Hierarchical information clustering by means of topologically embedded graphs

We introduce a graph-theoretic approach to extract clusters and hierarchies in complex data-sets in an unsupervised and deterministic manner, without the use of any prior information. This is achieved by building topologically embedded networks containing the subset of most significant links and analyzing the network structure. For a planar embedding, this method provides both the intra-cluster hierarchy, which describes the way clusters are composed, and the inter-cluster hierarchy which describes how clusters gather together. We discuss performance, robustness and reliability of this method by first investigating several artificial data-sets, finding that it can outperform significantly other established approaches. Then we show that our method can successfully differentiate meaningful clusters and hierarchies in a variety of real data-sets. In particular, we find that the application to gene expression patterns of lymphoma samples uncovers biologically significant groups of genes which play key-roles in diagnosis, prognosis and treatment of some of the most relevant human lymphoid malignancies

Comparative analyses of CTCF and BORIS occupancies uncover two distinct classes of CTCF binding genomic regions.

BackgroundCTCF and BORIS (CTCFL), two paralogous mammalian proteins sharing nearly identical DNA binding domains, are thought to function in a mutually exclusive manner in DNA binding and transcriptional regulation.ResultsHere we show that these two proteins co-occupy a specific subset of regulatory elements consisting of clustered CTCF binding motifs (termed 2xCTSes). BORIS occupancy at 2xCTSes is largely invariant in BORIS-positive cancer cells, with the genomic pattern recapitulating the germline-specific BORIS binding to chromatin. In contrast to the single-motif CTCF target sites (1xCTSes), the 2xCTS elements are preferentially found at active promoters and enhancers, both in cancer and germ cells. 2xCTSes are also enriched in genomic regions that escape histone to protamine replacement in human and mouse sperm. Depletion of the BORIS gene leads to altered transcription of a large number of genes and the differentiation of K562 cells, while the ectopic expression of this CTCF paralog leads to specific changes in transcription in MCF7 cells.ConclusionsWe discover two functionally and structurally different classes of CTCF binding regions, 2xCTSes and 1xCTSes, revealed by their predisposition to bind BORIS. We propose that 2xCTSes play key roles in the transcriptional program of cancer and germ cells

Distribution of tissue progenitors within the shield region of the zebrafish gastrula

The zebrafish has emerged as an important model system for the experimental analysis of vertebrate development because it is amenable to genetic analysis and because its optical clarity allows the movements and the differentiation of individual cells to be followed in vivo. In this paper, we have sought to characterize the spatial distribution of tissue progenitors within the outer cell layers of the embryonic shield region of the early gastrula. Single cells were labeled by iontophoretic injection of fluorescent dextrans. Subsequently, we documented their position with respect to the embryonic shield and their eventual fates. Our data show that progenitor cells of the neural, notochordal, somitic and endodermal lineages were all present within the embryonic shield region, and that these progenitors were arranged as intermingled populations. Moreover, close to the midline, there was evidence for significant biases in the distribution of neural and notochord progenitors between the layers, suggesting some degree of radial organization within the zebrafish embryonic shield region. The distributions of tissue progenitors in the zebrafish gastrula differ significantly from those in amphibians; this bears not only on interpretations of mutant phenotypes and in situ staining patterns, but also on our understanding of morphogenetic movements during gastrulation and of neural induction in the zebrafish

Differential Hox expression in murine embryonic stem cell models of normal and malignant hematopoiesis

The Hox family are master transcriptional regulators of developmental processes, including hematopoiesis. The Hox regulators, caudal homeobox factors (Cdx1-4), and Meis1, along with several individual Hox proteins, are implicated in stem cell expansion during embryonic development, with gene dosage playing a significant role in the overall function of the integrated Hox network. To investigate the role of this network in normal and aberrant, early hematopoiesis, we employed an in vitro embryonic stem cell differentiation system, which recapitulates mouse developmental hematopoiesis. Expression profiles of Hox, Pbx1, and Meis1 genes were quantified at distinct stages during the hematopoietic differentiation process and compared with the effects of expressing the leukemic oncogene Tel/PDGFR;2. During normal differentiation the Hoxa cluster, Pbx1 and Meis1 predominated, with a marked reduction in the majority of Hox genes (27/39) and Meis1 occurring during hematopoietic commitment. Only the posterior Hoxa cluster genes (a9, a10, a11, and a13) maintained or increased expression at the hematopoietic colony stage. Cdx4, Meis1, and a subset of Hox genes, including a7 and a9, were differentially expressed after short-term oncogenic (Tel/PDGFR;2) induction. Whereas Hoxa4-10, b1, b2, b4, and b9 were upregulated during oncogenic driven myelomonocytic differentiation. Heterodimers between Hoxa7/Hoxa9, Meis1, and Pbx have previously been implicated in regulating target genes involved in hematopoietic stem cell (HSC) expansion and leukemic progression. These results provide direct evidence that transcriptional flux through the Hox network occurs at very early stages during hematopoietic differentiation and validates embryonic stem cell models for gaining insights into the genetic regulation of normal and malignant hematopoiesis

Fighting viral infections and virus-driven tumors with cytotoxic CD4+ T cells

CD4+ T cells have been and are still largely regarded as the orchestrators of immune responses, being able to differentiate into distinct T helper cell populations based on differentiation signals, transcription factor expression, cytokine secretion, and specific functions. Nonetheless, a growing body of evidence indicates that CD4+ T cells can also exert a direct effector activity, which depends on intrinsic cytotoxic properties acquired and carried out along with the evolution of several pathogenic infections. The relevant role of CD4+ T cell lytic features in the control of such infectious conditions also leads to their exploitation as a new immunotherapeutic approach. This review aims at summarizing currently available data about functional and therapeutic relevance of cytotoxic CD4+ T cells in the context of viral infections and virus-driven tumors

A search for genes modulated by interleukin-6 alone or with interleukin-1β in HepG2 cells using differential display analysis

AbstractInterleukin-1 and interleukin-6 are principal cytokines involved in regulation of expression of acute-phase proteins. In the joint action of both cytokines IL-1 can suppress or enhance the IL-6-dependent induction of gene expression. Here, we report changes in the transcriptome profile of HepG2 cells exposed to IL-6 alone, or IL-1 and IL-6. Cytokine-responsive genes were identified by differential display analysis. Validation of observed changes in the transcript level was carried out using the slot blot method. Out of 88 cDNA species modulated by IL-6, only 38 represent different known genes whereas 18 clones match genomic clones in NCBI data with hypothetical cDNA sequences (the remaining 32 clones showed no homology with the database or represented several clones of the same gene). In the experiments with HepG2 cells prestimulated for 3 h with IL-1 and then stimulated with IL-6, 43 cDNA fragments were amplified. Twenty-three of them represent known genes while 10 clones have inserts matching hypothetical cDNA sequences in NCBI data. The identified transcripts modulated by IL-6 or both cytokines in HepG2 cells code for intracellular proteins of various function. The largest groups represent genes engaged in metabolism, protein synthesis and signaling pathways. Among all genes identified as differentially regulated under stimulation by IL-6, or IL-1/IL-6, six were detected in both types of stimulation. None of the typical genes coding for plasma acute phase proteins was identified in our experiments. This indicates that differential display cannot be used to characterize the profile of a given transcriptome. On the other hand, it is a useful technique for detection of new genes responding to IL-6 alone or IL-6 in combination with IL-1

The Immune System

Modern biotherapy has been in use for some 30 years. The first types of biotherapy were nonspecific stimulators of the immune response, but advances in genetic engineering are allowing the mass production of pure biological products which are now being tested as pharmaceutical agents. Biotherapy connotes the administration of products (1) that are coded by the mammalian genome; (2) that modify the expression of mammalian genes; or (3) that stimulate the immune system. In this chapter the discussion of the immune system will be limited primarily to topics relevant to cancer or autoimmune diseases. Because understanding the new biological agents requires an understanding of both the immune response and the molecular basis of oncogenesis, this chapter first presents a summary of the structure and function of the immune system. Following a discussion of immune responses, and the cells involved in these responses, will be a discussion on the current concepts of oncogenesis, particularly oncogenes and growth factors. Because research efforts are beginning to identify many biological proteins as having a role in autoimmune and other diseases, a brief introduction to autoimmune diseases is also included at the end of the chapter

Functional characterization of ARID1A mutations in follicular lymphoma

Background: Follicular lymphoma (FL) is one of the most common malignant lymphomas worldwide and the most common form of indolent lymphoma. FL is highly heterogeneous from both the clinical and molecular point of view. It remains a clinical challenge since advanced-stage disease is still considered incurable, and patients ultimately present with relapsed or re-fractory disease. Molecularly, FL is characterized by highly recurrent genetic mutations in genes coding for epigenetic modifiers. ARID1A mutations are among the most frequent mutations in FL (~10-20 % at the time of diagnosis). ARID1A mutations are a component of the prognostic clinic-genetic risk model m7-FLIPI (Pastore, Jurinovic et al. 2015). These mutations are primari-ly disruptive and result in protein haplodeficiency. Functionally, ARID1A is part of a SWI/SNF complex, which controls chromatin accessibility and is involved in numerous processes, includ-ing gene expression. Aim: Functionally characterize ARID1A mutations in representative human FL model systems. Methods: I used established and primary FL-like cell lines that harbor the hallmark t(14;18) translocation with or without heterozygous or homozygous ARID1A mutations (introduced by CRISPR/Cas9) or knock-down (by shRNA). I applied complementary omics approaches (RNA-Seq and ATAC-Seq) and functional assays to untangle the consequences of ARID1A loss in these FL model systems. Results: ARID1A loss profoundly altered gene expression. Across three cell lines, we observed consistent down-regulation of genes involved in cell cycle regulation and apoptosis pathways upon ARID1A loss. In functional experiments, I could show that ARID1A mutant clones are characterized by significantly slower cell proliferation and increased formation of anaphase bridges. Next, I demonstrated that ARID1A loss results in decreased FAS levels and lower sensitivity to FASLG-induced apoptosis. We discovered the underlying molecular mechanism through ad-vanced bioinformatics analyses and functional experiments. Briefly, ARID1A loss does not di-rectly affect FAS expression. Still, it results in reduced DNA accessibility and expression of the co-transcription factor RUNX3, thereby hindering RUNX3-ETS1 cooperativity and ETS1-induced FAS expression, which promotes a functionally and clinically relevant immune-evasive pheno-type. Finally, RNA-Seq analysis indicated that ARID1A loss alters the plasma membrane and cyto-skeleton functions, as well as the overall abundance of ligands and receptors. Ex vivo co-cultures of FL-like cells with T cells suggested impaired immune synapse formation with CD4 T lymphocytes upon ARID1A loss. Discussion and conclusion: Overall, our analyses provide novel insights into the functional consequences of ARID1A mutations in FL, most notably promoting immune evasion. A better understanding of mutation-specific biology, including its impact on interactions within the tumor microenvironment, holds promise for improved patient stratification and the development of personalized treatment approaches