MicroRNAs in CD4(+) T cell subsets are markers of disease risk and T cell dysfunction in individuals at risk for type 1 diabetes.

MicroRNAs (miRNAs) regulate T cell development and function and the disruption of miRNAs in natural regulatory CD4(+) FOXP3(+) T cells (nTreg) leads to autoimmune disease in mice. To investigate miRNA expression in relation to autoimmune disease risk in humans we sequenced them in purified CD4(+) T cell subsets from individuals at high risk of type 1 diabetes (pre-T1D), as well as other healthy individuals. Differences in miRNA expression patterns were observed between specific T cell subsets and, within subsets, between pre-T1D and healthy individuals. Compared to healthy, naive CD4(+) T cells in pre-T1D displayed 32 differentially expressed miRNAs, potentially a template for altered miRNA expression in effector memory T cells in T1D. Naive nTreg in pre-T1D displayed two differentially expressed miRNAs, Let-7c and miR-15a. In contrast, nTreg activated in vivo displayed a large number of differentially expressed miRNAs, revealing a pro-inflammatory and FOXP3-repressive signature. Differential expression of specific miRNAs was also a signpost to altered T cell function. For example, in pre-T1D, increased expression of miR-26a in nTreg activated in vivo or in vitro was associated with decreased expression of its target, the histone methyltransferase EZH2. Chemical inhibition of EZH2 decreased the number of activated naïve nTreg and their expression of nTreg signature genes FOXP3 and TIGIT. Our findings demonstrate that miRNAs differentially expressed in CD4(+) T cell subsets are markers of risk and T cell dysfunction in T1D.

A microRNA expression atlas of mouse dendritic cell development.

Dendritic cells (DCs) are sentinel cells of the immune system and are essential for inducing a proper immune response. The mechanisms driving the development of DCs are not fully understood. Although the roles of cytokines and transcription factors have been a major focus, there is now substantial interest in the role of microRNAs (miRNAs). miRNAs are small RNAs that regulate gene expression by targeting messenger RNAs for translational repression and ultimately degradation. By means of deep sequencing, we have assembled a comprehensive and quantitative resource of miRNA expression during DC development. We show that mature DCs and their hematopoietic progenitors can be distinguished based on miRNA expression profiles. On the other hand, we show that functionally distinct conventional and plasmacytoid DC subsets are indistinguishable based on miRNA profile. In addition, we identify differences between ex vivo purified conventional DCs and their in vitro Flt3L-generated counterparts. This miRNA expression atlas will provide a valuable resource for the study of miRNAs in DC development and function.

Deaf-1 regulates epithelial cell proliferation and side-branching in the mammary gland.

BACKGROUND: The transcription factor DEAF-1 has been identified as a high affinity binding partner of the LIM-only protein LMO4 that plays important roles in mammary gland development and breast cancer. Here we investigated the influence of DEAF-1 on human and mouse mammary epithelial cells both in vitro and in vivo and identified a potential target gene. RESULTS: Overexpression of DEAF-1 in human breast epithelial MCF10A cells enhanced cell proliferation in the mammary acini that develop in 3D cultures. To investigate the effects of Deaf-1 on mammary gland development and oncogenesis, we generated MMTV-Deaf-1 transgenic mice. Increased ductal side-branching was observed in young virgin mammary glands, accompanied by augmented cell proliferation. In addition, the ratio of the progesterone receptor isoforms PRA and PRB, previously implicated in regulating ductal side-branching, was altered. Affymetrix gene profiling studies revealed Rac3 as a potential target gene and quantitative RT-PCR analysis confirmed that Rac3 was upregulated by Deaf-1 in immortalized mouse mammary epithelial cells. Furthermore, MMTV-Deaf-1 transgenic mammary glands were found to have elevated levels of Rac3 mRNA, suggesting that it is a bona fide target. CONCLUSION: We have demonstrated that overexpression of Deaf-1 enhances the proliferation of human breast epithelial cells in vitro and mouse epithelial cells in vivo. Transgenic mammary glands overexpressing Deaf-1 exhibited a modest side-branching phenotype, accompanied by an increase in the number of BrdU-positive cells and a decrease in the proportion of PRA-expressing cells. Although proliferation was enhanced in Deaf-1 transgenic mice, overexpression of this gene was not sufficient to induce the formation of mammary tumors. In addition, our studies identified Rac3, encoding a small Rho-like GTPase, as a potential target of Deaf-1 in mouse mammary epithelial cells.

Identification of Pax5 target genes in early B cell differentiation.

The transcription factor Pax5 is essential for B cell commitment in the mouse, where it represses lineage-inappropriate gene expression while simultaneously activating the B cell gene expression program. In this study we have performed a global gene expression screen of wild-type and Pax5-deficient pro-B cells in an attempt to identify the crucial Pax5 targets in early B lymphopoiesis. These studies have identified 109 Pax5 targets comprising 61% activated and 39% repressed genes. Interestingly, Pax5 directly regulates the genes encoding a number of transcription factors that are required at the pre-B cell stage of differentiation, including Irf8, Spib, and Ikzf3 (Aiolos), suggesting that a key function of Pax5 is to activate secondary transcription factors that further reinforce the B cell program. Pax5 is also required for the expression of many genes known to be involved in adhesion and signaling, indicating that Pax5 modulates the homing and or migration properties of B cell progenitors. Finally, Pax5 also represses a cohort of genes that are involved in multiple biological processes, many of which are not typically associated with B cells. These include the repression of the adhesion molecule Embigin, which is expressed in bone marrow progenitors, T cells, and myeloid cells but is specifically repressed by Pax5 in B cells.

Signal regulatory protein molecules are differentially expressed by CD8- dendritic cells.

A normalized subtracted gene expression library was generated from freshly isolated mouse dendritic cells (DC) of all subtypes, then used to construct cDNA microarrays. The gene expression profiles of the three splenic conventional DC (cDC) subsets were compared by microarray hybridization and two genes encoding signal regulatory protein beta (Sirpbeta1 and Sirpbeta4) molecules were identified as differentially expressed in CD8(-) cDC. Genomic sequence analysis revealed a third Sirpbeta member localized in the same gene cluster. These Sirpbeta genes encode cell surface molecules containing extracellular Ig domains and short intracytoplasmic domains that have a charged amino acid in the transmembrane region which can potentially interact with ITAM-bearing molecules to mediate signaling. Indeed, we demonstrated interactions between Sirpbeta1 and beta2 with the ITAM-bearing signaling molecule Dap12. Real-time PCR analysis showed that all three Sirpbeta genes were expressed by CD8(-) cDC, but not by CD8(+) cDC or plasmacytoid pre-DC. The related Sirpalpha gene showed a similar expression profile on cDC subtypes but was also expressed by plasmacytoid pre-DC. The differential expression of Sirpalpha and Sirpbeta1 molecules on DC was confirmed by staining with mAbs, including a new mAb recognizing Sirpbeta1. Cross-linking of Sirpbeta1 on DC resulted in a reduction in phagocytosis of Leishmania major parasites, but did not affect phagocytosis of latex beads, perhaps indicating that the regulation of phagocytosis by Sirpbeta1 is a ligand-dependent interaction. Thus, we postulate that the differential expression of these molecules may confer the ability to regulate the phagocytosis of particular ligands to CD8(-) cDC.

affylmGUI: a graphical user interface for linear modeling of single channel microarray data.

SUMMARY: affylmGUI is a graphical user interface (GUI) to an integrated workflow for Affymetrix microarray data. The user is able to proceed from raw data (CEL files) to QC and pre-processing, and eventually to analysis of differential expression using linear models with empirical Bayes smoothing. Output of the analysis (tables and figures) can be exported to an HTML report. The GUI provides user-friendly access to state-of-the-art methods embodied in the Bioconductor software repository. AVAILABILITY: affylmGUI is an R package freely available from http://www.bioconductor.org. It requires R version 1.9.0 or later and tcl/tk 8.3 or later and has been successfully tested on Windows 2000, Windows XP, Linux (RedHat and Fedora distributions) and Mac OS/X with X11. Further documentation is available at http://bioinf.wehi.edu.au/affylmGUI CONTACT: keith@wehi.edu.au.

limmaGUI: a graphical user interface for linear modeling of microarray data.

SUMMARY: limmaGUI is a graphical user interface (GUI) based on R-Tcl/Tk for the exploration and linear modeling of data from two-color spotted microarray experiments, especially the assessment of differential expression in complex experiments. limmaGUI provides an interface to the statistical methods of the limma package for R, and is itself implemented as an R package. The software provides point and click access to a range of methods for background correction, graphical display, normalization, and analysis of microarray data. Arbitrarily complex microarray experiments involving multiple RNA sources can be accomodated using linear models and contrasts. Empirical Bayes shrinkage of the gene-wise residual variances is provided to ensure stable results even when the number of arrays is small. Integrated support is provided for quantitative spot quality weights, control spots, within-array replicate spots and multiple testing. limmaGUI is available for most platforms on the which R runs including Windows, Mac and most flavors of Unix. AVAILABILITY: http://bioinf.wehi.edu.au/limmaGUI.