PROGRAM IN GENOMICS OF DIFFERENTIATION
The Program in Genomics of Differentiation(PGD) combines the former Laboratory of Mammalian Genes and Development (LMGD), the former Laboratory of Molecular Genetics (LMG), and the former Laboratory of Molecular Growth Regulation (LMGR). The PGD brings together investigators working in a broad range of areas in genetics, genomics, and molecular and developmental biology. Subject areas include genetic control of developmental and physiological processes in model organisms from viruses to mammals, with emphasis on the mouse and zebrafish as model systems. The PGD uses wild-type and gene-altered mice to study embryonic and adult stem cells, pattern formation, immune development, genomic imprinting, cell proliferation, and chromatin-mediated gene regulation. The zebrafish is subject to study by genetic, molecular, and imaging tools to investigate the control of early embryogenesis and of morphogenesis, focusing on development of the vascular system and the brain.
Sohyun Ahn’s Unit on Developmental Neurogenetics is interested in the cellular and genetic mechanisms underpinning neural stem cell specification and lineage decisions. Using advanced genetic approaches in mice, the unit is currently investigating the differential requirement for activating and repressing effectors in the sonic hedgehog signaling pathway and the effectors’ roles in the maintenance, proliferation, and differentiation of neural stem cells both in vivo and in vitro. To develop an understanding of the elusive role of adult neurogenesis, the unit is also developing a novel in vivo reporter system to mark and follow the formation of neural circuits generated by neural stem cell–derived granule neurons in the hippocampal dentate gyrus.
Michael Cashel, who heads the Section on Molecular Regulation, has studied the mechanism of general metabolic control in bacteria; the small molecule guanosine tetra/pentaphosphate, or (p)ppGpp, mediates such control. Recently, the section demonstrated that (p)ppGpp is a key regulator of overall growth control in Escherichia coli. Further, the section studied the role of the protein DksA and its relationship to (p)ppGpp in regulating the activity of RNA polymerase.
Ajay Chitnis and colleagues in the Section on Neural Developmental Dynamics study how neurons differentiate in distinct patterns in the various compartments of the zebrafish nervous system. Previously, the section described the role of Zic genes in defining boundaries in the neural plate, adjacent to which cells differentiate as neurons. In the past year, the section characterized a fish-specific gene, Zic6, that has been lost in frogs, birds, and mammals, helping to elucidate the evolution of the Zic genes. In a related project, the section studied the role of the FERM-domain protein Mosaic eyes (Moe) in establishing Wnt signaling centers, adjacent to which neurogenic domains are established in the hindbrain. Further, the section used computer simulations to visualize the dynamics of the genetic network in order to understand how the signaling centers are established.
David Clark and his colleagues in the Section on Chromatin and Gene Expression study gene regulation in yeast. Gene activation must occur in the presence of nucleosomes, which are capable of blocking transcription. Blocked transcription is relieved by (1) chromatin-remodeling complexes such as the SWI/SNF complex that uses ATP to slide nucleosomes along DNA and (2) nucleosome-modifying complexes that contain enzymes such as histone acetylases, which modify histones to allow recognition by other factors. Clark and colleagues developed a system that involves purified plasmid chromatin in order to investigate the role of chromatin structure in gene activation. They found that gene activation correlates with large-scale movements and conformational changes of nucleosomes over entire genes. The section is now investigating (1) the mechanism of nucleosome remodeling by the SWI/SNF complex; (2) cell cycle–dependent regulation of yeast core histone genes by the Spt10p activator; and (3) whether the DNA integrase of human foamy virus is a sequence-specific DNA-binding protein.
Robert Crouch, who leads the Section on Formation of RNA, studies RNases H, which are enzymes that degrade the RNA component of RNA/DNA hybrids. Two types of RNases H exist in most organisms; type 1 is structurally and functionally related to an essential RNase H of the HIV-AIDS virus. A collaborative effort, which led to determination of the structure of the RNase H domain of human RNase H1 in complex with an RNA/DNA, yielded significant insights into the mechanism of replication of the HIV viral genome. Type 2 RNases H of humans and budding yeast comprise three subunits, one of which carries a sequence that helps recruit other proteins to DNA replication/repair complexes. Studies on RNase H2 are providing information on induction of the innate immune response of individuals with Aicardi-Goutières syndrome, an autosomal recessive encephalopathy caused by defective RNase H2.
Igor Dawid and the Section on Developmental Biology study mechanisms of embryogenesis in frogs and zebrafish, with an emphasis on gene discovery by forward genetics, DNA microarray technology, and expression profiling in order to identify genes that play a role in the regulation of embryonic development. The section studies the molecular control of gastrulation movements in the frog, leading to identification of a factor that mediates the rearrangement of the cytoskeleton in response to an extracellular signal. Another project investigated the specification of the neural crest, which gives rise to a wide variety of tissues, and identified two factors that play a role in the neural crest system: (1) a transmembrane protein that is required for the specification of the neural crest at early stages and (2) a transcription factor with a role in chondrogenesis during the formation of the craniofacial skeleton.
Melvin DePamphilis, who heads the Section on Eukaryotic Gene Regulation, studies the regulation of genome duplication and gene expression during mammalian development. The section established the basic features of the “ORC cycle” that restricts initiation of DNA replication. The largest subunit (Orc1) regulates association of the stable ORC(2–5) core complex with replication origins in vivo, and the cell cycle regulates Orc1 activity. The section further studied two transcription factors expressed in early mouse embryos. Targeted deletion showed that one factor, Tead2, is not essential for mouse embryogenesis, whereas the other factor, Tead4, is required for preimplantation development as well as for specification of the trophectoderm lineage in the mouse embryo.
Bruce Howard and the Human Genetics Section focus on higher-order chromatin structure, particularly how defects in the maintenance of such structures may underlie common developmental disorders and age-related diseases. The section uses random genome sampling, customized search algorithms for comparisons of annotated genomes, and genomics-based high-throughput approaches to detect and map age-related areas of chromatin remodeling.
Judy Kassis, who heads the Section on Gene Expression, is studying the mechanism of gene silencing by the Polycomb group genes (PcG) in Drosophila and the nature of the DNA elements responsible for silencing. Recent work shows that several proteins, most notably Sp1/Klf-type and Pho and Pho-like zinc-finger DNA-binding proteins, are required for functional silencing through sequences called Polycomb Response Elements (PREs). After studying the binding-site requirements in one particular PRE in detail, the section is now investigating whether different PREs use the same or different DNA-binding proteins. In related studies, the section found that sequences overlapping with or closely linked to PREs mediate interactions with distantly located enhancers. The section recently conducted a genetic screen to isolate new PcG genes and is in the process of characterizing a new member of this group.
Jim Kennison and the Section on Drosophila Gene Regulation study the genomics of pattern differentiation in Drosophila, particularly the function of homeotic genes in specifying segmental identities. Genetic screens have allowed Kennison to isolate a series of genes that control expression and function of homeotic genes and to characterize the proteins encoded by several of these genes. Recently, the section identified new genes that regulate the homeotic gene Sex combs reduced and defined cis-acting regulatory sequences in Sex combs reduced that are required for correct expression, leading to the interesting conclusion that two genetic elements that are about 70 kb apart must be in cis to maintain repression of the gene after the end of embryogenesis. The section studied nuclear import and export of the homeodomain protein Prospero. Caliban, a protein whose human homologue has been implicated in colon and lung cancer, controls such import/export activity. Drosophila lacking Caliban protein are more susceptible to tumor formation following irradiation.
Judith Levin and colleagues in the Section on Viral Gene Regulation study HIV-1 replication and reverse transcription, with an emphasis on the role of the viral nucleocapsid protein (NC). NC functions as a nucleic acid chaperone that destabilizes highly structured DNA or RNA intermediates in the viral DNA-synthetic pathway. The section found that the local structure at nucleation sites for annealing, rather than the overall thermostability of the nucleic acid partners, is a major determinant of NC’s chaperone activity. A related project concerns the human cytidine deaminase APOBEC3G, which blocks HIV-1 replication in the absence of the viral protein Vif. Using highly purified APOBEC3G for assays of individual steps in reverse transcription, the section demonstrated that APOBEC3G inhibits all reverse transcriptase–catalyzed DNA elongation reactions, but neither RNase H activity nor NC’s ability to promote annealing. Inhibition is deaminase-independent and determined by critical differences in the nucleic acid–binding properties of APOBEC3G, NC, and reverse transcriptase.
Paul Love, who heads the Section on Cellular and Developmental Biology, studies the development of the mammalian hematopoietic system. An area of particular interest is signal transduction molecules and pathways that regulate T cell maturation in the thymus. Current projects focus on generating transgenic and conditional deletion mutants to evaluate the importance of T cell antigen receptor signaling at specific stages of T cell development. The section is also using microarray gene profiling to identify molecules important for fine-tuning the T cell signaling response in maturing T lymphocytes. A recently initiated project focuses on the role of LIM domain–binding protein-1 (Ldb1) in hematopoiesis. Studies have revealed a critical function for Ldb1 in regulating the self-renewal/differentiation cell fate decision in hematopoietic stem cells, suggesting that Ldb1-nucleated transcription complexes may control maintenance of lineage-specific stem cells.
Richard Maraia and the Section on Molecular and Cell Biology study the synthesis and post-transcriptional processing pathways for tRNAs and other small RNAs and how they affect cell proliferation, growth, and development. The section focuses on transcription by RNA polymerase III and on the RNA-binding phosphoprotein known as La. Although La protein is an autoantigen in Sjögren’s syndrome, systemic lupus, and neonatal lupus, it functions in normal cells in tRNA production in the nucleus and in the metabolism of mRNAs that encode ribosome subunits and translation factors in the cytoplasm, thereby contributing to the protein-synthetic capacity of cells during growth and development. The section uses genetics, cell and structural biology, and biochemistry techniques in model systems that include yeast, human tissue culture cells, and gene-altered mice.
Keiko Ozato and colleagues in the Section on Molecular Genetics of Immunity study transcription factors and chromatin-binding proteins that control the development of innate immunity. IRF8, a DNA-binding protein previously cloned by this section, is essential for the development of cells that provide immediate resistance against infection, namely, macrophages and dendritic cells (DCs). DCs produce large amounts of type I interferons and are critical for the establishment of innate immunity and ensuing adaptive immunity. In differentiated DCs, IRF8 activates a diverse set of genes involved in host defense, including PML and type I interferon genes. In a separate project, the section studies the bromodomain protein Brd4, which binds to acetylated chromatin during interphase and mitosis and is implicated in the maintenance of transcriptional memory across cell division. Treatment of cells with antimitotic drugs temporarily abrogates the interaction of Brd4 with chromatin, even though the drugs have no effect on histone acetylation, indicating that Brd4-chromatin interactions are controlled by mitotic stress signals and that Brd4 protects cells against mitotic stress and is required for accurate cell division.
Karl Pfeifer, who heads the Section on Genomic Imprinting, and his colleagues examine the regulated expression and biological function of a cluster of imprinted genes on the distal end of mouse chromosome 7. The section’s gene expression studies focus on the Igf2/H19 gene cluster. The researchers identified a 2.4 kb DNA element called the H19ICR (for H19 imprinting control region) that is both necessary and sufficient to imprint the locus. The section recently described ICR’s role in organizing higher-order chromosome structures and long-range DNA interactions that determine the monoallelic expression patterns of both Igf2 and H19. The section also continues to develop and analyze mouse strains carrying ion channel mutations that pheno-copy cardiac disorders associated with this gene cluster in humans.
Tom Sargent and colleagues in the Section on Vertebrate Development study TFAP2, a central transcription factor in epidermal and neural crest development in Xenopus, and three downstream targets: Inca, PCNS, and MyosinX. Each is required at different stages for normal cranial neural crest (CNC) development, ranging from the initial migration out of the neural tube (PCNS, MyosinX) to final condensation and differentiation into cranial cartilage and bone (Inca). Inca and PCNS also play important roles in the morphogenesis of mesoderm during gastrulation. Inca is a novel signal transduction molecule that modulates RhoGTPase pathways by interacting with p21-activated kinase. PCNS, a protocadherin, appears to modify cell-cell and cell-substrate adhesion via a mechanism that differs from that of other protocadherins. The section is studying MyosinX, which has been previously linked to mitotic spindle and filopodial formation, for its role in the development of the CNC.
The overall research objective of Brant Weinstein, who heads the Section on Vertebrate Organogenesis, is to understand how networks of blood and lymphatic vessels arise during vertebrate embryogenesis. Mechanisms of vessel formation are a subject of intense clinical interest because of the roles played by blood and lymphatic vessels in cancer and ischemia. The zebrafish is uniquely suited for studying vessel formation, notably because of its optically clear embryo that facilitates high-resolution imaging of vessels in living animals. The laboratory developed confocal microangiography, compiled an atlas of vascular anatomy of the developing zebrafish, developed numerous vascular-specific transgenic fish lines, and established high-resolution in vivo imaging of zebrafish blood vessels, all of which make it possible to elucidate a pathway of artery specification, establish a role for neuronal guidance factors in vascular patterning, illuminate vascular tube formation in vivo, and identify a lymphatic vascular system in the zebrafish.
Robert Weisberg and his colleagues in the Section on Microbial Genetics study the control of gene expression in bacteria and bacterial viruses. Their major focus has been the mechanism of transcription antitermination, a process that activates the expression of genes preceded by terminators. The section showed that antitermination depends on a specific structure of the nascent RNA that interacts with elongating RNA polymerase. In addition, the section investigated the mechanistic relation between antitermination and suppression of transcriptional pausing. The studies have permitted a more precise definition of the site on RNA polymerase that binds to the antiterminator RNA and to reject a proposed antitermination mechanism. In a separate project, the section annotated the genome sequence of bacteriophage B40-8, whose host is Bacteroides fragilis, a frequently pathogenic human commensal bacterium. Phylogenetic comparisons and experimental data suggest that DNA packaging and the distribution of promoters and terminators in the virus do not resemble those of other viruses.
Heiner Westphal’s Section on Mammalian Molecular Genetics focuses on developmental controls exerted by LIM-homeodomain (LIM-HD) transcription factors as the mouse embryo enters the phase of post-gastrulation assembly of organ systems. Mounting evidence suggests that the LIM-HD proteins play a key role in lineage specification by activating downstream genes that define the ground state of organ identity. The assembly of newly formed cell lineages into complex tissue structures depends on precise patterns of cell migration and cell-cell communication that involve all major signaling pathways. The transcriptional activity of LIM-HD proteins is mediated by two families of obligatory cofactors, termed Ldb and Ssdp, that become part of the LIM-HD complex controlling target gene transcription. The section has analyzed different scenarios of tissue assembly in the developing mouse embryo and discovered that an intricate interplay between transcriptional regulation and secreted signal exchanges accompanies organogenesis.
8. PROGRAM IN MOLECULAR MEDICINE
Tracey Rouault, MD, Program Director
Researchers in the Program in Molecular Medicine seek to understand mechanisms of disease through analysis of mouse models and research on human patients. Abnormalities of metal homeostasis are under investigation, particularly the role of iron misregulation in the pathogenesis of neurodegeneration and refractory anemias and the role of copper transport abnormalities in Menkes’ disease and related conditions.
Stephen Kaler’s Unit on Pediatric Genetics works on three main projects: (1) copper transport disorders, including X-linked recessive Menkes’ disease and its allelic variant, occipital horn syndrome; (2) the PHACES syndrome of midline developmental abnormalities; and (3) the platelet glycoprotein adhesion complex GPIbalpha-GPIbbeta-GPIX. In the case of Menkes’ disease, the section has been investigating the effects of early diagnosis and treatment by conducting a clinical trial; the section evaluates patient materials using cellular and molecular bench methods. The investigators also studied a female child with PHACES syndrome and documented skewed X chromosome inactivation in the family. In conjunction with the pronounced female gender bias in this condition, the results suggest inheritance of the syndrome as an X-linked lethal trait. The group also continues to pursue characterization of the platelet glycoprotein GP1b beta, attempting to express and purify the protein for crystallographic confirmation of its proposed structure and for generation of neutralizing antibodies that may be active in modulating the platelet adhesion response in patients at risk for thromboembolic events.
Tracey Rouault’s Section on Human Iron Metabolism uses mouse models and tissue culture to study mammalian iron metabolism. Rouault previously identified and characterized two major cytosolic iron-regulatory proteins (IRPs). Targeted deletion of each IRP in mice revealed that misregulation of iron metabolism owing to loss of IRP2 causes functional iron deficiency, erythropoietic protoporphyria, anemia, and neurodegeneration in animals. The section also focuses on mammalian iron-sulfur cluster assembly because of its relevance to IRP1 regulation. Researchers characterized numerous mammalian genes involved in iron-sulfur cluster synthesis and developed in vitro and in vivo methods to assess cluster biogenesis. The section’s discoveries may promote understanding and treatment of neurodegenerative diseases, especially Parkinson’s disease and Friedreich ataxia, and hematologic disorders such as refractory anemias and erythropoietic protoporphyria.
