Mechanisms of Igf2/H19 Imprinting: DNA Methylation, Chromatin and Long-Distance Gene Regulation

Sasaki, Hidetada; Ishihara, Kazuhíko; Kato, Reiko

doi:10.1093/oxfordjournals.jbchem.a022661

Cited by 106 publications

(83 citation statements)

References 39 publications

(30 reference statements)

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“…Changes in the methylation pattern of the IGF2͞H19 locus are associated with Beckwith-Wiedemann syndrome and Wilms tumor. The methylation pattern in this region has been described and examined by several authors and, therefore, provides an ideal test case (17)(18)(19)(20). Vu et al (19) performed detailed bisulfite-sequencing examinations in several tissues of adult and fetal specimens.…”

Section: Resultsmentioning

confidence: 99%

Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry

Ehrich

Nelson

Stanssens

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

744

647

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Methylation is one of the major epigenetic processes pivotal to our understanding of carcinogenesis. It is now widely accepted that there is a relationship between DNA methylation, chromatin structure, and human malignancies. DNA methylation is potentially an important clinical marker in cancer molecular diagnostics. Understanding epigenetic modifications in their biological context involves several aspects of DNA methylation analysis. These aspects include the de novo discovery of differentially methylated genes, the analysis of methylation patterns, and the determination of differences in the degree of methylation. Here we present a previously uncharacterized method for high-throughput DNA methylation analysis that utilizes MALDI-TOF mass spectrometry (MS) analysis of base-specifically cleaved amplification products. We use the IGF2͞H19 region to show that a single base-specific cleavage reaction is sufficient to discover methylation sites and to determine methylation ratios within a selected target region. A combination of cleavage reactions enables the complete evaluation of all relevant aspects of DNA methylation, with most CpGs represented in multiple reactions. We successfully applied this technology under high-throughput conditions to quantitatively assess methylation differences between normal and neoplastic lung cancer tissue samples from 48 patients in 47 genes and demonstrate that the quantitative methylation results allow accurate classification of samples according to their histopathology. lung cancer ͉ MALDI-TOF MS ͉ epigenetics

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Section: Resultsmentioning

confidence: 99%

Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry

Ehrich

Nelson

Stanssens

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

744

647

View full text Add to dashboard Cite

show abstract

“…Igf2 and H19 imprinting is directed by epigenetic modifications in the differentially methylated region (DMR) of the imprinting control region (ICR) located between these two adjacent genes (1,9,19,21,29,30). The binding of CTCF to the unmethylated maternal ICR creates a physical boundary, blocking the interaction of downstream enhancers with the remote Igf2 promoters and silencing the maternal allele (4,13,15).…”

mentioning

confidence: 99%

CTCF Regulates Allelic Expression of Igf2 by Orchestrating a Promoter-Polycomb Repressive Complex 2 Intrachromosomal Loop

Qiu

et al. 2008

Molecular and Cellular Biology

174

193

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CTCF is a zinc finger DNA-binding protein that regulates the epigenetic states of numerous target genes. Using allelic regulation of mouse insulin-like growth factor II (Igf2) as a model, we demonstrate that CTCF binds to the unmethylated maternal allele of the imprinting control region (ICR) in the Igf2/H19 imprinting domain and forms a long-range intrachromosomal loop to interact with the three clustered Igf2 promoters. Polycomb repressive complex 2 is recruited through the interaction of CTCF with Suz12, leading to allelespecific methylation at lysine 27 of histone H3 (H3-K27) and to suppression of the maternal Igf2 promoters. Targeted mutation or deletion of the maternal ICR abolishes this chromatin loop, decreases allelic H3-K27 methylation, and causes loss of Igf2 imprinting. RNA interference knockdown of Suz12 also leads to reactivation of the maternal Igf2 allele and biallelic Igf2 expression. CTCF and Suz12 are coprecipitated from nuclear extracts with antibodies specific for either protein, and they interact with each other in a two-hybrid system. These findings offer insight into general epigenetic mechanisms by which CTCF governs gene expression by orchestrating chromatin loop structures and by serving as a DNA-binding protein scaffold to recruit and bind polycomb repressive complexes.The transcriptional regulator CCCTC-binding factor (CTCF) is a highly conserved 11-zinc-finger nuclear protein that controls the expression of a number of genes via chromatin insulation or enhancer blocking (for reviews, see references 5, 8, 23, and 28). CTCF silences genes by binding to sites within promoters, silencers, and insulators through the use of different combinations of zinc fingers (20). More than 15,000 CTCFbinding sites have been identified throughout the genome (16).The role of CTCF as an insulator regulating the imprinting of Igf2 and H19 has been extensively studied. Igf2 and H19 imprinting is directed by epigenetic modifications in the differentially methylated region (DMR) of the imprinting control region (ICR) located between these two adjacent genes (1,9,19,21,29,30). The binding of CTCF to the unmethylated maternal ICR creates a physical boundary, blocking the interaction of downstream enhancers with the remote Igf2 promoters and silencing the maternal allele (4,13,15). When this ICR is deleted (35) or mutated (32, 34), the maternal Igf2 allele is expressed, leading to biallelic expression. In addition, CTCF has recently been shown to act as a tethering protein, serving as a molecular glue to secure long-range intrachromosomal (17) and interchromosomal (18) interactions.By chromosome configuration capture (3C) methodology, it has been shown that CTCF participates in the formation of a long-range chromosomal loop to the upstream Igf2 DMRs when it is bound to the maternal ICR (17,42,21). This model suggests that CTCF may not only function as a physical insulator but also actively participate in the regulation of the imprinted Igf2 allele. We were interested in learning how CTCF mediates the suppressi...

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“…On the paternal allele, the ICR becomes methylated, thereby preventing H19 expression. However, this methylated ICR prevents CTCF from binding and hence allows Igf2 expression from a downstream enhancer (13,20). To our knowledge, CTCF has not been reported to regulate H19 expression.…”

mentioning

confidence: 85%

“…These genes are part of a well characterized cluster that contains the paternally expressed Igf2 and maternally expressed H19. Both genes share enhancers and are asynchronously regulated at a site called the imprinting control region (ICR) 3 (13). Loss of imprinting of both Igf2 and H19 has been observed in hepatocellular carcinoma (HCC) tissues and cell lines (14,15).…”

mentioning

confidence: 99%

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Prohibitin 1 Regulates the H19-Igf2 Axis and Proliferation in Hepatocytes

Ramani

Mavila

et al. 2016

Journal of Biological Chemistry

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Edited by Xiao-Fan WangProhibitin 1 (PHB1) is a mitochondrial chaperone that regulates cell growth. Phb1 knock-out mice exhibit liver injury and hepatocellular carcinoma (HCC). Phb1 knock-out livers show induction of tumor growth-associated genes, H19 and insulinlike growth factor 2 (Igf2). These genes are controlled by the imprinting control region (ICR) containing CCCTC-binding transcription factor (CTCF)-binding sites. Because Phb1 knockout mice exhibited induction of H19 and Igf2, we hypothesized that PHB1-mediated regulation of the H19-Igf2 axis might control cell proliferation in normal hepatocytes. H19 and Igf2 were induced (8 -20-fold) in 3-week-old Phb1 knock-out livers, in Phb1 siRNA-treated AML12 hepatocytes (2-fold), and HCC cell lines when compared with control. Phb1 knockdown lowered CTCF protein in AML12 by ϳ30% when compared with control. CTCF overexpression lowered basal H19 and Igf2 expression by 30% and suppressed Phb1 knockdown-mediated induction of these genes. CTCF and PHB1 co-immunoprecipitated and colocalized on the ICR element, and Phb1 knockdown lowered CTCF ICR binding activity. The results suggest that PHB1 and CTCF cooperation may control the H19-Igf2 axis. Human HCC tissues with high levels of H19 and IGF2 exhibited a 40 -50% reduction in PHB1 and CTCF expression and their ICR binding activity. Silencing Phb1 or overexpressing H19 in the mouse HCC cell line, SAMe-D, induced cell growth. Blocking H19 induction prevented Phb1 knockdown-mediated growth, whereas H19 overexpression had the reverse effect. Interestingly H19 silencing induced PHB1 expression. Taken together, our results demonstrate that the H19-Igf2 axis is negatively regulated by CTCF-PHB1 cooperation and that H19 is involved in modulating the growthsuppressive effect of PHB1 in the liver.

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Mechanisms of Igf2/H19 Imprinting: DNA Methylation, Chromatin and Long-Distance Gene Regulation

Cited by 106 publications

References 39 publications

Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry

Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry

CTCF Regulates Allelic Expression of Igf2 by Orchestrating a Promoter-Polycomb Repressive Complex 2 Intrachromosomal Loop

Prohibitin 1 Regulates the H19-Igf2 Axis and Proliferation in Hepatocytes

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