The E3 ubiquitin-protein ligase UHRF1 promotes adipogenesis and limits fibrosis by suppressing GPNMB-mediated TGF-ß signaling.

The E3 ubiquitin-ligase UHRF1 is an epigenetic regulator coordinating DNA methylation and histone modifications. However, little is known about how it regulates adipogenesis or metabolism. In this study, we discovered that UHRF1 is a key regulatory factor for adipogenesis, and we identified the altered molecular pathways that UHRF1 targets. Using CRISPR/Cas9-based knockout strategies, we discovered the whole transcriptomic changes upon UHRF1 deletion. Bioinformatics analyses revealed that key adipogenesis regulators such PPAR-γ and C/EBP-α were suppressed, whereas TGF-ß signaling and fibrosis markers were upregulated in UHRF1-depleted differentiating adipocytes. Furthermore, UHRF1-depleted cells showed upregulated expression and secretion of TGF-ß1, as well as the glycoprotein GPNMB. Treating differentiating preadipocytes with recombinant GPNMB led to an increase in TGF-ß protein and secretion levels, which was accompanied by an increase in secretion of fibrosis markers such as MMP13 and a reduction in adipogenic conversion potential. Conversely, UHRF1 overexpression studies in human cells demonstrated downregulated levels of GPNMB and TGF-ß, and enhanced adipogenic potential. In conclusion, our data show that UHRF1 positively regulates 3T3-L1 adipogenesis and limits fibrosis by suppressing GPNMB and TGF-ß signaling cascade, highlighting the potential relevance of UHRF1 and its targets to the clinical management of obesity and linked metabolic disorders.

Silencing of ANKRD12 circRNA induces molecular and functional changes associated with invasive phenotypes.

BACKGROUND: Circular RNAs (circRNAs) that form through non-canonical backsplicing events of pre-mRNA transcripts are evolutionarily conserved and abundantly expressed across species. However, the functional relevance of circRNAs remains a topic of debate. METHODS: We identified one of the highly expressed circRNA (circANKRD12) in cancer cell lines and characterized it validated it by Sanger sequencing, Real-Time PCR. siRNA mediated silencing of the circular junction of circANKRD12 was followed by RNA Seq analysis of circANKRD12 silenced cells and control cells to identify the differentially regulated genes. A series of cell biology and molecular biology techniques (MTS assay, Migration analysis, 3D organotypic models, Real-Time PCR, Cell cycle analysis, Western blot analysis, and Seahorse Oxygen Consumption Rate analysis) were performed to elucidate the function, and underlying mechanisms involved in circANKRD12 silenced breast and ovarian cancer cells. RESULTS: In this study, we identified and characterized a circular RNA derived from Exon 2 and Exon 8 of the ANKRD12 gene, termed here as circANKRD12. We show that this circRNA is abundantly expressed in breast and ovarian cancers. The circANKRD12 is RNase R resistant and predominantly localized in the cytoplasm in contrast to its source mRNA. We confirmed the expression of this circRNA across a variety of cancer cell lines and provided evidence for its functional relevance through downstream regulation of several tumor invasion genes. Silencing of circANKRD12 induces a strong phenotypic change by significantly regulating cell cycle, increasing invasion and migration and altering the metabolism in cancer cells. These results reveal the functional significance of circANKRD12 and provide evidence of a regulatory role for this circRNA in cancer progression. CONCLUSIONS: Our study demonstrates the functional relevance of circANKRD12 in various cancer cell types and, based on its expression pattern, has the potential to become a new clinical biomarker.

Genotyping-by-sequencing identifies date palm clone preference in agronomics of the State of Qatar.

Understanding the genetic diversity in a crop population is key to its targeted breeding for desired traits, such as higher yields, better fruit quality and resistance to disease and changing climates. Date fruits represent a major crop in the Middle East and are key to achieving future food independence in arid countries like Qatar. We previously determined the genome of the date palm Phoenix dactylifera and showed that date palm trees world-wide divide into two distinct subpopulations of Eastern and Western origins. Here we applied a resource of SNPs from 179 commercially available date fruits to assess the genetic diversity of date palm trees grown in the State of Qatar. We found that palm trees in Qatar are mainly of Eastern origin, and that their genetic diversity doesn't associate with regions of the State. Together with targeted genetic assays, our resource can be used in the future for date palm cultivar identification, to aid selecting suitable cultivars for targeted breeding, to improve a country's date palm genetic diversity, and to certify the origin of date fruits and trees.

Polymorphism at expressed DQ and DR loci in five common equine MHC haplotypes.

The polymorphism of major histocompatibility complex (MHC) class II DQ and DR genes in five common equine leukocyte antigen (ELA) haplotypes was determined through sequencing of mRNA transcripts isolated from lymphocytes of eight ELA homozygous horses. Ten expressed MHC class II genes were detected in horses of the ELA-A3 haplotype carried by the donor horses of the equine bacterial artificial chromosome (BAC) library and the reference genome sequence: four DR genes and six DQ genes. The other four ELA haplotypes contained at least eight expressed polymorphic MHC class II loci. Next generation sequencing (NGS) of genomic DNA of these four MHC haplotypes revealed stop codons in the DQA3 gene in the ELA-A2, ELA-A5, and ELA-A9 haplotypes. Few NGS reads were obtained for the other MHC class II genes that were not amplified in these horses. The amino acid sequences across haplotypes contained locus-specific residues, and the locus clusters produced by phylogenetic analysis were well supported. The MHC class II alleles within the five tested haplotypes were largely non-overlapping between haplotypes. The complement of equine MHC class II DQ and DR genes appears to be well conserved between haplotypes, in contrast to the recently described variation in class I gene loci between equine MHC haplotypes. The identification of allelic series of equine MHC class II loci will aid comparative studies of mammalian MHC conservation and evolution and may also help to interpret associations between the equine MHC class II region and diseases of the horse.

Altered expression pattern of circular RNAs in primary and metastatic sites of epithelial ovarian carcinoma.

Recently, a class of endogenous species of RNA called circular RNA (circRNA) has been shown to regulate gene expression in mammals and their role in cellular function is just beginning to be understood. To investigate the role of circRNAs in ovarian cancer, we performed paired-end RNA sequencing of primary sites, peritoneal and lymph node metastases from three patients with stage IIIC ovarian cancer. We developed an in-house computational pipeline to identify and characterize the circRNA expression from paired-end RNA-Seq libraries. This pipeline revealed thousands of circular isoforms in Epithelial Ovarian Carcinoma (EOC). These circRNAs are enriched for potentially effective miRNA seed matches. A significantly larger number of circRNAs are differentially expressed between tumor sites than mRNAs. Circular and linear expression exhibits an inverse trend for many cancer related pathways and signaling pathways like NFkB, PI3k/AKT and TGF-ß typically activated for mRNA in metastases are inhibited for circRNA expression. Further, circRNAs show a more robust expression pattern across patients than mRNA forms indicating their suitability as biomarkers in highly heterogeneous cancer transcriptomes. The consistency of circular RNA expression may offer new candidates for cancer treatment and prognosis.

A Genome-Wide Survey of Date Palm Cultivars Supports Two Major Subpopulations in Phoenix dactylifera.

The date palm (Phoenix dactylifera L.) is one of the oldest cultivated trees and is intimately tied to the history of human civilization. There are hundreds of commercial cultivars with distinct fruit shapes, colors, and sizes growing mainly in arid lands from the west of North Africa to India. The origin of date palm domestication is still uncertain, and few studies have attempted to document genetic diversity across multiple regions. We conducted genotyping-by-sequencing on 70 female cultivar samples from across the date palm-growing regions, including four Phoenix species as the outgroup. Here, for the first time, we generate genome-wide genotyping data for 13,000-65,000 SNPs in a diverse set of date palm fruit and leaf samples. Our analysis provides the first genome-wide evidence confirming recent findings that the date palm cultivars segregate into two main regions of shared genetic background from North Africa and the Arabian Gulf. We identify genomic regions with high densities of geographically segregating SNPs and also observe higher levels of allele fixation on the recently described X-chromosome than on the autosomes. Our results fit a model with two centers of earliest cultivation including date palms autochthonous to North Africa. These results adjust our understanding of human agriculture history and will provide the foundation for more directed functional studies and a better understanding of genetic diversity in date palm.

A first genetic map of date palm (Phoenix dactylifera) reveals long-range genome structure conservation in the palms.

BACKGROUND: The date palm is one of the oldest cultivated fruit trees. It is critical in many ways to cultures in arid lands by providing highly nutritious fruit while surviving extreme heat and environmental conditions. Despite its importance from antiquity, few genetic resources are available for improving the productivity and development of the dioecious date palm. To date there has been no genetic map and no sex chromosome has been identified. RESULTS: Here we present the first genetic map for date palm and identify the putative date palm sex chromosome. We placed ~4000 markers on the map using nearly 1200 framework markers spanning a total of 1293 cM. We have integrated the genetic map, derived from the Khalas cultivar, with the draft genome and placed up to 19% of the draft genome sequence scaffolds onto linkage groups for the first time. This analysis revealed approximately ~1.9 cM/Mb on the map. Comparison of the date palm linkage groups revealed significant long-range synteny to oil palm. Analysis of the date palm sex-determination region suggests it is telomeric on linkage group 12 and recombination is not suppressed in the full chromosome. CONCLUSIONS: Based on a modified genotyping-by-sequencing approach we have overcome challenges due to lack of genetic resources and provide the first genetic map for date palm. Combined with the recent draft genome sequence of the same cultivar, this resource offers a critical new tool for date palm biotechnology, palm comparative genomics and a better understanding of sex chromosome development in the palms.

A custom 148 gene-based resequencing chip and the SNP explorer software: new tools to study antibody deficiency.

Hyper-IgM syndrome and Common Variable Immunodeficiency are heterogeneous disorders characterized by a predisposition to serious infection and impaired or absent neutralizing antibody responses. Although a number of single gene defects have been associated with these immune deficiency disorders, the genetic basis of many cases is not known. To facilitate mutation screening in patients with these syndromes, we have developed a custom 300-kb resequencing array, the Hyper-IgM/CVID chip, which interrogates 1,576 coding exons and intron-exon junction regions from 148 genes implicated in B-cell development and immunoglobulin isotype switching. Genomic DNAs extracted from patients were hybridized to the array using a high-throughput protocol for target sequence amplification, pooling, and hybridization. A Web-based application, SNP Explorer, was developed to directly analyze and visualize the single nucleotide polymorphism (SNP) annotation and for quality filtering. Several mutations in known disease-susceptibility genes such as CD40LG, TNFRSF13B, IKBKG, AICDA, as well as rare nucleotide changes in other genes such as TRAF3IP2, were identified in patient DNA samples and validated by direct sequencing. We conclude that the Hyper-IgM/CVID chip combined with SNP Explorer may provide a cost-effective tool for high-throughput discovery of novel mutations among hundreds of disease-relevant genes in patients with inherited antibody deficiency.