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Program in Genomics of Differentiation

Director: Brant M. Weinstein, PhD

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The Program in 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 brain. Using conditional gene ablation approaches in the mouse, the Unit demonstrated a requirement of Gli3, a major negative regulator of Shh pathway, in neural progenitors in developing neocortex and in establishment of the postnatal neurogenic niche in the subventricular zone of the lateral ventricle. The Unit is currently characterizing additional signaling cues provided by the neurogenic niche that control the behavior of neural stem cells. Another project revealed that spatial and temporal genetic lineages of dopamine neuron precursors determine diversity among dopamine neurons.

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, found many years ago to function as a second messenger in bacteria and plants, where it couples global regulation of gene expression to nutrient availability. Recently, another laboratory discovered that worms, flies, and humans have genes for highly specific, structural homologs of bacterial ppGpp hydrolases. This raises the question as to whether ppGpp itself is present in animals, where ppGpp is exceedingly difficult to detect biochemically. Therefore, the group is embarking on a search for an animal ppGpp synthetase by screening cDNA developmental libraries from fruit flies for genes that complement growth defects of bacterial ppGpp mutants. The presence of ppGpp seems likely because either deletion of the fly hydrolase or overexpression of a bacterial ppGpp synthetase similarly regulate fly development and global gene expression in a manner reminiscent of ppGpp behavior in lower organisms. The Section also has a special interest in the details of bacterial mechanisms by which ppGpp either activates or inhibits RNA polymerase transcription of specific promoters. These effects are based upon intrinsic kinetic properties of specific promoters, without the help of sequence recognition proteins. The studies are important because ppGpp is necessary for virulence-gene expression in most bacterial pathogens.

Ajay Chitnis and colleagues in the Section on Neural Developmental Dynamics are examining how the posterior lateral line system is built in the zebrafish nervous system. The lateral line is a mechanosensory system that consists of sensory organs called neuromasts, which are distributed in a stereotypic pattern over the surface of the zebrafish. Development of hair cells in the lateral line neuromasts is remarkably similar to that of hair cells in the human ear. Furthermore, the mechanisms that guide migration of the lateral line primordium as it deposits neuromasts under the skin, are remarkably similar to those that determine migration of metastatic cancer cells. The goal of the laboratory is to define the genetic regulatory network that coordinates cell fate and morphogenesis in the lateral line system and to build computational models, based on these studies, to understand how this relatively simple sensory system in zebrafish builds itself.

David Clark and his colleagues in the Section on Chromatin and Gene Expression 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 this chromatin block, eukaryotic cells possess chromatin-remodeling and histone-modifying complexes. The laboratory uses Next-Generation paired-end sequencing to determine nucleosome positions genome-wide in the yeast Saccharomyces cerevisiae and finds that canonical nucleosomes are not uniquely positioned with respect to the DNA. In contrast, the specialized centromeric nucleosomes are perfectly positioned. Gene activation correlates with large-scale loss of nucleosomes and re-positioning of the remaining nucleosomes over entire genes.

Robert Crouch, who leads the Section on Formation of RNA, studies RNases H, enzymes that degrade RNA in RNA/DNA hybrids. Failure to degrade RNA in DNA can lead to loss of mitochondrial DNA, detrimental DNA recombination, and severe neurological defects. Type I RNase H is structurally and functionally related to an essential RNase H of the HIV-AIDS virus and could be a target for HIV drug therapy. In humans, defective Type II RNase H can result in Aicardi-Goutières syndrome (AGS), an encephalopathy that mimics in utero viral infection. A collaborative effort determined the structure of human RNase H2, a three-subunit protein, permitting localization in the 3-D structure of all presently known AGS mutations. Human RNase H2 can remove single ribonucleotides misincorporated in DNA as well as longer stretches of RNA/DNA hybrids, thereby helping to maintain genome stability.

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 the formation of the caudal region and the neural crest in the zebrafish embryo. The Wnt target gene cdx4 controls development of caudal tissues and is essential for hematopoiesis. Regulation of cdx4 by Wnt signaling was shown to be modulated by E4f1 through dissociation of the Tcf3 repressor complex. This mechanism assures robustness of the graded expression of cdx4 in the caudal body region. In a second project, the group studied the regulation of neural crest specification by the BTB domain protein Kctd15. Kctd15 inhibits neural crest formation and is believed to act to assure separation of the neural crest and pre-placodal domains in the zebrafish embryo.

Mel DePamphilis, who heads the Section on Eukaryotic DNA Replication, studies the control of DNA replication and gene expression during mammalian development. His current work focuses on three questions: how genome duplication is restricted to once per cell division in proliferating cells; how these mechanisms are circumvented during mammalian development to allow some cells to differentiate into viable, nonproliferating polyploid cells; and how these mechanisms are related to human pathologies such as cancer. The Section recently discovered a novel role for the protein geminin in preventing DNA re-replication during mitosis and showed that suppression of geminin could be used to kill cancer cells without harming normal cells. The Section is currently using high-throughput screening technology to look for anti-geminin drugs and for other genes essential for proliferation of cancer cells, but not for normal cells. In addition, the group discovered novel roles for the transcription factor TEAD4 in regulating energy homeostasis during preimplantation development aa well as for the CDK inhibitors p57/Kip2 and p21/Cip1 and the DNA damage–response protein kinase CHK1 in triggering differentiation of trophoblast stem cells into trophoblast giant cells.

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. The group pays special attention 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. The group develops and implements bioinformatic tools to facilitate human genome annotation as well as pattern recognition/comparison searches of next-generation sequencing data. Together, these unbiased genomics approaches will be 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. The laboratory uses two approaches to understand PRE function. First, the group identifies DNA sequences required for PRE function and has made considerable progress towards identifying the complex array of DNA binding proteins required for the activity of one PRE. Second, in an effort to understand the role of PREs in the context of other regulatory DNA, the Section studies PRE activity at the PcG target gene engrailed. The DNA that regulates engrailed expression stretches over 70kb. The Section's recent results indicate that the chromatin structure set up by the PcG proteins may facilitate the activity of some engrailed enhancers.

Jim Kennison, who heads the Section on Drosophila Gene Regulation, studies the genomics of pattern differentiation in Drosophila. 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. New trans-acting factors that maintain proper repression of the homeotic genes have been identified in screens for new recessive mutants in somatic clones. While characterizing the essential genes within two regions that span about 1% of the Drosophila genome, the group also identified the first gene desert in Drosophila, a region of 55kb with many evolutionarily conserved DNA sequences, but with no apparent function under laboratory conditions.

Judith Levin and colleagues in the Section on Viral Gene Regulation study the molecular aspects of HIV-1 replication. A major focus is the critical role of the viral nucleocapsid protein (NC), a nucleic acid chaperone that remodels nucleic acid structures and is required for almost all steps in reverse transcription. The Section demonstrated that the Gag precursor (through its NC domain) also has chaperone activity, although at high concentrations, Gag severely inhibits DNA elongation. Based on this finding, the group proposed a novel roadblock mechanism for regulating reverse transcription. Ongoing functional analysis of HIV-1 capsid protein has revealed the importance of the interdomain linker region for proper core assembly and stability. Additionally, the group is investigating the molecular properties and three-dimensional structure of human APOBEC3A, a restriction factor that blocks LINE-1 retrotransposition.

The Section on Cellular and Developmental Biology, led by Paul Love, studies mammalian hematopoietic development. A main area of research conerns T lymphocyte development, particularly signal transduction molecules and pathways that regulate T cell maturation in the thymus. The group revealed a critical function for T cell antigen receptor (TCR) signaling in controlling key developmental events essential for the prevention of autoimmunity and recently described a previously unknown T cell–specific protein, Themis, which functions to sustain TCR signaling during T cell development. Additional studies identified an essential function for the nuclear adapter LIM domain protein-1 in hematopoietic stem cell maintenance and erythropoiesis.

Richard Maraia, who heads the Section of Molecular and Cell Biology, studies RNA metabolism, with continued interest in tRNA biogenesis. Efforts focus on RNA 3′-end formation by RNA polymerase III and the RNA 3′-end–stabilizing protein La. The Section's recent data suggest that La-related protein-4 (LARP4) binds to 3′ poly(A) and stabilizes mRNAs. Another focus is on Tit1p, an anticodon loop–modification enzyme that modifies a small subset of tRNAs, and its effects on the subset of mRNAs with cognate codon bias. The Section uses genetics coupled with genome-wide profiling and this-generation sequencing, cell and structural biology, and biochemistry in model systems that include yeast, mammalian cultured cells, and gene-altered mice.

Keiko Ozato heads the Section on Molecular Genetics of Immunity. The Section studies gene regulation in innate immunity, focusing on the role of chromatin. The group showed that the transcription factor IRF8 plays a central role in eliciting innate anti-pathogen resistance in macrophages and dendritic cells. The group's recent analyses show that pathogen stimulation alters SUMO modification of numerous nuclear proteins leading to a global shift in gene expression patterns in macrophages. The study also found that SUMO modification is an important mechanism by which to control IRF8 transcriptional activity. The group previously showed that the bromodomain protein BRD4 binds to acetylated chromatin and the elongation factor P-TEFb to stimulate transcription. Studying interferon (IFN) stimulation as a model for innate immunity, the Section showed that IFN triggers the recruitment of BRD4 to IFN–stimulated genes and that this recruitment is the primary event that initiates productive elongation. The Section's subsequent work with this model revealed that transcription leads to a large-scale exchange of chromatin in the IFN–stimulated genes, replacing the standard histone H3 with the variant H3.3. The exchange led to the formation of a lasting mark signifying earlier transcription of IFN–stimulated genes.

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 role of non-coding RNAs in establishing these chromosomal structures. The group establishes mouse models for human diseases associated with genetic lesions in the region. Current studies focus on conditional gain of function and conditional loss of function of the cardiac calsequestrin 2 gene.

Tom Sargent, who heads the Section on Vertebrate Development, and colleagues study TFAP2–regulated genes in the cranial neural crest (CNC). In previous years, the group investigated MyosinX, which encodes a non-muscle motor protein, and Inka, a novel protein that promotes actin stress-fiber formation and inhibits microtubule acetylation, probably through interactions with pak4 (p21-activated kinase 4), a Rho-GTPase effector molecule. More recently, the focus has shifted to the functions of pak4 itself in zebrafish development, where it behaves as a maternal effect gene and is required for somite morphogenesis, molecular signaling of early blood cell development, and other functions. The group is also developing imaging strategies for monitoring cell-cell signaling in living zebrafish and is using a mutant version of the homeobox gene dlx3, corresponding to a human genetic disorder, to probe neural crest development, again in zebrafish.

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 now 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. Weinstein and his colleagues also 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 (HD) 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 continues 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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