Integration of microRNA signatures of distinct mammary epithelial cell types with their gene expression and epigenetic portraits.

INTRODUCTION: MicroRNAs (miRNAs) have been implicated in governing lineage specification and differentiation in multiple organs; however, little is known about their specific roles in mammopoiesis. We have determined the global miRNA expression profiles of functionally distinct epithelial subpopulations in mouse and human mammary tissue, and compared these to their cognate transcriptomes and epigenomes. Finally, the human miRNA signatures were used to interrogate the different subtypes of breast cancer, with a view to determining miRNA networks deregulated during oncogenesis. METHODS: RNA from sorted mouse and human mammary cell subpopulations was subjected to miRNA expression analysis using the TaqMan MicroRNA Array. Differentially expressed (DE) miRNAs were correlated with gene expression and histone methylation profiles. Analysis of miRNA signatures of the intrinsic subtypes of breast cancer in The Cancer Genome Atlas (TCGA) database versus those of normal human epithelial subpopulations was performed. RESULTS: Unique miRNA signatures characterized each subset (mammary stem cell (MaSC)/basal, luminal progenitor, mature luminal, stromal), with a high degree of conservation across species. Comparison of miRNA and transcriptome profiles for the epithelial subtypes revealed an inverse relationship and pinpointed key developmental genes. Interestingly, expression of the primate-specific miRNA cluster (19q13.4) was found to be restricted to the MaSC/basal subset. Comparative analysis of miRNA signatures with H3 lysine modification maps of the different epithelial subsets revealed a tight correlation between active or repressive marks for the top DE miRNAs, including derepression of miRNAs in Ezh2-deficient cellular subsets. Interrogation of TCGA-identified miRNA profiles with the miRNA signatures of different human subsets revealed specific relationships. CONCLUSIONS: The derivation of global miRNA expression profiles for the different mammary subpopulations provides a comprehensive resource for understanding the interplay between miRNA networks and target gene expression. These data have highlighted lineage-specific miRNAs and potential miRNA-mRNA networks, some of which are disrupted in neoplasia. Furthermore, our findings suggest that key developmental miRNAs are regulated by global changes in histone modification, thus linking the mammary epigenome with genome-wide changes in the expression of genes and miRNAs. Comparative miRNA signature analyses between normal breast epithelial cells and breast tumors confirmed an important linkage between luminal progenitor cells and basal-like tumors.

A mouse model of harlequin ichthyosis delineates a key role for Abca12 in lipid homeostasis.

Harlequin Ichthyosis (HI) is a severe and often lethal hyperkeratotic skin disease caused by mutations in the ABCA12 transport protein. In keratinocytes, ABCA12 is thought to regulate the transfer of lipids into small intracellular trafficking vesicles known as lamellar bodies. However, the nature and scope of this regulation remains unclear. As part of an original recessive mouse ENU mutagenesis screen, we have identified and characterised an animal model of HI and showed that it displays many of the hallmarks of the disease including hyperkeratosis, loss of barrier function, and defects in lipid homeostasis. We have used this model to follow disease progression in utero and present evidence that loss of Abca12 function leads to premature differentiation of basal keratinocytes. A comprehensive analysis of lipid levels in mutant epidermis demonstrated profound defects in lipid homeostasis, illustrating for the first time the extent to which Abca12 plays a pivotal role in maintaining lipid balance in the skin. To further investigate the scope of Abca12's activity, we have utilised cells from the mutant mouse to ascribe direct transport functions to the protein and, in doing so, we demonstrate activities independent of its role in lamellar body function. These cells have severely impaired lipid efflux leading to intracellular accumulation of neutral lipids. Furthermore, we identify Abca12 as a mediator of Abca1-regulated cellular cholesterol efflux, a finding that may have significant implications for other diseases of lipid metabolism and homeostasis, including atherosclerosis.

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.

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.