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The interplay between glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) is central to maintain energy homeostasis. It remains to be determined whether there is a mechanism governing metabolic fluxes based on substrate availability in microenvironments. Here we show that menin is a key transcription factor regulating the expression of OXPHOS and glycolytic genes in cancer cells and primary tumors with poor prognosis. A group of menin-associated proteins (MAPs), including KMT2A, MED12, WAPL, and GATA3, is found to restrain menin&rsquo, s full function in this transcription regulation. shRNA knockdowns of menin and MAPs result in reduced ATP production with proportional alterations of cellular energy generated through glycolysis and OXPHOS. When shRNA knockdown cells are exposed to metabolic stress, the dual functionality can clearly be distinguished among these metabolic regulators. A MAP can negatively counteract the regulatory mode of menin for OXPHOS while the same protein positively influences glycolysis. A close-proximity interaction between menin and MAPs allows transcriptional regulation for metabolic adjustment. This coordinate regulation by menin and MAPs is necessary for cells to rapidly adapt to fluctuating microenvironments and to maintain essential metabolic functions.

Triple negative breast cancer (TNBC) refers to tumors that do not express clinically significant levels of estrogen and progesterone receptors, and lack membrane overexpression or gene amplification of ErbB-2 tyrosine kinase receptor. Transcriptome and proteome heterogeneity of TNBC poses a major challenge to precision medicine. Gene expression analyses have categorized TNBC into distinct molecular subtypes. Up to 78% of clinical TNBCs belong to the basal-like (BL) subtype. Here we found ErbB-2 in an unanticipated scenario: the nucleus of TNBC (NErbB-2). Our study on ErbB-2 alternative splicing, using a PCR-sequencing approach combined with RNA interference, revealed that BL TNBC cells express the canonical ErbB-2 (WTErbB-2), encoded by transcript 1, and the non-canonical isoform c, encoded by alternative transcript 3 (T3). The latter was not previously reported in normal or malignant cells. To characterize the isoform c we designed siRNAs targeting T3 (T3 siRNAs), which silenced up to 93% of said isoform. Transfection of T3 siRNAs into BL cells expressing only isoform c or both isoform c and WTErbB-2 was sufficient to decrease cell proliferation. Intratumoral injections of T3 siRNAs into mice bearing BL TN tumors also blocked in vivo growth. To explore whether isoform c growth-promoting effect is due to its functions as a transcriptional regulator, we performed RNA-seq in BL cells expressing only this isoform. We identified a set of genes differentially regulated in BL cells where we evicted isoform c from the nucleus, as compared to control cells. In the up-regulated group, we found enrichment of pro-apoptotic and tumor suppressor genes and in the down-regulated one, genes involved in proliferation and stemness. We used gene set enrichment analysis (GSEA) to identify the biological processes associated with these isoform c-regulated genes. We found a pronounced enrichment of gene sets related to apoptosis, activation of DNA damage pathways and cell cycle arrest in response to eviction of nuclear isoform c. GSEA also revealed negative regulation of gene sets involved in cell motility, cellular differentiation and growth pathways in BL cells lacking nuclear isoform c expression. These results suggest that NErbB-2 function modulates tumor growth and promotes a metastatic phenotype in TNBC. Furthermore, our clinical findings identified NErbB-2 as an independent predictor of shorter OS (HR 2.54; 95% CI 1.22-5.28; P = 0.013), DFS (HR 2.91; 95% CI 1.44-5.87; P = 0.003), and DMFS (HR 2.59; 95% CI 1.20-5.60; P = 0.015) in 99 TN primary tumors. Our discoveries challenge the present scenario of drug development for personalized BC medicine that focuses on wild-type proteins, which conserve the canonical domains and are located in their classical cellular compartments, highlighting the potential of NErbB-2 isoforms as novel therapeutic targets and clinical biomarkers in TNBC.

Metabolic reprogramming in cancer cells not only sustains bioenergetic and biosynthetic needs but also influences transcriptional programs, yet how chromatin regulatory networks are rewired by altered metabolism remains elusive. Here we investigate genome-scale chromatin remodeling in response to 2-hydroxyglutarate (2HG) oncometabolite using single-cell assay for transposase accessible chromatin with sequencing (scATAC-seq). We find that 2HG enantiomers differentially disrupt exquisite control of epigenome integrity by limiting α-ketoglutarate (αKG)-dependent DNA and histone demethylation, while enhanced cell-to-cell variability in the chromatin regulatory landscape is most evident upon exposure to L2HG enantiomer. Despite the highly heterogeneous responses, 2HG largely recapitulates two prominent hallmarks of the breast cancer epigenome, i.e., global loss of 5-hydroxymethylcytosine (5hmC) and promoter hypermethylation, particularly at tumor suppressor genes involved in DNA damage repair and checkpoint control. Single-cell mass cytometry further demonstrates downregulation of BRCA1, MSH2 and MLH1 in 2HG-responsive subpopulations, along with acute reversal of chromatin remodeling upon withdrawal. Collectively, this study provides a molecular basis for metabolism-epigenome coupling and identifies metabolic vulnerabilities imposed on the breast cancer epigenome.

Additional file 1: Figure S1. The expression profile of breast tumors with PAM50 subtypes and normal controls of the 96 amplification-associated transcription coupling (ATC) loci from the five regions on 17q (A, 17q11.1-q11.2; B, 17q12-q21.2; C, 17q21.2-q21.31; D, 17q21.31-q21.33; E, 17q22-q24.1). The locations of the tumors of different subtypes are displayed by the solid lines with designated colors on the top of the heat map. Figure S2. Circos plots of inter- and intra- chromosomal rearrangements related to three 17q regions. Figure S3. Nanopore sequencing of inter-chromosomal rearrangements involving the 17q23 region. a. 17q23-associated rearrangements identified in MCF-7. Rearrangement frequencies were determined using previously generated whole-genome mate-pair sequencing data. b. Nanopore sequencing of the MCF-7 genome. A schematic flow chart (left) indicates the principle of Nanopore long-read sequencing. Unique molecular barcodes were incorporated into amplicons individually by PCR to enable multiplex sequencing of samples and the resultant reactions were then pooled. After end repair and A-tail reactions, leader and the hairpin adapters, each containing a motor protein (orange), were ligated to the end prepared DNA, followed by His-tag purification. On the MinION device, DNA molecules are pulled through a protein pore (gray) with motor proteins, producing 2D reads, which were consensus calls of the combined template and complement strands (red). PCR amplicons (right) spanning the 18 chromosomal breakpoints between chromosomes 17q23 and 20q13 were individually barcoded and pooled together for sequencing. The image of the desktop sequencing process on the MinION device (left bottom) was captured in our laboratory. c. Screen captures of representative NCBI-BLAST search outputs. DNA sequences from the 17q23 and 20q13 regions are shown in orange and blue, respectively.

Purpose: Little is known about adaptive selection of tumor cells transiting from in situ proliferation to distant colonization through blood circulation. This study used genomic, transcriptomic, and biophysical analyses at single-cell levels to explore biological and physical properties of circulating tumor cells (CTCs) undergoing hemodynamic stress. The aims are trying to understand how CTCs strive to survive in the bloodstream and to develop strategies for therapeutic intervention. Experimental Design: We compared genomic profiles of CTCs in blood and primary tumor cells shed in urine of prostate cancer patients to identify amplified regions preferentially retained in CTCs. Single-cell RNA-seq was used to confirm amplification-associated overexpression of genes in CTCs. Results: Among 261 recurrently amplified genomic regions in the analysis, 70 were found predominantly shown in CTCs relative to primary tumor cells. In line with the results at the genomic level, the transcriptomic data of CTCs demonstrate a great amount of cells showing high expression levels in the oxidative phosphorylation (OXPHOS) pathway compared to other hallmark gene sets. The finding suggests that tumor cells with these pre-existing genomic alterations were adaptively selected for transcription reprogramming during blood circulation. Specifically, amplified genes associated with OXPHOS were exploited by CTCs for alternative fuels. As to the upstream of this transcription event, we found the expression of MEN1, encoding menin known to form a transcription factor complex with the mixed-lineage leukemia protein (MLL) in prostate cancer cells, was positively correlated with 11 of the 14 OXPHOS loci in the Cancer Genomic Atlas prostate cancer cohort. In vitro assay showed that MEN1 knockdown by shRNA resulted in attenuation of both mRNA and protein expression of OXPHOS loci in PC-3 cells. Taken together pre-existing amplification of OXPHOS loci can be used as a transcription apparatus by menin for metabolic reprogramming of tumor cells in response to harsh microenvironments in the bloodstream. Conclusions: Single-cell profiling identified signaling pathways that are crucial for CTCs to survive in the bloodstream. The finding suggests that a metabolic shift from Warburg to OXPHOS metabolism can be associated with a hybrid mesenchymal-epithelial phenotype of CTCs. Moreover, the study demonstrates the feasibility of routinely conducting single-cell analysis of exfoliated tumor cells for minimally invasive monitoring of disease progression and treatment response in prostate cancer patients Citation Format: Chun-Lin Lin, Tan Xi, Chia-Nung Hung, Pawel A. Osmulski, Chih-Wei Chou, Meizhen Chen, Chiou-Miin Wang, Kohzoh Mitsuya, Nameer Kirma, Maria E. Gaczynska, Chun-Liang Chen, Tim H.-M. Huang. Amplification-associated upregulation of genes involved in oxidative phosphorylation for disseminated prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2766.

The inflammatory and metabolic derangements of obesity in pregnant women generate an adverse intrauterine environment, increase pregnancy complications and adverse fetal outcomes and program the fetus for obesity and metabolic syndrome in later life. We hypothesized that epigenetic modifications in placenta including altered DNA methylation/hydroxymethylation may mediate these effects. Term placental villous tissue was collected following cesarean section from lean (prepregnancy BMI30) women. Genomic DNA was isolated, methylated and hydroxymethylated DNA immunoprecipitated and hybridized to the NimbleGen 2.1M human DNA methylation array. Intermediate metabolites in placental tissues were measured by HPLC-ESI-MS, ascorbate levels by reverse phase HPLC and gene expression by RT-PCR. Differentially methylated and hydroxymethylated regions occurred across the genome, with a 21% increase in methylated but a 31% decrease in hydroxymethylated regions in obese vs lean groups. Whereas increased methylation and decreased methylation was evident around transcription start sites of multiple genes in the GH/CSH and PSG gene clusters on chromosomes 17 and 19 in other areas there was no relationship. Increased methylation was associated with decreased expression only for some genes in these clusters. Biological pathway analysis revealed the 262 genes which showed reciprocal differential methylation/ hydroxymethylation were enriched for pregnancy, immune response and cell adhesion-linked processes. We found a negative relationship for maternal BMI but a positive relationship for ascorbate with α-ketoglutarate a metabolite that regulates ten eleven translocase (TET) which mediates DNA methylation. We provide evidence for the obese maternal metabolic milieu being linked to an altered DNA methylome that may affect placental gene expression in relation to adverse outcomes.

Genomic imprinting causes parental origin-specific monoallelic gene expression through differential DNA methylation established in the parental germ line. However, the mechanisms underlying how specific sequences are selectively methylated are not fully understood. We have found that the components of the PIWI-interacting RNA (piRNA) pathway are required for de novo methylation of the differentially methylated region (DMR) of the imprinted mouse Rasgrf1 locus, but not other paternally imprinted loci. A retrotransposon sequence within a noncoding RNA spanning the DMR was targeted by piRNAs generated from a different locus. A direct repeat in the DMR, which is required for the methylation and imprinting of Rasgrf1, served as a promoter for this RNA. We propose a model in which piRNAs and a target RNA direct the sequence-specific methylation of Rasgrf1.

Human Placental Trophoblasts: Impact of Maternal Nutrition explores the vital roles of trophoblasts play in fetal growth and pregnancy, giving you new insight into the modulation of placental trophoblast functions by nutrients. It also reviews the role of fatty acids, folic acids, and specific vitamins in this aspect. The book highlights the critic

Igf2 and H19 are reciprocally imprinted genes on mouse distal chromosome 7. They share several regulatory elements, including a differentially methylated region (DMR) upstream of H19 that is paternally methylated throughout development. The cis-acting sequence requirements for targeting DNA methylation to the DMR remain unknown; however, it has been suggested that direct tandem repeats near DMRs could be involved. Previous studies of the imprinted Rasgrf1 locus demonstrate indeed that a direct repeat element adjacent to a DMR is responsible for establishing paternal allele-specific methylation at the DMR and therefore allelic expression of the Rasgrf1 transcript. We identified a prominent and conserved direct tandem repeat 1 kb upstream of the H19 DMR and proposed that it played a similar role in imprinted regulation of H19. To test our hypothesis, we generated mice harboring a 1.7-kb targeted deletion of the direct repeat element and analyzed fetal growth, allelic expression, and methylation within the Igf2-H19 region. Surprisingly the deletion had no effect on imprinting. These results together with deletions of other repeats close to imprinted genes suggest that direct repeats may not be important for the targeting of methylation at the majority of imprinted loci and that the Rasgrf1 locus may be an exception to this rule.

Russell-Silver syndrome (RSS) is a form of congenital short stature characterized by severe growth retardation and variable dysmorphic features. In some RSS individuals, alterations in imprinted genes may be involved because approximately 7% of sporadic patients have been observed to have maternal uniparental disomy (mUPD) of chromosome 7. RSS patients with structural abnormalities of chromosome 7 have also been described. In these individuals the chromosome rearrangement could disrupt the balance of imprinted genes, contribute to a recessive form of RSS, or lead to haploinsufficiency of a crucial developmental gene product. Because the mechanism and molecular defects on chromosome 7 causing RSS are still unknown, we tested our collection of 77 RSS families for mUPD7 and were able to identify three new cases. We also characterized two RSS patients with de novo cytogenetic abnormalities involving the short arm of chromosome 7. One had a partial duplication [46, XX, dup(7)(p12 p14)] and the second contained a paracentric inversion [46, XY, inv(7)(p14 p21)]. Fluorescence in situ hybridization (FISH) mapping revealed that the breakpoints on 7p14 were localized to the same novel gene, C7orf10, which encompasses >700 kb of DNA. We also identified other transcription units from this immediate region, but all seem to be biallelically expressed when using a somatic cell hybrid assay.

Genomic imprinting is the phenomenon by which the two alleles of certain genes are differentially expressed according to their parental origin. Extensive analysis of allelic expression at multiple imprinted loci in a normal population has not performed so far. In the present study, we examined the allelic expression pattern of three imprinted genes in a panel of 262 Japanese normal individuals. We observed differences in the extent of maintenance of allele-specific expression of the three genes. The allelic expression of small nuclear ribonucleoprotein N (SNRPN) was stringently regulated while that of multimembrane-spanning polyspecific transporter-like gene 1 (IMPT1) showed a large degree of variation. Significant biallelic expression of insulin-like growth factor II (IGF2) was observed in about 10% of normal individuals. Our findings add to the accumulating evidence for variable allelic expression at multiple loci in a normal human population. This epigenetic heterogeneity can be a stable trait and potentially influence individual phenotypes.

As an in vitro assay system for the identification of human imprinted genes, a library of human/mouse A9 monochromosomal hybrids containing a single, intact bsr-tagged human chromosome of known parental origin, derived from normal human fibroblasts, has been previously generated by microcell-mediated chromosome transfer (MMCT). To supplement this assay system, we constructed additional 700 A9 monochromosomal hybrids, using a pSTneo or pPGKneo selection marker. To validate the A9 hybrids, we screened them with chromosome-specific polymorphic markers, and identified the hybrids containing either human chromosome 6, 7, 14, 18, or 21 of known parental origin. Matching paternal and maternal chromosome pairs of A9 hybrids were identified for chromosomes 6, 7, 14, and 18. The paternal-specific expression of ZAC (zinc finger protein, which regulates apoptosis and cell cycle arrest) and HYMAI (hydatidiform mole-associated and imprinted transcript), and the maternal-specific methylation of a CpG island within an imprinted domain on human chromosome 6q24, were maintained in A9 hybrids. For an example, we profiled the expression of expressed sequence tags (ESTs) and the methylation of CpG islands in the 300-kb imprinted domain around 6q24, which may be associated with cancers and transient neonatal diabetes mellitus (TNDM). Thus, the 700 A9 hybrids should be useful for various aspects of imprinting studies.

Loss of paternal gene expression at the imprinted domain on proximal human chromosome 15 causes Prader-Willi syndrome (PWS), a complex multiple-anomaly disorder involving variable mental retardation, hyperphasia leading to obesity and infantile hypotonia with failure to thrive. Although numerous paternally expressed transcripts have been identified that reside in the candidate region, the individual contributions to the development of PWS have not been firmly established. Recent studies of mouse models carrying a cytogenetic deletion suggest that paternal deficiency of the SNRPN-IPW interval is critical for perinatal lethality of potential relevance to PWS. Here we determined the allelic expression profiles of a total of 118 cDNA clones using monochromosomal hybrids retaining either a paternal or maternal human chromosome 15. Our results demonstrated a preponderance of unusual transcripts lacking protein-coding potential that were expressed exclusively from the paternal copy of the critical interval. This interval was also found to encompass a large direct repeat (DR) cluster displaying a potentially active chromatin conformation of paternal origin, as suggested by enhanced sensitivity to nuclease digestion. Database searches revealed an unexpected organization of tandemly repeated consensus elements, all of which possessed well-defined box C and D sequences characteristic of small nucleolar RNAs (snoRNAs). Southern blot analysis further demonstrated a considerable degree of phylogenetic conservation of the DR locus in the genomes of all mammalian species tested, but not in chicken, Xenopus and Drosophila. These findings imply a potential direct contribution of the DR locus, representing a cluster of multiple snoRNA genes, to certain phenotypic features of PWS.

Preterm birth involves the interaction of societal and environmental factors potentially modulating the length of gestation via the epigenome. An established form of epigenetic regulation is DNA methylation where promoter hypermethylation is associated with gene repression. We hypothesized we would find differences in DNA methylation in the myometrium of women with preterm labor of different phenotypes versus normal term labor. Myometrial tissue was obtained at cesarean section at term with or without labor, preterm without labor, idiopathic preterm labor, and twin gestations with labor. Genomic DNA was isolated, and samples in each group were combined and analyzed on a NimbleGen 2.1M human DNA methylation array. Differences in methylation from −8 to +3 kb of transcription start sites of 22 contraction-associated genes were determined. Cytosine methylation was not present in CpG islands of any gene but was present outside of CpG islands in shores and shelves in 19 genes. No differential methylation was found across the tissue groups for six genes (PTGES3L, PTGER2, PTGER4, PTGFRN, ESR2, and GJA1). For 13 genes, differential methylation occurred in several patterns between tissue groups. We find a correlation between hypomethylation and increased mRNA expression of PTGES/mPGES-1, indicating potential functional relevance of methylation, but no such correlation for PTGS2/COX-2, suggesting other regulatory mechanisms for PTGS2 at labor. The majority of differential DNA methylation of myometrial contraction-associated genes with different labor phenotypes occurs outside of CpG islands in gene promoters, suggesting that the entirety of DNA methylation across the genome should be considered.

We have constructed mouse A9 hybrids containing a single normal human chromosome 15, via microcell-mediated chromosome transfer. Cytogenetic and DNA-polymorphic analyses identified mouse A9 hybrids that contained either a paternal or maternal human chromosome 15. Paternal specific expression of the known imprinted genes SNRPN (small nuclear ribonucleoprotein-associated polypeptide N gene) and IPW (imprinted gene in the Prader-Willi syndrome region) was maintained in the A9 hybrids. Using this system, we first demonstrated that human GABAAreceptor subunit genes, GABRB3 , GABRA5 and GABRG3 , were expressed exclusively from the paternal allele and that E6-AP (E6-associated protein or UBE3A ) was biallelically expressed. Moreover, the 5' portion of the GABRB3 gene was found to be hypermethylated on the paternal allele. Our data imply that GABAAreceptor subunit genes are imprinted and are possible candidates for Prader-Willi syndrome, and that this human monochromosomal hybrid system enables the efficient analysis of imprinted loci.

DNA methyltransferase (cytosine-5) 1 (Dnmt1) is the principal enzyme responsible for maintenance of CpG methylation and is essential for the regulation of gene expression, silencing of parasitic DNA elements, genomic imprinting and embryogenesis. Dnmt1 is needed in S phase to methylate newly replicated CpGs occurring opposite methylated ones on the mother strand of the DNA, which is essential for the epigenetic inheritance of methylation patterns in the genome. Despite an intrinsic affinity of Dnmt1 for such hemi-methylated DNA, the molecular mechanisms that ensure the correct loading of Dnmt1 onto newly replicated DNA in vivo are not understood. The Np95 (also known as Uhrf1 and ICBP90) protein binds methylated CpG through its SET and RING finger-associated (SRA) domain. Here we show that localization of mouse Np95 to replicating heterochromatin is dependent on the presence of hemi-methylated DNA. Np95 forms complexes with Dnmt1 and mediates the loading of Dnmt1 to replicating heterochromatic regions. By using Np95-deficient embryonic stem cells and embryos, we show that Np95 is essential in vivo to maintain global and local DNA methylation and to repress transcription of retrotransposons and imprinted genes. The link between hemi-methylated DNA, Np95 and Dnmt1 thus establishes key steps of the mechanism for epigenetic inheritance of DNA methylation.

Human chromosome 15q11-q13 involves a striking imprinted gene cluster of more than 2 Mb that is concomitant with multiple neurological disorders manifested by Prader-Willi syndrome (PWS) and Angelman syndrome (AS). PWS and AS patients with imprinting mutation have microdeletions, which share a 4.3 kb short region of overlap (SRO) at the 5' end of the paternal SNURF-SNRPN gene in PWS, or on the maternal allele, which shares a 880 bp SRO located at the 35 kb upstream of the SNURF-SNRPN promoter in AS. Recent studies have revealed an essential role of PWS-SRO in the postzygotic maintenance of the appropriate epigenotype on the paternal chromosome. For AS-SRO, however, there is insufficient experimental evidence exists to determine the direct functions. Here we show that the complete deletion of AS-SRO does not cause any anomalies of imprinted gene expression or DNA methylation on the mutated human chromosome 15, further supporting the idea that AS-SRO is dispensable for post implantation imprint maintenance. This implies that AS-SRO is not essential for the robust epigenotype preservation in somatic cells.

Imprinted genes are expressed from only one of the parental chromosomes and are marked epigenetically by DNA methylation and histone modifications1,2,3,4,5,6,7,8,9. The imprinting center 2 (IC2) on mouse distal chromosome 7 is flanked by several paternally repressed genes, with the more distant ones imprinted exclusively in the placenta. We found that most of these genes lack parent-specific DNA methylation, and genetic ablation of methylation does not lead to loss of their imprinting in the trophoblast (placenta). The silent paternal alleles of the genes are marked in the trophoblast by repressive histone modifications (dimethylation at Lys9 of histone H3 and trimethylation at Lys27 of histone H3), which are disrupted when IC2 is deleted, leading to reactivation of the paternal alleles. Thus, repressive histone methylation is recruited by IC2 (potentially through a noncoding antisense RNA) to the paternal chromosome in a region of at least 700 kb and maintains imprinting in this cluster in the placenta, independently of DNA methylation. We propose that an evolutionarily older imprinting mechanism limited to extraembryonic tissues was based on histone modifications, and that this mechanism was subsequently made more stable for use in embryonic lineages by the recruitment of DNA methylation.

Genomic imprinting in mammals marks the parental alleles in gametes, resulting in differential gene expression in offspring. A number of epigenetic features are associated with imprinted genes. These include differential DNA methylation, histone acetylation and methylation, subnuclear localization and DNA replication timing. While DNA methylation has been shown to be necessary both for establishment and maintenance of imprinting, the connections with the other types of epigenetic marking systems are not clear. Specifically, it is not known whether the other marking systems, either on their own or in conjunction with DNA methylation, are required for imprinting. Here we show that in the mouse mutant Minute (Mnt) the Igf2-H19 locus acquires a paternal methylation imprint in the maternal germline. DNA methylation of the H19 DMR is established in oogenesis, maintained during postzygotic development on the maternal allele, and erased in primordial germ cells. The fact that a paternal type methylation imprint can also be established in the maternal germline indicates that trans-acting factors that target methylation to this imprinted region are likely to be the same in both germlines. Surprisingly, however, asynchrony of DNA replication of the locus is maintained despite the altered expression and methylation imprint of Igf2 and H19. These results show clearly that replication asynchrony of this region is neither the determinant factor for, nor a consequence of, epigenetic modifications that are critical for genomic imprinting. Replication asynchrony may thus be regulated differently from methylation imprints and have a separate function.

Epigenetic asymmetry between parental genomes and embryonic lineages exists at the earliest stages of mammalian development. The maternal genome in the zygote is highly methylated in both its DNA and its histones and most imprinted genes have maternal germline methylation imprints. The paternal genome is rapidly remodelled with protamine removal, addition of acetylated histones, and rapid demethylation of DNA before replication. A minority of imprinted genes have paternal germline methylation imprints. Methylation and chromatin reprogramming continues during cleavage divisions, but at the blastocyst stage lineage commitment to inner cell mass (ICM) or trophectoderm (TE) fate is accompanied by a dramatic increase in DNA and histone methylation, predominantly in the ICM. This may set up major epigenetic differences between embryonic and extraembryonic tissues, including in X–chromosome inactivation and perhaps imprinting. Maintaining epigenetic asymmetry appears important for development as asymmetry is lost in cloned embryos, most of which have developmental defects, and in particular an imbalance between extraembryonic and embryonic tissue development.

MEST is one of the imprinted genes in human. With the assistance of our integration map and the complete sequence in the registry, we mapped a total of 16 genes/transcripts at the 1.5-Mb MEST-flanking region at 7q32. This region has been suggested to form an imprinted gene cluster, because MEST and its three flanking genes/transcripts (MESTIT1, CPA4, and COPG2IT1) were reported to be imprinted, although two (TSGA14 and COPG2) were shown to escape imprinting. In this study, 10 other genes/transcripts were examined for their imprinting status in human fetal tissues. The results indicated that 8 genes/transcripts (NRF1, UBE2H, HSPC216, KIAA0265, FLJ14803, CPA2, CPA1, and DKFZp667F0312) were expressed biallelically. The imprinting status of two (TSGA13 and CPA5) was not conclusive, because of their weak and/or tissue-specific expression and inconstant results. These findings provided evidence that only 4 of the 16 genes/transcripts located to the region show monoallelic expression, while others are not involved in imprinting. Therefore, it is less likely that the MEST-flanking 7q32 region forms a large imprinted domain.

Loss of imprinting (LOI) of the insulin-like growth factor 2 (IGF2) and H19 genes on human chromosome 11 has been found not only in childhood tumors but also in common adult cancers including colorectal cancer. Recently, a transcript called LIT1 (long QT intronic transcript 1) has been identified within the KvLQT1 locus on chromosome 11. LIT1 is expressed preferentially from the paternal allele and is transcribed in most human tissues. LOI of LIT1 was found in a considerable number of Beckwith-Wiedemann syndrome (BWS) patients, suggesting that it is associated with the etiology of BWS. Since LOI of IGF2 was observed in association with overexpression of IGF2 in colorectal cancer in our previous study, we examined the status of genomic imprinting of LIT1 and H19 in comparison with IGF2 in colorectal cancer. We examined 44 surgically dissected colorectal cancer tissues. Ten of them represented informative cases for LIT1. None of these patients exhibited loss of heterozygosity (LOH) of LIT1, and LOI of LIT1 was observed in 4 of the 10 (40%) informative patients, but not in non-cancerous tissues. Neither LOH nor LOI of H19 was observed. LOI of IGF2 was observed in 4 of 18 (22%) informative patients. These results suggest that LOI of LIT1 is frequently observed in colorectal cancer and may be a useful marker for diagnosis of colorectal cancer.

Lack of a maternal contribution to the genome at the imprinted domain on proximal chromosome 15 causes Angelman syndrome (AS) associated with neurobehavioral anomalies that include severe mental retardation, ataxia and epilepsy. Although AS patients have infrequent mutations in the gene encoding an E6-AP ubiquitin ligase required for long-term synaptic potentiation (LTP), most cases are attributed to de novo maternal deletions of 15q11-q13. We report here that a novel maternally expressed gene, ATP10C, maps within the most common interval of deletion and that ATP10C expression is virtually absent from AS patients with imprinting mutations, as well as from patients with maternal deletions of 15q11-q13.

Human chromosome 11p15.5 harbors an intriguing imprinted gene cluster of 1 Mb. This imprinted domain is implicated in a wide variety of malignancies and Beckwith-Wiedemann syndrome (BWS). Recently, several lines of evidence have suggested that the BWS-associated imprinting cluster consists of separate chromosomal domains. We have previously identified LIT1, a paternally expressed antisense RNA within the KvLQT1 locus through a positional screening approach using human monochromosomal hybrids. KvLQT1 encompasses the translocation breakpoint cluster in BWS and patients exhibit frequent loss of maternal methylation at the LIT1 CpG island, implying a regulatory role for the LIT1 locus in coordinate control of the imprinting cluster. Here we generated modified human chromosomes carrying a targeted deletion of the LIT1 CpG island using recombination-proficient chicken DT40 cells. Consistent with the prediction, this mutation abolished LIT1 expression on the paternal chromosome, accompanied by activation of the normally silent paternal alleles of multiple imprinted loci at the centromeric domain including KvLQT1 and p57(KIP2). The deletion had no effect on imprinting of H19 located at the telomeric end of the cluster. Our findings demonstrate that the LIT1 CpG island can act as a negative regulator in cis for coordinate imprinting at the centromeric domain, thereby suggesting a role for the LIT1 locus in a BWS pathway leading to functional inactivation of p57(KIP2). Thus, the targeting and precise modification of human chromosomal alleles using the DT40 cell shuttle system can be used to define regulatory elements that confer long-range control of gene activity within chromosomal domains.

Disrupted imprinting is implicated in certain tumorigenesis. Since aberrant methylation has been described for a majority of microsatellite instability (MSI)-positive sporadic colorectal cancers, we have investigated alteration to the imprinting in 55 sporadic colorectal cancers with or without MSI. Loss of imprinting (LOI) of IGF2 and PEG1/MEST was observed in 42% and 35% of informative cancers, respectively. H19 expression was not detected in 24% of informative cancers. SNRPN and NDN retained monoallelic expression in all the cancers examined. These findings indicate no simultaneous disruption of the imprinted genes. LOI of IGF2 and PEG1/MEST was also observed in colorectal mucosa from almost all the patients with LOI in tumor tissue. Moreover, MSI-positive colorectal cancers exhibit LOI of IGF2 with a high frequency compared to MSI-negative cancers (P=0.013). These observations, consistent with a previous report, establish an association between LOI of IGF2 and MSI in colorectal cancers and provide insight into susceptibility of tumor development.

Mammalian imprinted genes are frequently arranged in clusters on particular chromosomes. The imprinting cluster on human chromosome 11p15 is associated with Beckwith-Wiedemann syndrome (BWS) and a variety of human cancers. To clarify the genomic organization of the imprinted cluster, an extensive screen for differentially expressed transcripts in the 11p15 region was performed using monochromosomal hybrids with a paternal or maternal human chromosome 11. Here we describe an imprinted antisense transcript identified within the KvLQT1 locus, which is associated with multiple balanced chromosomal rearrangements in BWS and an additional breakpoint in embryonal rhabdoid tumors. The transcript, called LIT1 (long QT intronic transcript 1), was expressed preferentially from the paternal allele and produced in most human tissues. Methylation analysis revealed that an intronic CpG island was specifically methylated on the silent maternal allele and that four of 13 BWS patients showed complete loss of maternal methylation at the CpG island, suggesting that antisense regulation is involved in the development of human disease. In addition, we found that eight of eight Wilms' tumors exhibited normal imprinting of LIT1 and five of five tumors displayed normal differential methylation at the intronic CpG island. This contrasts with five of six tumors showing loss of imprinting of IGF2. We conclude that the imprinted gene domain at the KvLQT1 locus is discordantly regulated in cancer from the imprinted domain at the IGF2 locus. Thus, this positional approach using human monochromosomal hybrids could contribute to the efficient identification of imprinted loci in humans.

Genomic imprinting plays a fundamental role in cancer and some hereditary diseases, including Beckwith–Wiedemann syndrome (BWS), a disorder of prenatal overgrowth and predisposition to embryonal malignancies such as Wilms tumor. We have previously shown that the K V LQT1 gene on chromosomal band 11p15 is imprinted, with expression of the maternal allele, and that the maternal allele is disrupted in rare BWS patients with balanced germ-line chromosomal rearrangements. We now show that an antisense orientation transcript within K V LQT1, termed LIT1 (long QT intronic transcript 1) is expressed normally from the paternal allele, from which K V LQT1 transcription is silent, and that in the majority of patients with BWS, LIT1 is abnormally expressed from both the paternal and maternal alleles. Eight of sixteen informative BWS patients (50%) showed biallelic expression, i.e., loss of imprinting (LOI) of LIT1. Similarly, 21 of 36 (58%) BWS patients showed loss of maternal allele-specific methylation of a CpG island upstream of LIT1. Surprisingly, LOI of LIT1 was not linked to LOI of insulin-like growth factor II (IGF2), which was found in 2 of 10 (20%) BWS patients, even though LOI of IGF2 occurs frequently in Wilms and other tumors, and in some patients with BWS. Thus, LOI of LIT1 is the most common genetic alteration in BWS. We propose that 11p15 harbors two imprinted gene domains—a more centromeric domain including K V LQT1 and p57 KIP2 , alterations in which are more common in BWS, and a more telomeric domain including IGF2, alterations in which are more common in cancer.

The Wilms' tumor suppressor gene ( WT1 ) was previously identified as being imprinted, with frequent maternal expression in human placentae and fetal brains. We examined the allele-specific expression of WT1 in cultured human fibroblasts from 15 individuals. Seven of 15 fibroblast lines were heterozygous for polymorphic alleles, and the expression patterns were variable, i.e., equal, unequal or monoallelic paternal expression in three, two and two cases, respectively. Exclusive paternal expression of WT1 was also shown in non-cultured peripheral lymphocytes from the latter two individuals. The allele-specific expression profiles of other imprinted genes, IGF2 and H19, on human chromosome 11 were constant and consistent with those in other tissues. Our unexpected observations of paternal or biallelic expression of WT1 in fibroblasts and lymphocytes, together with the previous findings of maternal or biallelic expression in placentae and brains, suggest that the allele-specific regulatory system of WT1 is unique and may be controlled by a putative tissue- and individual-specific modifier.

We have isolated human homologues of the imprinted mouse gene, U2af1-rs1. Two different types of cDNAs and three distinct genomic DNAs belonging to different groups were isolated. We have identified chromosomal genes corresponding to each cDNA by restriction mapping and sequencing. Using both a panel of rodent/human somatic cell hybrids and fluorescence in situ hybridization, group 1 and group 2 genes were mapped to chromosome 5q22 and chromosome Xp22.1, respectively. We designated group 1 and group 2 genes as human U2AF1-RS1 and U2AF1-RS2, respectively, because these genes corresponded to mouse U2af1-rs1 (chromosome 11) and U2af1-rs2 (chromosome X), which we also isolated and mapped. Amino acid sequences of human U2AF1-RS1 and U2AF-RS2 showed significant homology to U2AF small subunit. The group 3 gene, designated as U2AF1-RS3, of which the cDNA has not yet been isolated, was mapped to chromosome 19p13.2.

For identification of the chromosome carrying cellular senescence-inducing activity, normal human chromosome 2, 3, 6, 7, 9, 11, or 12 tagged with a selectable marker gene (neo) was introduced into the human cervical carcinoma cell line SiHa via microcell-mediated chromosome transfer. Seventy-six percent (158/207) of the G418-resistant clones obtained by the transfer of chromosome 2 showed a remarkable change in morphology (cells were flat), and 93% (147/158) of them ceased to divide (senesced) prior to 6-9 population doublings, whereas most of the clones generated by the transfer of other chromosomes exhibited a morphology similar to that of the parental cells and continued to grow. Chromosome analyses suggested that cells which escaped from senescence contained only a small fragment derived from the transferred chromosome 2, whereas the transferred chromosomes were apparently intact in most of the continuously growing microcell hybrids with introduction of other chromosomes. These results indicate that the normal human chromosome 2 carries a gene or genes that induce cellular senescence in SiHa cells.

We have isolated and mapped 75 new DNA markers including 52 restriction fragment length polymorphism (RFLP) markers on human chromosome 3. Clones were mapped by nonisotopic in situ hybridization, in which discrete fluorescent signals can be detected on prometaphase R-banded chromosomes. Thirty-seven markers were mapped to each arm of chromosome 3, and one was localized to the centromere. Five markers defined variable number of tandem repeat (VNTR) loci. Although the 75 clones were scattered throughout the chromosome, they were concentrated in the R-positive bands. This physical map of chromosome 3 will contribute to the characterization of the chromosomal and molecular aberrations involved in renal cell carcinoma, small-cell lung cancer, and other malignancies and in single-gene disorders such as von Hippel-Lindau disease and autosomal dominant retinitis pigmentosa.

Biological responses to environmental effects are mediated through epigenetic changes such as chemical modifications of the histone tails and DNA (5-cytosine) methylation. We report that dietary protein restriction in pregnant mice can alter histone modifications in the promoter region of the Igf2 gene and cause a two-fold repression in promoter specific Igf2 transcription in the liver of the fetus. Suppression of Igf2 is accompanied with low birth weight of the pups born to the protein-restricted dams. Our results provide new information about the epigenetic aspects of early life programming and will improve our understanding about the developmental origins of adult diseases.

Imprinted gene(s) on human chromosome 7 are thought to be involved in Russell-Silver syndrome (RSS), based on the fact that approximately 10% of patients have maternal uniparental disomy of chromosome 7. However, involvement of the known imprinted genes (GRB10 at 7p12, PEG10 at 7q21.3 and MEST at 7q32) in RSS has yet to be established. To screen for new imprinted genes, we are initially using somatic cell hybrids containing a paternal or maternal human chromosome 7. Transcripts located between D7S530 and D7S649 (a 1.5 Mb interval encompassing MEST ) were subjected to RT-PCR analysis using somatic cell hybrids. One transcript named MESTIT1 (for MEST intronic transcript 1) reproducibly showed paternal-specific expression. Upon further analysis, we found MESTIT1 to be (1) paternally (and not maternally) expressed in all fetal tissues and fibroblasts examined, (2) to be located in an intron of one of the two isoforms of MEST but transcribed in the opposite direction, (3) to be composed of at least two exons without any significant open reading frame, and (4) to exist as a 4.2 kb transcript in many fetal and adult tissues. We could also identify two isoforms of the mouse Mest gene as observed in humans, but it is still unknown if a murine ortholog of MESTIT1 exists. We also examined the imprinting status of MEST isoforms as a first step in assessing whether MESTIT1 might influence the allelic expression pattern of the sense transcript. MEST isoform 1 was determined to be exclusively expressed from the paternal allele in all fetal tissues and cell lines examined, whereas MEST isoform 2 was only preferentially expressed from the paternal allele in a tissue/cell-type-specific manner. Our results suggest that MESTIT1 is a paternally expressed non-coding RNA that may be involved in the regulation of MEST expression during development. MESTIT1 (also known as PEG1-AS) is now the third independent transcript (with MEST and COPG2IT1) identified at human chromosome 7q32 demonstrating paternal chromosome-specific expression.