Revisiting Global Gene Expression Analysis

Gene expression analysis is a widely used and powerful method for investigating the transcriptional behavior of biological systems, for classifying cell states in disease, and for many other purposes. Recent studies indicate that common assumptions currently embedded in experimental and analytical practices can lead to misinterpretation of global gene expression data. We discuss these assumptions and describe solutions that should minimize erroneous interpretation of gene expression data from multiple analysis platforms.National Institutes of Health (U.S.) (grant NIH HG002668)National Institutes of Health (U.S.) (grant NIH CA146445)American Cancer Society (Postdoctoral Fellowship PF-11-042-01-DMC)Swedish Research Council (Postdoctoral Fellowship VR-B0086301

Master Transcription Factors Determine Cell-Type-Specific Responses to TGF-β Signaling

Transforming growth factor beta (TGF-β) signaling, mediated through the transcription factors Smad2 and Smad3 (Smad2/3), directs different responses in different cell types. Here we report that Smad3 co-occupies the genome with cell-type-specific master transcription factors. Thus, Smad3 occupies the genome with Oct4 in embryonic stem cells (ESCs), Myod1 in myotubes, and PU.1 in pro-B cells. We find that these master transcription factors are required for Smad3 occupancy and that TGF-β signaling largely affects the genes bound by the master transcription factors. Furthermore, we show that induction of Myod1 in nonmuscle cells is sufficient to redirect Smad3 to Myod1 sites. We conclude that cell-type-specific master transcription factors determine the genes bound by Smad2/3 and are thus responsible for orchestrating the cell-type-specific effects of TGF-β signaling.American Gastroenterological AssociationNational Institutes of Health (U.S.) (NIH grant DK090122)Canadian Institutes of Health ResearchNational Institutes of Health (U.S.) (NIH grant HG002668

Regulation of Nuclear Hormone Receptors by MYCN-Driven miRNAs Impacts Neural Differentiation and Survival in Neuroblastoma Patients

MYCN amplification and MYC signaling are associated with high-risk neuroblastoma with poor prognosis. Treating these tumors remains challenging, although therapeutic approaches stimulating differentiation have generated considerable interest. We have previously shown that the MYCN-regulated miR-17 similar to 92 cluster inhibits neuroblastoma differentiation by repressing estrogen receptor alpha. Here, we demonstrate that this microRNA (miRNA) cluster selectively targets several members of the nuclear hormone receptor (NHR) superfamily, and we present a uniqueNHRsignature associated with the survival of neuroblastoma patients. We found that suppressing glucocorticoid receptor (GR) expression in MYCN-driven patient and mouse tumors was associated with an undifferentiated phenotype and decreased survival. Importantly, MYCN inhibition and subsequent reactivation of GR signaling promotes neural differentiation and reduces tumor burden. Our findings reveal a key role for the miR-17 similar to 92-regulated NHRs in neuroblastoma biology, thereby providing a potential differentiation approach for treating neuroblastoma patients

Genome-wide localization of small molecules

A vast number of small-molecule ligands, including therapeutic drugs under development and in clinical use, elicit their effects by binding specific proteins associated with the genome. An ability to map the direct interactions of a chemical entity with chromatin genome-wide could provide important insights into chemical perturbation of cellular function. Here we describe a method that couples ligand-affinity capture and massively parallel DNA sequencing (Chem-seq) to identify the sites bound by small chemical molecules throughout the human genome. We show how Chem-seq can be combined with ChIP-seq to gain unique insights into the interaction of drugs with their target proteins throughout the genome of tumor cells. These methods will be broadly useful to enhance understanding of therapeutic action and to characterize the specificity of chemical entities that interact with DNA or genome-associated proteins.National Institutes of Health (U.S.) (Grant HG002668)National Institutes of Health (U.S.) (Grant CA109901)National Institutes of Health (U.S.) (Grant CA146445

Global Transcriptional and Translational Repression in Human-Embryonic-Stem-Cell-Derived Rett Syndrome Neurons

Rett syndrome (RTT) is caused by mutations of MECP2, a methyl CpG binding protein thought to act as a global transcriptional repressor. Here we show, using an isogenic human embryonic stem cell model of RTT, that MECP2 mutant neurons display key molecular and cellular features of this disorder. Unbiased global gene expression analyses demonstrate that MECP2 functions as a global activator in neurons but not in neural precursors. Decreased transcription in neurons was coupled with a significant reduction in nascent protein synthesis and lack of MECP2 was manifested as a severe defect in the activity of the AKT/mTOR pathway. Lack of MECP2 also leads to impaired mitochondrial function in mutant neurons. Activation of AKT/mTOR signaling by exogenous growth factors or by depletion of PTEN boosted protein synthesis and ameliorated disease phenotypes in mutant neurons. Our findings indicate a vital function for MECP2 in maintaining active gene transcription in human neuronal cells.National Institutes of Health (U.S.) (Grant R01-HG002668)National Cancer Institute (U.S.) (P30-CA14051)David H. Koch Institute for Integrative Cancer Research at MITKathy and Curt Marble Cancer Research FundNational Institutes of Health (U.S.) (Grant HD 045022)National Institutes of Health (U.S.) (Grant R37-CA084198)Simons FoundationEuropean Leukodystrophy Associatio