Program in Genomics of Differentiation
Igor B.H. Dawid, PhD, Program Director
The Program in Genomics of Differentiation comprises three laboratories. The Laboratory of Mammalian Genes and Development generates gene-altered mice to study embryonic and adult stem cells, pattern formation, T cell development, and genomic imprinting. The Laboratory of Molecular Genetics studies the regulation of gene expression and the genetic control of developmental and physiological processes in model organisms from bacteria to vertebrate animals. The Laboratory of Molecular Growth Regulation conducts research on the control of cell proliferation, DNA replication, epigenetic gene regulation, regulation of the immune system, gene expression during early embryogenesis, chromatin-mediated gene silencing, and transcription of small RNA–encoding genes.
Laboratory of Mammalian Genes and Development
Using advanced mouse genetics, Sohyun Ahn and colleagues in the Unit on Developmental Neurogenetics study the molecular and genetic mechanisms by which neural stem cells (NSC) are specified and regulated. They investigate the role of Sonic hedgehog (Shh) signaling and its downstream effectors in the developing and mature nervous system. The Unit uses microarray gene expression analysis to identify genes specific to Shh-responding NSCs in adult mouse brain. The Unit recently generated novel transgenic mouse lines, first, to study how NSC-generated newborn granule neurons in the dentate gyrus of the hippocampus form functional circuits and, second, to understand the elusive role of adult neurogenesis.
Paul Love and the Section on Cellular and Developmental Biology focus on T lymphocyte development, particularly signal transduction molecules and pathways that regulate T cell maturation in the thymus. The Section discovered an important difference in the subunit composition and ignaling potential of the antigen receptors (TCR) expressed on two distinct classes of T cells. In studying molecules that control T cell migration and trafficking, the researchers found a critical function for the chemokine receptor CCR9 in regulating the migration of developing T cells to and within the thymus. Additional studies have focused on the role of LIM-domain proteins and the partner LIM-domain binding protein in the regulation of T cells.
Karl Pfeifer and the Section on Genomic Imprinting examine the regulated expression and biological function of a cluster of imprinted genes on the distal end of mouse chromosome 7. In particular, the Section focuses on the Igf2/H19 gene cluster, for which it has characterized a 2.4 kb DNA element called the H19ICR that is both necessary and sufficient to imprint the locus. The Section recently described the ICR’s role in organizing higher-order chromosome structures and long-range DNA interactions that determine the mono-allelic expression patterns of both Igf2 and H19. Current work emphasizes the identification of proteins that interact with the ICR. The Section also continues to develop and analyze mouse models carrying ion channel mutations that phenocopy cardiac disorders associated with this gene cluster in humans.
Heiner Westphal and colleagues in the Section on Mammalian Molecular Genetics use a loss-of-function approach to study the function of LIM-homeodomain (LIM-HD) proteins and associated co-factors. They investigate the regulation of target gene transcription by oligomeric complexes of LIM-HD, Ldb, and Ssdp proteins and the role of these processes in tissue patterning and organ formation in the mouse embryo, specifically in brain, limb, and heart development. A novel aspect of the work concerns the role of the LIM-HD transcriptional machinery in embryonic stem (ES) cell activation, the reprogramming of somatic cells to an induced pluripotent stem (iPS) cell state, and maintenance and activation of stem cells in the adult organism.
Laboratory of Molecular Genetics
Mike Cashel and the Section on Molecular Regulation have described the guanine nucleotide analogue ppGpp as a regulator of RNA synthesis in E. coli. Throughout the microbial and plant kingdoms, ppGpp acts as a regulator of global gene expression in response to stress. Regulation by ppGpp does not operate on transcription initiation but rather interacts with secondary channel proteins that non-covalently modify RNA polymerase to alter transcription kinetics. The Section studied new ppGpp regulatory roles in terms of (1) transcription factors GreA and GreB, (2) the small RNA GraL emerging from the GreA leader transcript, (3) an adenine nucleotide analogue (AppppA) with known functions in eukaryotic DNA replication, (4) TraR encoded by the episome governing conjugational recombination, and (5) intermediary metabolism.
Ajay Chitnis and colleagues in the Section on Neural Developmental Dynamics study the formation of the nervous system in zebrafish, particularly neuronal versus non-neuronal fate determination and how different regions assume different structural and functional identities. The Section examined proteins interacting with Mindbomb (Mib), a known regulator of Notch signaling, which is required for proper specification of neuronal differentiation. The study revealed that the protein Mosaic eyes (Moe) is a Mib-interacting protein; the Section is now investigating Moe’s role in modulating Notch signaling.
Robert Crouch, who leads the Section on Formation of RNA, studies RNases H, which are 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 the RNase H domain of human RNase H1 in complex with an RNA/DNA hybrid, yielding significant insight into the mechanism of replication of the HIV viral genome. Translation initiation of Rnaseh1 mRNA occurs from two distinct sites, leading to synthesis of mitochondrial and nuclear isoforms of RNase H1. Studies on type II RNase H, an enzyme consisting of three subunits, provide information on Aicardi-Goutières syndrome, an encephalopathy that mimics in utero viral infection.
Igor Dawid in the Section on Developmental Biology studies early development in the frog and zebrafish. In one project, the Section identified a guanine nucleotide exchange factor named WGEF that is a component of the Wnt-PCP signaling pathway involved in the regulation of cell movements in frog gastrulation. Another project identified Lrig3, a transmembrane protein that plays a role in the formation of the neural crest by modulating the Fgf signaling pathway. The Section showed that Lrig3 keeps Fgf signaling within an optimal range required for normal neural crest development.
Judith Kassis, who heads the Section on Gene Expression, studies the mechanism of gene silencing by the Polycomb group genes (PcG) in Drosophila as well as the nature of DNA elements responsible for such silencing. Several proteins, most notably an Sp1/Klf-type protein and the Pho and Pholike proteins, are required for functional silencing through sequences called Polycomb Response Elements (PRE). The Section has characterized the sequences responsible for Sp1/KLF binding in a particular PRE. Investigators studied recruitment of the silencing protein complex to the PRE, showing that Pho and Pho-like play largely redundant roles in the recruitment process. In addition, studies analyzed the transcriptional regulation of the Engrailed gene and the role played by the Engrailed PRE in such regulation.
Jim Kennison, who heads the Section on Drosophila Gene Regulation, studies the genomics of pattern differentiation in Drosophila. The Section showed that the previously identified verthandi gene encodes a subunit of the cohesin complex, which is required for proper segregation of sister chromatids during anaphase. The finding that verthandi is required for transcription of homeotic genes suggests that cohesion may also play a fundamental role in developmental gene regulation. The Section has 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 and colleagues in the Section on Viral Gene Regulation study the molecular aspects of HIV-1 replication and reverse transcription. Nucleocapsid protein (NC) is a nucleic acid chaperone that destabilizes highly structured nucleic acid reverse transcription intermediates. NC’s chaperone activity, together with RNase H cleavage, ensures the specificity of plus-strand DNA synthesis. The Section demonstrated that human APOBEC3G, an anti–HIV cytidine deaminase, inhibits all reverse transcriptase–catalyzed DNA elongation reactions. Inhibition is deaminase-independent and is determined by differences among the nucleic acid–binding properties of A3G, NC, and reverse transcriptase. Studies on the HIV-1 capsid protein demonstrated that mutations in the linker region result in abnormal core structure and loss of infectivity.
Tom Sargent and colleagues in the Section on Vertebrate Development investigate TFAP2-regulated genes in the cranial neural crest (CNC), focusing on those encoding MyosinX, which is a non-muscle motor protein, and Inca. The Section has shown that both genes are necessary for normal craniofacial development in Xenopus and, in the case of Inca, in zebrafish. Inca, a novel protein that promotes actin stress-fiber formation and inhibits microtubule acetylation, is required for gastrulation. Inca inhibits p21-activated kinase 4 (PAK4), which is a Rho-GTPase signaling factor. Under certain circumstances, Inca can regulate signal transduction through the ERK/MAPK. Loss of MyosinX expression inhibits CNC cell migration in Xenopus. Live confocal imaging revealed co-localization of MyosinX and beta3 integrin and disruption of filopodia in MyosinX-knockdown embryos.
Brant Weinstein in the Section on Vertebrate Organogenesis studies blood and lymphatic vessel formation during vertebrate embryogenesis. Vessel formation is of intense clinical interest because of the roles played by blood and lymphatic vessels in cancer and ischemia. Using the zebrafish, the Section developed a widely used confocal microangiography method, compiled an atlas of zebrafish vasculature, developed numerous vascular-specific transgenic lines, and pioneered methods for high-resolution in vivo imaging of blood vessels. The Section 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; it also identified the zebrafish’s lymphatic vascular system. Current studies use genetic screening and imaging to examine cues directing vascular patterning and morphogenesis, regulation of vascular integrity, and assembly of the lymphatic system.
Robert Weisberg and colleagues in the Section on Microbial Genetics explore the control of gene expression in bacteria and bacterial viruses, with a focus on the regulation of transcript elongation. The Section showed that newly synthesized transcripts can control transcriptional pausing by binding to RNA polymerase molecules. Given that control of pausing is an important component of many regulatory networks and permits targeting of individual pauses, the Section’s discovery may have widespread application. In a separate project, the Section determined and annotated the genome of bacteriophage B40-8. The host of this phage is Bacteroides fragilis, a human commensal bacterium that is frequently pathogenic. Phylogenetic comparisons and experimental data suggest that the distribution of promoters and terminators and the mechanism of translation initiation in this virus do not resemble those of other viruses.
Laboratory of Molecular Growth Regulation
David Clark and colleagues in the Unit on Chromatin and Gene Expression study gene activation resulting in the formation of an initiation complex by RNA polymerase II and transcription. These events 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 Unit has developed a model system involving native plasmid chromatin purified from the yeast Saccharomyces cerevisiae to investigate the role of chromatin structure in gene activation. The studies revealed that activation correlates with large-scale movements of nucleosomes and conformational changes within nucleosomes over entire genes.
Melvin DePamphilis in the Section on Eukaryotic Gene Regulation studies the control of gene expression and DNA replication during early mouse embryogenesis, particularly the differentiation of trophoblast stem (TS) cells and ES cells. One project investigates cell-cycle control and the role of the origin recognition complex (ORC) in mammalian cell DNA replication. Orc1 is activated by ubiquitination and phosphorylation during the M- to G1-phase transition. The bromo-adjacent homology (BAH) domain in human Orc1 promotes association of the ORC(2–5) core complex with chromatin and is thus involved in origin recognition. In an additional project, the Section and collaborators showed that the transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development.
A major effort of the Human Genetics Section, led by Bruce Howard, focuses on higher-order chromatin structure, particularly how defects in the maintenance of such structures (or failures in programmed transitions, especially in the perinatal period) may underlie common developmental disorders and age-related diseases. Random genome sampling, customized search algorithms for comparisons of annotated genomes, and genomics-style high-throughput approaches facilitate the detection and mapping of age-related areas of chromatin remodeling.
Richard Maraia, who heads the Section on Molecular and Cell Biology, studies the biogenesis pathways for tRNAs and other small RNAs and how they affect cell proliferation, growth, and development. Efforts focus on transcription termination by RNA polymerase III and the RNAbinding phosphoprotein La. The La protein is an autoantigen found in patients with Sjögren’s syndrome, systemic lupus, and neonatal lupus. It functions in tRNA production and in the translation of mRNAs encoding ribosome subunits and growth-related factors. 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 investigate transcription factors and chromatin binding proteins that control the development of innate immunity. The Section showed that IRF8 drives the development of plasmacytoid dendritic cells (DC) and CD8α+ DCs, subsets that produce type I interferons (IFN) and IL-12. These cytokines confer antimicrobial activities. IRF8 contributes to high IFN production in DCs by amplifying the feedback phase of transcription. The Section 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.