Seeing the forest through the trees: prioritising potentially functional interactions from Hi-C.

Eukaryotic genomes are highly organised within the nucleus of a cell, allowing widely dispersed regulatory elements such as enhancers to interact with gene promoters through physical contacts in three-dimensional space. Recent chromosome conformation capture methodologies such as Hi-C have enabled the analysis of interacting regions of the genome providing a valuable insight into the three-dimensional organisation of the chromatin in the nucleus, including chromosome compartmentalisation and gene expression. Complicating the analysis of Hi-C data, however, is the massive amount of identified interactions, many of which do not directly drive gene function, thus hindering the identification of potentially biologically functional 3D interactions. In this review, we collate and examine the downstream analysis of Hi-C data with particular focus on methods that prioritise potentially functional interactions. We classify three groups of approaches: structural-based discovery methods, e.g. A/B compartments and topologically associated domains, detection of statistically significant chromatin interactions, and the use of epigenomic data integration to narrow down useful interaction information. Careful use of these three approaches is crucial to successfully identifying potentially functional interactions within the genome.Ning Liu, Wai Yee Low, Hamid Alinejad, Rokny, Stephen Pederson, Timothy Sadlon, Simon Barry, and James Bree

Robust, reversible gene knockdown using a single lentiviral short hairpin RNA vector

Manipulation of gene expression is an invaluable tool to study gene function in vitro and in vivo. The application of small inhibitory RNAs to knock down gene expression provides a relatively simple, elegant, but transient approach to study gene function in many cell types as well as in whole animals. Short hairpin structures (shRNAs) are a logical advance as they can be expressed continuously and are hence suitable for stable gene knockdown. Drug-inducible systems have now been developed; however, application of the technology has been hampered by persistent problems with low or transient expression, leakiness or poor inducibility of the short hairpin, and lack of reversibility. We have developed a robust, versatile, single lentiviral vector tool that delivers tightly regulated, fully reversible, doxycycline-responsive knockdown of target genes (FOXP3 and MYB), using single short hairpin RNAs. To demonstrate the capabilities of the vector we targeted FOXP3 because it plays a critical role in the development and function of regulatory T cells. We also targeted MYB because of its essential role in hematopoiesis and implication in breast cancer progression. The versatility of this vector is hence demonstrated by knockdown of distinct genes in two biologically separate systems.Cheryl Y. Brown, Timothy Sadlon, Tessa Gargett, Elizabeth Melville, Rui Zhang, Yvette Drabsch, Michael Ling, Craig A. Strathdee, Thomas J. Gonda, and Simon C. Barr

Primordial Germ Cell Specification from Embryonic Stem Cells

Background: Primordial germ cell (PGC) specification is the first crucial step in germ line development. However, owing to significant challenges regarding the in vivo system, such as the complex cellular environment and potential problems with embryo manipulation, it is desirable to generate embryonic stem (ES) cells that are capable of overcoming these aforementioned limitations in order to provide a potential in vitro model to recapitulate the developmental processes in vivo. Methodology and Principal Findings: Here, we studied the detailed process of PGC specification from stella-GFP ES cells. We first observed the heterogeneous expression of stella in ES cells. However, neither Stella-positive ES cells nor Stellanegative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors. Second, we derived PGCs from ES cells using two differentiation methods, namely the attachment culture technique and the embryoid body (EB) method. Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method. Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM) that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation

Heme-Oxygenases during Erythropoiesis in K562 and Human Bone Marrow Cells

In mammalian cells, heme can be degraded by heme-oxygenases (HO). Heme-oxygenase 1 (HO-1) is known to be the heme inducible isoform, whereas heme-oxygenase 2 (HO-2) is the constitutive enzyme. Here we investigated the presence of HO during erythroid differentiation in human bone marrow erythroid precursors and K562 cells. HO-1 mRNA and protein expression levels were below limits of detection in K562 cells. Moreover, heme was unable to induce HO-1, at the protein and mRNA profiles. Surprisingly, HO-2 expression was inhibited upon incubation with heme. To evaluate the physiological relevance of these findings, we analyzed HO expression during normal erythropoiesis in human bone marrow. Erythroid precursors were characterized by lack of significant expression of HO-1 and by progressive reduction of HO-2 during differentiation. FLVCR expression, a recently described heme exporter found in erythroid precursors, was also analyzed. Interestingly, the disruption in the HO detoxification system was accompanied by a transient induction of FLVCR. It will be interesting to verify if the inhibition of HO expression, that we found, is preventing a futile cycle of concomitant heme synthesis and catabolism. We believe that a significant feature of erythropoiesis could be the replacement of heme breakdown by heme exportation, as a mechanism to prevent heme toxicity

FOXP3 and FOXP3-regulated microRNAs suppress SATB1 in breast cancer cells

The transcription factor FOXP3 has been identified as a tumour suppressor in the breast and prostate epithelia, but little is known about its specific mechanism of action. We have identified a feed-forward regulatory loop in which FOXP3 suppresses the expression of the oncogene SATB1. In particular, we demonstrate that SATB1 is not only a direct target of FOXP3 repression, but that FOXP3 also induces two miRs, miR-7 and miR-155, which specifically target the 3′-UTR of SATB1 to further regulate its expression. We conclude that FOXP3-regulated miRs form part of the mechanism by which FOXP3 prevents the transformation of the healthy breast epithelium to a cancerous phenotype. Approaches aimed at restoring FOXP3 function and the miRs it regulates could help provide new approaches to target breast cancer.N McInnes, TJ Sadlon, CY Brown, S Pederson, M Beyer, JL Schultze, S McColl, GJ Goodall and SC Barr

Environmental determinants of islet autoimmunity (ENDIA): a pregnancy to early life cohort study in children at-risk of type 1 diabetes

Members of ENDIA Study Group: Peter Baghurst, Simon Barry, Jodie Dodd, Maria Makrides for the University of Adelaide.BACKGROUND The incidence of type 1 diabetes has increased worldwide, particularly in younger children and those with lower genetic susceptibility. These observations suggest factors in the modern environment promote pancreatic islet autoimmunity and destruction of insulin-producing beta cells. The Environmental Determinants of Islet Autoimmunity (ENDIA) Study is investigating candidate environmental exposures and gene-environment interactions that may contribute to the development of islet autoimmunity and type 1 diabetes. METHODS/DESIGN ENDIA is the only prospective pregnancy/birth cohort study in the Southern Hemisphere investigating the determinants of type 1 diabetes in at-risk children. The study will recruit 1,400 unborn infants or infants less than six months of age with a first-degree relative (i.e. mother, father or sibling) with type 1 diabetes, across five Australian states. Pregnant mothers/infants will be followed prospectively from early pregnancy through childhood to investigate relationships between genotype, the development of islet autoimmunity (and subsequently type 1 diabetes), and prenatal and postnatal environmental factors. ENDIA will evaluate the microbiome, nutrition, bodyweight/composition, metabolome-lipidome, insulin resistance, innate and adaptive immune function and viral infections. A systems biology approach will be used to integrate these data. Investigation will be by 3-monthly assessments of the mother during pregnancy, then 3-monthly assessments of the child until 24 months of age and 6-monthly thereafter. The primary outcome measure is persistent islet autoimmunity, defined as the presence of autoantibodies to one or more islet autoantigens on consecutive tests. DISCUSSION Defining gene-environment interactions that initiate and/or promote destruction of the insulin-producing beta cells in early life will inform approaches to primary prevention of type 1 diabetes. The strength of ENDIA is the prospective, comprehensive and frequent systems-wide profiling from early pregnancy through to early childhood, to capture dynamic environmental exposures that may shape the development of islet autoimmunity. TRIAL REGISTRATION Australia New Zealand Clinical Trials Registry ACTRN12613000794707.Megan AS Penno, Jennifer J Couper, Maria E Craig, Peter G Colman, William D Rawlinson, Andrew M Cotterill, Timothy W Jones, Leonard C Harrison and ENDIA Study Grou

Identification of a second heparin binding domain in human complement factor H

Complement factor H (fH) regulates activation of the alternative pathway of C, reducing the amount of C3b deposited on sialic acid-rich surfaces. Heparin binding has been used as a model for examining the sialic acid- binding characteristics of fH. We have previously shown thai of the 20 short consensus repeat (SCR) modules of fH, SCR 7 contains an important heparin binding site, but other SCRs also play a role in heparin binding. To localize the other sites, we prepared recombinant truncated and SCR deletion mutants of fH and tested them by heparin-agarose affinity chromatography. The 5 C- terminal SCRs were found to contain a heparin binding site as an SCR 7 deletion mutant of the N terminal 15 SCRs did not bind heparin, but a construct consisting of SCRs 16-20 was shown to bind heparin. Double deletion of SCRs 7 and 20 fH abrogated binding to heparin, indicating that SCR 20 contains a heparin binding site. This finding was confirmed with the observation that attachment of SCR 20 to a group of nonbinding SCRs produced a heparin-binding protein. A protein consisting of SCRs 19 and 20 did not bind heparin, whereas SCRs 18-20 did, indicating that, although SCR 20 contains a heparin binding site, at least two nonspecific adjacent SCRs are required. fH-related protein-3 (FHR-3) possesses an SCR homologous to SCR 7 of fH and bound heparin, whereas FHR-4, which lacks such an SCR, did not. Thus, fH contains two separate heparin binding sites which are located in SCRs 7 and 20.Timothy K. Blackmore, Jens Hellwage, Tania A. Sadlon, Naomi Higgs, Peter F. Zipfel, Helena M. Ward, and David L. Gordo

3DFAACTS-SNP: using regulatory T cell-specific epigenomics data to uncover candidate mechanisms of type 1 diabetes (T1D) risk

Background: Genome-wide association studies (GWAS) have enabled the discovery of single nucleotide polymorphisms (SNPs) that are significantly associated with many autoimmune diseases including type 1 diabetes (T1D). However, many of the identified variants lie in non-coding regions, limiting the identification of mechanisms that contribute to autoimmune disease progression. To address this problem, we developed a variant filtering workflow called 3DFAACTS-SNP to link genetic variants to target genes in a cell-specific manner. Here, we use 3DFAACTS-SNP to identify candidate SNPs and target genes associated with the loss of immune tolerance in regulatory T cells (Treg) in T1D. Results: Using 3DFAACTS-SNP, we identified from a list of 1228 previously fine-mapped variants, 36 SNPs with plausible Treg-specific mechanisms of action. The integration of cell type-specific chromosome conformation capture data in 3DFAACTS-SNP identified 266 regulatory regions and 47 candidate target genes that interact with these variant-containing regions in Treg cells. We further demonstrated the utility of the workflow by applying it to three other SNP autoimmune datasets, identifying 16 Treg-centric candidate variants and 60 interacting genes. Finally, we demonstrate the broad utility of 3DFAACTS-SNP for functional annotation of all known common (> 10% allele frequency) variants from the Genome Aggregation Database (gnomAD). We identified 9376 candidate variants and 4968 candidate target genes, generating a list of potential sites for future T1D or other autoimmune disease research. Conclusions: We demonstrate that it is possible to further prioritise variants that contribute to T1D based on regulatory function, and illustrate the power of using cell type-specific multiomics datasets to determine disease mechanisms. Our workflow can be customised to any cell type for which the individual datasets for functional annotation have been generated, giving broad applicability and utility.Ning Liu, Timothy Sadlon, Ying Y. Wong, Stephen Pederson, James Breen, and Simon C. Barr

Pneumococcal histidine triad proteins are regulated by the Zn(2+)-dependent repressor AdcR and inhibit complement deposition through the recruitment of complement factor H

The pneumococcal histidine triad (Pht) proteins are a recently recognized family of surface proteins, comprising 4 members: PhtA, PhtB, PhtD, and PhtE. They are being promoted for inclusion in a multicomponent pneumococcal protein vaccine currently under development, but to date, their biological functions and their relative contributions to pathogenesis have not been clarified. In this study, the involvement of these proteins in pneumococcal virulence was investigated in murine models of sepsis and pneumonia by using defined, nonpolar mutants of the respective genes in Streptococcus pneumoniae D39. In either challenge model, mutagenesis of all 4 genes was required to completely abolish virulence relative to the wild-type, suggesting significant functional redundancy among Pht proteins. The in vivo expression of pht genes was significantly up-regulated in the nasopharynx and lungs compared with blood. We provide unequivocal molecular evidence for Zn²⁺-dependent, AdcR-mediated, regulation of pht gene expression by real-time reverse transcriptase-polymerase chain reaction, Western blotting, and electrophoretic mobility-shift assays. We also present the first direct evidence for the biological function of this protein family by demonstrating that Pht proteins are required for inhibition of complement deposition on the pneumococcal surface through the recruitment of complement factor H.—Ogunniyi, A. D., Grabowicz, M., Mahdi, L. K., Cook, J., Gordon, D. L., Sadlon, T. A., Paton, J. C. Pneumococcal histidine triad proteins are regulated by the Zn²⁺-dependent repressor AdcR and inhibit complement deposition through the recruitment of complement factor H.Abiodun D. Ogunniyi, Marcin Grabowicz, Layla K. Mahdi, Jan Cook, David L. Gordon, Tania A. Sadlon and James C. Pato

Development of CD4(+)CD25(+)FoxP3(+) regulatory T cells from cord blood hematopoietic progenitor cells

Adult stem cells are capable of generating all of the cells of the hematopoietic system, and this process is orchestrated in part by the interactions between these cells and the stroma. T cell progenitors emerge from the stem cell compartment and migrate to the thymus, where their terminal differentiation and maturation occur, and it is during this phase that selection shapes the immune repertoire. Notch ligands, including Delta-like 1 (DL1), play a critical role in this lymphoid differentiation. To mimic this in vitro, stroma-expressing DL1 have been used to generate CD4(+)CD8(+) double-positive and single-positive T cells from hematopoietic stem/progenitor cells. This system provides a robust tool to investigate thymopoiesis; however, its capacity to generate regulatory T cells (Tregs) has yet to be reported. Natural Tregs (nTregs) develop in the thymus and help maintain immune homeostasis and have potential clinical use as a cell therapy for modulation of autoimmune disease or for transplant tolerization. Here, we describe for the first time the development of a population of CD4(+)CD25(+) CD127(lo)FoxP3(+) cells that emerge in coculture of cord blood (CB) CD34(+) progenitors on OP9-DL1 stroma. These hematopoietic progenitor-derived CD4(+)CD25(+) Tregs have comparable suppressor function with CB nTregs in vitro. The addition of IL-2 to the coculture enhanced the expansion and survival of this population significantly. This manipulable culture system, therefore, generates functional Tregs and provides a system to elucidate the mechanism of Treg development.Jonathon F. Hutton, Tessa Gargett, Timothy J. Sadlon, Suzanne Bresatz, Cheryl Y. Brown, Heddy Zola, M. Frances Shannon, Richard J. D’Andrea and Simon C. Barr