National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2015 Annual Report of the Division of Intramural Research

Program in Cellular Regulation and Metabolism

Director: Alan G. Hinnebusch, PhD

The seven investigators in this program, all belonging to the Laboratory of Gene Regulation and Development (LGRD), share a common interest in using genetically tractable model organisms and a combination of genetics, biochemistry, cell biology, and structural biology to elucidate the molecular basis of processes fundamentally important in cell biology or animal development. Three groups employ yeast as a model system, with two studying molecular mechanisms of transcriptional and translational control of gene expression in budding yeast, and a third studying transposition of mobile elements in fission yeast. Two groups employ the fruit fly Drosophila to investigate development and function of neural circuits, including synaptic specificity in the visual system and assembly and growth of synapses at the neuromuscular junction. The anuran Xenopus laevis serves as a model system for the work of the remaining two groups, one of which focuses on spindle assembly and chromosome segregation and the roles of covalent protein modification by SUMO in regulating these events, and the other on transcriptional programming during the postembryonic formation of adult stem cells.

The Section on Cell Cycle Regulation, headed by Mary Dasso, studies cellular pathways that assure the integrity of genomic transmission during each cell division in higher eukaryotes, particularly mechanisms of mitotic chromosome segregation. The group showed that proteins previously described for their functions in interphase nuclear-cytoplasmic protein trafficking are critical for correct spindle assembly and mitotic progression and is studying the details of these functions, as well as mitotic roles of the SUMO protein modification pathway. The laboratory recently described the function of IRBIT, a novel inhibitor of ribonucleotide reductase (RNR). RNR supplies deoxynucleotide triphosphates for DNA replication, and its uncontrolled activity is associated with malignant transformation and tumor cell growth. Members of the Section showed that IRBIT is essential for proper cycle progression and genomic integrity, and they are currently exploring IRBIT's physiological activities and regulation.

The Section on Protein Biosynthesis, headed by Thomas Dever, is characterizing the structure and function of translation factors and stress-responsive eIF2α kinases that control cellular protein synthesis. Recently, the group showed that the baculovirus pseudokinase PK2 inhibits an insect eIF2a kinase through a proposed lobe-swapping mechanism. Members of the Section also identified an eIF2a–docking motif conserved among cellular and viral proteins that targets protein phosphatase PP1 to dephosphorylate eIF2a. They demonstrated that the hypusine-containing protein eIF5A promotes translation elongation by facilitating the reactivity of poor substrates like proline, and they characterized a human X-linked intellectual disability syndrome caused by a mutation in translation factor eIF2γ.

Members of the Section on Nutrient Control of Gene Expression, headed by Alan Hinnebusch, study fundamental mechanisms of transcriptional and translational control of gene expression in budding yeast. Recently, they demonstrated conformational rearrangements in the small (40S) ribosomal subunit that accompany transition from the scanning to start codon selection stages of translation initiation, implicated the 40S protein uS7 directly in this process, and showed that the DEAD–box helicase Ded1/Ddx3 selectively promotes initiation on mRNAs with structured 5′ untranslated regions. They established that Rli1/ABCE1 is required in vivo for ribosome recycling, the last step of protein synthesis, and blocks aberrant reinitiation by unrecycled ribosomes in 3′ untranslated regions of all mRNAs. They also demonstrated that the chromatin remodeling complex SWI/SNF, histone acetyltransferase complex SAGA, and Hsp70 co-chaperone Ydj1 functionally cooperate in evicting promoter nucleosomes genome-wide and selectively stimulating transcription of the most highly expressed genes in cells.

The Section on Neuronal Connectivity, headed by Chi-Hon Lee, investigates the assembly and function of chromatic circuits in Drosophila. The group combines imaging and genetic approaches to determine the mechanisms by which optic lobe neurons elaborate dendrites in stereotypic patterns to synapse with appropriate partners. Members of the Section recently identified several molecular cues that control different aspects of dendritic patterning, including dendritic receptive field sizes and planar projection directions. They demonstrated that dendritic patterning defects lead to the formation of erroneous synaptic connections. To study visual functions, they mapped the visual circuits involved in innate and learned color-driven behaviors and identified the neuro-pathways from photoreceptors to peripheral and to higher visual centers.

Henry Levin heads the Section on Eukaryotic Transposable Elements, which analyzes the integration of LTR retrotransposons, retroviruses, and DNA transposons into the chromosomes of host cells. Recently, the laboratory developed new methods of deep sequencing that yield ultra-dense profiles of integration. Dense maps of retrotransposon Tf1 integration in Schizosaccharomyces pombe demonstrated that the promoters of stress-response genes are actively targeted. In other studies, dense maps of HIV-1 integration in cultured human cells reveal that cancer-related genes are among the most frequently disrupted. In a surprising turn, the laboratory found that HIV-1 integration is directed by the host factor LEDGF to highly spliced genes. The laboratory also adapted the Hermes transposable element from the housefly to generate maps of unprecedented density in S. pombe. The profiles of Hermes integration yielded comprehensive sets of essential genes and genes with roles in heterochromatin formation.

The Unit on Cellular Communication, headed by Mihaela Serpe, investigates molecular mechanisms that regulate synapse development. The group focuses on glutamatergic synapses and uses the Drosophila neuromuscular junction (NMJ) model system. Recently, the group identified Neto (Neuropillin and Tolloid-like) as an essential component of the NMJ ionotropic glutamate receptor (iGluR) complexes required for clustering of these receptors at synaptic sites. The laboratory established that Neto (1) engages in extracellular interactions that stabilize iGluRs at synaptic sites and trigger postsynaptic differentiation, (2) mediates intracellular interactions that anchor postsynaptic density components and sculpt iGluRs postsynaptic composition, and (3) modulates iGluRs' function, but not their assembly or surface delivery.

The Section on Molecular Morphogenesis, headed by Yun-Bo Shi, uses the Xenopus model to study the gene-regulatory mechanisms controlled by thyroid hormone receptor (TR) that establish the postembryonic developmental program in vertebrates. Using the TALEN-mediated gene knockdown approach, members of the Section revealed novel functions of TRα in development, i.e., regulating premetamorphic tadpole growth rate and controlling the timing of metamorphosis. They further demonstrated that the histone methyltransferase Dot1L is a direct TR target gene and is required for premetamorphic tadpole growth and survival but not embryogenesis. They also identified and characterized candidate thyroid hormone–regulated genes involved in adult intestinal stem cell development during metamorphosis.

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