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PubMed1998

Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2.

Differentially methylated sequences associated with imprinted genes are proposed to control genomic imprinting. A 2-kb region located 5' to the imprinted mouse H19 gene is hypermethylated on the inactive paternal allele throughout development. To determine whether this differentially methylated domain (DMD) is required for imprinted expression at the endogenous locus, we have generated mice harboring a 1.6-kb targeted deletion of the DMD and assayed for allelic expression of H19 and the linked, oppositely imprinted Igf2 gene. H19 is activated and Igf2 expression is reduced when the DMD deletion is paternally inherited; conversely, upon maternal transmission of the mutation, H19 expression is reduced and Igf2 is activated. Consistent with the DMD's hypothesized role of setting up the methylation imprint, the mutation also perturbs allele-specific methylation of the remaining H19 sequences. In conclusion, these experiments show that the H19 hypermethylated 5' flanking sequences are required to silence paternally derived H19. Additionally, these experiments demonstrate a novel role for the DMD on the maternal chromosome where it is required for the maximal expression of H19 and the silencing of Igf2. Thus, the H19 differentially methylated sequences are required for both H19 and Igf2 imprinting.

Alleles↗
PubMed2005

A genetic approach to cancer epigenetics.

In over 20 years since the discovery of altered methylation in cancer, many epigenetic alterations have been found in human cancer, including global and specific gene hypomethylation, hypermethylation, altered chromatin marks, and loss of genomic imprinting. Cancer epigenetics has been limited by questions of cause and effect, since epigenetic changes can arise secondary to the cancer process and its associated widespread changes in gene expression. Furthermore, mutations in the DNA methylation machinery have not been observed in tumors, whereas they have been for chromatin modification. To address the issue of human cancer etiology, we have taken a genetic approach to cancer epigenetics. One line of investigation has been on the disorder Beckwith-Wiedemann syndrome (BWS). We have found that loss of imprinting (LOI) of the autocrine growth factor gene IGF2 and of the untranslated antisense RNA LIT1, within the K(V)LQT1 gene, account for most cases of BWS, and that cancer risk is specifically associated with LOI of IGF2. Wilms' tumors, both in BWS and in the general population, involve LOI leading to an expansion of nephrogenic precursor cells. We have also developed an animal model for the role of LOI of IGF2 in cancer, showing that it cooperates with Apc mutations to increase cancer frequency, consistent with human data suggesting a severalfold increased cancer risk for this common epigenetic variant in the adult population. These data suggest that a major component of cancer risk involves epigenetic changes in normal cells that increase the probability of cancer after genetic mutation. They suggest a model of cancer prevention that involves the epigenetic analysis of normal cells for risk stratification and cancer prevention strategies.

Animals↗
PubMed1999

Use of green fluorescent protein to detect expression of an endopolygalacturonase gene of Colletotrichum lindemuthianum during bean infection.

The 5' noncoding region of clpg2, an endopolygalacturonase gene of the bean pathogen Colletotrichum lindemuthianum, was fused to the coding sequence of a gene encoding a green fluorescent protein (GFP), and the construct was introduced into the fungal genome. Detection of GFP accumulation by fluorescence microscopy examination revealed that clpg2 was expressed at the early stages of germination of the conidia and during appressorium formation both in vitro and on the host plant.

Colletotrichum↗
PubMed2002

Dnmt1 overexpression causes genomic hypermethylation, loss of imprinting, and embryonic lethality.

Biallelic expression of Igf2 is frequently seen in cancers because Igf2 functions as a survival factor. In many tumors the activation of Igf2 expression has been correlated with de novo methylation of the imprinted region. We have compared the intrinsic susceptibilities of the imprinted region of Igf2 and H19, other imprinted genes, bulk genomic DNA, and repetitive retroviral sequences to Dnmt1 overexpression. At low Dnmt1 methyltransferase levels repetitive retroviral elements were methylated and silenced. The nonmethylated imprinted region of Igf2 and H19 was resistant to methylation at low Dnmt1 levels but became fully methylated when Dnmt1 was overexpressed from a bacterial artificial chromosome transgene. Methylation caused the activation of the silent Igf2 allele in wild-type and Dnmt1 knockout cells, leading to biallelic Igf2 expression. In contrast, the imprinted genes Igf2r, Peg3, Snrpn, and Grf1 were completely resistant to de novo methylation, even when Dnmt1 was overexpressed. Therefore, the intrinsic difference between the imprinted region of Igf2 and H19 and of other imprinted genes to postzygotic de novo methylation may be the molecular basis for the frequently observed de novo methylation and upregulation of Igf2 in neoplastic cells and tumors. Injection of Dnmt1-overexpressing embryonic stem cells in diploid or tetraploid blastocysts resulted in lethality of the embryo, which resembled embryonic lethality caused by Dnmt1 deficiency.

Alleles↗
PubMed2002

Analysis of sequence upstream of the endogenous H19 gene reveals elements both essential and dispensable for imprinting.

Imprinting of the linked and oppositely expressed mouse H19 and Igf2 genes requires a 2-kb differentially methylated domain (DMD) that is located 2 kb upstream of H19. This element is postulated to function as a methylation-sensitive insulator. Here we test whether an additional sequence 5' of H19 is required for H19 and Igf2 imprinting. Because repetitive elements have been suggested to be important for genomic imprinting, the requirement of a G-rich repetitive element that is located immediately 3' to the DMD was first tested in two targeted deletions: a 2.9-kb deletion (Delta D MD Delta G) that removes the DMD and G-rich repeat and a 1.3-kb deletion (Delta G) removing only the latter. There are also four 21-bp GC-rich repetitive elements within the DMD that bind the insulator-associated CTCF (CCCTC-binding factor) protein and are implicated in mediating methylation-sensitive insulator activity. As three of the four repeats of the 2-kb DMD were deleted in the initial 1.6-kb Delta DMD allele, we analyzed a 3.8-kb targeted allele (Delta 3.8kb-5'H19), which deletes the entire DMD, to test the function of the fourth repeat. Comparative analysis of the 5' deletion alleles reveals that (i) the G-rich repeat element is dispensable for imprinting, (ii) the Delta DMD and Delta DMD Delta G alleles exhibit slightly more methylation upon paternal transmission, (iii) removal of the 5' CTCF site does not further perturb H19 and Igf2 imprinting, suggesting that one CTCF-binding site is insufficient to generate insulator activity in vivo, (iv) the DMD sequence is required for full activation of H19 and Igf2, and (v) deletion of the DMD disrupts H19 and Igf2 expression in a tissue-specific manner.

Alleles↗
PubMed2005

Functional characterization of a novel Ku70/80 pause site at the H19/Igf2 imprinting control region.

The imprinted expression of the H19 and Igf2 genes in the mouse is controlled by an imprinting control center (ICR) whose activity is regulated by parent-of-origin differences in methylation. The only protein that has been implicated in ICR function is the zinc-finger protein CTCF, which binds at multiple sites within the maternally inherited ICR and is required to form a chromatin boundary that inhibits Igf2 expression. To identify other proteins that play a role in imprinting, we employed electrophoresis mobility shift assays to identify two novel binding sites within the ICR. The DNA binding activity was identified as the heterodimer Ku70/80, which binds nonspecifically to free DNA ends. The sites within the ICR bind Ku70/80 in a sequence-specific manner and with higher affinity than previously reported binding sites. The binding required the presence of Mg(2+), implying that the sequence is a pause site for Ku70/80 translocation from a free end. Chromatin immunoprecipitation assays were unable to confirm that Ku70/80 binds to the ICR in vivo. In addition, mutation of these binding sites in the mouse did not result in any imprinting defects. A genome scan revealed that the binding site is found in LINE-1 retrotransposons, suggesting a possible role for Ku70/80 in transposition.

Animals↗
PubMed2006

Developmental profile of H19 differentially methylated domain (DMD) deletion alleles reveals multiple roles of the DMD in regulating allelic expression and DNA methylation at the imprinted H19/Igf2 locus.

The differentially methylated domain (DMD) of the mouse H19 gene is a methylation-sensitive insulator that blocks access of the Igf2 gene to shared enhancers on the maternal allele and inactivates H19 expression on the methylated paternal allele. By analyzing H19 DMD deletion alleles H19DeltaDMD and H19Delta3.8kb-5'H19 in pre- and postimplantation embryos, we show that the DMD exhibits positive transcriptional activity and is required for H19 expression in blastocysts and full activation of H19 during subsequent development. We also show that the DMD is required to establish Igf2 imprinting by blocking access to shared enhancers when Igf2 monoallelic expression is initiated in postimplantation embryos and that the single remaining CTCF site of the H19DeltaDMD allele is unable to provide this function. Furthermore, our data demonstrate that sequence outside of the DMD can attract some paternal-allele-specific CpG methylation 5' of H19 in preimplantation embryos, although this methylation is not maintained during postimplantation in the absence of the DMD. Finally, we report a conditional allele floxing the 1.6-kb sequence deleted from the H19DeltaDMD allele and demonstrate that the DMD is required to maintain repression of the maternal Igf2 allele and the full activity of the paternal Igf2 allele in neonatal liver.

Alleles↗
PubMed2001

Imprinting and deviation from Mendelian transmission ratios.

Deviations from a Mendelian 1:1 transmission ratio have been observed in human and mouse chromosomes. With few exceptions, the underlying mechanism of the transmission-ratio distortion remains obscure. We tested a hypothesis that grandparental-origin dependent transmission-ratio distortion is related to imprinting and possibly results from the loss of embryos which carry imprinted genes with imprinting marks that have been incorrectly reset. We analyzed transmission of alleles in four regions of the human genome that carry imprinted genes presumably critical for normal embryonic growth and development: 11p15.5 (H19, IGF2, HASH2, etc.), 11p13 (WT1), 7p11-12 (GRB10), and 6q25-q27 (IGF2R), among the offspring of 31 three-generation Centre d'Etude de polymorphism Humain (CEPH) families. Deviations from expected 1:1 ratios were found in the maternal chromosomes for regions 11p15.5, 11p13, and 6q25-27 and in the paternal chromosomes for regions 11p15 and 7p11-p12. The likelihood of the results was assessed empirically to be statistically significant (p = 0.0008), suggesting that the transmission ratios in the imprinted regions significantly deviated from 1:1. We did not find deviations from a 1:1 transmission ratio in imprinted regions that are not crucial for embryo viability (13q14 and 15q11-q13). The analysis of a larger set of 51 families for the 11p15.5 region suggests that there is heterogeneity among the families with regard to the transmission of 11p15.5 alleles. The results of this study are consistent with the hypothesis that grandparental-origin dependent transmission-ratio distortion is related to imprinting and embryo loss.

Alleles↗
PubMed1997

Modulation of Igf2 genomic imprinting in mice induced by 5-azacytidine, an inhibitor of DNA methylation.

The adjacent genes, insulin-like growth factor 2 (Igf2) and H19, are imprinted in both mouse and human. While Igf2 is expressed from the paternal allele, H19 is transcribed exclusively from the maternal allele. To explore the underlying mechanism of Igf2 and H19 imprinting, we studied the effect of DNA demethylation on allelic expression by injecting mice with the demethylating agent 5-azacytidine (5-aza-C). We observed a > or = 2-fold increase in the abundance of Igf2 mRNA in liver from treated mice compared with that of control mice. There was no significant change in Igf2 or H19 expression in brain. In the 5-aza-C-treated mice, there was dramatic modulation of Igf2 imprinting. In some tissues, Igf2 was expressed biallelically, while in other tissues, the paternal allele was silenced and the normally imprinted maternal allele was expressed, an example of allelic switching. There was no change in the normal biallelic pattern of Igf2 expression in brain. H19, on the other hand, remained imprinted in all tissues in mice treated with 5-aza-C. These results provide the first example of a pharmacological manipulation of genomic imprinting of an endogenous gene in vivo and further implicate DNA methylation as an important factor in maintaining the differential allelic expression of the Igf2 gene.

Animals↗
PubMed1986

Restriction fragment-length polymorphisms of class II gene sequences in mice expressing minor structural variants of I-Ak and I-Ap.

Serologic and structural analyses of the I-A molecules expressed among a large collection of wild mouse-derived H-2 haplotypes has led to the definition of "families" of I-A alleles which encode antigenically similar molecules that are identical in more than 90% of their tryptic peptides. Two of these families, denoted the I-Ak and I-Ap families, consist of 10 I-A alleles which encode I-A molecules whose structures are closely related to either I-Ap or I-Ak. The evolutionary relationships of the I-A alleles in these families were assessed by a molecular analysis of their genomic structures. The A alpha and A beta alleles within these I-A families were compared by analysis of restriction fragment-length polymorphisms (RFLP) detected at high stringency by Southern blot hybridization with DNA probes specific for either A alpha or A beta. The polymorphic restriction enzyme sites detected in this survey were distributed over more than 7 kb of genomic DNA surrounding each gene. Because both A alpha and A beta are encoded by about 700 bp of exon DNA, the majority of the restriction enzyme sites assayed by this RFLP analysis reflect polymorphisms in noncoding regions. The DNA sequence homologies of these alleles were estimated from the RFLP results with seven restriction endonucleases by calculating the fraction homologous value as defined previously. The results indicate that evolutionarily dissimilar I-A alleles can encode I-A molecules with very similar structures. The five I-A alleles in the I-Ak family could be divided into two discrete groups, denoted K1 and K2, on the basis of their restriction fragment (RF) genotypes. The RF genotypes of alleles within each group shared more than 80% of the restriction fragments for both A and A beta. In contrast, the RF genotypes of alleles in group K1 differed extensively from those in group K2, indicating that alleles in these separate groups may not be evolutionarily closely related. These observations suggest that gene conversion or intragenic recombinational events may have been involved in the evolution of groups K1 and K2 in the I-Ak family. The RF genotypes of alleles in the I-Ap family demonstrated a close evolutionary relationship among all but two of the alleles. These two alleles encoded I-A molecules whose structures were the least related to I-Ap of any of the alleles in the I-Ap family.(ABSTRACT TRUNCATED AT 400 WORDS)

Alleles↗
PubMed1996

Heritability of X chromosome--inactivation phenotype in a large family.

One of the two X chromosomes in each somatic cell of normal human females becomes inactivated very early in embryonic development. Although the inactivation of an X chromosome in any particular somatic cell of the embryonic lineage is thought to be a stochastic and epigenetic event, a strong genetic influence on this process has been described in the mouse. We have attempted to uncover evidence for genetic control of X-chromosome inactivation in the human by examining X chromosome-inactivation patterns in 255 females from 36 three-generation pedigrees, to determine whether this quantitative character exhibits evidence of heritability. We have found one family in which all seven daughters of one male and the mother of this male have highly skewed patterns of X-chromosome inactivation, suggesting strongly that this quantitative character is controlled by one or more X-linked genes in some families.

Alleles↗
PubMed1997

Sequence of the 3' end of the simian hemorrhagic fever virus genome.

SHFV is a member of a new virus family which includes the genus arterivirus. We have cloned and sequenced 6,314 nt from the 3' end of the SHFV genome. This sequence encompasses nine complete ORFs which is three additional ORFs as compared to the other arteriviruses. We have numbered these ORFs 2a, 2b, 3, 4, 5, 6, 7, 8 and 9. At the 5' end of this sequence is a partial ORF (ORF 1b) of 1590 nt and at the 3' end is a poly(A) tract preceded by a 76 nt noncoding region. The coding capacity for each of the SHFV ORFs as well as the potential mass, pI and number of N-linked glycosylation sites for each of the encoded peptides was determined.

Amino Acid Sequence↗
PubMed1996

A new P element subfamily from Drosophila tristis, D. ambigua, and D. obscura.

A new P element subfamily, designated T-type, was found in the genomes of the three closely related species Drosophila ambigua, Drosophila obscura, and Drosophila tristis. The subfamily comprises both full-sized and internally deleted P elements. The T-type element of D. ambigua is longer than the canonical P elements owing to a 300-bp insertion in the 3' noncoding region. Tandemly arranged T-type elements were detected in D. ambigua and D. tristis. The overall structure of T-type elements resembles that of the Drosophila melanogaster P element and the termini are formed by perfect inverted repeats of 33 bp. However, none of the elements studied so far have intact reading frames. Sequence comparisons with other P element subfamilies from the obscura group indicate that the T-type elements are most closely related to the terminally truncated P homologues of Drosophila guanche and Drosophila subobscura. Therefore they can be considered as the lineage-specific P transposons of the obscura group. Furthermore, this finding indicates that the clustered P homologues of D. guanche and D. subobscura must be derived from transpositionally active P elements rather than from an immobile genomic sequence.

Animals↗
PubMed2003

Rates of DNA duplication and mitochondrial DNA insertion in the human genome.

The hundreds of mitochondrial pseudogenes in the human nuclear genome sequence (numts) constitute an excellent system for studying and dating DNA duplications and insertions. These pseudogenes are associated with many complete mitochondrial genome sequences and through those with a good fossil record. By comparing individual numts with primate and other mammalian mitochondrial genome sequences, we estimate that these numts arose continuously over the last 58 million years. Our pairwise comparisons between numts suggest that most human numts arose from different mitochondrial insertion events and not by DNA duplication within the nuclear genome. The nuclear genome appears to accumulate mtDNA insertions at a rate high enough to predict within-population polymorphism for the presence/absence of many recent mtDNA insertions. Pairwise analysis of numts and their flanking DNA produces an estimate for the DNA duplication rate in humans of 2.2 x 10(-9) per numt per year. Thus, a nucleotide site is about as likely to be involved in a duplication event as it is to change by point substitution. This estimate of the rate of DNA duplication of noncoding DNA is based on sequences that are not in duplication hotspots, and is close to the rate reported for functional genes in other species.

Animals↗
PubMed2025

Identification and external validation of a prognostic signature based on myeloid-derived suppressor cells-related LncRNAs to evaluate survival prognosis and treatment efficacy in invasive breast carcinoma.

BACKGROUND: Originating in the hematopoietic tissue, myeloid-derived suppressor cells (MDSCs) significantly contribute to tumor-related immunological processes. However, their relationship with long noncoding RNAs (lncRNAs) and breast cancer remains incompletely understood. In this study, we introduced MDSCs-associated lncRNAs as novel prognostic biomarkers to assess outcomes in patients with invasive breast carcinoma (BRCA). METHODS: Information regarding BRCA cases, including clinical and genomic details, was obtained from the TCGA repository. Predictive indicators were discovered, and their reliability underwent thorough verification. A clinically useful nomogram was developed following application-based validation. Additional investigations encompassed functional analysis, TMB assessment, TME profiling, immunotherapy efficacy forecasting, and drug sensitivity testing along with target identification. Long non-coding RNA expression was measured using reverse transcription quantitative PCR. RESULTS: A risk stratification model incorporating eight MDSCs-related lncRNAs effectively predicted patient outcomes. Kaplan-Meier (K-M) survival analysis clearly indicated a much worse prognosis among patients classified as high-risk (p&#xa0;<&#xa0;0.001). The nomogram accurately forecasted overall survival (OS). Analysis of functional enrichment revealed that pathways associated with epithelial cells showed activity among patients at higher risk. Characterization of the tumor microenvironment showed increased immune cell presence in those classified as low-risk. Conversely, individuals with greater risk displayed higher tumor mutational burden. TIDE and IPS analyses indicated superior immunotherapy responsiveness in the low-risk BRCA subgroup. Among 47 drugs with notable IC50 variations, Ribociclib, PD173074, KU-55933, NU7441, and nutlin-3a exhibited lower IC50 values within the low-risk group, whereas Lapatinib demonstrated greater efficacy among the high-risk group. Moreover, 10 potential therapeutic agents and their targets were predicted for high-risk patients. RT-qPCR validation confirmed the robustness of the model. CONCLUSIONS: We successfully verified a new model of molecular markers of MDSCs-related lncRNAs, offering critical insights for predicting outcomes and guiding therapeutic decisions in BRCA cases.

Bioinformatics↗
PubMed2026

Lipid Metabolism-related lncRNA Model Identifies AC026412.3 as a Driver of Fatty Acid &#x3b2;-oxidation in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Dysregulated lipid metabolism contributes to hepatocellular carcinoma (HCC) progression, but the prognostic value and mechanistic roles of lipid metabolism-related long noncoding RNAs (LRLs) remain insufficiently characterized. This study aimed to construct and validate an LRL-based prognostic model and to investigate the biological function and metabolic mechanism of AC026412.3 in HCC. METHODS: Transcriptomic and clinical data from the The Cancer Genome Atlas Liver Hepatocellular Carcinoma cohort were analyzed to identify LRLs based on their correlation with curated lipid metabolism genes. Differential expression, univariate Cox, least absolute shrinkage and selection operator (LASSO), and multivariate Cox analyses were performed to construct a prognostic signature, which was evaluated using Kaplan-Meier survival and time-dependent receiver operating characteristic (ROC) analyses. Functional enrichment analyses Gene Ontology [GO], Kyoto Encyclopedia of Genes and Genomes [KEGG] and gene set enrichment analysis [GSEA], mutation profiling, tumor mutational burden, immune infiltration estimation, and consensus clustering were applied to characterize associated features. A key LRL was identified through integrated bioinformatic screening and prioritization. Its biological role was assessed by quantitative reverse transcription polymerase chain reactionq (RT-PCR), western blotting, BODIPY staining, colony formation, Transwell assays, and xenograft models. RNA sequencing followed by pathway enrichment analysis was conducted to explore underlying mechanisms. RESULTS: A three-LRL signature (AL031985.3, NRAV, and AC026412.3) stratified HCC patients into distinct risk groups with significantly different survival outcomes and demonstrated independent prognostic value. AC026412.3 was markedly upregulated in HCC and associated with poor prognosis. Functional assays demonstrated that AC026412.3 promoted proliferation, invasion, and tumor growth while reducing lipid accumulation. Mechanistically, AC026412.3 upregulated solute carrier family 22 member 5 (SLC22A5), enhanced fatty acid &#x3b2;-oxidation, and increased adenosine triphosphate (ATP) production, thereby driving metabolic reprogramming. CONCLUSIONS: This study establishes a robust LRL-based prognostic model and identifies AC026412.3 as a key regulator of lipid metabolic reprogramming via the SLC22A5-fatty acid &#x3b2;-oxidation axis, highlighting its potential as a biomarker and therapeutic target in HCC.

HCC↗