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Nuclear reprogramming of somatic cells into a pluripotent status has the potential to create patient-specific pluripotent stem cells (iPSCs) for regenerative medicine. Currently, however, the epigenetic mechanisms underlying this pluripotent reprogramming are poorly understood. To delineate this epigenetic regulatory network, we utilized a chromatin RNA in situ reverse transcription sequencing (CRIST-seq) approach to identify long noncoding RNAs (lncRNAs) embedded in the 3-dimensional intrachromosomal architecture of stem cell core factor genes. By combining CRIST-seq and RNA-seq, we identified Osilr9 (Oct4-Sox2 interacting lncRNA 9) as a pluripotency-associated lncRNA. Osilr9 expression was associated with the status of stem cell pluripotency in reprogramming. Using shRNA knockdown, we showed that this lncRNA was required for the optimal maintenance of stem cell pluripotency. Overexpression of Osilr9 induced robust activation of endogenous stem cell core factor genes in fibroblasts. Osilr9 participated in the formation of the intrachromosomal looping required for maintenance of pluripotency. After binding to the Oct4 promoter, Osilr9 recruited the DNA demethylase TET1, leading to promoter demethylation. These data demonstrate that Osilr9 is a critical chromatin epigenetic modulator that coordinates the promoter activity of core stem cell factor genes, highlighting the critical role of pluripotency-associated lncRNAs in stem cell pluripotency and reprogramming.

Background A specific 3-dimensional intrachromosomal architecture of core stem cell factor genes is required to reprogram a somatic cell into pluripotency. As little is known about the epigenetic readers that orchestrate this architectural remodeling, we used a novel chromatin RNA in situ reverse transcription sequencing (CRIST-seq) approach to profile long noncoding RNAs (lncRNAs) in the Oct4 promoter. Results We identify Platr10 as an Oct4 - Sox2 binding lncRNA that is activated in somatic cell reprogramming. Platr10 is essential for the maintenance of pluripotency, and lack of this lncRNA causes stem cells to exit from pluripotency. In fibroblasts, ectopically expressed Platr10 functions in trans to activate core stem cell factor genes and enhance pluripotent reprogramming. Using RNA reverse transcription-associated trap sequencing (RAT-seq), we show that Platr10 interacts with multiple pluripotency-associated genes, including Oct4, Sox2, Klf4, and c-Myc, which have been extensively used to reprogram somatic cells. Mechanistically, we demonstrate that Platr10 helps orchestrate intrachromosomal promoter-enhancer looping and recruits TET1, the enzyme that actively induces DNA demethylation for the initiation of pluripotency. We further show that Platr10 contains an Oct4 binding element that interacts with the Oct4 promoter and a TET1-binding element that recruits TET1. Mutation of either of these two elements abolishes Platr10 activity. Conclusion These data suggest that Platr10 functions as a novel chromatin RNA molecule to control pluripotency in trans by modulating chromatin architecture and regulating DNA methylation in the core stem cell factor network.

Formation of a pluripotency-specific chromatin network is a critical event in reprogramming somatic cells into pluripotent status. To characterize the regulatory components in this process, we used ‘chromatin RNA in situ reverse transcription sequencing’ (CRIST-seq) to profile RNA components that interact with the pluripotency master gene Oct4. Using this approach, we identified a novel nuclear lncRNA Oplr16 that was closely involved in the initiation of reprogramming. Oplr16 not only interacted with the Oct4 promoter and regulated its activity, but it was also specifically activated during reprogramming to pluripotency. Active expression of Oplr16 was required for optimal maintenance of pluripotency in embryonic stem cells. Oplr16 was also able to enhance reprogramming of fibroblasts into pluripotent cells. RNA reverse transcription-associated trap sequencing (RAT-seq) indicated that Oplr16 interacted with multiple target genes related to stem cell self-renewal. Of note, Oplr16 utilized its 3′-fragment to recruit the chromatin factor SMC1 to orchestrate pluripotency-specific intrachromosomal looping. After binding to the Oct4 promoter, Oplr16 recruited TET2 to induce DNA demethylation and activate Oct4 in fibroblasts, leading to enhanced reprogramming. These data suggest that Oplr16 may act as a pivotal chromatin factor to control stem cell fate by modulating chromatin architecture and DNA demethylation.

Long noncoding RNAs (lncRNAs) can regulate the activity of target genes by participating in the organization of chromatin architecture. We have devised a “chromatin-RNA in situ reverse transcription sequencing” (CRIST-seq) approach to profile the lncRNA interaction network in gene regulatory elements by combining the simplicity of RNA biotin labeling with the specificity of the CRISPR/Cas9 system. Using gene-specific gRNAs, we describe a pluripotency-specific lncRNA interacting network in the promoters of Sox2 and Pou5f1, two critical stem cell factors that are required for the maintenance of pluripotency. The promoter-interacting lncRNAs were specifically activated during reprogramming into pluripotency. Knockdown of these lncRNAs caused the stem cells to exit from pluripotency. In contrast, overexpression of the pluripotency-associated lncRNA activated the promoters of core stem cell factor genes and enhanced fibroblast reprogramming into pluripotency. These CRIST-seq data suggest that the Sox2 and Pou5f1 promoters are organized within a unique lncRNA interaction network that determines the fate of pluripotency during reprogramming. This CRIST approach may be broadly used to map lncRNA interaction networks at target loci across the genome.

Chromatin looping is key to gene regulation, yet no broadly applicable methods to selectively modify chromatin loops have been described. We have engineered a method for chromatin loop reorganization using CRISPR-dCas9 (CLOuD9) to selectively and reversibly establish chromatin loops. We demonstrate the power of this technology to selectively modulate gene expression at targeted loci., Chromatin looping plays an important role in gene regulation and the ability to manipulate loops would aid in understanding how this occurs. Here the authors present CLOuD9, a system that uses dimerized Cas9 complexes to selectively and reversibly establish chromatin loops.

Pluripotent stem cells have broad applications in regenerative medicine and offer ideal models for understanding the biological process of embryonic development and specific diseases. Studies suggest that the self-renewal and multi-lineage differentiation of stem cells are regulated by a complex network consisting of transcription factors, chromatin regulators, signaling factors, and non-coding RNAs. It is of great interest to identify RNA regulatory factors that determine the fate of stem cells. Long non-coding RNA (lncRNA), a class of non-coding RNAs with more than 200 bp in length, has been shown to act as essential epigenetic regulators of stem cell pluripotency and specific lineage commitment. In this review, we focus on recent research progress related to the function and epigenetic mechanisms of lncRNA in determining the fate of stem cells, particularly pluripotency maintenance and lineage-specific differentiation. We discuss the role of the Oct4 and Sox2 promoter-interacting lncRNA as identified by Chromatin RNA In Situ reverse Transcription sequencing (CRIST-seq). Further understanding of their potential actions will provide a basis for the development of regenerative medicine for clinical application. This work offers comprehensive details and better understanding of the role of lncRNA in determining the fate of stem cells and paves the way for clinical stem cell applications.

// Xin Zhao 1 , Xiaoliang Liu 1 , Guanjun Wang 1 , Xue Wen 1 , Xiaoying Zhang 1 , Andrew R. Hoffman 2, * , Wei Li 1, * , Ji-Fan Hu 1, 2, * , Jiuwei Cui 1, * 1 Cancer Center, The First Hospital of Jilin University, Changchun, Jilin 130021, China 2 Stanford University Medical School, Palo Alto Veterans Institute for Research, Palo Alto, CA 94304, USA * These authors are senior authors of this work Correspondence to: Jiuwei Cui, email: cuijiuwei@vip.qq.com Wei Li, email: jdyylw@163.com Ji-Fan Hu, email: jifan@stanford.edu Keywords: cancer stem cells, epigenetics, genomic imprinting, IGF2, intrachromosomal looping Received: September 28, 2015 Accepted: May 13, 2016 Published: June 02, 2016 ABSTRACT Insulin-like growth factor II ( IGF2 ) is maternally imprinted in most tissues, but the epigenetic regulation of the gene in cancer stem cells (CSCs) has not been defined. To study the epigenetic mechanisms underlying self-renewal, we isolated CSCs and non-CSCs from colon cancer (HT29, HRT18, HCT116), hepatoma (Hep3B), breast cancer (MCF7) and prostate cancer (ASPC) cell lines. In HT29 and HRT18 cells that show loss of IGF2 imprinting (LOI), IGF2 was biallelically expressed in the isolated CSCs. Surprisingly, we also found loss of IGF2 imprinting in CSCs derived from cell lines HCT116 and ASPC that overall demonstrate maintenance of IGF2 imprinting. Using chromatin conformation capture (3C), we found that intrachromosomal looping between the IGF2 promoters and the imprinting control region (ICR) was abrogated in CSCs, in parallel with loss of IGF2 imprinting in these CSCs. Loss of imprinting led to increased IGF2 expression in CSCs, which have a higher rate of colony formation and greater resistance to chemotherapy and radiotherapy in vitro . These studies demonstrate that IGF2 LOI is a common feature in CSCs, even when the stem cells are derived from a cell line in which the general population of cells maintain IGF2 imprinting. This finding suggests that aberrant IGF2 imprinting may be an intrinsic epigenetic control mechanism that enhances stemness, self-renewal and chemo/radiotherapy resistance in cancer stem cells.

Genetic variants from a considerable number of susceptibility loci have been identified in association with cancer risk, but their interaction with epidemiologic factors in lung cancer remains to be defined. We sought to establish a forecasting model for identifying individuals with high-risk of lung cancer by combing gene single-nucleotide polymorphisms with epidemiologic factors. Genotyping and clinical data from 500 lung cancer cases and 500 controls were used for developing the logistic regression model. We found that lung cancer was associated with telomerase reverse transcriptase (TERT) rs2736100 single-nucleotide polymorphism. The TERT rs2736100 model was still significantly associated with lung cancer risk when combined with environmental and lifestyle factors, including lower education, lower BMI, COPD history, heavy cigarettes smoking, heavy cooking emission, and dietary factors (over-consumption of meat and deficiency in fish/shrimp, vegetables, dairy products, and soybean products). These data suggest that combining TERT SNP and epidemiologic factors may be a useful approach to discriminate high and low-risk individuals for lung cancer.

The aim of the present study was to explore the underlying mechanism of microRNA‑338‑3p (miR‑338‑3p) in lung cancer cell (A549) invasion and proliferation. A microarray assay showed that miR‑338‑3p upregulated 216 and downregulated 147 genes in A549 cells, and the differentially expressed genes were enriched for several signaling pathways, including AKT and β‑catenin signaling pathways. Western blotting results showed that phosphorylated (p)‑AKT at Ser473 and at Thr308 and p‑β‑catenin at Ser552 were downregulated. Inhibiting AKT signaling pathway decreased β‑catenin signaling pathway. Overexpression of β‑catenin promoted the invasion of A549 cells. In addition, miR‑338‑3p showed inhibitory effect on the primary tumor developed from KrasG12D mice. Together, the data of the present study support that miR‑338‑3p inhibits lung cancer cell invasion and proliferation by downregulating AKT/β‑catenin signaling pathway. The present findings supported the potential use of miR‑338‑3p in the treatment of lung cancer.

In animal cells, mitochondria are the primary powerhouses and metabolic factories. They also contain genomes and can produce mitochondrial-specific nucleic acids and proteins. To maintain homeostasis of the entire cell, an intense cross-talk between mitochondria and the nucleus, mediated by encoded noncoding RNAs (ncRNAs), as well as proteins, is required. Long ncRNAs (lncRNAs) contain characteristic structures, and they are involved in the regulation of almost every stage of gene expression, as well as being implicated in a variety of disease states, such as cancer. In the coordinated signaling system, several lncRNAs, transcribed in the nucleus but residing in mitochondria, play a key role in regulating mitochondrial functions or dynamics. For example, RMRP, a component of the mitochondrial RNase MRP, is important for mitochondrial DNA replication and RNA processing, and the steroid receptor RNA activator, SRA, is a key modulator of hormone signaling and is present in both the nucleus and mitochondria. Some RNA-binding proteins maybe play a role in the lncRNAs transport system, such as HuR, GRSF1, SHARP, SLIRP, PPR, and PNPASE. Furthermore, a series of nuclear DNA-encoded lncRNAs were implicated in mitochondria-mediated apoptosis, mitochondrial bioenergetics and biosynthesis, and glutamine metabolism. The mitochondrial genome can also encode a set of lncRNAs, and they are divided into three categories: (1) lncND5, lncND6, and lncCyt b RNA; (2) chimeric mitochondrial DNA-encoded lncRNAs; and (3) putative mitochondrial DNA-encoded lncRNAs. It has been reported that the mitochondrial DNA-encoded lncRNAs appear to operate in the nucleus. The molecular mechanisms underlying trafficking of the mitochondrial DNA-encoded lncRNAs to the nucleus in mammals are only now beginning to emerge. In conclusion, both nuclear- and mitochondrial DNA-encoded lncRNAs mediate an intense intercompartmental cross-talk, which opens a rich field for investigation of the mechanism underlying the intercompartmental coordination and the maintenance of whole cell homeostasis.

RUNX1, a master regulator of hematopoiesis, is the most commonly perturbed target of chromosomal abnormalities in hematopoietic malignancies. The t(8;21) translocation is found in 30-40% of cases of acute myeloid leukemia (AML). Recent whole-exome sequencing also reveals mutations and deletions of RUNX1 in some solid tumors. We describe a RUNX1-intragenic long noncoding RNA RUNXOR that is transcribed as unspliced transcript from an upstream overlapping promoter. RUNXOR was upregulated in AML samples and in response to Ara-C treatment in vitro. RUNXOR utilizes its 3'-terminal fragment to directly interact with the RUNX1 promoter and enhancers and participates in the orchestration of an intrachromosomal loop. The 3' region of RUNXOR also participates in long-range interchromosomal interactions with chromatin regions that are involved in multiple RUNX1 translocations. These data suggest that RUNXOR noncoding RNA may function as a previously unidentified candidate component that is involved in chromosomal translocation in hematopoietic malignancies.

A long noncoding RNA directly builds an intrachromosomal interaction complex to establish allele-specific transcriptional gene silencing over a large chromosomal domain., Kcnq1ot1 is a long noncoding ribonucleic acid (RNA; lncRNA) that participates in the regulation of genes within the Kcnq1 imprinting domain. Using a novel RNA-guided chromatin conformation capture method, we demonstrate that the 5′ region of Kcnq1ot1 RNA orchestrates a long-range intrachromosomal loop between KvDMR1 and the Kcnq1 promoter that is required for maintenance of imprinting. PRC2 (polycomb repressive complex 2), which participates in the allelic repression of Kcnq1, is also recruited by Kcnq1ot1 RNA via EZH2. Targeted suppression of Kcnq1ot1 lncRNA prevents the creation of this long-range intrachromosomal loop and causes loss of Kcnq1 imprinting. These observations delineate a novel mechanism by which an lncRNA directly builds an intrachromosomal interaction complex to establish allele-specific transcriptional gene silencing over a large chromosomal domain.

Targeting tumor-specific gene abnormalities has become an attractive approach in developing therapeutics to treat cancer. Overexpression of Bcl2 and mutations of p53 represent two of the most common molecular defects in tumors. In the nucleus, p53 induces cell cycle arrest, while it interacts with Bcl2 outside of the nucleus to regulate signal pathways involved in apoptosis. To potentiate antitumor activity, we tested a "double target" approach to antitumor therapy by combining H101, a recombinant oncolytic adenovirus that targets the inactive p53 in tumors, with a small interfering RNA (siBCL2) that targets Bcl2. In cell culture, the combined treatment significantly enhanced apoptosis and cytotoxicity as compared with treatment with either H101 or siBCL2 alone. In animals carrying tumor xenographs, combined H101 and siBCL2 treatment significantly inhibited tumor growth and prolonged survival. At the end of the study, all animals in the combined therapy group survived and two of the five animals showed complete eradication of their tumors. Interestingly, siBCL2 treatment increased H101 viral replication in both treated cells and tumor tissues. Simultaneously targeting two tumor-specific gene abnormalities using an oncolytic adenovirus and siRNA potentiates total antitumor activity.

TRAG-3, originally identified as a taxol resistance-associated gene from an ovarian carcinoma cell line, is upregulated in many human tumors. Like many tumor antigens, TRAG-3 mRNA is not detectable or is expressed at very low levels in normal fetal and adult human tissues except for testis, where TRAG-3 mRNA transcripts are detected abundantly. TRAG-3 mRNA is frequently overexpressed in tumors but is rarely detected in adjacent normal tissues. To delineate the transcriptional regulation of this tumor antigen, we cloned and sequenced the TRAG-3 promoter. A 539-base pair fragment upstream of the initiation site, which contains two unusual CT repeat stretches, was sufficient to drive the maximum activity of a luciferase reporter gene. Sodium bisulfite sequencing of genomic DNA revealed that the amount of DNA methylation in exon 2 and in the promoter regions is inversely correlated with gene expression. In normal tissues, TRAG-3 is hypermethylated and is thus transcriptionally silenced. In those tumors where TRAG-3 is actively transcribed, the TRAG-3 promoter and exon 2 are hypomethylated. Treatment of a TRAG-3-silenced cell line H23 with the demethylating reagent 5-aza-cytosine reduced DNA methylation and induced TRAG-3 expression in a dose-dependent manner. These results indicate that DNA demethylation is an important epigenetic mechanism that regulates the TRAG-3 tumor antigen in human tumors.

The mouse insulin-like growth factor II (Igf2) and H19 genes are located adjacent to each other on chromosome 7q11-13 and are reciprocally imprinted. It is believed that the allelic expression of these two genes is regulated by the binding of CTCF insulators to four parent-specific DNA methylation sites in an imprinting control center (ICR) located between these two genes. Although monoallelically expressed in peripheral tissues, Igf2 is biallelically transcribed in the CNS. In this study, we examined the allelic DNA methylation and CTCF binding in the Igf2/H19 imprinting center in CNS, hypothesizing that the aberrant CTCF binding as one of the mechanisms leads to biallelic expression of Igf2 in CNS. Using hybrid F1 mice (M. spretus males x C57BL/6 females), we showed that in CNS, CTCF binding sites in the ICR were methylated exclusively on the paternal allele, and CTCF bound only to the unmethylated maternal allele, showing no differences from the imprinted peripheral tissues. Among three other epigenetic modifications examined, histone H3 lysine 9 methylation correlated well with Igf2 allelic expression in CNS. These results suggest that CTCF binding to the ICR alone is not sufficient to insulate the Igf2 maternal promoter and to regulate the allelic expression of the gene in the CNS, thus challenging the aberrant CTCF binding as a common mechanism for lack of Igf2 imprinting in CNS. Further studies should be focused on the identification of factors that are involved in histone methylation and CTCF-associated factors that may be needed to coordinate Igf2 imprinting.

The adjacent insulin-like growth factor 2 (IGF2) and H19 genes are imprinted in most normal human tissues, but imprinting is often lost in tumors. The mechanisms involved in maintenance of imprinting (MOI) and loss of imprinting (LOI) are unresolved. We show here that osteosarcoma (OS) tumors with IGF2/H19 MOI exhibit allele-specific differential methylation of a CTCF-binding site upstream of H19. LOI of IGF2 or H19 in OS occurs in a mutually exclusive manner, and occurs with monoallelic expression of the other gene. Bisulfite sequencing reveals IGF2 LOI occurs with biallelic CpG methylation of the CTCF-binding site, while H19 LOI occurs with biallelic hypomethylation of this site. Our data demonstrate that IGF2 LOI and H19 LOI are accompanied by reciprocal methylation changes at a critical CTCF-binding site. We propose a model in which incomplete gain or loss of methylation at this CTCF-binding site during tumorigenesis explains the complex and often conflicting expression patterns of IGF2 and H19 in tumors.

Dysregulation of the insulin-like growth factor type I receptor (IGF1R) has been implicated in the progression and therapeutic resistance of malignancies. In acute myeloid leukemia (AML) cells, IGF1R is one of the most abundantly phosphorylated receptor tyrosine kinases, promoting cell growth through the PI3K/Akt signaling pathway. However, little is known regarding the molecular mechanisms underlying IGF1R gene dysregulation in cancer. We discovered a novel intragenic long noncoding RNA (lncRNA) within the IGF1R locus, named IRAIN, which is transcribed in an antisense direction from an intronic promoter. The IRAIN lncRNA was expressed exclusively from the paternal allele, with the maternal counterpart being silenced. Using both reverse transcription-associated trap and chromatin conformation capture assays, we demonstrate that this lncRNA interacts with chromatin DNA and is involved in the formation of an intrachromosomal enhancer/promoter loop. Knockdown of IRAIN lncRNA with shRNA abolishes this intrachromosomal interaction. In addition, IRAIN was downregulated both in leukemia cell lines and in blood obtained from high-risk AML patients. These data identify IRAIN as a new imprinted lncRNA that is involved in long-range DNA interactions.

The insulin-like growth factor type I receptor (IGF1R) is frequently dysregulated in breast cancers, yet the molecular mechanisms are unknown. A novel intragenic long non-coding RNA (lncRNA) IRAIN within the IGF1R locus has been recently identified in haematopoietic malignancies using RNA-guided chromatin conformation capture (R3C). In breast cancer tissues, we found that IRAIN lncRNA was transcribed from an intronic promoter in an antisense direction as compared to the IGF1R coding mRNA. Unlike the IGF1R coding RNA, this non-coding RNA was imprinted, with monoallelic expression from the paternal allele. In breast cancer tissues that were informative for single nucleotide polymorphism (SNP) rs8034564, there was an imbalanced expression of the two parental alleles, where the 'G' genotype was favorably imprinted over the 'A' genotype. In breast cancer patients, IRAIN was aberrantly imprinted in both tumours and peripheral blood leucocytes, exhibiting a pattern of allele-switch: the allele expressed in normal tissues was inactivated and the normally imprinted allele was expressed. Epigenetic analysis revealed that there was extensive DNA demethylation of CpG islands in the gene promoter. These data identify IRAIN lncRNA as a novel imprinted gene that is aberrantly regulated in breast cancer.

Background Targeted gene silencing is an important approach in both drug development and basic research. However, the selection of a potent suppressor has become a significant hurdle to implementing maximal gene inhibition for this approach. We attempted to construct a ‘super suppressor’ by combining the activities of two suppressors that function through distinct epigenetic mechanisms. Results Gene targeting vectors were constructed by fusing a GAL4 DNA-binding domain with a epigenetic suppressor, including CpG DNA methylase Sss1, histone H3 lysine 27 methylase vSET domain, and Kruppel-associated suppression box (KRAB). We found that both Sss1 and KRAB suppressors significantly inhibited the expression of luciferase and copGFP reporter genes. However, the histone H3 lysine 27 methylase vSET did not show significant suppression in this system. Constructs containing both Sss1 and KRAB showed better inhibition than either one alone. In addition, we show that KRAB suppressed gene expression by altering the histone code, but not DNA methylation in the gene promoter. Sss1, on the other hand, not only induced de novo DNA methylation and recruited Heterochromatin Protein 1 (HP1a), but also increased H3K27 and H3K9 methylation in the promoter. Conclusions Epigenetic studies can provide useful data for the selection of suppressors in constructing therapeutic vectors for targeted gene silencing.

The mouse Gnas gene encodes an important signal transduction protein, the α subunit of the stimulatory G protein, G s . In humans, partial deficiency of G s α, the α subunit of G s , results in the hormone-resistance syndrome pseudohypoparathyroidism type 1a. The mouse Gnas (and the human GNAS1 ) locus is transcribed from three promoter regions. Transcripts from P1, which encode Nesp55, are derived from the maternal allele only. Transcripts from P2 encode Xlαs and are derived only from the paternal allele, while transcripts from P3 encode the α subunit and are from both parental alleles. The close proximity of reciprocal imprinting suggests the presence of important putative imprinting elements in this region. In this report, we demonstrate that the reciprocal imprinting occurs in normal tissues of interspecific ( Mus spretus × C57BL/6) mice. Transcripts from P1 are most abundant in CNS (pons and medulla) in contrast to the more ubiquitous expression from P2 and P3. In the P1–P2 genomic region, we have identified an antisense transcript that starts 2.2 kb upstream of the P2 exon and spans the P1 region. While the P1 transcript is derived from the maternal allele, the P1-antisense ( Gnas-as ) is derived only from the paternal allele in most but not all tissues. Although both the Nesp55 region and the Gnas-as transcripts are present in cerebral cortex, adrenal, and spleen, Gnas-as is abundant in some tissues in which transcription from the Nesp55 region is negligible. Furthermore, the Nesp55 region transcripts remain strictly imprinted in tissues that lack Gnas-as. Our results suggest that multiple imprinting elements, including the unique Gnas-as, regulate the allelic expression of the Nesp55 region sense transcript.

p73, a proposed tumor suppressor, shares significant amino acid sequence homology with p53. However, p73 is rarely mutated in tumors but it has been suggested that p73 is monoallelically expressed in some tissues. This latter feature would predispose p73 to gene inactivation because a single genetic 'hit' or the loss of the expressed parental allele would leave the cell without p73 activity. We examined the allelic expression of p73 in normal fetal tissues and in ovarian cancer and Wilms' tumor. We found that p73 was biallelically expressed in all fetal tissues, except in brain, where differential expression of the two parental alleles was observed. Biallelic expression of p73 was also observed in paired samples of ovary cancer and Wilms' tumor. Loss of heterozygosity of p73 occurred at relatively low rates in tumors: one of 11 informative samples (9.1%) of ovarian cancer and two of 19 (10.1%) Wilms' tumors. These data demonstrate that p73 is biallelically expressed in most tissues, thus excluding genomic imprinting as a molecular mechanism to predispose to allelic inactivation of p73 in human tumors.

Two models have been proposed to account for the molecular mechanism underlying genomic imprinting of the insulin-like growth factor II receptor gene (Igf2r): expression-competition and promoter DNA methylation. To examine which model best explains the regulation of Igf2r imprinting, we examined the allelic expression of endogenous Igf2r sense and antisense RNAs in mice. In peripheral tissues, Igf2r sense and antisense RNAs show a reciprocal pattern of imprinting and DNA methylation between the two parental alleles: the sense RNA is monoallelically expressed only from the maternal promoter which is unmethylated in region 1, and the antisense RNA is derived solely from the paternal promoter which is unmethylated in region 2. The paternal promoter of sense Igf2r and the maternal promoter of antisense Igf2r are hypermethylated and are transcriptionally suppressed. In CNS, the genomic imprinting of Igf2r sense and antisense RNAs is uncoupled: both parental promoters of Igf2r gene coding for sense RNA are unmethylated and are biallelically used for transcription. In contrast, antisense RNA of Igf2r is derived only from the paternal allele that is unmethylated in region 2, while the methylated maternal allele is silent. Uncoupling of genomic imprinting of Igf2r sense and antisense RNAs in CNS correlates with DNA methylation of the appropriate promoter region, thus favoring the model of DNA methylation over that of antisense as the chief regulator of Igf2r genomic imprinting.

Insulin-like growth factor II (Igf2) is maternally imprinted in normal tissues with only the paternal copy of the gene being transcribed, whereas the contiguous gene H19 is paternally imprinted. Dysregulation of IGF2 imprinting is commonly observed in Wilms' tumor and other human tumors. Previous work comparing promoter-specific imprinting of human and mouse Igf2 suggested the presence of a cis element upstream of Igf2 that regulates or maintains the imprinting of three downstream promoters. To explore the molecular mechanism of maintenance of genomic imprinting, we targeted the region between insulin 2 and Igf2, where the cis imprint maintenance element (IME) resides in mouse fibroblasts. In those clones in which the targeting vector was randomly integrated into the genome, mouse Igf2 remained imprinted. However, when the targeted region containing the IME was deleted by homologous recombination, whether from the paternal or maternal allele, activation of the imprinted maternal allele of Igf2 was observed. In addition, there was a loss of H19 imprinting when the IME was deleted. The requirement of IME from both parental alleles for the maintenance of genomic imprinting thus suggests the importance of a spatial structure of DNA around Igf2 and H19. Modifications in the IME, like abnormal methylation in Wilms' tumors, may represent a novel mechanism for loss of genomic imprinting.

Aberrant imprinting of the insulin-like growth factor II (IGF2) gene is a molecular hallmark of many tumors. Reactivation of the normally suppressed maternal allele leads to upregulation of the growth factor that promotes tumor growth. However, the mechanisms underlying the loss of imprinting (LOI) remain poorly defined. We examined the epigenotypes at the gene promoters that control IGF2 allelic expression. Using chromatin immunoprecipitation, we found that in cells characterized by maintenance of IGF2 imprinting, three IGF2 promoters were differentially modified, with the suppressed allele heavily methylated at histone H3K27 while the active allele was unmethylated. In the LOI tumors, however, both alleles were unmethylated, and correspondingly there was no binding of SUZ12, the docking factor of the polycomb repressive complex 2 (PRC2), and of the zinc finger-containing transcription factor (CTCF) that recruits the PRC2. Using chromatin conformation capture, we found that the CTCF-orchestrated intrachromosomal loop between the IGF2 promoters and the imprinting control region was abrogated in cells with LOI. SUZ12, which docks the PRC2 to IGF2 promoters for H3K27 methylation, was downregulated in LOI cells. These data reveal a new epigenetic control pathway related to the loss of IGF2 imprinting in tumors.

Toll-like receptors (TLRs) are pivotal components of the innate immune response. Activation of the innate immune system and subsequent chronic low-grade inflammation are thought to be involved in the pathogenesis of atherosclerosis and type 2 diabetes. In the study, we genotyped TLRs gene polymorphisms, including TLR2 Arg677Trp and Arg753Gln, TLR4 Asp299Gly and Thr399Ile, TLR9-1486T/C and -1237T/C. The frequencies of TT, TC and CC genotype of TLR9-1486T/C mutation were 39.6%, 45.8% and 14.6%, respectively; the frequencies of T allele and C allele were 62.5% and 37.5%. However, neither of these parameters was statistically significant among study groups. In addition, we were surprised to find that the commonly reported TLR SNPs in the Western countries, like TLR2 Arg677Trp or Arg753Gln, TLR4 Asp299Gly or Thr399Ile and TLR9-1237T/C, were not polymorphic at all in all study subjects. In conclusion, our data suggests that TLR2 Arg677Trp or Arg753Gln, TLR4 Asp299Gly or Thr399Ile and TLR9-1237T/C polymorphisms have low frequency and TLR9-1486T/C polymorphism may not be a suitable marker in predicting the susceptibility to type 2 diabetes or coronary artery disease in the Chinese Han population.

Study question What are the implications of multiple alterations of the forkhead box L2 (FOXL2) gene in blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) patients? Summary answer A multi-mutation of FOXL2, consisting of the expansion of the polyalanine tract from 14 to 24 residues (FOXL2-Ala24), an novel Y186C substitution from c.557A>G, and a synonymous variant (c.505G>A), had a cumulative effect on ovarian phenotypes in BPES patients. What is known already Mutations in FOXL2, a gene encoding a forkhead transcription factor cause BPES. Overall, the expansion of the polyalanine tract of FOXL2 from 14 to 24 residues (FOXL2-Ala24) accounts for 30% of intragenic mutations. Study design, size, duration In this study, patients from seven BPES families and six sporadic cases were included. Participants/materials, setting, methods We conducted an extensive clinical, hormonal and functional study in 20 patients carrying the expansion of the polyalanine tract of FOXL2 associated with BPES. A multi-mutation of FOXL2 was detected in one BPES family that showed more severe BPES symptoms. Subcellular localization and transactivation studies were performed for the constructs of FOXL2-Ala24, Y186C and FOXL2-Ala24-Y186C. Main results We described the first multi-mutation of FOXL2 (c. [672_701dup30; 557A>G]) that leads to the polyalanine expansion of +10 residues (FOXL2-Ala24) combined with an Y186C substitution and a synonymous variant in a Chinese BPES family. This multi-mutation genotype was associated with more serious BPES clinical manifestations and the development of esotropia in the right eye. In in vitro studies, the multi-mutation affected the function of FOKL2 on the StAR promoter and DK3, and induced more aggressive aggregation and mislocalization of FOXL2 protein. The synonymous variant, while not affecting amino acid coding, causes a change in the RNA stem-loop structure. Limitations, reasons for caution The multi-mutation of FOXL2 was detected in one BPES family and it needs to be validated further by more BPES subjects. Wider implications of the findings The results of our study contribute new insights into the research field of BPES caused by the multi-mutation of FOXL2. Study funding/competing interests This study was supported by Shanghai Leading Academic Discipline Project (Grant number S30205) and Shanghai Jiao Tong University School of Medicine Doctor Innovation Fund (Grant number 201131). The authors have no competing interests to declare.

There is a need for more effective treatments for uveal melanoma. The recombinant oncolytic adenovirus H101 replicates specifically in p53-depleted tumor cells, and has been approved for use by the Chinese State Food and Drug Administration. However, this treatment is associated with subsequent remission. Transfection of uveal melanoma cells with a small interfering RNA against Notch1 (siNotch1) effectively suppressed Notch1 expression, resulting in significant cell growth inhibition when combined with H101 treatment. Combined treatment with siNotch1 and H101 (H101-Notch1-siRNA) greatly enhanced apoptosis and cell cycle arrest in vitro as compared to treatment with H101 or siNotch1 alone. For in vivo treatments, the combined treatment of siNotch1 and H101 showed remarkable tumor growth inhibition and prolonged mouse survival in the OCM1 xenograft model. We predict that Notch pathway deregulation could be a feature of uveal melanoma, and could be a therapeutic target, especially if p53 is concurrently targeted.

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 allele-specific 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.

7018 Background: RUNX1, a master regulator of hematopoiesis, is the most commonly perturbed target of chromosomal abnormalities in hematopoietic malignancies. About 30%–40% of acute myeloid leukemi...

Gene transcription may be regulated by remote enhancer or insulator regions through chromosome looping. Using a modification of chromosome conformation capture (3C) and fluorescence in situ hybridization, we found that one allele of the insulin-like growth factor 2 ( Igf2 )/ H19 imprinting control region (ICR) on chromosome 7 colocalized with one allele of Wsb1/Nf1 on chromosome 11. Omission of CCCTC-binding factor (CTCF) or deletion of the maternal ICR abrogated this association and altered Wsb1/Nf1 gene expression. These findings demonstrate that CTCF mediates an interchromosomal association, perhaps by directing distant DNA segments to a common transcription factory, and the data provide a model for long-range allele-specific associations between gene regions on different chromosomes that suggest a framework for DNA recombination and RNA trans-splicing.

Recent studies suggest that IVF and assisted reproduction technologies (ART) may result in abnormal genomic imprinting, leading to an increased frequency of Angelman syndrome (AS) and Beckwith–Weidemann syndrome (BWS) in IVF children. To learn how ART might alter the epigenome, we examined morulas and blastocysts derived from C57BL/6J X M. spretus F1 mice conceived in vivo and in vitro and determined the allelic expression of four imprinted genes: Igf2, H19, Cdkn1c and Slc221L. IVFderived mouse embryos that were cultured in human tubal fluid (HTF) (Quinn’s advantage) media displayed a high frequency of aberrant H19 imprinting, whereas in vivo and IVF embryos showed normal maternal expression of Cdkn1c and normal biallelic expression of Igf2 and Slc221L. Embryonic stem (ES) cells derived from IVF blastocysts also showed abnormal Igf2/H19 imprinting. Allele-specific bisulphite PCR reveals abnormal DNA methylation at a CCCTC-binding factor (CTCF) site in the imprinting control region (ICR), as the normally unmethylated maternal allele acquired a paternal methylation pattern. Chromatin immunoprecipitation (ChIP) assays indicate an increase of lysine 4 methylation (dimethyl Lys4-H3) on the paternal chromatin and a gain in lysine 9 methylation (trimethyl Lys9-H3) on the maternal chromatin at the same CTCF-binding site. Our results indicate that de novo DNA methylation on the maternal allele and allele-specific acquisition of histone methylation lead to aberrant Igf2/ H19 imprinting in IVF-derived ES cells. We suggest that ART, which includes IVF and various culture media, might cause imprinting errors that involve both aberrant DNA methylation and histone methylation at an epigenetic switch of the Igf2-H19 gene region.

Activation and suppression of gene transcription is tightly controlled by epigenetic modifications. The imprinted Gnas1 gene region contains closely juxtaposed maternally expressed (Nesp) and paternally expressed (Nespas, Gnasxl, Exon 1A) transcripts, providing a unique opportunity to study how epigenetic modifications change in nucleosomes from active to silenced promoters. Using 30 polymorphic sites across the Gnas1 gene region in (C57BL/6JxMus spretus) F(1) mice and chromatin immunoprecipitation (ChIP) assays we identified two allelic switch regions (ASRs) that mark boundaries of epigenetic information. We show that activating signals (histone acetylation and methylation of H3 Lys4) and silencing signals (histone methylation of H3 Lys9 and DNA methylation) segregate independently across the ASRs and suggest that these ASRs allow the transcriptional elongation to proceed through the silenced domain of nearby imprinted promoters. We discuss these findings in light of recent progress in the conceptualization of nucleosome remodeling during transcriptional elongation and in the development of histone code.

Gene single nucleotide polymorphisms (SNPs) have been extensively studied in association with development and prognosis of various malignancies. However, the potential role of genetic polymorphisms of cancer stem cell (CSC) marker genes with respect to cancer risk has not been examined. We conducted a case-control study involving a total of 1000 subjects (500 lung cancer patients and 500 age-matched cancer-free controls) from northeastern China. Lung cancer risk was analyzed in a logistic regression model in association with genotypes of four lung CSC marker genes (CD133, ALDH1, Musashi-1, and EpCAM). Using univariate analysis, the Musashi-1 rs2522137 GG genotype was found to be associated with a higher incidence of lung cancer compared with the TT genotype. No significant associations were observed for gene variants of CD133, ALDH1, or EpCAM. In multivariate analysis, Musashi-1 rs2522137 was still significantly associated with lung cancer when environmental and lifestyle factors were incorporated in the model, including lower BMI; family history of cancer; prior diagnosis of chronic obstructive pulmonary disease, pneumonia, or pulmonary tuberculosis; occupational exposure to pesticide; occupational exposure to gasoline or diesel fuel; heavier smoking; and exposure to heavy cooking emissions. The value of the area under the receiver-operating characteristic (ROC) curve (AUC) was 0.7686. To our knowledge, this is the first report to show an association between a Musashi-1 genotype and lung cancer risk. Further, the prediction model in this study may be useful in determining individuals with high risk of lung cancer.

The two contiguous IGF2 (human insulin-like growth factor II) and H19 genes are reciprocally imprinted in both human and mouse. In most tissues, IGF2 is transcribed only from the paternal chromosome while H19 is transcribed only from the maternal allele. The presence of a differential methylation region (DMR) on the two parental alleles at the 5' flanking region of H19 has been proposed to constitute the gametic imprint, which controls the reciprocal allelic expression of the two genes. Using bisulfite genomic sequencing, we have assessed the methylation status of cytosine (including 154 CpG sites) in six CpG-rich regions of the human IGF2-H19 genes. In a CpG island near promoter P3 of the IGF2 gene, more than 99.8% of all cytosines were converted to thymidine by sodium bisulfite mutagenesis, indicating that none of the CpGs was methylated. In the IGF2 exon 8-9 region, mosaic methylation of 56 CpG sites was observed in fetal tissues and in adult blood DNA. In contrast to the mosaic methylation of IGF2, the allelic methylation of the human H19 DMR was uniform. In the CpG region located 2 kb upstream (-2362 to -1911) of the H19 transcription site, all 25 CpG sites were completely methylated on only one parental allele. Uniform allele-specific methylation was also observed in the CpG island proximal to the H19 promoter (-711 to -290) with complete methylation of all 25 CpG sites in one parental allele. In contrast, the CpG region in the H19 promoter (-292 to +15) was mosaically methylated in all tissues. In addition, cytosine was methylated at three CpNpG and GpNpC sites on the top DNA strand and one CpNpG site on the bottom DNA strand from the fetal brain. The cytosines at CpG sites were methylated on both DNA strands (symmetric methylation) while cytosines at the CpNpG and GpNpC sites were methylated on only one DNA strand (asymmetric methylation). The asymmetric methylation was associated with tissue-specific disruption of H19 genomic imprinting in fetal brain.

Imprinted genes may be expressed uniparentally in a tissue- and development-specific manner. The insulin-like growth factor II receptor gene (Igf2r), one of the first imprinted genes to be identified, is an attractive candidate for studying the molecular mechanism of genomic imprinting because it is transcribed monoallelically in the mouse but biallelically in humans. To identify the factors that control genomic imprinting, we examined allelic expression of Igf2r at different ages in interspecific mice. We found that Igf2r is not always monoallelically expressed. Paternal imprinting of Igf2r is maintained in peripheral tissues, including liver, kidney, heart, spleen, intestine, bladder, skin, bone, and skeletal muscle. However, in central nervous system (CNS), Igf2r is expressed from both parental alleles. Southern analysis of the Igf2r promoter (region 1) revealed that, outside of the CNS where Igf2r is monoallelically expressed, the suppressed paternal allele is fully methylated while the expressed maternal allele is completely unmethylated. In CNS, however, both parental alleles are unmethylated in region 1. The importance of DNA methylation in the maintenance of the genomic imprint was also confirmed by the finding that Igf2r imprinting was relaxed by 5-azacytidine treatment. The correlation between genomic imprinting and allelic Igf2r methylation in CNS and other tissues thus suggests that the epigenetic modification in the promoter region may function as one of the major factors in maintaining the monoallelic expression of Igf2r.

Imprinting of the insulin-like growth factor II gene (IGF-II) is conserved in human, rat, and mouse. In human liver and chondrocytes, IGF-II transcripts from promoter hP1 are always derived from both parental alleles, while transcripts from promoters hP2-hP4 are from one parental allele. To examine the promoter-specific imprinting pattern of mouse IGF-II, we examined IGF-II expression in F1 generation mice derived from crossing M. spretus with M. musculus using a novel BsaA1 polymorphism in mouse IGF-II exon 6. There was maintenance of maternal IGF-II imprinting in all non-central nervous system (CNS) tissues in the F1 generation animals. However, there was biallelic expression of the IGF-II gene in CNS. Allelic expression of each IGF-II promoter transcript was examined by full-length cDNA amplification with promoter-specific primers. In every tissue in which IGF-II was imprinted, IGF-II transcripts were derived from paternal promoters mP1-mP3, while the maternal allele was suppressed. In the CNS, however, promoters mP1-mP3 of the imprinted maternal allele became activated, leading to the biallelic expression of IGF-II. Moreover, the expression of IGF-II from each parental allele differed in various CNS regions. In leptomeninges, mP1-mP3 drive IGF-II expression predominantly from the paternal allele, while in some CNS regions, the promoter transcripts were primarily from the maternal allele. The coordinate regulation of mouse IGF-II promoters suggests the presence of an upstream imprinting complex controlling IGF-II imprinting.(ABSTRACT TRUNCATED AT 250 WORDS)

SummaryGeneration of induced pluripotent stem cells (iPSCs) by defined factors is an extremely inefficient process, because there is a strong epigenetic block preventing cells from achieving pluripotency. Here we report that virally expressed factors bound to the promoters of their target genes to the same extent in both iPSCs and unreprogrammed cells (URCs). However, expression of endogenous pluripotentcy genes was observed only in iPSCs. Comparison of local chromatin structure of the OCT4 locus revealed that there was a cohesin-complex-mediated intrachromosomal loop that juxtaposes a downstream enhancer to the gene’s promoter, enabling activation of endogenous stemness genes. None of these long-range interactions were observed in URCs. Knockdown of the cohesin-complex gene SMC1 by RNAi abolished the intrachromosomal interaction and affected pluripotency. These findings highlight the importance of the SMC1-orchestrated intrachromosomal loop as a critical epigenetic barrier to the induction of pluripotency.