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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 20 principle investigators, the PGD is the largest program in the NICHD DIR, encompassing several 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, 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. One project revealed that the duration of Shh signaling contributes to diversity among dopamine neurons. Combining the power of genetics with other in vivo manipulation techniques, the Unit demonstrated a requirement for Gli3, a major negative regulator of the Shh pathway, in neural progenitors in developing neocortex and in establishing the postnatal neurogenic niche in the subventricular zone of the lateral ventricle. Through comprehensive gene expression profiling experiments, the Unit also identified additional regulatory signals presented by other cells in the niche, signals that dictate the behavior of neural stem cells. Insights gained through these studies will allow the development of cell type–specific strategies for the recruitment of endogenous neural stem cells during regeneration or disease repairs.

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. The group 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 analogue 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, the Section turned its attention the possibility that ppGpp–like regulation is present in animal cells. This follows findings by others of highly specific, structural homologues of bacterial ppGpp hydrolases from humans together with evidence of a developmental role for these enzymes in fruit flies and their ability to complement bacterial ppGpp mutant functions. The Section recently revealed that animal hydrolases exhibit degenerate substrate specificity, cleaving not only ppGpp but also related analogs such as ppApp and an ATP derivative (pppApp). 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 by direct effects upon the intrinsic kinetic properties of promoters, without the help of sequence recognition proteins. The studies are important because ppGpp is necessary for virulence-gene expression in most bacterial pathogens, potentially providing unique paradigms for vaccines and new antibiotics.

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 gene-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 the 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 found 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, severe neurological defects, and a general loss of genome integrity. 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 uterine viral infection. In eukaryotes, type II RNase H2 has two distinct activities: removal of single ribonucleotides misincorporated in DNA and of longer stretches of RNA/DNA hybrids, thereby helping to maintain genome stability. Studies on mice and yeast defective in RNase H2 are providing insights into the contribution of this protein to genome integrity.

Igor Dawid, who heads the Section for Developmental Biology, and colleagues study early development in the frog Xenopus and the zebrafish. Recent efforts focused on the regulation of the formation of the neural crest and the pancreas, and on applying TALENs for targeted gene disruption in frogs and fish. TALENs are a novel class of specific DNA-binding hybrid proteins with nuclease activity, with high promise as tools for targeted mutagenesis. The group participated in a collaborative study to adapt TALEN technology to Xenopus, demonstrating its effectiveness in the system. In analyzing neural crest development, the group studied the role of the BTB domain protein Kctd15. Kctd15 inhibits neural crest formation, and its action is believed to assure separation of the neural crest and pre-placodal domains in the zebrafish embryo. A focus of the group's efforts is the relationship of Kctd15 inhibition to its interaction with the transcription factor AP-2. Pancreas development in Xenopus was studied in a collaborative project that focused on the critical role of the hhex gene in this process.

Mel DePamphilis, who heads the Section on Eukaryotic DNA Replication, studies the control of DNA replication and gene expression during mammalian development. The Section's current work focuses on three questions: how genome duplication is restricted to once per cell division in proliferating cells; how these mechanisms can be circumvented in order to selectively kill cancer cells; and how these mechanisms are circumvented during normal mammalian development to allow some cells to differentiate into viable, nonproliferating, polyploid cells. Recently, members of the Section discovered that one of the proteins (Geminin) involved in preventing DNA from re-replicating prior to mitosis is essential to prevent DNA re-replication and apoptosis in cells derived from human cancers, but not in cells derived from normal tissues. They are pursuing this discovery by investigating the effects of Geminin ablation in mice, using high-throughput screening to identify all genes essential for restricting genome duplication in cancer cells to once per cell division, and by identifying small molecules that selectively target these genes. Such molecules would be useful in cancer chemotherapy as well as in research into the regulation of cell proliferation. To understand how normal cells escape the restrictions on genome duplication, they are investigating how trophoblast stem cells exit their mitotic cell cycle and differentiate into the polyploid giant cells essential for both implantation and placentation.

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) 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, such 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 made considerable progress towards describing the complex array of DNA–binding proteins required for the activity of PREs. 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 PcG proteins may facilitate the activity of some engrailed enhancers. Finally, the group recently found that increasing cohesin binding stability counteracts PcG repression.

Jim Kennison, studies the genomics of pattern differentiation in Drosophila. His group defined both cis-acting regulatory sequences and trans-acting factors required to maintain activation and/or repression of the the Sex combs reduced homeotic gene. After embryogenesis, genetic elements about 70 kb apart in the Sex combs reduced gene must be in cis to maintain proper repression. The elements appear to correspond to clusters of Polycomb group response elements (PREs). New trans-acting factors that act through the PREs have been identified in genetic screens for recessive mutations that fail to maintain repression in somatic clones. The group also analyzed two regions that span about 1% of the Drosophila genome, identifying all the genes required for zygotic viability or male fertility. The studies 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 recently demonstrated that the three RNA removal reactions that occur during viral DNA synthesis rely on a common mechanism based on requirements for the helix-destabilizing activity of NC, which promotes strand displacement, and for RNase H. In other work, functional analysis of the HIV-1 capsid protein revealed that the interdomain linker region, a short flexible peptide connecting the N- and C-terminal domains, is essential for proper core assembly and stability. The group is also investigating the molecular properties and three-dimensional structure of human APOBEC3A, a deoxycytidine deaminase that is part of the cellular innate immune response to viral pathogens such as HIV.

The Section on Cellular and Developmental Biology, led by Paul Love, studies mammalian hematopoietic development. A main area of research concerns 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. The group 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.

Todd Macfarlan's Unit on Mammalian Epigenome Reprogramming was established in July, 2012. The Unit uses a multidisciplinary approach that combines mouse genetics, molecular biology, biochemistry, cell biology, multi-dimensional imaging, and next-generation sequencing approaches to explore mechanisms of gene regulation and epigenetic inheritance during embryo development. Furthermore, the Macfarlan group is taking advantage of genetically engineered embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, which can be differentiated into specific cell types to model early cell fate decisions. The cells can be grown in very large numbers to allow biochemical interrogation of the epigenome.

Richard Maraia, who heads the Section of Molecular and Cell Biology, studies RNA metabolism, with a continued interest in tRNA. Efforts focus on tRNA 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 of the Section's work is on Tit1p, a tRNA anticodon loop–modification enzyme, and its effects on the specific activity of its substrate tRNAs in decoding during translation and its more global effects on the subsets of mRNAs with cognate codon bias. The studies led to a new view of the genetic information potential of the redundancy component of the genetic code. 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, which studies gene regulation in innate immunity, with an emphasis on the role of chromatin. Current research centers on three nuclear proteins, IRF8, BRD4, and histone H3.3. IRF8 is a DNA–binding transcription factor that directs the development of macrophages and dendritic cells, the cell type principally responsible for innate immunity. As such, IRF8 is critically required for eliciting early anti-pathogen resistance. BRD4 binds to acetylated chromatin through the bromodomain, as well as to the elongation factor P-TEFb through the C-terminal domain, and controls transcription of many cellular and viral genes. The histone variant H3.3, also known as a replacement histone, is incorporated into chromatin in conjunction with transcription. There is mounting recognition of the significance of the replacement histones, given that it has become clear that nucleosomes are subject to destabilization and eviction during transcription and repair. However, the activity of H3.3 in transcription activation, particularly during immune responses, is not well understood. Studying interferon (IFN)–stimulated genes as a model, the group showed that IFN triggers the recruitment of BRD4 to IFN–stimulated genes and that the recruitment is the primary event initiating productive elongation. 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, which creates a lasting epigenetic mark on previously expressed IFN–stimulated genes. To further study the activity of these proteins in vivo, the group recently generated novel knock-in and conditional knockout mice for Irf8, Brd4, and H3f3a/b and began studying their activity in various immune cells.

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 the 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 the development of the cranial neural crest (CNC) in zebrafish. They are using an inducible tissue-specific transgenic approach to express specific regulatory molecules in the developing CNC cells of live fish embryos. For example, they are using a dominant negative mutant version of the human homeobox gene DLX3, mutations of which correspond to the hereditary disorder tricho-dento-osseous syndrome, to test for the function of dlx genes in craniofacial development of the fish. They are also using this approach to express dominant negative growth factor receptors in zebrafish CNC to evaluate the role of cell-cell signaling pathways in craniofacial morphogenesis. In addition, the group investigated the function of pak4 (p21-activated kinase 4), a Rho-GTPase effector molecule that was identified earlier as an interaction partner for Inca, a novel NC–regulatory factor initially identified by this laboratory. Knockdown experiments showed pak4 to be a maternal-effect gene that is required for multiple aspects of organogenesis, including formation of myeloid cells (macrophages and granulocytes) and morphogenesis of axial muscles.

Brant Weinstein's Section on Vertebrate Organogenesis studies blood and lymphatic vessel formation during vertebrate embryogenesis. Vessel formation is of intense clinical interest given 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. The 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 cell (iPSC) state. The project focuses on generating patient-specific iPSC 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 iPSC 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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