Howard Hughes Medical Institute

About: Howard Hughes Medical Institute is a nonprofit organization based out in Chevy Chase, Maryland, United States. It is known for research contribution in the topics: Gene & RNA. The organization has 20371 authors who have published 34677 publications receiving 5247143 citations. The organization is also known as: HHMI & hhmi.org.

Detection and quantification of rare mutations with massively parallel sequencing

Abstract: The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Although massively parallel sequencing instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System (“Safe-SeqS”), are (i) assignment of a unique identifier (UID) to each template molecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are considered mutant (“supermutants”) only if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

The role of β-arrestins in the termination and transduction of G-protein-coupled receptor signals

Abstract: beta-Arrestins are versatile adapter proteins that form complexes with most G-protein-coupled receptors (GPCRs) following agonist binding and phosphorylation of receptors by G-protein-coupled receptor kinases (GRKs). They play a central role in the interrelated processes of homologous desensitization and GPCR sequestration, which lead to the termination of G protein activation. beta-arrestin binding to GPCRs both uncouples receptors from heterotrimeric G proteins and targets them to clathrin-coated pits for endocytosis. Recent data suggest that beta-arrestins also function as GPCR signal transducers. They can form complexes with several signaling proteins, including Src family tyrosine kinases and components of the ERK1/2 and JNK3 MAP kinase cascades. By recruiting these kinases to agonist-occupied GPCRs, beta-arrestins confer distinct signaling activities upon the receptor. beta-arrestin-Src complexes have been proposed to modulate GPCR endocytosis, to trigger ERK1/2 activation and to mediate neutrophil degranulation. By acting as scaffolds for the ERK1/2 and JNK3 cascades, beta-arrestins both facilitate GPCR-stimulated MAP kinase activation and target active MAP kinases to specific locations within the cell. Thus, their binding to GPCRs might initiate a second wave of signaling and represent a novel mechanism of GPCR signal transduction.

Genetic and pharmacological disruption of the TEAD–YAP complex suppresses the oncogenic activity of YAP

Abstract: The Hippo tumor suppressor pathway restricts organ size in Drosophila and mammals by antagonizing the oncoprotein Yki/YAP (Zeng and Hong 2008; Pan 2010; Zhao et al. 2010; Halder and Johnson 2011). Central to the Hippo pathway is a kinase cascade leading from the protein kinase Hpo/Mst to Yki/YAP. The Hippo kinase cascade, in turn, is regulated by a complex network of proteins, which most notably includes the Neurofibromin 2 (NF2)/Merlin tumor suppressor. Consistent with the critical role of Hippo signaling in normal tissue homeostasis, the YAP oncoprotein is overexpressed or hyperactivated in a wide spectrum of human cancers due to YAP locus amplification or genetic/epigenetic inactivation of upstream tumor suppressors. Small molecule inhibitors of YAP will not only provide important tools for pharmacological manipulation of Hippo signaling, but also bear tremendous potential for developing therapeutic drugs against human cancers caused by defective Hippo signaling. As a transcriptional coactivator, YAP has been reported to bind to several DNA-binding transcription factors (for review, see Pan 2010). Among the reported YAP partners, the TEAD/TEF transcription factors are best characterized (Vassilev et al. 2001; Chen et al. 2010; Li et al. 2010). Genetic studies in Drosophila revealed an interesting property of its single TEAD ortholog, Scalloped (Sd): While Sd is required for tissue overgrowth driven by hyperactivated Yki, Sd (but not Yki) is largely dispensable for normal tissue growth (Huang et al. 2005; Wu et al. 2008). Thus, Sd/TEAD may belong to a growing list of genes that contribute to “non-oncogene addiction”—genes that are not mutated in cancers but are critically required for cancer growth (Luo et al. 2009). The dispensability of Sd for normal growth in Drosophila suggests that the mammalian TEAD factors may be ideal targets for selective inhibition of oncogenic growth driven by YAP hyperactivation with minimal effects on normal tissue homeostasis. Although previous studies have shown that the TEAD factors are required for YAP's oncogenic activity in cell cultures (Zhao et al. 2008), whether the TEAD factors (or any of the other reported YAP partners) are required for YAP-mediated tumorigenesis has not been determined in intact mammalian tissues. It also remains to be seen whether inhibition of the mammalian TEAD factors, like loss of Drosophila Sd, has minimal impact on normal tissue homeostasis and physiology. Such information will shed light on the “therapeutic window” of pharmacological strategies aimed at disrupting the TEAD–YAP complex as a selective means against YAP-driven tumorigenesis. Using a combination of genetic suppression in transgenic mice and discovery of lead compounds with in vitro and in vivo activities, we provide here proof of principle that inhibiting TEAD–YAP interactions is a promising and pharmacologically viable strategy against the YAP oncoprotein.

Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells

Abstract: Induced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here we show that iPSCs obtained from mouse fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Notably, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and altered differentiation capacity. These observations may influence ongoing attempts to use iPSCs for disease modeling and could also be exploited in potential therapeutic applications to enhance differentiation into desired cell lineages.