Skeletal muscle in aged mice reveals extensive transformation of muscle gene expression.

BACKGROUND: Aging leads to decreased skeletal muscle function in mammals and is associated with a progressive loss of muscle mass, quality and strength. Age-related muscle loss (sarcopenia) is an important health problem associated with the aged population. RESULTS: We investigated the alteration of genome-wide transcription in mouse skeletal muscle tissue (rectus femoris muscle) during aging using a high-throughput sequencing technique. Analysis revealed significant transcriptional changes between skeletal muscles of mice at 3 (young group) and 24 (old group) months of age. Specifically, genes associated with energy metabolism, cell proliferation, muscle myosin isoforms, as well as immune functions were found to be altered. We observed several interesting gene expression changes in the elderly, many of which have not been reported before. CONCLUSIONS: Those data expand our understanding of the various compensatory mechanisms that can occur with age, and further will assist in the development of methods to prevent and attenuate adverse outcomes of aging.

Cisd2 deficiency drives premature aging and causes mitochondria-mediated defects in mice.

CISD2, the causative gene for Wolfram syndrome 2 (WFS2), is a previously uncharacterized novel gene. Significantly, the CISD2 gene is located on human chromosome 4q, where a genetic component for longevity maps. Here we show for the first time that CISD2 is involved in mammalian life-span control. Cisd2 deficiency in mice causes mitochondrial breakdown and dysfunction accompanied by autophagic cell death, and these events precede the two earliest manifestations of nerve and muscle degeneration; together, they lead to a panel of phenotypic features suggestive of premature aging. Our study also reveals that Cisd2 is primarily localized in the mitochondria and that mitochondrial degeneration appears to have a direct phenotypic consequence that triggers the accelerated aging process in Cisd2 knockout mice; furthermore, mitochondrial degeneration exacerbates with age, and the autophagy increases in parallel to the development of the premature aging phenotype. Additionally, our Cisd2 knockout mouse work provides strong evidence supporting an earlier clinical hypothesis that WFS is in part a mitochondria-mediated disorder; specifically, we propose that mutation of CISD2 causes the mitochondria-mediated disorder WFS2 in humans. Thus, this mutant mouse provides an animal model for mechanistic investigation of Cisd2 protein function and help with a pathophysiological understanding of WFS2.

An H2A histone isotype regulates estrogen receptor target genes by mediating enhancer-promoter-3'-UTR interactions in breast cancer cells.

A replication-dependent histone H2A isotype, H2ac, is upregulated in MCF-7 cells and in estrogen receptor-positive clinical breast cancer tissues. Cellular depletion of this H2A isotype leads to defective estrogen signaling, loss of cell proliferation and cell cycle arrest at G0/G1 phase. H2ac mediates regulation of estrogen receptor target genes, particularly BCL2 and c-MYC, by recruiting estrogen receptor alpha through its HAR domain and facilitating the formation of a chromatin loop between the promoter, enhancer and 3'-untranslated region of the respective genes. These findings reveal a new role for histone isotypes in the regulation of gene expression in cancer cells, and suggest that these molecules may be targeted for anti-cancer drug discovery.

Integrated analyses to reconstruct microRNA-mediated regulatory networks in mouse liver using high-throughput profiling.

BACKGROUND: MicroRNAs (miRNAs) simultaneously target many transcripts through partial complementarity binding, and have emerged as a key type of post-transcriptional regulator for gene expression. How miRNA accomplishes its pleiotropic effects largely depends on its expression and its target repertoire. Previous studies discovered thousands of miRNAs and numerous miRNA target genes mainly through computation and prediction methods which produced high rates of false positive prediction. The development of Argonaute cross-linked immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq) provides a system to effectively determine miRNA target genes. Likewise, the accuracy of dissecting the transcriptional regulation of miRNA genes has been greatly improved by chromatin immunoprecipitation of the transcription factors coupled with sequencing (ChIP-Seq). Elucidation of the miRNA target repertoire will provide an in-depth understanding of the functional roles of microRNA pathways. To reliably reconstruct a miRNA-mediated regulatory network, we established a computational framework using publicly available, sequence-based transcription factor-miRNA databases, including ChIPBase and TransmiR for the TF-miRNA interactions, along with miRNA-target databases, including miRTarBase, TarBase and starBase, for the miRNA-target interactions. We applied the computational framework to elucidate the miRNA-mediated regulatory network in the Mir122a⁻/⁻ mouse model, which has an altered transcriptome and progressive liver disease. RESULTS: We applied our computational framework to the expression profiles of miRNA/mRNA of Mir122a⁻/⁻ mutant mice and wild-type mice. The miRNA-mediated network involves 40 curated TFs contributing to the aberrant expression of 65 miRNAs and 723 curated miRNA target genes, of which 56% was found in the differentially-expressed genes of Mir122a--mice. Hence, the regulatory network disclosed previously-known and also many previously-unidentified miRNA-mediated regulations in mutant mice. Moreover, we demonstrate that loss of imprinting at the chromosome 12qF1 region is associated with miRNA overexpression in human hepatocellular carcinoma and stem cells, suggesting initiation of precancerous changes in young mice deficient in miR-122. A group of 9 miRNAs was found to share miR-122 target genes, indicating synergy between miRNAs and target genes by way of multiplicity and cooperativity. CONCLUSIONS: The study provides significant insight into miRNA-mediated regulatory networks. Based on experimentally verified data, this network is highly reliable and effective in revealing previously-undetermined disease-associated molecular mechanisms. This computational framework can be applied to explore the significant TF-miRNA-miRNA target interactions in any complex biological systems with high degrees of confidence.

Protein domain repetition is enriched in Streptococcal cell-surface proteins.

Tandem repetition of domain in protein sequence occurs in all three domains of life. It creates protein diversity and adds functional complexity in organisms. In this work, we analyzed 52 streptococcal genomes and found 3748 proteins contained domain repeats. Proteins not harboring domain repeats are significantly enriched in cytoplasm, whereas proteins with domain repeats are significantly enriched in cytoplasmic membrane, cell wall and extracellular locations. Domain repetition occurs most frequently in S. pneumoniae and least in S. thermophilus and S. pyogenes. DUF1542 is the highest repeated domain in a single protein, followed by Rib, CW_binding_1, G5 and HemolysinCabind. 3D structures of 24 repeat-containing proteins were predicted to investigate the structural and functional effect of domain repetition. Several repeat-containing streptococcal cell surface proteins are known to be virulence-associated. Surface-associated tandem domain-containing proteins without experimental functional characterization may be potentially involved in the pathogenesis of streptococci and deserve further investigation.

Overlapping high-resolution copy number alterations in cancer genomes identified putative cancer genes in hepatocellular carcinoma.

UNLABELLED: Recurrent cancer genome aberrations are indicators of residing crucial cancer genes. Although recent advances in genomic technologies have led to a global view of cancer genome aberrations, the identification of target genes and biomarkers from the aberrant loci remains difficult. To facilitate searches of cancer genes in human hepatocellular carcinoma (HCC), we established a comprehensive protocol to analyze copy number alterations (CNAs) in cancer genomes using high-density single nucleotide polymorphism arrays with unpaired reference genomes. We identified common HCC genes by overlapping the shared aberrant loci in multiple cell lines with functional validation and clinical implications. A total of 653 amplicons and 57 homozygous deletions (HDs) were revealed in 23 cell lines. To search for novel HCC genes, we overlapped aberrant loci to uncover 6 HDs and 126 amplicons shared by at least two cell lines. We selected two novel genes, fibronectin type III domain containing 3B (FNDC3B) at the 3q26.3 overlapped amplicon and solute carrier family 29 member 2 (SLC29A2) at the 11q13.2 overlapped amplicon, to investigate their aberrations in HCC tumorigenesis. Aberrant up-regulation of FNDC3B and SLC29A2 occurred in multiple HCC data sets. Knockdown of these genes in amplified cells decreased cell proliferation, anchorage-independent growth, and tumor formation in xenograft models. Importantly, up-regulation of SLC29A2 in HCC tissues was significantly associated with advanced stages (P = 0.0031), vascular invasion (P = 0.0353), and poor patient survival (P = 0.0325). Overexpression of FNDC3B or SLC29A2 in unamplified HCC cells promoted cell proliferation through activation of the signal transducer and activator of transcription 3 signaling pathway. CONCLUSION: A standardized genome-wide CNA analysis protocol using data from user-generated or public domains normalized with unpaired reference genomes has been established to facilitate high-throughput detection of cancer genes as significant target genes and biomarkers for cancer diagnosis and therapy.

Pediatric primary central nervous system germ cell tumors of different prognosis groups show characteristic miRNome traits and chromosome copy number variations.

BACKGROUND: Intracranial pediatric germ cell tumors (GCTs) are rare and heterogeneous neoplasms and vary in histological differentiation, prognosis and clinical behavior. Germinoma and mature teratoma are GCTs that have a good prognosis, while other types of GCTs, termed nongerminomatous malignant germ cell tumors (NGMGCTs), are tumors with an intermediate or poor prognosis. The second group of tumors requires more extensive drug and irradiation treatment regimens. The mechanisms underlying the differences in incidence and prognosis of the various GCT subgroups are unclear. RESULTS: We identified a distinct mRNA profile correlating with GCT histological differentiation and prognosis, and also present in this study the first miRNA profile of pediatric primary intracranial GCTs. Most of the differentially expressed miRNAs were downregulated in germinomas, but miR-142-5p and miR-146a were upregulated. Genes responsible for self-renewal (such as POU5F1 (OCT4), NANOG and KLF4) and the immune response were abundant in germinomas, while genes associated with neuron differentiation, Wnt/beta-catenin pathway, invasiveness and epithelial-mesenchymal transition (including SNAI2 (SLUG) and TWIST2) were abundant in NGMGCTs. Clear transcriptome segregation based on patient survival was observed, with malignant NGMGCTs being closest to embryonic stem cells. Chromosome copy number variations (CNVs) at cytobands 4q13.3-4q28.3 and 9p11.2-9q13 correlated with GCT malignancy and clinical risk. Six genes (BANK1, CXCL9, CXCL11, DDIT4L, ELOVL6 and HERC5) within 4q13.3-4q28.3 were more abundant in germinomas. CONCLUSIONS: Our results integrate molecular profiles with clinical observations and provide insights into the underlying mechanisms causing GCT malignancy. The genes, pathways and microRNAs identified have the potential to be novel therapeutic targets.

MicroRNA-122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma.

UNLABELLED: MicroRNAs (miRNAs), which are inhibitors of gene expression, participate in diverse biological functions and in carcinogenesis. In this study, we show that liver-specific microRNA-122 (miR-122) is significantly down-regulated in liver cancers with intrahepatic metastasis and negatively regulates tumorigenesis. Restoration of miR-122 in metastatic Mahlavu and SK-HEP-1 cells significantly reduced in vitro migration, invasion, and anchorage-independent growth as well as in vivo tumorigenesis, angiogenesis, and intrahepatic metastasis in an orthotopic liver cancer model. Because an inverse expression pattern is often present between an miRNA and its target genes, we used a computational approach and identified multiple miR-122 candidate target genes from two independent expression microarray datasets. Thirty-two target genes were empirically verified, and this group of genes was enriched with genes regulating cell movement, cell morphology, cell-cell signaling, and transcription. We further showed that one of the miR-122 targets, ADAM17 (a disintegrin and metalloprotease 17) is involved in metastasis. Silencing of ADAM17 resulted in a dramatic reduction of in vitro migration, invasion, in vivo tumorigenesis, angiogenesis, and local invasion in the livers of nude mice, which is similar to that which occurs with the restoration of miR-122. CONCLUSION: Our study suggests that miR-122, a tumor suppressor microRNA affecting hepatocellular carcinoma intrahepatic metastasis by angiogenesis suppression, exerts some of its action via regulation of ADAM17. Restoration of miR-122 has a far-reaching effect on the cell. Using the concomitant down-regulation of its targets, including ADAM17, a rational therapeutic strategy based on miR-122 may prove to be beneficial for patients with hepatocellular carcinoma.

A novel regulatory event-based gene set analysis method for exploring global functional changes in heterogeneous genomic data sets.

BACKGROUND: Analyzing gene expression data by assessing the significance of pre-defined gene sets, rather than individual genes, has become a main approach in microarray data analysis and this has promisingly derive new biological interpretations of microarray data. However, the detection power of conventional gene list or gene set-based approaches is limited on highly heterogeneous samples, such as tumors. RESULTS: We developed a novel method, the regulatory event-based Gene Set Analysis (eGSA), which considers not only the consistently changed genes but also every gene regulation (event) of each sample to overcome the detection limit. In comparison with conventional methods, eGSA can detect functional changes in heterogeneous samples more precisely and robustly. Furthermore, by utilizing eGSA, we successfully revealed novel functional characteristics and potential mechanisms of very early hepatocellular carcinoma (HCC). CONCLUSION: Our study creates a novel scheme to directly target the major cellular functional changes in heterogeneous samples. All potential regulatory routines of a functional change can be further analyzed by the regulatory event frequency. We also provide a case study on early HCCs and reveal a novel insight at the initial stage of hepatocarcinogenesis. eGSA therefore accelerates and refines the interpretation of heterogeneous genomic data sets in the absence of gene-phenotype correlations.

Frequent down-regulation of hRAB37 in metastatic tumor by genetic and epigenetic mechanisms in lung cancer.

We looked for the involvement tumor suppressor gene (TSG) in lung cancer in 17q25 region by loss of heterozygosity analysis and 5'/3' RACE and identified a candidate gene named human RAB37 (hRAB37), which encodes a small GTPase. The Ras-GTPase superfamily functions as important regulators including membrane trafficking and cytoskeletal organization. Therefore, we further examined the mRNA expression and promoter/exon1 hypermethylation of hRAB37 gene in paired normal and tumor lung tissue from 71 non-small cell lung cancer (NSCLC) patients. Low hRAB37 mRNA expression occurred in 47.9% (34/71) of patient and promoter/exon1 hypermethylation of hRAB37 was found in 57.7% (41/71) of patients. Low mRNA expression of hRAB37 was significantly associated with their promoter/exon1 hypermethylation. Importantly, a reduction in hRAB37 mRNA expression and promoter/exon1 hypermethylation was found to be significantly associated with lung metastatic patients as compared to non-metastatic patients. 5-Aza-2-deoxycytidine treatment of a highly metastatic cell line showed demethylation and re-expression of the hRAB37 gene and coincided with reduced migration. Knockdown of hRAB37 in low metastasis cell line led to a significant increase in cell migration. Our findings demonstrated that hRAB37 small GTPase and acts as a metastasis-related TSG in lung cancer. Promoter/exon1 methylation is the predominant mechanism in down-regulation of the hRAB37, and can serve as a potential prediction biomarker of NSCLC progression.

Signature Evaluation Tool (SET): a Java-based tool to evaluate and visualize the sample discrimination abilities of gene expression signatures.

BACKGROUND: The identification of specific gene expression signature for distinguishing sample groups is a dominant field in cancer research. Although a number of tools have been developed to identify optimal gene expression signatures, the number of signature genes obtained is often overly large to be applied clinically. Furthermore, experimental verification is sometimes limited by the availability of wet-lab materials such as antibodies and reagents. A tool to evaluate the discrimination power of candidate genes is therefore in high demand by clinical researchers. RESULTS: Signature Evaluation Tool (SET) is a Java-based tool adopting the Golub's weighted voting algorithm as well as incorporating the visual presentation of prediction strength for each array sample. SET provides a flexible and easy-to-follow platform to evaluate the discrimination power of a gene signature. Here, we demonstrated the application of SET for several purposes: (1) for signatures consisting of a large number of genes, SET offers the ability to rapidly narrow down the number of genes; (2) for a given signature (from third party analyses or user-defined), SET can re-evaluate and re-adjust its discrimination power by selecting/de-selecting genes repeatedly; (3) for multiple microarray datasets, SET can evaluate the classification capability of a signature among datasets; and (4) by providing a module to visualize the prediction strength for each sample, SET allows users to re-evaluate the discrimination power on mis-grouped or less-certain samples. Information obtained from the above applications could be useful in prognostic analyses or clinical management decisions. CONCLUSION: Here we present SET to evaluate and visualize the sample-discrimination ability of a given gene expression signature. This tool provides a filtration function for signature identification and lies between clinical analyses and class prediction (or feature selection) tools. The simplicity, flexibility and brevity of SET could make it an invaluable tool for marker identification in clinical research.

easyExon--a Java-based GUI tool for processing and visualization of Affymetrix exon array data.

BACKGROUND: Alternative RNA splicing greatly increases proteome diversity and thereby contribute to species- or tissue-specific functions. The possibility to study alternative splicing (AS) events on a genomic scale using splicing-sensitive microarrays, including the Affymetrix GeneChip Exon 1.0 ST microarray (exon array), has appeared very recently. However, the application of this new technology is hindered by the lack of free and user-friendly software devoted to these novel platforms. RESULTS: In this study we present a Java-based freeware, easyExon http://microarray.ym.edu.tw/easyexon, to process, filtrate and visualize exon array data with an analysis pipeline. This tool implements the most commonly used probeset summarization methods as well as AS-orientated filtration algorithms, e.g. MIDAS and PAC, for the detection of alternative splicing events. We include a biological filtration function according to GO terms, and provide a module to visualize and interpret the selected exons and transcripts. Furthermore, easyExon can integrate with other related programs, such as Integrate Genome Browser (IGB) and Affymetrix Power Tools (APT), to make the whole analysis more comprehensive. We applied easyExon on a public accessible colon cancer dataset as an example to illustrate the analysis pipeline of this tool. CONCLUSION: EasyExon can efficiently process and analyze the Affymetrix exon array data. The simplicity, flexibility and brevity of easyExon make it a valuable tool for AS event identification in genomic research.

MicroRNA-23a/27a/24-2 cluster promotes gastric cancer cell proliferation synergistically.

Previous studies have indicated that certain microRNAs (miRNAs/miRs) function as either tumor suppressors or oncogenes in human cancer. The present study identified the miR-23a/27a/24-2 cluster, containing miR-23, miR-27a and miR-24, as an oncogene in gastric cancer. The expression of the miR-23a/27a/24-2 cluster was upregulated in clinical gastric cancer tissues. Transfection with inhibitors of miR-23a, miR-27a, or miR-24, either independently or together, repressed in vitro colony formation and in vivo tumor formation. The miR23a/27a/24-2 cluster inhibitors repressed the growth of gastric cancer cells in a synergistic manner. In addition, treatment with lower doses of the miRNA inhibitor mixture induced the formation of apoptotic bodies. According to computational predictions using TargetScan, suppressor of cytokine-induced signaling 6 (SOCS6) was identified as one of the downstream target genes of the miR-23a/27a/24-2 cluster. The expression of SOCS6 was significantly lower in tumor tissues than in matched normal tissues (P<0.01) and was associated with poor survival (P<0.00001). Taken together, these results strongly suggested that the miR-23a/27a/24-2 cluster may mediate the progression of gastric cancer through the suppression of SOCS6 expression. The present study also provides a novel molecular target for the development of an anti-gastric cancer agent.

ArrayFusion: a web application for multi-dimensional analysis of CGH, SNP and microarray data.

UNLABELLED: ArrayFusion annotates conventional CGH results and various types of microarray data from a range of platforms (cDNA, expression, exon, SNP, array-CGH and ChIP-on-chip) and converts them into standard formats which can be visualized in genome browsers (Affymetrix Integrated Genome Browser and GBrowse in the HapMap Project). Converted files can then be imported simultaneously into a single genome browser to benefit a collective interpretation between different array results. ArrayFusion therefore provides a new type of tool facilitating the integration of CGH and array results to provide new experimental directions. AVAILABILITY: http://microarray.ym.edu.tw/tools/arrayfusion

Correlated 5-Hydroxymethylcytosine (5hmC) and Gene Expression Profiles Underpin Gene and Organ-Specific Epigenetic Regulation in Adult Mouse Brain and Liver.

BACKGROUND: DNA methylation is an epigenetic mechanism essential for gene regulation and vital for mammalian development. 5-hydroxymethylcytosine (5hmC) is the first oxidative product of the TET-mediated 5-methylcytosine (5mC) demethylation pathway. Aside from being a key intermediate in cytosine demethylation, 5hmC may have potential regulatory functions with emerging importance in mammalian biology. METHODS: Here, we investigate the global 5hmC enrichment in five brain structures, including cerebellum, cerebral cortex, hippocampus, hypothalamus and thalamus, as well as liver tissues from female and male adult mice by using chemical capture-based technique coupled with next-generation sequencing. At the same time, we carried out total RNA sequencing (RNA-seq) to analyze the transcriptomes of brain regions and liver tissues. RESULTS: Our results reveal preferential 5hmC enrichment in the gene bodies of expressed genes, and 5hmC levels of many protein-coding genes are positively correlated with RNA expression intensity. However, more than 75% of genes with low or no 5hmC enrichment are genes encode for mitochondrial proteins and ribosomal proteins despite being actively transcribed, implying different transcriptional regulation mechanisms of these housekeeping genes. Brain regions developed from the same embryonic structures have more similar 5hmC profiles. Also, the genic 5hmC enrichment pattern is highly tissue-specific, and 5hmC marks genes involving in tissue-specific biological processes. Sex chromosomes are mostly depleted of 5hmC, and the X inactive specific transcript (Xist) gene located on the X chromosome is the only gene to show sex-specific 5hmC enrichment. CONCLUSIONS: This is the first report of the whole-genome 5hmC methylome of five major brain structures and liver tissues in mice of both sexes. This study offers a comprehensive resource for future work of mammalian cytosine methylation dynamics. Our findings offer additional evidence that suggests 5hmC is an active epigenetic mark stably maintained after the global reprogramming event during early embryonic development.

An H2A Histone Isotype, H2ac, Associates with Telomere and Maintains Telomere Integrity.

Telomeres are capped at the ends of eukaryotic chromosomes and are composed of TTAGGG repeats bound to the shelterin complex. Here we report that a replication-dependent histone H2A isotype, H2ac, was associated with telomeres in human cells and co-immunoprecipitates with telomere repeat factor 2 (TRF2) and protection of telomeres protein 1 (POT1), whereas other histone H2A isotypes and mutations of H2ac did not bind to telomeres or these two proteins. The amino terminal basic domain of TRF2 was necessary for the association with H2ac and for the recruitment of H2ac to telomeres. Depletion of H2ac led to loss of telomeric repeat sequences, the appearance of dysfunctional telomeres, and chromosomal instability, including chromosomal breaks and anaphase bridges, as well as accumulation of telomere-associated DNA damage factors in H2ac depleted cells. Additionally, knockdown of H2ac elicits an ATM-dependent DNA damage response at telomeres and depletion of XPF protects telomeres against H2ac-deficiency-induced G-strand overhangs loss and DNA damage response, and prevents chromosomal instability. These findings suggest that the H2A isotype, H2ac, plays an essential role in maintaining telomere functional integrity.

Regulation of IL-20 Expression by Estradiol through KMT2B-Mediated Epigenetic Modification.

Cytokines are low molecular weight regulatory proteins, or glycoproteins, with both tumor-promoting and inhibitory effects on breast cancer growth. Different cytokines play important roles in breast cancer initiation and progression. Here, we show that of the 39 interleukin (IL) genes, IL-20 is the only gene over-expressed in MCF-7 cells treated with estradiol (E2) and that induction of IL-20 expression by estrogen was epigenetically regulated. Methylation of histone H3K4 in the IL-20 promoter was shown to occur via the specific recruitment of KMT2B by estrogen receptor alpha (ERα), but not by other members of the mixed-lineage leukemia (MLL) family of histone methyltransferases. Depletion of KMT2B, or IL-20, disrupts estrogen signaling, attenuates cell proliferation, reduces colony formation, and results in cell cycle arrest. Furthermore, we demonstrated that KMT2B-mediated epigenetic modification also affected the expression of several ERα target genes. IL-20 and KMT2B expression were also associated with ERα-positive breast cancer tissues. We have revealed an important role for KMT2B in the epigenetic transcriptional regulation of cytokine IL-20, and other ERα-responsive genes, in breast cancer cells. Inhibition of IL-20 and KMT2B may have therapeutic benefits in ERα-positive breast cancer.

FNDC3B promotes cell migration and tumor metastasis in hepatocellular carcinoma.

Recurrence and metastasis are common in hepatocellular carcinoma (HCC) and correlate with poor prognosis. We investigated the role of fibronectin type III domain containing 3B (FNDC3B) in HCC metastasis. Overexpression of FNDC3B in HCC cell lines enhanced cell migration and invasion. On the other hand, knockdown of FNDC3B using short-hairpin RNA reduced tumor nodule formation in both intra- and extra-hepatic metastasis. High levels of FNDC3B were observed in metastatic HCCs and correlated with poor patient survival and shorter recurrence time. Mutagenesis and LC-MS/MS analyses showed that FNDC3B promotes cell migration by cooperating with annexin A2 (ANXA2). Furthermore, FNDC3B and ANXA2 expression correlated negatively with patient survival. Our results indicate that FNDC3B behaves like an oncogene by promoting cell migration. This suggests FNDC3B could serve as a biomarker and therapeutic target for HCC metastasis.

MAF1 represses CDKN1A through a Pol III-dependent mechanism.

MAF1 represses Pol III-mediated transcription by interfering with TFIIIB and Pol III. Herein, we found that MAF1 knockdown induced CDKN1A transcription and chromatin looping concurrently with Pol III recruitment. Simultaneous knockdown of MAF1 with Pol III or BRF1 (subunit of TFIIIB) diminished the activation and looping effect, which indicates that recruiting Pol III was required for activation of Pol II-mediated transcription and chromatin looping. Chromatin-immunoprecipitation analysis after MAF1 knockdown indicated enhanced binding of Pol III and BRF1, as well as of CFP1, p300, and PCAF, which are factors that mediate active histone marks, along with the binding of TATA binding protein (TBP) and POLR2E to the CDKN1A promoter. Simultaneous knockdown with Pol III abolished these regulatory events. Similar results were obtained for GDF15. Our results reveal a novel mechanism by which MAF1 and Pol III regulate the activity of a protein-coding gene transcribed by Pol II.

Hierarchical clustering of breast cancer methylomes revealed differentially methylated and expressed breast cancer genes.

Oncogenic transformation of normal cells often involves epigenetic alterations, including histone modification and DNA methylation. We conducted whole-genome bisulfite sequencing to determine the DNA methylomes of normal breast, fibroadenoma, invasive ductal carcinomas and MCF7. The emergence, disappearance, expansion and contraction of kilobase-sized hypomethylated regions (HMRs) and the hypomethylation of the megabase-sized partially methylated domains (PMDs) are the major forms of methylation changes observed in breast tumor samples. Hierarchical clustering of HMR revealed tumor-specific hypermethylated clusters and differential methylated enhancers specific to normal or breast cancer cell lines. Joint analysis of gene expression and DNA methylation data of normal breast and breast cancer cells identified differentially methylated and expressed genes associated with breast and/or ovarian cancers in cancer-specific HMR clusters. Furthermore, aberrant patterns of X-chromosome inactivation (XCI) was found in breast cancer cell lines as well as breast tumor samples in the TCGA BRCA (breast invasive carcinoma) dataset. They were characterized with differentially hypermethylated XIST promoter, reduced expression of XIST, and over-expression of hypomethylated X-linked genes. High expressions of these genes were significantly associated with lower survival rates in breast cancer patients. Comprehensive analysis of the normal and breast tumor methylomes suggests selective targeting of DNA methylation changes during breast cancer progression. The weak causal relationship between DNA methylation and gene expression observed in this study is evident of more complex role of DNA methylation in the regulation of gene expression in human epigenetics that deserves further investigation.