David Bentley - US grants

A major problem in developmental neurobiology is the question of how nerve cells are able to make specific connections. This capability depends on the ability of axonal growth cones to navigate accurately over prescribed routes. The purpose of this project is to understand, in a particularly advantageous "model" system, how such navigation occurs. In limb buds of embryonic grasshoppers, the first "pioneer" neurons project axons on a stereotyped path. We are studying what the guidance features are which mark the route, how these features are located by growth cones, and how location of the guidance features results in steering of the growth cones in appropriate directions. Currently available evidence indicates that the guidance features are a chain of "guidepost" cells whose location marks the route, and suggests that these cells may be located by undirected exploration of the cellular landscape by filopodia extended from growth cones. In the next project period, we intend to solidify the evidence for the guidepost hypothesis, to test the filopodial exploration hypothesis, and to explore certain hypotheses for growth cone steering. The normal behavior of these in vivo growth cones can be observed with fluorescently-tagged antibodies. We plan to extend to observations from fixed tissue with monitoring of growing cells and with pulse-labeling. The system can be experimentally manipulated by deletion of specific pioneer neurons or guidepost cells with photoinactivation of dye-injected cells, or with a UV microbeam. We hope to extend manipulation with laser-deletion of filopodia, and with cell-culture techniques which will enable tests of cell-surface properties, the role of adhesion, and the alternative hypotheses of filopodial exploration or diffusible factors for locating guidepost cells. Understanding how nerve cells make specific connections is medically important both for analysis of neuro-pathological conditions, and for restoring normal function after neural trauma.

To provide much needed information on the immune response of pneumococcal vaccine (PV) in older persons, we have designed a double-blind alternate-vaccine cohort study. We will enroll approximately 120 patients and residents, ages 50-79 and 80 plus, in nursing homes and approximately 120, age and sex matched, persons in apartments. We will compare their antibody responses following PV with those of 30 young adults. We will administer tetanus-diphtheria toxoid to a 20 percent sample of roommates to evaluate confounding effects of carriage, inapparent infection, etc. Measurements will include: type-specific antibody (total) to 12 of the 14 pneumococcal polysaccharides in the PV vaccine by RIA; immunoglobulin classes (IgG, IgA and IgM) distribution of antibody to types 3, 6 and 9 by ELISA and antibody to phosphorylcholine (PC), a major antigenic determinant of C-polysaccharide, by ELISA. Tetanus anti-toxin antibody levels will be measured by ELISA as a non-carbohydrate protein in vaccine control. Serum immunoglobulin levels will be determined to exclude a latent immunodeficiency. We will determine the amount, class and duration of type-specific antibody and the amount and duration of antibody to PC by comparing the pre-vaccination antibody levels (geometric means [GM]) versus the 1-month post-PV antibody levels (number [percent]) with 2-fold increase in GM antibody titer and number (percent) with 300 ng of antibody nitrogen/ml) versus the antibody levels at 6, 12 and 24-months post-PV. We will compare for age and sex within groups using the individual as his own control and compare between groups. We will identify individuals who respond poorly to PV by evaluating the predicted value of serum immunoglobulin and IgG subclass levels. Because nontype-specific antibody is more likely to occur following natural infection, we will also measure antibody to PC in post-pneumococcal pneumonia sera. The long-range objectives are to develop an effective strategy to prevent pneumococcal pneumonia in the elderly. If the current PV does not induce persistent protective antibody levels, then we will study new vaccines with the polysaccharides covalently coupled to protein carriers. If a response to PC occurs after pneumococcal pneumonia, and is correlated with protective type-specific antibody levels, a vaccine to induce antibody to this antigen would be an attractive alternative to a multi-valent capsular vaccine.

This is a conference grant proposal to NIA for an international scientific meeting, MiniSymposium: Aging, Immunity and Infection that will take place during the next biannual International Symposium on Infections in the Immunocompromised Host (ISIIH), Peebles, Scotland, UK, June 3-6, 1990. The specific aims of the proposal include: 1) direct costs to attract outstanding principal speakers, discussants and abstract presenters, 2) partial support for a unique international minisymposium to promote the interfacing of infectious disease with advances in cellular and molecular immunity and 3) partial support for publication of the Proceedings of the MiniSymposium. Long-range goals are to: 1) promote mutual collaborative efforts among U.S. basic science researchers and clinical investigators working in the field of aging, immune response and infection and their international colleagues, 2) encourage future applications to NIA regarding the pathogenesis of aging and the mechanisms of host response to infection and 3) integrate aging and the concept of the elderly as an immunocompromised host as a regular section in the biannual meetings of the ISIIH. The Co-leaders of the MiniSymposium are DW Bentley and NR Klinman. The MiniSymposium will focus on 3 major areas: cell-mediated immunity, humoral and molecular immunity and resistance to infection. The format and agenda will include invited speakers (Miller, Goidl and Murasko), discussants (Weigle, Klinman and Kauffman), 9 TBN presenters of extended abstracts who will be selected from 25 TBN presenters of abstracts for poster session and closing remarks (Weksler). The Proceedings of the MiniSymposium will be published in a dedicated issue of the journal, Aging: Immunology and Infectious Disease within five months of the MiniSymposium. The forum of the MiniSymposium, the international setting of the ISIIH and the outstanding participants will provide an ideal opportunity for interfacing basic immunology with infectious disease and defense mechanisms in the elderly.

9410068 Bentley Establishment of the appropriate connections between neurons in the developing nervous system depends upon the ability of neurons to migrate and to extend processes accurately to distant target cells. Effective guidance depends upon the ability of the tip of neuronal processes (the nerve growth cone) to detect guidance information and to respond by appropriate direction change, or steering. The purpose of this research is to analyze the molecular mechanisms that underlie growth cone steering. These studies will investigate the pathfinding of a single neuron in the methathoracic limb bud of an insect to test the hypothesis that tyrosine phosphorylation is involved in the steering of neuronal growth cones. Antiphosphotyrosine antibodies will be injected into the growth cone and their effects on pathfinding will be determined. Tyrosine kinase and phosphatase genes will then be cloned and used to alter the function of the system. The results will be of fundamental importance in understanding the processes that are involved in establishing connections between nerve cells during development, in vertebrates as well as invertebrates.

mRNA production requires transcription by pol II and processing of the primary transcript by a set of proteins which carry out capping, splicing and cleavage/polyadenylation. Transcripts mae in vivo by polymerases other than pol II are not processed correctly into mature mRNA. A mechanism clearly exists to couple transcription by pol II with the specific RNA processing events responsible for maturation of mRNA. Although the coupling mechanism is of fundamental importance, the details of how it works are almost completely unknown. The objective of this proposal is to lean how the coupling mechanism works. This work may help elucidate how the splicing, 3' processing, and stability of mRNAs are regulated under normal conditions and how they become mis-regulated in the disease state. One approach taken is to compare processing of transcripts made by different mutated forms of RNA polymerase. This approach led to the identification of a key player in coupling: the conserved C-terminal domain (CTD) of the pol II large subunit. A follow-up strategy that has been taken is to identify protein-protein interactions between the CTD and RNA processing and factors in vitro and then to test their functional significance in vivo. This work is based on the hypothesis that interactions between processing factors and the CTD are responsible for the functional coupling between transcription and processing. According to this idea, "mRNA factory" complexes containing pol II and RNA processing factors may carry out both transcription and processing in the nucleus. A series of biochemical and genetic experiments is proposed to test this idea. The focus is on the role of the CTD in processing of the 5' and 3' ends of the mRNA molecule. Specific aims of this work are: 1. To test if the association between the cleavage/polyadenylation factor, CstF, and the CTD is necessary for mRNA 3' processing in vivo. 2. To determine if the CTD is sufficient as well as necessary for efficient 5' and 3' processing. 3. To determine if 3' processing is dependent on the CTD in the genetically amenable organism Saccharomyces cerevisiae. 4. To characterize the interactions between the CTD and the capping enzymes and to determine the functions of these interactions in vivo.

The synthesis of messenger RNA is the primary event in gene expression and is central to the life of cells. mRNA production requires transcription by pol II and processing of the primary transcript by a set of proteins which carry out capping, splicing and cleavage/polyadenylation. Transcripts made in by polymerases other than pol II are not processed correctly into mature mRNA. A network of protein:protein contacts in the cell nucleus exists to couple transcription by pol II with the specific RNA processing events responsible for maturation of mRNA. The objective of this proposal is to elucidate how proteins communicate to achieve coupling of pol II transcription with RNA packaging and processing using biochemical and genetic approaches in mammalian cells, frog oocytes and budding yeast. Our research is testing the idea that mRNA is made by a "factory" complex containing RNA pol II and RNA processing factors which contact it through a repetitive protein domain called the CTD. This model has changed the way we think about proteins which were once thought to operate independently but are now thought to be co-ordinated with one another in the nucleus. One important functional consequence of the integration between transcription and processing that we are beginning to uncover is that transcription factors can regulate the efficiency of RNA processing and conversely processing factors can potentially regulate transcription. This work may help elucidate how the transcription and processing of mRNAs are regulated under normal conditions and how they become mis-regulated in the disease state. Defects in splicing resulting of pre-mRNA's are responsible for a large fraction of all inherited diseases. The specific aims of this work are: 1. To determine how mammalian capping, splicing and cleavage polyadenylation factors associate with pol II elongation complexes on genes in vivo. 2. To determine the extent to which introns are excised co-transcriptionally and what factors affect the efficiency of co-transcriptional splicing. 3. To test the CTD code hypothesis by determining the structural features of the CTD that contribute to co- transcriptional pre-mRNA processing, mRNA export and hnRNP packaging.

DESCRIPTION (provided by applicant): The control of gene expression at the level of transcription by RNA polymerase II is central to the life of the healthy cell and is frequently corrupted in disease. During each stage of the transcription cycle, initiation, elongation and termination, the polymerase must negotiate with a DNA template that is packaged with histones into chromatin. Access by the transcription machinery to the DNA is regulated at one level through modifications of the histones on their tails. We recently identified a new class of enzymes in yeast and mammals that modify the histone tails by removing methyl groups at Lysine 4 on histone H3 (Appendix 3). This new class of demethylase enzymes belong to a family called JARID1 with one member in yeast and four in mammals. They are almost certainly targeted to individual genes to regulate their expression but we still do not know which genes they are targeted to. In budding yeast we have found that sporulation, a major program of highly regulated changes in gene expression, is severely disrupted when the JARID1 homologue (Yjr119c) is mutated. Sporulation in yeast is an excellent model for regulated gene activity during development in higher organisms. By studying when and how the sporulation program is disrupted, we aim to uncover the mechanism by which H3K4 demethylation contributes to proper control of gene activity in a complex developmental program. During elongation, pol II faces the challenge of ploughing its way through chromatin. How elongating pol II interacts with chromatin is a major open question. We found that rapid changes in H3K4 methylation occur in yeast in response to acute inhibition of transcription elongation ("transcriptional stress") (Appendix 1) and are investigating whether this mechanism is conserved in multicellular animals. Perhaps the least well understood segment of the pol II transcription cycle is termination, the process by which pol II is informed that it has reached the end of the gene and is then released from the DNA template. We proposed a new model for how termination is triggered (Appendix 2) and will investigate how this final step in the transcription cycle is controlled by properties of RNA polymerase II and associated termination factors.

[unreadable] DESCRIPTION (provided by applicant): The control of gene expression at the level of transcription by RNA polymerase II is central to the life of the healthy cell and is frequently corrupted in disease. During each stage of the transcription cycle, initiation, elongation and termination, the polymerase must negotiate with a DNA template that is packaged with histones into chromatin. Access by the transcription machinery to the DNA is regulated at one level through modifications of the histones on their tails. We recently identified a new class of enzymes in yeast and mammals that modify the [unreadable] histone tails by removing methyl groups at Lysine 4 on histone H3 (Appendix 3). This new class of demethylase enzymes belong to a family called JARID1 with one member in yeast and four in mammals. They are almost certainly targeted to individual genes to regulate their expression but we still do not know which genes they are targeted to. In budding yeast we have found that sporulation, a major program of highly regulated changes in gene expression, is severely disrupted when the JARID1 homologue (Yjr119c) is mutated. Sporulation in yeast is an excellent model for regulated gene activity during development in higher organisms. By studying when and how the sporulation program is disrupted, we aim to uncover the mechanism by which H3K4 demethylation contributes to proper control of gene activity in a complex developmental program. During elongation, pol II faces the challenge of ploughing its way through chromatin. How elongating pol II interacts with chromatin is a major open question. We found that rapid changes in H3K4 methylation occur in yeast in response to acute inhibition of transcription elongation ("transcriptional stress") (Appendix 1) and are investigating whether this mechanism is conserved in multicellular animals. Perhaps the least well understood segment of the pol II transcription cycle is termination, the process by which pol II is informed that it has reached the end of the gene and is then released from the DNA template. We proposed a new model for how termination is triggered (Appendix 2) and will investigate how this final step in the transcription cycle is controlled by properties of RNA polymerase II and associated termination factors. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A hallmark of cancer is that it is a disease of gene misexpression. This application addresses how gene expression in cancer is disrupted by aberrant maturation of mRNA 3' ends where the poly (A) tail is added. Most human genes have multiple poly (A) sites and, as a result, making choices between alternative poly (A) sites is an almost ubiquitous event in gene expression. In recent groundbreaking work by other labs, it was reported that alternative poly (A) site choice is commonly disrupted in cancer cells. This is important because poly(A) site choice can have a large impact on protein expression by dictating what sequences are included in an mRNA's 3' untranslated region (UTR). 3' UTR sequences are the principle targets for microRNAs and RNA binding proteins that regulate mRNA stability and translation efficiency. For example mRNAs will evade these controls if a poly (A) site is chosen that cuts off these target sequences to produce an abbreviated 3'UTR. We still lack comprehensive information about which genes are affected by abnormal poly(A) site selection in cancer cells and whether this process is regulated during tumor evolution. Furthermore little is known about the basis for how one poly(A) site is selected over another for processing. What might be going wrong in cancer cells to divert mRNA 3' end processing from the correct poly (A) sites is very much a mystery. We propose to tackle two broad fundamental questions: 1) What genes are affected by aberrant poly(A) site choice in cancer cells during tumor progression, adaptation to hypoxia, and the response to therapeutics? and 2) What is the molecular basis for the corruption of mRNA 3' end formation in cancer cells?. We will use a genome-wide RNA-seq and ChIP-seq strategies to answer these questions using human breast and lung cancer cells.

? DESCRIPTION (provided by applicant): The production of messenger RNA (mRNA) is the primary event in gene expression where genetic information is transferred from the gene's DNA into a disposable RNA copy. Corruption of this process is a hallmark of many diseases including cancer. mRNA synthesis requires not only making an RNA transcript but maturation of that transcript by 5' capping, excision of introns and splicing of exons and 3' end formation by cleavage/polyadenylation. The mRNA is also packaged with RNA binding proteins that facilitate its maturation and ultimate export to the cytoplasm as a messenger ribonucleoprotein particle (mRNP). The mRNA processing and packaging steps that radically transform the primary transcript occur largely co-transcriptionally; that is to say the substrate of mRNA processing and packaging factors is the growing nascent RNA that is extruded by an RNA polymerase II (pol II) molecule at rates of 0.5-4.5 kilobases/min. The goal of this proposal is to understand mRNP biosynthesis in its co- transcriptional context by focusing not on the mature mRNA products but on the nascent transcripts and how their transformation is affected by the process of transcription elongation that grows RNA chains. Our working model is that synthesis and processing of a mRNA precursor are carried out in an integrated fashion within a dynamic 'mRNA factory' complex that includes both RNA polymerase and processing factors some of which make direct contacts with the pol II C-terminal domain (CTD). We use genetic and genomic approaches to ask how ongoing transcription and mRNA maturation are coupled with one another in space through recruitment of factors to the `mRNA factory' and in time through kinetic coupling mechanisms to achieve efficient and accurate production of fully formed mRNP's. In this proposal we will address these questions: 1) How does pol II transcription elongation affect processing of the nascent transcript? 2) How are nascent transcripts folded, and how is folding affected by pol II transcription elongation? 3) How does transcription elongation affect RNA binding protein association with nascent transcripts? 4) How does elongation rate affect phosphorylation of the RNA pol II CTD and recruitment of processing factors to the transcription elongation complex? 5) How does co-transcriptional RNA processing affect elongation and chromatin structure?

Project Summary/Abstract A signature feature of Down syndrome (DS) cells with trisomy 21 (21T) is misexpression of thousands of genes on all chromosomes but the underlying mechanism that accounts for this paradoxical situation is a mystery. Widespread gene misregulation is almost certainly an important causal factor in many of the clinical challenges faced by individuals with DS such as intellectual disability, heart defects, leukemia, and accelerated senescence with early onset Alzheimer's disease. We will test the hypothesis that trisomy 21 causes changes how RNA polymerase II (pol II) works in fundamental ways that have consequences for expression of the thousands of genes that are transcribed by this enzyme complex. We will map for the first time the global landscape of pol II transcriptional activity by nascent RNA sequencing of isogenic euploid and 21T iPSCs before and after neuronal differentiation. We will also test whether three fundamental steps common to the mechansim of transcription at all genes are altered by 21T: a) promoter proximal pausing, b) elongation speed and c) termination at the 3' ends of genes. This idea is a plausible one based on recent work from our lab and others showing that 1) pausing and termination are regulated in response to stress stimuli that are likely mediated throught the actions of kinases and phosphatases acting on components of the pol II transcription apparatus 2) the DYRK1A kinase, encoded within the Down syndrome critical region of chromosome 21, phosphorylates RNA pol II directly. A substantial body of research demonstrates that DYRK1A is over- expressed, and that stress-inducible signalling pathways are chroniclaly activated in DS. As a result pol II itself or associated elongation and termination factors could be abnormally modified in ways that cause widespread transcriptional changes. We will address these questions: 1. How is the genome-wide landscape of pol II transcription affected by trisomy 21? 2. How is ?promoter proximal pausing? affected by trisomy 21? 3. How is transcription elongation rate affected by trisomy 21? 4. How is transcriptional pausing within genes affected by trisomy 21? 5. How is genome-wide pol II transcription affected by DYRK1A copy number? 6. How is in vivo pol II CTD phosphorylation affected by DYRK1A?