Spy1 role in mammary gland development and tumorigenesis

Spy1 is a cell cycle activator, known to mediate cell cycle progression through an atypical activation of the cyclin dependent kinases. To understand the development and progression of breast cancer it is essential to elucidate the mechanisms and interactions of normal regulators of breast development. This study demonstrates that endogenous Spy1 protein and mRNA levels are tightly regulated during normal mammary gland development; being expressed during proliferative stages and downregulated at the onset of lactation. This appears to be regulated, in part, through the oncogene c-Myc and the MAPK signaling pathway. Importantly, we show that aberrant expression of the Spy1 protein prevents normal differentiation and results in disrupted morphology of the gland as well as tumorigenesis. Collectively this work has revealed a novel molecular mechanism regulating normal developmental processes in the breast and has provided evidence that the Spy1 protein may also be implicated in the development of breast cancer

Novel Regulators of Somatic Cell Reprogramming

Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by expression of defined embryonic factors. My thesis is focused on exploring the mechanisms underlying reprogramming using a secondary mouse embryonic fibroblast model that forms iPS cells with high efficiency upon inducible expression of Oct4, Klf4, c-Myc and Sox2. My analyses of the temporal changes in gene expression reveal that reprogramming is a multi-step process characterized by initiation, maturation and stabilization phases. Using functional RNAi screening, I discovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. I showed that MET induction was linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs. These studies thus defined a multi-step mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming. Next I focused on the two later phases of reprogramming, maturation and stabilization. I showed the stabilization phase and acquisition of pluripotency is dependent on removal of transgene expression late in the maturation phase. Clonal analysis of reprogramming cells revealed subsets of stabilization competent (SC) versus stabilization incompetent (SI) cells. SC clones robustly entered the pluripotent state upon transgene withdrawal in the late, but not early maturation phase, whereas SI clones failed to reprogram at either stage. Transcriptome profiling by RNA-Seq revealed that SC clones acquire a competency gene expression signature late in the maturation phase. Functional RNAi screening of SC signature genes further identified regulators of transition to the stabilization phase, while screening of the same signature in iPS cells revealed a distinct subset of genes required for maintenance of pluripotency. These studies reveal that the acquisition and subsequent maintenance of pluripotency are controlled by distinct molecular networks and uncover a novel regulatory program that is required for transition to transgene independence.Ph

Correction of pathology in mice displaying Gaucher disease type 1 by a clinically-applicable lentiviral vector

This study evaluates a clinically applicable lentiviral vector for treatment of Gaucher disease type 1. Hematopoietic stem cells transduced with the vector and transplanted into a mouse model successfully halted or reversed pathology. These data were used as proof-of-concept for regulatory filing enabling the commencement of an international phase 1/2 clinical trial

MBNL proteins repress ES-cell-specific alternative splicing and reprogramming

Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators. Alternative splicing represents a widely acting mode of gene regulation, yet its role in regulating ES-cell pluripotency and differentiation is poorly understood. Here we identify the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between ES cells and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ES-cell-like alternative splicing pattern for approximately half of these events, whereas overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in the forkhead family transcription factor FOXP1 that controls pluripotency. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming.Canadian Institutes of Health Research (grant)Ontario Research FoundationStem Cell Network (Canada)National Institutes of Health (U.S.) (grant R33MH087908)University of Toronto (Open Fellowship)Ontario Stem Cell InitiativeHuman Frontier Science Program (Strasbourg, France)European Union (Marie Curie Actions

MBNL proteins repress ES-cellspecific alternative splicing and reprogramming. Nature 498: 241–245. doi: 10.1038/nature12270 PMID: 23739326

Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators A core set of transcription factors that includes OCT4 (also called POU5F1), NANOG and SOX2, together with specific microRNAs and long non-coding RNAs, control the expression of genes required for the establishment and maintenance of ES-cell pluripotency To identify such factors, we used high-throughput RNA sequencing (RNA-seq) data to define human and mouse cassette alternative exons that are differentially spliced between ES cells and induced pluripotent stem cells (iPSCs), and diverse differentiated cells and tissues, referred to below as 'ES-cell-differential alternative splicing'. A splicing code analysis 17 was then performed to identify cis-elements that may promote or repress these exons. The RNA-seq data used to profile alternative splicing were also used to detect human and mouse splicing factor genes that are differentially expressed between ES cells/iPSCs and non-ES cells/tissues. By integrating these data sources, we sought to identify differentially expressed splicing regulators with defined binding sites that match cis-elements predicted by the code analysis to function in ES-cell-differential alternative splicing. We identified 181 human and 103 mouse ES-cell-differential alternative splicing events, with comparable proportions of exons that are $25% more included or more skipped in ES cells versus the other profiled cells and tissues 216 ; hypergeometric test). The human and mouse ES-cell-differential alternative splicing events are significantly enriched in genes associated with the cytoskeleton (for example, DST, ADD3), plasma membrane (for example, DNM2, ITGA6) and kinase activity (for example, CASK, MARK2 and MAP2K7) (Supplementary The splicing code analysis revealed that motifs corresponding to consensus binding sites of the conserved MBNL proteins are the most strongly associated with ES-cell-differential alternative splicing in human and mouse. The presence of MBNL motifs in downstream flanking intronic sequences is associated with exon skipping in ES cells, whereas their presence in upstream flanking intronic sequences is associated with exon inclusion in ES cells From RNA-seq expression profiling of 221 known or putative splicing factors, 11 genes showed significant differential expression between human ES cells/iPSCs and other cells and tissues (Bonferronicorrected P , 0.05, Wilcoxon rank-sum test