Gene expression and splicing alterations analyzed by high throughput RNA sequencing of chronic lymphocytic leukemia specimens.

BackgroundTo determine differentially expressed and spliced RNA transcripts in chronic lymphocytic leukemia specimens a high throughput RNA-sequencing (HTS RNA-seq) analysis was performed.MethodsTen CLL specimens and five normal peripheral blood CD19+ B cells were analyzed by HTS RNA-seq. The library preparation was performed with Illumina TrueSeq RNA kit and analyzed by Illumina HiSeq 2000 sequencing system.ResultsAn average of 48.5 million reads for B cells, and 50.6 million reads for CLL specimens were obtained with 10396 and 10448 assembled transcripts for normal B cells and primary CLL specimens respectively. With the Cuffdiff analysis, 2091 differentially expressed genes (DEG) between B cells and CLL specimens based on FPKM (fragments per kilobase of transcript per million reads and false discovery rate, FDR q < 0.05, fold change >2) were identified. Expression of selected DEGs (n = 32) with up regulated and down regulated expression in CLL from RNA-seq data were also analyzed by qRT-PCR in a test cohort of CLL specimens. Even though there was a variation in fold expression of DEG genes between RNA-seq and qRT-PCR; more than 90 % of analyzed genes were validated by qRT-PCR analysis. Analysis of RNA-seq data for splicing alterations in CLL and B cells was performed by Multivariate Analysis of Transcript Splicing (MATS analysis). Skipped exon was the most frequent splicing alteration in CLL specimens with 128 significant events (P-value <0.05, minimum inclusion level difference >0.1).ConclusionThe RNA-seq analysis of CLL specimens identifies novel DEG and alternatively spliced genes that are potential prognostic markers and therapeutic targets. High level of validation by qRT-PCR for a number of DEG genes supports the accuracy of this analysis. Global comparison of transcriptomes of B cells, IGVH non-mutated CLL (U-CLL) and mutated CLL specimens (M-CLL) with multidimensional scaling analysis was able to segregate CLL and B cell transcriptomes but the M-CLL and U-CLL transcriptomes were indistinguishable. The analysis of HTS RNA-seq data to identify alternative splicing events and other genetic abnormalities specific to CLL is an added advantage of RNA-seq that is not feasible with other genome wide analysis

<i>STAG2 </i>mutations reshape the cohesin-structured spatial chromatin architecture to drive gene regulation in acute myeloid leukemia

Cohesin shapes the chromatin architecture, including enhancer-promoter interactions. Its components, especially STAG2, but not its paralog STAG1, are frequently mutated in myeloid malignancies. To elucidate the underlying mechanisms of leukemogenesis, we comprehensively characterized genetic, transcriptional, and chromatin conformational changes in acute myeloid leukemia (AML) patient samples. Specific loci displayed altered cohesin occupancy, gene expression, and local chromatin activation, which were not compensated by the remaining STAG1-cohesin. These changes could be linked to disrupted spatial chromatin looping in cohesin-mutated AMLs. Complementary depletion of STAG2 or STAG1 in primary human hematopoietic progenitors (HSPCs) revealed effects resembling STAG2-mutant AML-specific changes following STAG2 knockdown, not invoked by the depletion of STAG1. STAG2-deficient HSPCs displayed impaired differentiation capacity and maintained HSPC-like gene expression. This work establishes STAG2 as a key regulator of chromatin contacts, gene expression, and differentiation in the hematopoietic system and identifies candidate target genes that may be implicated in human leukemogenesis

Epigenetic alterations affecting hematopoietic regulatory networks as drivers of mixed myeloid/lymphoid leukemia

Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias.</p

Update of the FANTOM web resource: from mammalian transcriptional landscape to its dynamic regulation

The international Functional Annotation Of the Mammalian Genomes 4 (FANTOM4) research collaboration set out to better understand the transcriptional network that regulates macrophage differentiation and to uncover novel components of the transcriptome employing a series of high-throughput experiments. The primary and unique technique is cap analysis of gene expression (CAGE), sequencing mRNA 5′-ends with a second-generation sequencer to quantify promoter activities even in the absence of gene annotation. Additional genome-wide experiments complement the setup including short RNA sequencing, microarray gene expression profiling on large-scale perturbation experiments and ChIP–chip for epigenetic marks and transcription factors. All the experiments are performed in a differentiation time course of the THP-1 human leukemic cell line. Furthermore, we performed a large-scale mammalian two-hybrid (M2H) assay between transcription factors and monitored their expression profile across human and mouse tissues with qRT-PCR to address combinatorial effects of regulation by transcription factors. These interdependent data have been analyzed individually and in combination with each other and are published in related but distinct papers. We provide all data together with systematic annotation in an integrated view as resource for the scientific community (http://fantom.gsc.riken.jp/4/). Additionally, we assembled a rich set of derived analysis results including published predicted and validated regulatory interactions. Here we introduce the resource and its update after the initial release

Carboxypeptidase-M is regulated by lipids and CSFs in macrophages and dendritic cells and expressed selectively in tissue granulomas and foam cells

Granulomatous inflammations, characterized by the presence of activated macrophages (MAs) forming epithelioid cell (EPC) clusters, are usually easy to recognize. However, in ambiguous cases the use of a MA marker that expresses selectively in EPCs may be needed. Here, we report that carboxypeptidase-M (CPM), a MA-differentiation marker, is preferentially induced in EPCs of all granuloma types studied, but not in resting MAs. As CPM is not expressed constitutively in MAs, this allows utilization of CPM-immunohistochemistry in diagnostics of minute granuloma detection when dense non-granulomatous MAs are also present. Despite this rule, hardly any detectable CPM was found in advanced/active tubercle caseous disease, albeit in early tuberculosis granuloma, MAs still expressed CPM. Indeed, in vitro both the CPM-protein and -mRNA became downregulated when MAs were infected with live mycobacteria. In vitro, MA-CPM transcript is neither induced remarkably by interferon-γ, known to cause classical MA activation, nor by IL-4, an alternative MA activator. Instead, CPM is selectively expressed in lipid-laden MAs, including the foam cells of atherosclerotic plaques, xanthomatous lesions and lipid pneumonias. By using serum, rich in lipids, and low-density lipoprotein (LDL) or VLDL, CPM upregulation could be reproduced in vitro in monocyte-derived MAs both at transcriptional and protein levels, and the increase is repressed under lipid-depleted conditions. The microarray analyses support the notion that CPM induction correlates with a robust progressive increase in CPM gene expression during monocyte to MA maturation and dendritic cell (DC) differentiation mediated by granulocyte–MA-colony-stimulating factor+IL-4. M-CSF alone also induced CPM. These results collectively indicate that CPM upregulation in MAs is preferentially associated with increased lipid uptake, and exposure to CSF, features of EPCs, also. Therefore, CPM-immunohistochemistry is useful for granuloma and foam MA detections in tissue sections. Furthermore, the present data offer CPM for the first time to be a novel marker and cellular player in lipid uptake and/or metabolism of MAs by promoting foam cell formation

Chitinase 3-like 1 expression by human (MG63) osteoblasts in response to lysophosphatidic acid and 1,25-Dihydroxyvitamin D3

Chitinase 3-like 1, otherwise known as YKL-40, is a secreted glycoprotein purported to have a role in extracellular matrix metabolism. The first mammalian cell type found to express YKL-40 was the human osteosarcoma-derived osteoblast, MG63. In that first study the active vitamin D3 metabolite, 1,25-dihydroxycholecalciferol (1,25D), stimulated YKL-40 expression, thereby indicating that a vital factor for skeletal health promoted YKL-40 synthesis by bone forming cells. However, when these MG63 cells were exposed to 1,25D they were also exposed to serum, a rich source of the pleiotropic lipid mediator, lysophosphatidic acid (LPA). Given that 1,25D is now known to co-operate with selected growth factors, including LPA, to influence human osteoblast differentiation we hypothesised that 1,25D and LPA may work together to stimulate osteoblast YKL-40 expression. Herein we report that 1,25D and LPA synergistically promote YKL-40 expression by MG63 cells. Inhibitors targeting AP1, MEK, Sp1 and STAT3 blunted the expression of both alkaline phosphatase and YKL-40 by MG63 cells in response to co-stimulation with 1,25D and LPA. Other ligands of the vitamin D receptor also co-operated with LPA in driving YKL-40 mobilisation. Collectively our findings highlight another important role of 1,25D and LPA in the regulation of human osteoblast function

Bcl-2 Inhibits the Innate Immune Response during Early Pathogenesis of Murine Congenital Muscular Dystrophy

Laminin α2 (LAMA2)-deficient congenital muscular dystrophy is a severe, early-onset disease caused by abnormal levels of laminin 211 in the basal lamina leading to muscle weakness, transient inflammation, muscle degeneration and impaired mobility. In a Lama2-deficient mouse model for this disease, animal survival is improved by muscle-specific expression of the apoptosis inhibitor Bcl-2, conferred by a MyoD-hBcl-2 transgene. Here we investigated early disease stages in this model to determine initial pathological events and effects of Bcl-2 on their progression. Using quantitative immunohistological and mRNA analyses we show that inflammation occurs very early in Lama2-deficient muscle, some aspects of which are reduced or delayed by the MyoD-hBcl-2 transgene. mRNAs for innate immune response regulators, including multiple Toll-like receptors (TLRs) and the inflammasome component NLRP3, are elevated in diseased muscle compared with age-matched controls expressing Lama2. MyoD-hBcl-2 inhibits induction of TLR4, TLR6, TLR7, TLR8 and TLR9 in Lama2-deficient muscle compared with non-transgenic controls, and leads to reduced infiltration of eosinophils, which are key death effector cells. This congenital disease model provides a new paradigm for investigating cell death mechanisms during early stages of pathogenesis, demonstrating that interactions exist between Bcl-2, a multifunctional regulator of cell survival, and the innate immune response

SeesawPred: A Web Application for Predicting Cell-fate Determinants in Cell Differentiation

Abstract Cellular differentiation is a complex process where a less specialized cell evolves into a more specialized cell. Despite the increasing research effort, identification of cell-fate determinants (transcription factors (TFs) determining cell fates during differentiation) still remains a challenge, especially when closely related cell types from a common progenitor are considered. Here, we develop SeesawPred, a web application that, based on a gene regulatory network (GRN) model of cell differentiation, can computationally predict cell-fate determinants from transcriptomics data. Unlike previous approaches, it allows the user to upload gene expression data and does not rely on pre-compiled reference data sets, enabling its application to novel differentiation systems. SeesawPred correctly predicted known cell-fate determinants on various cell differentiation examples in both mouse and human, and also performed better compared to state-of-the-art methods. The application is freely available for academic, non-profit use at http://seesaw.lcsb.uni.lu

Phylogenetic and Preliminary Phenotypic Analysis of Yeast PAQR Receptors: Potential Antifungal Targets

Proteins belonging to the Progestin and AdipoQ Receptor (PAQR) superfamily of membrane bound receptors are ubiquitously found in fungi. Nearly, all fungi possess two evolutionarily distinct paralogs of PAQR protein, which we have called the PQRA and PQRB subtypes. In the model fungus Saccharomyces cerevisiae, these subtypes are represented by the Izh2p and Izh3p proteins, respectively. S. cerevisiae also possesses two additional PQRA-type receptors called Izh1p and Izh4p that are restricted to other species within the “Saccharomyces complex”. Izh2p has been the subject of several recent investigations and is of particular interest because it regulates fungal growth in response to proteins produced by plants and, as such, represents a new paradigm for interspecies communication. We demonstrate that IZH2 and IZH3 gene dosage affects resistance to polyene antifungal drugs. Moreover, we provide additional evidence that Izh2p and Izh3p negatively regulate fungal filamentation. These data suggest that agonists of these receptors might make antifungal therapeutics, either by inhibiting fungal development or by sensitizing fungi to the toxic effects of current antifungal therapies. This is particularly relevant for pathogenic fungi such as Candida glabrata that are closely related to S. cerevisiae and contain the same complement of PAQR receptors