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.

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

Gaucher disease type 1 (GD1) is an inherited lysosomal disorder with multisystemic effects in patients. Hallmark symptoms include hepatosplenomegaly, cytopenias, and bone disease with varying degrees of severity. Mutations in a single gene, glucosidase beta acid 1 (GBA1), are the underlying cause for the disorder, resulting in insufficient activity of the enzyme glucocerebrosidase, which in turn leads to a progressive accumulation of the lipid component glucocerebroside. In this study, we treat mice with signs consistent with GD1, with hematopoietic stem/progenitor cells transduced with a lentiviral vector containing an RNA transcript that, after reverse transcription, results in codon-optimized cDNA that, upon its integration into the genome encodes for functional human glucocerebrosidase. Five months after gene transfer, a highly significant reduction in glucocerebroside accumulation with subsequent reversal of hepatosplenomegaly, restoration of blood parameters, and a tendency of increased bone mass and density was evident in vector-treated mice compared to non-treated controls. Furthermore, histopathology revealed a prominent reduction of Gaucher cell infiltration after gene therapy. The vector displayed an oligoclonal distribution pattern but with no sign of vector-induced clonal dominance and a typical lentiviral vector integration profile. Cumulatively, our findings support the initiation of the first clinical trial for GD1 using the lentiviral vector described here.

A late transition in somatic cell reprogramming requires regulators distinct from the pluripotency network.

Reprogramming of somatic cells to a pluripotent state via expression of Oct4, Klf4, Myc, and Sox2 is a multistep process involving phased changes in gene expression. Here, we focus on the later stages of reprogramming, termed maturation and stabilization. We show that the stabilization phase and the acquisition of pluripotency are dependent on the removal of transgene expression late in the maturation phase. Clonal analysis of cells undergoing reprogramming revealed subsets of stabilization-competent (SC) and stabilization-incompetent (SI) cells. SC clones acquire a competency gene-expression signature late in the maturation phase. Functional analysis of SC signature genes identified enhancers of the transition to the stabilization phase and a distinct subset of genes required for the maintenance of pluripotency. Thus, the acquisition and maintenance of pluripotency are regulated by distinct molecular networks, and a specific regulatory program not previously implicated in reprogramming is required for the transition to transgene independence.

Looking into the black box: insights into the mechanisms of somatic cell reprogramming.

The dramatic discovery that somatic cells could be reprogrammed to induced pluripotent stem cells (iPSCs), by the expression of just four factors, has opened new opportunities for regenerative medicine and novel ways of modeling human diseases. Extensive research over the short time since the first iPSCs were generated has yielded the ability to reprogram various cell types using a diverse range of methods. However the duration, efficiency, and safety of induced reprogramming have remained a persistent limitation to achieving a robust experimental and therapeutic system. The field has worked to resolve these issues through technological advances using non-integrative approaches, factor replacement or complementation with microRNA, shRNA and drugs. Despite these advances, the molecular mechanisms underlying the reprogramming process remain poorly understood. Recently, through the use of inducible secondary reprogramming systems, researchers have now accessed more rigorous mechanistic experiments to decipher this complex process. In this review we will discuss some of the major recent findings in reprogramming, pertaining to proliferation and cellular senescence, epigenetic and chromatin remodeling, and other complex cellular processes such as morphological changes and mesenchymal-to-epithelial transition. We will focus on the implications of this work in the construction of a mechanistic understanding of reprogramming and discuss unexplored areas in this rapidly expanding field.

Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming.

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expression of defined embryonic factors. However, little is known of the molecular mechanisms underlying the reprogramming process. Here we explore somatic cell reprogramming by exploiting a secondary mouse embryonic fibroblast model that forms iPSCs with high efficiency upon inducible expression of Oct4, Klf4, c-Myc, and Sox2. Temporal analysis of gene expression revealed that reprogramming is a multistep process that is characterized by initiation, maturation, and stabilization phases. Functional analysis by systematic RNAi screening further uncovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. We show that this is linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs that are key regulators of MET. These studies thus define a multistep mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming. PAPERCLIP:

The Spy1/RINGO family represents a novel mechanism regulating mammary growth and tumorigenesis.

Spy1A is a unique cell cycle activator known to mediate cell cycle progression and override the DNA damage response. This study focused on determining the role of this protein on postnatal mammary gland morphogenesis and neoplasia. Herein, we show that Spy1A levels are tightly regulated during mammary gland development and that ectopic expression stimulates precocious development and results in disrupted morphology of the gland. This follows the same trend as the oncogene c-Myc, and we show that Spy1A expression is regulated downstream of c-Myc signaling. Importantly, we show that overexpression of Spy1A accelerates tumorigenesis in vivo. Collectively, this work is the first report that the Spy1/RINGO family of proteins may play an essential role in regulating both normal and abnormal growth processes in the breast.