transposon-mediated insertional mutagenesis to occur. From the resulting leukaemias/lymphomas that developed in these mice, we identified nine loci that are potentially associated with tumour formation in the context of Trp53 heterozygosity, including AB041803 and the Jun dimerization protein 2 (Jdp2). We show that Jdp2 transcriptionally regulates the Trp53 promoter, via an atypical AP-1 site, and that Jdp2 expression negatively regulates Trp53 expression levels. This study is the first to identify a genetic mechanism for tumour formation in the context of Trp53 heterozygosity

Mapping and characterisation of genomic binding sites of the chromatin barrier protein VEZF1

VEZF1 is a highly conserved transcription factor that is restricted to vertebrates. A chicken homologue of this protein, BGP1 has been recently identified to function at the HS4 insulator element demarcating the 5' boundary of the chicken β-globin domain. BGP1 binding sites are required for chromatin barrier activity and are associated with protection from de novo DNA methylation. Gene targeting experiments in mice have also revealed crucial roles for Vezf1 in vascular and lymphatic development. Work described in my thesis utilises a ChIP-chip approach to map VEZF1 binding sites across 1% of the human genome, known as the ENCODE regions, in K562 erythroid cells. It was found that VEZF1 preferentially targets gene regulatory elements and promoters of actively transcribed genes in particular. A significant proportion of intergenic sites were identified at the boundary regions between active and repressive chromatin. This indicates that these may harbour insulator activities akin to HS4. In addition, a subset of binding sites were found to bind VEZF1 in tissue specific manner being restricted to haemopoietic/ erythroid lineages. Depletion of VEZF1 levels in K562 cells results in specific downregulation of alpha- and beta-globin gene RNA levels, whilst expression of more than 20 other gene targets bound by VEZF1 remains unchanged. Findings presented in this thesis provide a global view of VEZF1 mediated functions in the genome and highlight its potential roles in regulating endothelial and haemopoietic gene expression