Kun Sun

Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments

Significance Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA, we obtained a bird’s eye view of the identities and contributions of these tissues to the circulating DNA pool. The tissue contributors and their relative proportions are identified by a bioinformatics deconvolution process that draws reference from DNA methylation signatures representative of each tissue type. We validated this approach in pregnant women, cancer patients, and transplant recipients. This method also allows one to identify the tissue of origin of genomic aberrations observed in plasma DNA. This approach has numerous research and diagnostic applications in prenatal testing, oncology, transplantation monitoring, and other fields. Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma.

Noninvasive detection of cancer-associated genome-wide hypomethylation and copy number aberrations by plasma DNA bisulfite sequencing

Significance Genome-wide hypomethylation is frequently observed in cancers. In this study, we showed that genome-wide hypomethylation analysis in plasma using shotgun massively parallel bisulfite sequencing is a powerful general approach for the detection of multiple types of cancers. This approach is particularly attractive because high sensitivity and specificity can be achieved using low sequence depth, which is practical diagnostically. This approach can also be used for monitoring patients following treatment. The same sequencing data can be further used for detecting cancer-associated copy number aberrations at no additional costs. One could thus combine plasma hypomethylation and copy number analyses in a synergistic manner for further enhancing detection sensitivity or specificity. We explored the detection of genome-wide hypomethylation in plasma using shotgun massively parallel bisulfite sequencing as a marker for cancer. Tumor-associated copy number aberrations (CNAs) could also be observed from the bisulfite DNA sequencing data. Hypomethylation and CNAs were detected in the plasma DNA of patients with hepatocellular carcinoma, breast cancer, lung cancer, nasopharyngeal cancer, smooth muscle sarcoma, and neuroendocrine tumor. For the detection of nonmetastatic cancer cases, plasma hypomethylation gave a sensitivity and specificity of 74% and 94%, respectively, when a mean of 93 million reads per case were obtained. Reducing the sequencing depth to 10 million reads per case was found to have no adverse effect on the sensitivity and specificity for cancer detection, giving respective figures of 68% and 94%. This characteristic thus indicates that analysis of plasma hypomethylation by this sequencing-based method may be a relatively cost-effective approach for cancer detection. We also demonstrated that plasma hypomethylation had utility for monitoring hepatocellular carcinoma patients following tumor resection and for detecting residual disease. Plasma hypomethylation can be combined with plasma CNA analysis for further enhancement of the detection sensitivity or specificity using different diagnostic algorithms. Using the detection of at least one type of aberration to define an abnormality, a sensitivity of 87% could be achieved with a specificity of 88%. These developments have thus expanded the applications of plasma DNA analysis for cancer detection and monitoring.

iSeeRNA: identification of long intergenic non-coding RNA transcripts from transcriptome sequencing data

BackgroundLong intergenic non-coding RNAs (lincRNAs) are emerging as a novel class of non-coding RNAs and potent gene regulators. High-throughput RNA-sequencing combined with de novo assembly promises quantity discovery of novel transcripts. However, the identification of lincRNAs from thousands of assembled transcripts is still challenging due to the difficulties of separating them from protein coding transcripts (PCTs).ResultsWe have implemented iSeeRNA, a support vector machine (SVM)-based classifier for the identification of lincRNAs. iSeeRNA shows better performance compared to other software. A public available webserver for iSeeRNA is also provided for small size dataset.ConclusionsiSeeRNA demonstrates high prediction accuracy and runs several magnitudes faster than other similar programs. It can be integrated into the transcriptome data analysis pipelines or run as a web server, thus offering a valuable tool for lincRNA study.

LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration

Emerging studies document the roles of long non-coding RNAs (LncRNAs) in regulating gene expression at chromatin level but relatively less is known how they regulate DNA methylation. Here we identify an lncRNA, Dum (developmental pluripotency-associated 2 (Dppa2) Upstream binding Muscle lncRNA) in skeletal myoblast cells. The expression of Dum is dynamically regulated during myogenesis in vitro and in vivo. It is also transcriptionally induced by MyoD binding upon myoblast differentiation. Functional analyses show that it promotes myoblast differentiation and damage-induced muscle regeneration. Mechanistically, Dum was found to silence its neighboring gene, Dppa2, in cis through recruiting Dnmt1, Dnmt3a and Dnmt3b. Furthermore, intrachromosomal looping between Dum locus and Dppa2 promoter is necessary for Dum/Dppa2 interaction. Collectively, we have identified a novel lncRNA that interacts with Dnmts to regulate myogenesis.

Genome-wide survey by ChIP-seq reveals YY1 regulation of lincRNAs in skeletal myogenesis.

Skeletal muscle differentiation is orchestrated by a network of transcription factors, epigenetic regulators, and non‐coding RNAs. The transcription factor Yin Yang 1 (YY1) silences multiple target genes in myoblasts (MBs) by recruiting Ezh2 (Enhancer of Zeste Homologue2). To elucidate genome‐wide YY1 binding in MBs, we performed chromatin immunoprecipitation (ChIP)‐seq and found 1820 specific binding sites in MBs with a large portion residing in intergenic regions. Detailed analysis demonstrated that YY1 acts as an activator for many loci in addition to its known repressor function. No significant co‐occupancy was found between YY1 and Ezh2, suggesting an additional Ezh2‐independent function for YY1 in MBs. Further analysis of intergenic binding sites showed that YY1 potentially regulates dozens of large intergenic non‐coding RNAs (lincRNAs), whose function in myogenesis is underexplored. We characterized a novel muscle‐associated lincRNA (Yam‐1) that is positively regulated by YY1. Yam‐1 is downregulated upon differentiation and acts as an inhibitor of myogenesis. We demonstrated that Yam‐1 functions through in cis regulation of miR‐715, which in turn targets Wnt7b. Our findings not only provide the first genome‐wide picture of YY1 association in muscle cells, but also uncover the functional role of lincRNA Yam‐1.

Noninvasive Prenatal Methylomic Analysis by Genomewide Bisulfite Sequencing of Maternal Plasma DNA

BACKGROUNDnEpigenetic mechanisms play an important role in prenatal development, but fetal tissues are not readily accessible. Fetal DNA molecules are present in maternal plasma and can be analyzed noninvasively.nnnMETHODSnWe applied genomewide bisulfite sequencing via 2 approaches to analyze the methylation profile of maternal plasma DNA at single-nucleotide resolution. The first approach used maternal blood samples and polymorphic differences between the mother and fetus to analyze the fetal methylome across the genome. The second approach used the methylation profile of maternal blood cells and the fractional fetal DNA concentration in maternal plasma to deduce the placental methylomic profile from maternal plasma DNA-sequencing data.nnnRESULTSnBecause of the noninvasive nature of these approaches, we were able to serially assess the methylation profiles of fetal, placental, and maternal plasma with maternal blood samples collected in the first and third trimesters and after delivery. Gestation-related changes were observed. The fetal methylation profile deduced from maternal plasma data resembled that of the placental methylome, both on a genomewide level and per CpG site. Imprinted genes and differentially methylated regions were identified from the maternal plasma data. We demonstrated one potential clinical application of maternal plasma bisulfite sequencing with the successful detection of fetal trisomy 21.nnnCONCLUSIONSnWe successfully analyzed fetal and placental methylomes on a genomewide scale, noninvasively and serially. This development offers a powerful method for research, biomarker discovery, and clinical testing for pregnancy-related disorders.

A Novel YY1-miR-1 Regulatory Circuit in Skeletal Myogenesis Revealed by Genome-Wide Prediction of YY1-miRNA Network

microRNAs (miRNAs) are non-coding RNAs that regulate gene expression post-transcriptionally, and mounting evidence supports the prevalence and functional significance of their interplay with transcription factors (TFs). Here we describe the identification of a regulatory circuit between muscle miRNAs (miR-1, miR-133 and miR-206) and Yin Yang 1 (YY1), an epigenetic repressor of skeletal myogenesis in mouse. Genome-wide identification of potential down-stream targets of YY1 by combining computational prediction with expression profiling data reveals a large number of putative miRNA targets of YY1 during skeletal myoblasts differentiation into myotubes with muscle miRs ranking on top of the list. The subsequent experimental results demonstrate that YY1 indeed represses muscle miRs expression in myoblasts and the repression is mediated through multiple enhancers and recruitment of Polycomb complex to several YY1 binding sites. YY1 regulating miR-1 is functionally important for both C2C12 myogenic differentiation and injury-induced muscle regeneration. Furthermore, we demonstrate that miR-1 in turn targets YY1, thus forming a negative feedback loop. Together, these results identify a novel regulatory circuit required for skeletal myogenesis and reinforce the idea that regulatory circuitries involving miRNAs and TFs are prevalent mechanisms.

A Novel Wnt Regulatory Axis in Endometrioid Endometrial Cancer

The Protocadherin 10 (PCDH10) is inactivated often by promoter hypermethylation in various human tumors, but its possible functional role as a tumor suppressor gene is not established. In this study, we identify PCDH10 as a novel Wnt pathway regulatory element in endometrioid endometrial carcinoma (EEC). PCDH10 was downregulated in EEC tumor cells by aberrant methylation of its promoter. Restoring PCDH10 levels suppressed cell growth and triggered apoptosis in EEC cells and tumor xenografts. Gene expression profiling revealed as part of the transcriptomic changes induced by PCDH10 a reduction in levels of MALAT1, a long noncoding RNA, that mediated tumor suppression functions of PCDH10 in EEC cells. We found that MALAT1 transcription was regulated by Wnt/β-catenin signaling via TCF promoter binding and PCDH10 decreased MALAT1 by modulating this pathway. Clinically, MALAT1 expression was associated with multiple parameters in patients with EEC. Taken together, our findings establish a novel PCDH10-Wnt/β-catenin-MALAT1 regulatory axis that contributes to EEC development. Cancer Res; 74(18); 5103-17. ©2014 AACR.

Pax3/7BP Is a Pax7- and Pax3-Binding Protein that Regulates the Proliferation of Muscle Precursor Cells by an Epigenetic Mechanism

In mouse skeletal muscles, Pax7 uniquely marks muscle satellite cells and plays some important yet unknown functions at the perinatal stage. To elucidate its in vivo functions, we initiated a yeast two-hybrid screening to look for Pax7-interacting proteins and identified a previously uncharacterized Pax7- and Pax3-binding protein (Pax3/7BP). Pax3/7BP is a ubiquitously expressed nuclear protein, enriched in Pax7+ muscle precursor cells (MPCs), and serves as an indispensable adaptor for Pax7 to recruit the histone 3 lysine 4 (H3K4) methyltransferase (HMT) complex by bridging Pax7 and Wdr5. Knockdown of Pax3/7BP abolished the Pax3/7-associated H3K4 HMT activity and inhibited the proliferation of Pax7+ MPCs from young mice both in culture and in vivo. Id3 and Cdc20 were direct target genes of Pax7 and Pax3/7BP involved in the proliferation of Pax7+ MPCs. Collectively, our work establishes Pax3/7BP as an essential adaptor linking Pax3/7 with the H3K4 HMT to regulate the proliferation of MPCs.

Linc-YY1 promotes myogenic differentiation and muscle regeneration through an interaction with the transcription factor YY1

Little is known how lincRNAs are involved in skeletal myogenesis. Here we describe the discovery of Linc-YY1 from the promoter of the transcription factor (TF) Yin Yang 1 (YY1) gene. We demonstrate that Linc-YY1 is dynamically regulated during myogenesis in vitro and in vivo. Gain or loss of function of Linc-YY1 in C2C12 myoblasts or muscle satellite cells alters myogenic differentiation and in injured muscles has an impact on the course of regeneration. Linc-YY1 interacts with YY1 through its middle domain, to evict YY1/Polycomb repressive complex (PRC2) from target promoters, thus activating the gene expression in trans. In addition, Linc-YY1 also regulates PRC2-independent function of YY1. Finally, we identify a human Linc-YY1 orthologue with conserved function and show that many human and mouse TF genes are associated with lincRNAs that may modulate their activity. Altogether, we show that Linc-YY1 regulates skeletal myogenesis and uncover a previously unappreciated mechanism of gene regulation by lincRNA.