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Program in Genomics of Differentiation

Director: Brant M. Weinstein, PhD

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The Program on Genomics of Differentiation (PGD) is a diverse and highly interactive program in cellular, molecular, and developmental biology research within the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), Division of Intramural Research (DIR). With 19 primary investigators, the PGD is the largest program in the NICHD DIR, encompassing a variety of research areas, including developmental differentiation and patterning, chromatin dynamics and epigenetics, the immune system, the viral life cycle, DNA replication, gene regulation, and RNA metabolism. Program investigators perform research using a wide variety of models including viruses, bacteria, mammalian cell culture, yeast, fruit flies, zebrafish, frogs, and mice. Vertebrate models are a major focus of the program. The zebrafish is used as a model for analysis of embryonic development and organogenesis, as well as for modeling certain human conditions. Using genetics, genomics, and high-resolution imaging techniques, PGD investigators study cell-cell signaling and cellular behavior in early embryogenesis, formation and morphogenesis of the vascular system, cellular specification in the developing nervous system, and cellular and molecular mechanisms underlying behavior. The mouse provides another important vertebrate model. PGD investigators employ advanced gene-targeting and transgenic technologies to study genes that control mouse development, including transcriptional control in the early embryo, the role of Lim-homeobox genes and chromatin-binding proteins, mechanisms of genomic imprinting, regulation of immune cells, the development of the central and peripheral nervous systems, and the behavior of neural stem cells in the adult organism. In addition, the Program generates mouse models of a diverse array of human genetic disorders.

Sohyun Ahn, who heads the Unit on Developmental Neurogenetics, and colleagues continue to investigate the role of Sonic hedgehog (Shh) signaling and its downstream effectors in neurogenesis of the developing and mature brains. Using conditional gene ablation approaches in the mouse, the Unit demonstrated a requirement of Gli3, a major negative regulator of Shh pathway, in intermediate progenitors in developing neocortex. The Unit recently identified several genes that are specifically expressed in adult neural stem cells and is in the process of functional validation. An additional project is aimed at elucidating the origin of diversity among dopamine neurons and their circuit formation in the ventral midbrain.

Harold Burgess's Unit on Behavioral Neurogenetics studies the development and function of neural circuits required for motor control in larval zebrafish. Brainstem circuits, which control behavior in zebrafish larvae, represent the core of the movement control system in higher vertebrates and are impaired in numerous neurological disorders. The Unit applies computational analysis to high-speed video recordings of larvae challenged with distinct sensory stimuli to determine the function of identified brainstem neurons in transgenic fish. Burgess's group has generated novel transgenic fish lines to study how distinct cohorts of neurons in the serotonergic raphe nuclei modulate behavior. In addition, the group is currently mapping genetic mutations that impair normal modulation of the startle response and that phenocopy deficits in prepulse inhibition in schizophrenic patients.

Many years ago, Mike Cashel's Section on Molecular Regulation discovered the guanine nucleotide analog ppGpp, which functions as a second messenger in bacteria and plants to regulate global gene expression in response to many sources of nutritional and environmental stress. Regulation of transcription results in either negative or positive effects on initiation specificity by direct ppGpp binding to the RNA polymerase protein, unlike regulatory mechanisms for the cyclic nucleotide cAMP. The Section has special interest in synergistic effects on ppGpp transcription regulation of additional RNA polymerase–interacting proteins (GreA, GreB, DksA, and TraR). Studies currently focus on mechanisms of positive regulation by ppGpp because, for many bacterial pathogens, virulence gene expression frequently requires ppGpp.

Ajay Chitnis, who heads the Section on Neural Developmental Dynamics, and colleagues study the formation of the nervous system in zebrafish, in particular neuronal versus non-neuronal fate determination, and how distinct regions assume different structural and functional identities. The group studied proteins interacting with Mindbomb (Mib), a known regulator of Notch signaling, which is required for proper specification of neuronal differentiation. The study identified the protein Mosaic eyes (Moe) as a Mib-interacting protein. The group is now investigating Moe's role in modulating Notch signaling.

David Clark, who heads the Section on Chromatin and Gene Expression, and his colleagues study the role of chromatin structure in gene activation. Gene activation must occur in the presence of nucleosomes, which are compact structures capable of blocking transcription at every step. To circumvent the chromatin block, eukaryotic cells possess chromatin-remodeling and nucleosome-modifying complexes. The laboratory developed a model system in which native plasmid chromatin containing an activated or inactive gene is purified from yeast cells and analyzed using high-resolution mapping techniques. The studies showed that gene activation correlates with large-scale movements of nucleosomes and conformational changes within nucleosomes over entire genes, as well as with histone modifications.

Robert Crouch, who leads the Section on Formation of RNA, studies RNases H, enzymes that degrade RNA in RNA/DNA hybrids. Type I RNase H is structurally and functionally related to an essential RNase H of the HIV-AIDS virus. A collaborative effort determined the structure of a domain of human RNase H1 in complex with an RNA/DNA, providing an explanation for how the mammalian RNase H1 binds via a binding domain that provides additional functionality. Studies on type 2 RNase H, an enzyme composed of three subunits, provide information on Aicardi-Goutières syndrome, an encephalopathy that mimics in utero viral infection, and demonstrated that there is no direct correlation between enzymatic activity and the disorder.

Igor Dawid, who heads the Section for Developmental Biology, and colleagues study early development in the frog and zebrafish. Recent efforts focused on the regulation of early axis specification in zebrafish. The control of stability of a regulatory protein mediated by an E3 ubiquitin ligase was shown to be a critical step in this process. Another project illuminated the mechanism through which fibroblast growth factor (Fgf) signaling influences the establishment of laterality in the zebrafish. The effect of Fgf in this system is mediated by two novel factors, named Ier2 and Fibp1. Both factors are induced by Fgf and are required for the pathway that eventually leads to proper placement of visceral organs in the zebrafish larva.

Mel DePamphilis, who heads the Section on Eukaryotic DNA Replication, and his colleagues study the control of DNA replication and gene expression during early mouse embryogenesis, particularly the differentiation of trophoblast stem (TS) cells and embryonic stem cells. Current work focuses on the mechanisms that prevent proliferating cells from duplicating their genome more than once per cell division and how these mechanisms are circumvented during normal mammalian development to induce polyploidy in specific cell types. Recent discoveries include the unique role of the CDK inhibitor p57/Kip2 in triggering differentiation of TS cells into trophoblast giant cells and the regulation of p57 expression in TS cells by the DNA damage–response protein kinase CHK1. In proliferating cells, suppression of geminin (a protein unique to metazoa that suppresses re-replication of DNA during S-phase) can be used to kill cancer cells without harming normal cells, a discovery that resulted in a patent application for anti-geminin drugs in cancer therapy. Investigation of genes that regulate cell proliferation unexpectedly revealed a novel role for Dkkl1 in sperm development and fertilization and a role for Tead genes in cell proliferation.

A major effort of the Human Genetics Section, led by Bruce Howard, focuses on epigenome structure, including higher-order chromatin architecture and DNA methylation patterns. Special attention is paid to how defects in the maintenance of epigenetic structures (or failures in programmed transitions, especially in the perinatal period) may underlie common developmental disorders and age-related diseases. Bioinformatic tools are developed and implemented to facilitate human genome annotation and pattern recognition/comparison searches of chromatin immunoprecipitation data, as well as analyses of microarray and next-generation sequencing data. Together, these genomics approaches are invaluable for identifying new features of developmental and age-related of epigenome remodeling.

Judith Kassis, who heads the Section on Gene Expression, studies the mechanism of gene silencing by the Polycomb group genes (PcG) in Drosophila and the nature of DNA elements responsible for silencing (called Polycomb response elements or PREs). PREs are several hundred nucleotides in length and consist of binding sites for numerous proteins. Two approaches are taken to understand PRE function. First, DNA sequences required for PRE function are identified. This past year, two new PREs were characterized. Second, in an effort to understand the role of PREs in the context of other regulatory DNA, PRE activity at a target gene is studied. This past year, the group investigated enhancer-promoter communication and the role of PREs in engrailed regulation.

Jim Kennison, who heads the Section on Drosophila Gene Regulation, studies the genomics of pattern differentiation in Drosophila. The rhinoceros gene, which encodes a transcription factor that interacts with the von Hippel Lindau tumor suppressor, was shown to interact with the Brahma chromatin remodeling complex to regulate transcription of homeotic genes. Characterization of mutants in one of the gamma-tubulin genes also suggested a role for microtubule organizer complexes in transcriptional regulation. The group defined cis-acting regulatory sequences in the Sex combs reduced gene, indicating that, after the end of embryogenesis, two genetic elements about 70 kb apart in the Sex combs reduced gene must be in cis to maintain proper repression.

Judith Levin, who heads the Section on Viral Gene Regulation, and colleagues study the molecular aspects of HIV-1 replication and reverse transcription, focusing on the critical role of the nucleocapsid protein (NC), a nucleic acid chaperone that destabilizes highly structured nucleic acid reverse transcription intermediates. The Section demonstrated that the Gag precursor (through its NC domain) also has chaperone activity. However, unlike NC, Gag exhibits slow nucleic acid binding dissociation kinetics and, at high concentrations, Gag inhibits DNA elongation. The group proposed a novel roadblock mechanism for regulating reverse transcription. Studies on the HIV-1 capsid protein demonstrated that non-infectious linker mutants have detergent-sensitive cores with aberrant structure and abnormally low viral DNA synthesis in infected cells.

The Section on Cellular and Developmental Biology, led by Paul Love, focuses on T lymphocyte development, particularly signal transduction molecules and pathways that regulate T cell maturation in the thymus. The group revealed a key difference in the subunit composition and signaling potential of the antigen receptors (TCRs) expressed on two distinct classes of T cells. Recently, the group described a previously unknown T cell–specific protein, Themis, which functions during CD4 versus CD8 lineage choice of T cells and in their subsequent maturation. In studying molecules that control T cell migration and trafficking, the group discovered a critical function for the chemokine receptor CCR9 in regulating the migration of developing T cells to and within the thymus. Additional studies focused on the role of LIM domain proteins and the partner LDB in the regulation of T cells.

Richard Maraia, who heads the Section of Molecular and Cell Biology, studies RNA metabolism, with a continued interest in tRNA biogenesis. Efforts focus on 3′-end formation by RNA polymerase III and the RNA 3′-end stabilizing protein La. Human La protein is an autoantigen in autoimmune patients. The Section's new data suggest that La-related protein-4 (LARP4) stabilizes mRNAs. Another focus is on a tRNA anticodon-loop modification enzyme that confers resistance to rapamycin, an antitumor drug to which certain pediatric cancers develop resistance. The Section uses genetics coupled with genome-wide profiling and sequencing, cell and structural biology, and biochemistry in model systems that include yeast, mammalian cultured cells, and gene-altered mice.

Keiko Ozato, who heads the Section on Molecular Genetics of Immunity, and colleagues study transcription factors and chromatin-binding proteins that control the development of innate immunity. They showed that IRF8 drives the development of plasmacytoid dendritic cells (DCs) and CD8a+ DCs, subsets that produce type I interferons (IFNs) and IL-12. These cytokines confer anti-microbial activities. The group also showed that the bromodomain protein Brd4 binds to acetylated chromatin and is implicated in the maintenance of transcriptional memory. Further, Brd4 regulates cell cycle progression by binding to the promoters of many G1 genes to recruit P-TEFb, a kinase that triggers transcriptional elongation.

The Section on Genomic Imprinting, led by Karl Pfeifer, examines the regulated expression and the biological functions of a cluster of imprinted genes on the distal end of mouse chromosome 7. Imprinting is an unusual form of gene regulation in which expression of a gene depends on parental-specific epigenetic modifications of the chromosome. Imprinted loci are an excellent model system for studying how epigenetic mechanisms regulate development and how developmental processes establish the epigenome. The group identified and characterized a 2.4 kb element that organizes higher-order chromosomal structures and long-range DNA interactions across a 120 kb region. Current work focuses on the mechanisms for paternal-specific methylation of this element. The group established mouse models for human diseases associated with genetic lesions in the region. The studies have led to novel therapies for calsequestrin 2–deficient patients.

Tom Sargent, who heads the Section on Vertebrate Development, and colleagues study TFAP2-regulated genes in the cranial neural crest (CNC), focusing on MyosinX, which encodes a non-muscle motor protein, and Inka. The group showed that both genes are necessary for normal craniofacial development in Xenopus, and, in the case of Inka, in zebrafish. Inka is a novel protein that promotes actin stress fiber formation and inhibits microtubule acetylation, probably through interactions with p21-activated kinase 4, a Rho-GTPase signaling factor. Inka is also required for the normal function of inductive pathways during gastrulation. Under certain circumstances, Inka can also regulate signal transduction through the ERK/MAPK pathway. Loss of MyosinX expression inhibits CNC cell migration in Xenopus.

Brant Weinstein's Section on Vertebrate Organogenesis studies blood and lymphatic vessel formation during vertebrate embryogenesis. Vessel formation is of intense clinical interest because of the roles blood and lymphatic vessels play in cancer and ischemia. Using the zebrafish, the group developed a widely used confocal microangiography method, compiled an atlas of the vasculature, developed numerous vascular-specific transgenic lines, and pioneered methods for high-resolution in vivo imaging of blood vessels. The group discovered a novel pathway of artery specification, a role for neuronal guidance factors in vascular patterning, and a mechanism for vascular tube formation in vivo; further, Weinstein and his colleagues identified the lymphatic vascular system in zebrafish. Current studies use genetic screening, experimental analysis, and imaging to examine cues directing vascular patterning and morphogenesis, regulation of vascular integrity, and assembly of the lymphatic system.

Heiner Westphal and his Section on Mammalian Molecular Genetics study the function of members of the Lhx gene family, which encode LIM-homeodomain transcription factors. Transcription of target genes is regulated by oligomeric complexes involving individual Lhx gene products plus members of the Ldb and Ssdp families of transcriptional co-regulators. Over the years, the group has gained insights into the individual or combined actions of individual LIM-HD factors during patterning and organ formation in the developing mouse embryo. This work is being continued with a main focus on the involvement of Lhx genes in the development of the mouse brain. A novel aspect of the group's research concerns the reprogramming of somatic cells to an induced pluripotent stem (iPS) cell state. The project focuses on generating patient-specific iPS cell clones pertaining to a number of patient cohorts that are included in clinical protocols directed by members of the NIH intramural research community. Neuronal cells differentiated from these iPS clones will be of preeminent importance for subsequent experiments aimed at studying the basis of the neurological deficiencies observed in these patients.

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