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XLS file - 34K, KEGG Pathway Categories of Genes Highly Enriched in hESCs, HCCs and hPHs

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PDF file - 1.3MB, S1. Pearson correlation analysis of hESCs, HCCs and hPHS. S2. EHCC miRNAs are not capable of rescuing hepatic progenitor cell induction in Dgcr8 null mESCs during the in vitro differentiation process of mESCs along a hepatic lineage. S3. EHCC miRNAs cannot rescue the deficiency in hepatocyte differentiation in Dgcr8 null mESCs. S4. Experimental timeline of hPHs. S5. Global gene expression analysis between hPHs and hPHs expressing EHCC miRNAs. S6. Expression patterns of genes reported to be associated with HCCs, ESCs and hepatic progenitor stem cells following EHCC miRNA overexpression in hPHs

XLS file - 42K, KEGG Pathway Categories of Differentially Up-Regulated Genes Following EHCC miRNA Expression in hPHs

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This article traces the journey of two indigenous textile fibres—Kala cotton and Desi Oon—and the efforts of farmers, pastoralists, craft artisans and Khamir, to conserve, sustain and promote the natural and cultural heritage that have nourished these fibres. It recreates the revival of these fibres in Kutch, a semi-arid region of India, and unfolds the technical, cultural and creative challenges that have accompanied this process—from land to the loom. Large-scale shifts in Indian agriculture have marked a significant breakdown of the value loops and deep interdependent economies between farmers, spinners, weavers, dyers and tailors. First, in the nineteenth century, when the cultivation of short staple indigenous varieties of cotton shifted to long staple cotton, which could feed the mills of Britain; and then in the late twentieth century when a global push towards hybrid, genetically modified varieties of cotton strode over the farming of more resilient local cotton varieties. Similarly, across India, vast genetic resource of sheep, yak and camels, in particular, have, for centuries, been conserved and bred by the nomadic pastoral communities of these regions. Even two and a half decade ago, the animal’s fibre was used extensively by the pastoralists themselves and fetched them a market price for the wool that was almost equal to that of meat. However, with India becoming a major sheep meat producing country, accompanied by the relaxation of tariffs on wool imports in the early nineties, the Indian wool market has moved completely to softer, longer staple wool from New Zealand, Australia and the Middle East. Today, this is the only natural fibre in which India is deficient despite having the third largest number of sheep in the world! The process of conserving indigenous cotton seeds, sheep breeds, their fibres and a range of associated textile crafts, challenge all the essential premises on which the political economy of contemporary textiles rest. However hope comes in many forms—the intense climatic variabilities and growing vulnerabilities to climate change, is compelling farmers and pastoralists to revisit the choices they have been compelled to adopt by a globalized market economy. Their growing understanding and need to revive their local cotton seeds and animal breeds, which are more adaptable and resilient to climatic variabilities, hold out a tremendous opportunity to transform the landscape of indigenous textile fibres. Through her experiences, the author stitches together a story of localization wherein Khamir attempts to restore a lost heritage and the bonds between farmers, pastoralists, spinners, weavers and dyers.

Single-nucleotide polymorphism (SNP)-based non-invasive prenatal testing (NIPT) can currently predict a subset of submicroscopic abnormalities associated with severe clinical manifestations. We retrospectively analyzed the performance of SNP-based NIPT in 80 449 referrals for 22q11.2 deletion syndrome and 42 326 referrals for 1p36, cri-du-chat, Prader-Willi, and Angelman microdeletion syndromes over a 1-year period, and compared the original screening protocol with a revision that reflexively sequenced high-risk calls at a higher depth of read. The prevalence of these microdeletion syndromes was also estimated in the referral population. The positive predictive value of the original test was 15.7% for 22q11.2 deletion syndrome, and 5.2% for the other 4 disorders combined. With the revised protocol, these values increased to 44.2% for 22q11.2 and 31.7% for the others. The 0.33% false-positive rate (FPR) for 22q11.2 deletion syndrome decreased to 0.07% with the revised protocol. Similarly, the FPR for the other 4 disorders combined decreased from 0.56% to 0.07%. Minimal prevalences were estimated to be 1 in 1255 for 22q11.2 deletion syndrome and 1 in 1464 for 1p36, cri-du-chat, and Angelman syndromes combined. Our results show that these microdeletions are relatively common in the referral population, and that the performance of SNP-based NIPT is improved with high-depth resequencing.

Background The 22q11.2 deletion syndrome is a variably expressed disorder that can include cardiac, palate, and other physical abnormalities, immunodeficiency, and hypocalcemia. Because of the extreme variability in phenotype, there has been no available estimate of the total medical expenditure associated with the average case. Methods We have developed a model to estimate the cost from the time of diagnosis to age 20. Costs were based on patients seen at a specialty center but also considered those components of care expected to have been provided by external healthcare facilities. Expense was based on billed medical charges extracted from the electronic medical billing system for all patients with a diagnosis of DiGeorge or velocardiofacial syndrome from 1993–2015. Expenditures included maternal prenatal care directly related to an affected pregnancy, molecular/cytogenetic diagnosis, consultations, surgery, and/or other treatment and management. Most mental health services (except inpatient), therapy related to cognitive, behavioral, speech, pharmacy, and nonmedical costs (special education, vocational, respite, lost earnings) were not included. Results Data were available for 642 patients with 50.7% diagnosed prenatally or in the first year of life. The average cost for a patient was $727,178. Costs were highest for patients ascertained prenatally ($2,599,955) or in the first year of life ($1,043,096), those with cardiac abnormalities or referred for cardiac evaluation ($751,535), and patients with low T-cell counts ($1,382,222). Conclusion This study demonstrates that there are significant medical costs associated with 22q11.2 deletion syndrome.

Non-invasive prenatal testing (NIPT) has expanded its coverage beyond the common aneuploidies to include subchromosomal abnormalities such as the 22q11.2 deletion through the analysis of cell-free DNA. We previously reported a positive predictive value (PPV) of 18% and false-positive rate (FPR) of 0.38% for a single-nucleotide polymorphism (SNP)-based NIPT for fetal 22q11.2 deletion in a clinical cohort. Herein, we assess the performance of the test following methodology changes that include sequencing high-risk calls at high depth of read (HDOR) and raising the confidence threshold of the algorithm from 0.90 to 0.95. At the original confidence cut-off, a PPV of 42.3% upon reflex-sequencing of high-risk samples at a HDOR was reported. Raising the algorithm’s confidence threshold further increased the PPV to 52.4% and reduced the FPR to 0.07%, with no loss in test sensitivity. The PPV was 100% for high a priori risk cases, and 20% for low a priori risk cases. The improved assay performance documented with the updated methodology supports the efficacy of the SNP-based NIPT in the detection of fetal 22q11.2 deletion.

Background The 22q11.2 deletion syndrome is the most common microdeletion syndrome in livebirths, but data regarding its incidence in other populations is limited and also include ascertainment bias. This study was designed to determine the incidence of the 22q11.2 deletion in miscarriage samples sent for clinical molecular cytogenetic testing. Results Twenty-six thousand one hundred one fresh product of conception (POC) samples were sent to a CLIA- certified, CAP-accredited laboratory from April 2010–-May 2016 for molecular cytogenetic miscarriage testing using a single-nucleotide polymorphism (SNP)-based microarray platform. A retrospective review determined the incidence of the 22q11.2 deletion in this sample set. Fetal results were obtained in 22,451 (86%) cases, of which, 15 (0.07%) had a microdeletion in the 22q11.2 region (incidence, 1/1497). Of those, 12 (80%) cases were found in samples that were normal at the resolution of traditional karyotyping (i.e., had no chromosome abnormalities above 10 Mb in size) and three (20%) cases had additional findings (Trisomy 15, Trisomy 16, XXY). Ten (67%) cases with a 22q11.2 deletion had the common ~3 Mb deletion; the remaining 5 cases had deletions ranging in size from 0.65 to 1.5 Mb. A majority (12/15) of cases had a deletion on the maternally inherited chromosome. No significant relationship between maternal age and presence of a fetal 22q11.2 deletion was observed. Conclusions The observed incidence of 1/1497 for the 22q11.2 deletion in miscarriage samples is higher than the reported general population prevalence (1/4000–1/6000). Further research is needed to determine whether the 22q11.2 deletion is a causal factor for miscarriage.

The self-renewal capacity ascribed to embryonic stem cells (ESC) is reminiscent of cancer cell proliferation, raising speculation that a common network of genes may regulate these traits. A search for general regulators of these traits yielded a set of microRNAs for which expression is highly enriched in human ESCs and liver cancer cells (HCC) but attenuated in differentiated quiescent hepatocytes. Here, we show that these microRNAs promote hESC self-renewal, as well as HCC proliferation, and when overexpressed in normally quiescent hepatocytes, induce proliferation and activate cancer signaling pathways. Proliferation in hepatocytes is mediated through translational repression of Pten, Tgfbr2, Klf11, and Cdkn1a, which collectively dysregulates the PI3K/AKT/mTOR and TGFβ tumor suppressor signaling pathways. Furthermore, aberrant expression of these miRNAs is observed in human liver tumor tissues and induces epithelial–mesenchymal transition in hepatocytes. These findings suggest that microRNAs that are essential in normal development as promoters of ESC self-renewal are frequently upregulated in human liver tumors and harbor neoplastic transformation potential when they escape silencing in quiescent human hepatocytes. Mol Cancer Res; 10(7); 979–91. ©2012 AACR.

In mammalian cells, multiple cellular processes, including gene silencing, cell growth and differentiation, pluripotency, neoplastic transformation, apoptosis, DNA repair, and maintenance of genomic integrity, converge on the evolutionarily conserved protein KAP1, which is thought to regulate the dynamic organization of chromatin structure via its ability to influence epigenetic patterns and chromatin compaction. In this minireview, we discuss how KAP1 might execute such pleiotropic effects, focusing on genomic targeting mechanisms, protein-protein interactions, specific post-translational modifications of both KAP1 and associated histones, and transcriptome analyses of cells deficient in KAP1.

We compared 12 different cell populations, including embryonic stem cells before and during differentiation into embryoid bodies as well as various types of normal and tumor cells to determine if pluripotent versus differentiated cell types use different mechanisms to establish their transcriptome. We first identified genes that were not expressed in the 12 different cell populations and then determined which of them were regulated by histone methylation, DNA methylation, at the step of productive elongation, or by the inability to establish a preinitiation complex. For these experiments, we performed chromatin immunoprecipitation using antibodies to H3me3K27, H3me3K9, 5-methyl-cytosine, and POLR2A. We found that (1) the percentage of low expressed genes bound by POLR2A, H3me3K27, H3me3K9, or 5-methyl-cytosine is similar in all 12 cell types, regardless of differentiation or neoplastic state; (2) a gene is generally repressed by only one mechanism; and (3) distinct classes of genes are repressed by certain mechanisms. We further characterized two transitioning cell populations, 3T3 cells progressing from G0/G1 into S phase and mES cells differentiating into embryoid bodies. We found that the transient regulation through the cell cycle was achieved predominantly by changes in the recruitment of the general transcriptional machinery or by post-POLR2A recruitment mechanisms. In contrast, changes in chromatin silencing were critical for the permanent changes in gene expression in cells undergoing differentiation.

TRIM28 (KAP1) is upregulated in many cancers and has been implicated in both transcriptional activation and repression. Using chromatin immunoprecipitation and sequencing, we show that KAP1 binding sites fall into several categories, specifically, the 3' coding exons of zinc finger (ZNF) genes and promoter regions of ZNFs and other genes. The currently accepted model is that KAP1 is recruited to the genome via interaction of its N-terminal RBCC domain with KRAB ZNFs (KRAB domain containing ZNFs). To determine whether the interaction of KAP1 with KRAB ZNFs is the mechanism by which KAP1 is recruited to genomic binding sites, we analyzed stable cell lines that express tagged wild-type and mutant KAP1. Surprisingly, deletion of the RBCC domain abolished KAP1 binding to the 3' exons of ZNF genes but KAP1 binding to promoter regions was unaffected. Using KAP1 knockdown cells, we showed that the genes most responsive to KAP1 were not ZNF genes but instead were either indirect targets or had KAP1 bound 10 to 100 kb from the transcription start site. Therefore, our studies suggest that KAP1 plays a role distinct from transcriptional regulation at the majority of its strongest binding sites.

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome., National Human Genome Research Institute (U.S.), National Institutes of Health (U.S.)

The H3K9me3 histone modification is often found at promoter regions, where it functions to repress transcription. However, we have previously shown that 3′ exons of zinc finger genes (ZNFs) are marked by high levels of H3K9me3. We have now further investigated this unusual location for H3K9me3 in ZNF genes. Neither bioinformatic nor experimental approaches support the hypothesis that the 3′ exons of ZNFs are promoters. We further characterized the histone modifications at the 3′ ZNF exons and found that these regions also contain H3K36me3, a mark of transcriptional elongation. A genome-wide analysis of ChIP-seq data revealed that ZNFs constitute the majority of genes that have high levels of both H3K9me3 and H3K36me3. These results suggested the possibility that the ZNF genes may be imprinted, with one allele transcribed and one allele repressed. To test the hypothesis that the contradictory modifications are due to imprinting, we used a SNP analysis of RNA-seq data to demonstrate that both alleles of certain ZNF genes having H3K9me3 and H3K36me3 are transcribed. We next analyzed isolated ZNF 3′ exons using stably integrated episomes. We found that although the H3K36me3 mark was lost when the 3′ ZNF exon was removed from its natural genomic location, the isolated ZNF 3′ exons retained the H3K9me3 mark. Thus, the H3K9me3 mark at ZNF 3′ exons does not impede transcription and it is regulated independently of the H3K36me3 mark. Finally, we demonstrate a strong relationship between the number of tandemly repeated domains in the 3′ exons and the H3K9me3 mark. We suggest that the H3K9me3 at ZNF 3′ exons may function to protect the genome from inappropriate recombination rather than to regulate transcription.

The self-renewal capacity ascribed to hESCs is paralleled in cancer cell proliferation, suggesting that a common network of genes may facilitate the promotion of these traits. However, the molecular mechanisms that are involved in regulating the silencing of these genes as stem cells differentiate into quiescent cellular lineages remain poorly understood. Here, we show that a differentiated cell specific miR-122 exemplifies this regulatory attribute by suppressing the translation of a gene, Pkm2, which is commonly enriched in hESCs and liver cancer cells (HCCs), and facilitates self-renewal and proliferation. Through a series of gene expression analysis, we show that miR-122 expression is highly elevated in quiescent human primary hepatocytes (hPHs) but lost or attenuated in hESCs and HCCs, while an opposing expression pattern is observed for Pkm2. Depleting hESCs and HCCs of Pkm2, or overexpressing miR-122, leads to a common deficiency in self-renewal and proliferation. Likewise, during the differentiation process of hESCs into hepatocytes, a reciprocal expression pattern is observed between miR-122 and Pkm2. An examination of the genomic region upstream of miR-122 uncovered hyper-methylation in hESCs and HCCs, while the same region is de-methylated and occupied by a transcription initiating protein, RNA polymerase II (RNAPII), in hPHs. These findings indicate that one possible mechanism by which hESC self-renewal is modulated in quiescent hepatic derivatives of hESCs is through the regulatory activity of a differentiated cell-specific miR-122, and that a failure to properly turn “on” this miRNA is observed in uncontrollably proliferating HCCs.

Next-generation sequencing is revolutionizing the identification of transcription factor binding sites throughout the human genome. However, the bioinformatics analysis of large datasets collected using chromatin immunoprecipitation and high-throughput sequencing is often a roadblock that impedes researchers in their attempts to gain biological insights from their experiments. We have developed integrated peak-calling and analysis software (Sole-Search) which is available through a user-friendly interface and (i) converts raw data into a format for visualization on a genome browser, (ii) outputs ranked peak locations using a statistically based method that overcomes the significant problem of false positives, (iii) identifies the gene nearest to each peak, (iv) classifies the location of each peak relative to gene structure, (v) provides information such as the number of binding sites per chromosome and per gene and (vi) allows the user to determine overlap between two different experiments. In addition, the program performs an analysis of amplified and deleted regions of the input genome. This software is web-based and automated, allowing easy and immediate access to all investigators. We demonstrate the utility of our software by collecting, analyzing and comparing ChIP-seq data for six different human transcription factors/cell line combinations.

Poster presentation Advances in high-throughput technologies such as ChIPchip and the completion of human genomic sequences allow analysis of the mechanisms of gene regulation on a systems level. In this study, we have developed a computational genomics approach (ChIPModules) and a motif discovery approach (ChIPMotifs) to mine the ChIP-chip data. The ChIPModules approach begins with experimentally determined binding sites and integrates positional weight matrices, a comparative genomics approach, and statistical learning methods to identify transcriptional regulatory modules. Using E2F1 ChIP-chip data performed on ENCODE regions in both HeLa and MCF7 cells, we have identified five regulatory modules for E2F1. One of modules was validated by using ChIP-chip with arrays containing ~14,000 human promoters. The ChIPMotifs approach incorporates a bootstrap re-sampling method to statistically infer the optimal cutoff threshold for a position weight matrix (PWM) of a motif identified from ChIP-chip data by ab initio motif discovery programs. Using OCT4 ChIP-chip data, we developed an in vivo OCT4 PWM. We then used this PWM and our ChIPModules to identify transcription factors co-localizing with OCT4 in a testicular germ cell tumor (Ntera2 cells).