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Scientific Reports

2009:  Beutler Lab | Lazzerini Denchi Lab | Franc Lab 
2008:  Beutler Lab

Beutler Lab Scientific Report 2009

Understanding Immunity, Phenotype First, and One Gene at a Time
 
C.N. Arnold, M. Berger, A. Blasius, C. Boulton, K. Brandl, C. Domingo, X. Du, C. Eidenschenk, E. Hanley, H. Huang, K. Khovananth, P. Krebs, B. Layton, O. Milstein, E.M.Y. Moresco, N. Nelson, X. Li, E. Pirie, D. Popkin, C. Ross, O. Siggs, N. Smart, L. Sun, S. Won, Y. Xia and B. Beutler
 
Host resistance and the genetic approach
In recent decades we have come to see viruses as minute machines, programmed to parasitize the host and direct the synthesis of many copies of themselves.  All microbes might be regarded in the same way.  While larger and more complex than viruses, bacteria, fungi, and protozoan pathogens parasitize the host without volition:  they are programmed to do what they do.  While we mammalian hosts can think, feel, and hold opinions about microbes, we are machines as well.  Our immune system eliminates the great majority of infections before we become aware of them.  It acts autonomously, depending upon molecular sensors and biochemical pathways for response to infection that have yet to be fully deciphered.  While it has become traditional to speak of "innate" and "adaptive" immunity, it is clear that the two systems are connected at many levels:  so much so that it is more productive to analyze the two as a single system that mediates host defense.

Genetics is the principal tool used by our laboratory to analyze resistance to infection.  We also use genetics to understand why the immune system sometimes over-reacts, and why it sometimes reacts with self.  The first step is to know which proteins are indispensable for normal function:  resisting infection and enforcing tolerance to self.  Random germline mutagenesis can be used to compile the list.  By introducing mutations into the mouse genome with the alkylating agent N-ethyl-N-nitrosourea (ENU) and keeping surveillance over immune processes that interest us, we can find abnormalities, and use positional cloning to track them down.  A sometimes-difficult process follows in which one tries to understand how the critical proteins interact with one another in order to function.

To date the Beutler lab has created and preserved more than 267 phenotypes, mapped 164 of them to chromosomes, and identified the causative mutation in 142 cases (45 of these during the past year; see http://mutagenetix.scripps.edu for a detailed description of many of these mutations).  We have reason to believe that more than 1,000 proteins have non-redundant function in resisting infection by a single defined pathogen (mouse cytomegalovirus, or MCMV).  And we are beginning to see that many other proteins maintain immune homeostasis in the event of inadvertent infection.

The screens
Our laboratory monitors visible anomalies (changes in coat color, behavior, or development) that may present new models of human diseases.  We also deliberately screen for dysfunction in ten different categories of immunological competence in order to find mutations that disrupt resistance to infection, or immune homeostasis.  For example, we search for models of inflammatory colitis (as observed in humans with Crohn's disease or ulcerative colitis).  We look for abnormal macrophage responses to microbial stimuli.  And we look for abnormally enhanced or diminished responses to infection with MCMV.

At present, Katharina Brandl has identified 6 mutations that mimic inflammatory bowel disease (IBD) by screening for a model phenotype.  Four of these (Schlendrian, Phoebus, Pomona, and Liesgen) are being mapped for positional cloning at present.  She has also determined that a mutation called Woodrat, originally spotted because of its effect on pigmentation, causes IBD susceptibility by impairing a biochemical process known as the unfolded protein response.  Velvet (an epidermal growth factor receptor mutation) and Pococurante (a mutation of the immune adapter protein MyD88) do so as well.  Gradually, we have begun to assemble a picture of how IBD operates (Figure 1).  A future step will be to validate this picture by combining mutations to determine whether augmentation of the abnormal phenotype is observed as the model predicts.

Michael Berger has positionally cloned and extensively studied a mutation called Elektra that impairs both innate and adaptive immune function.  This mutation causes the death of immune cells just as they begin to multiply to fulfill their functions during an immune response.  While cell death is a normal part of the response to immune activation, it normally does not occur during infection, but after the infectious agent has been cleared.  Elektra points to a mechanism (and an essential protein) that prevents cell death from occurring prematurely.

Owen Siggs and Nengming Xiao have tracked down a mutation called Sinecure that is required for Toll-like receptors-key sensors of the immune system that inform us when infections are present-to signal properly.  Sinecure points to a protein utilized by several of the TLRs for transportation or assembly within the cell.  The function of this protein was not previously known, nor was a role in innate immunity suspected.

The Warmflash mutation, identified and positionally cloned by Karine Crozat and thereafter analyzed in collaboration with Celine Eidenschenk, points to a new signaling loop within dendritic cells:  key antigen-presenting cells of the immune system.  This loop permits proper communication between dendritic cells and natural killer (NK) cells, which are key effector lymphocytes that contain many different forms of viral infection.  If the loop is disrupted, the host cannot cope with infection by MCMV.  Warmflash gives us insight into how we normally survive certain kinds of viral infection, and provides certain hints as to how we might enhance our resistance.

Sometimes, visible or behavioral abnormalities give new insight into the operation of biological systems.  Amanda Blasius tackled two such mutations this year.  Moonlight, a mutation that caused white spots and generalized lightening of fur, was ascribed to a guanine nucleotide exchange factor known as Dock7.  Moonlight was also found to be allelic with Misty, a classical phenotype known for more than 60 years but never identified.  The two mutations suggest that Dock7 is required for the normal function and migration of melanocytes, pigment producing cells in the skin.  But the protein is probably not required for neurological function as speculated by others.   The Possum mutation, on the other hand, was detected because of its neurobehavioral phenotype.  It causes mice to "freeze" when gently handled:  a phenotype reminiscent of catalepsy.  Now identified, the mutation suggests a new role for a protein already known to be involved in sensory perception.  It now appears that the same protein has important functions both in the central and peripheral nervous system.

The pace of mutation finding has increased dramatically during the past year, with 45 new mutations identified since our previous report (as compared with 30 the previous year).  It will increase further still with advances in DNA sequencing technology, and with the ability to make identical copies of mutants through inducible pluripotent stem cell technology (currently being developed by Lei Sun and Xiaohong Li in our lab).  Gradually these mutations are permitting the elucidation of more and more immune functions.
 
NDEX/TERMS/KEY WORDS
Innate immunity, Toll-like receptors, TLRs, signaling, infection, positional cloning, forward genetics, mouse cytomegalovirus, resistome, apoptosis
 
AWARDS
2008:  Elected to the Institute of Medicine
2009:  Albany Medical Center Prize

Lazzerini Denchi Lab Scientific Report 2009

DNA Damage Response at Chromosome Ends
 
Eros Lazzerini Denchi, Ph.D., Assistant Professor
Keiji Okamoto, Ph.D., Research Associate
Beatriz Virgen, Research Technician
 
Because of their linear design, mammalian chromosomes are vulnerable and would be subject to fusion, degradation and recombination if it were not for telomeres; specialized nucleoprotein structures at their termini. The major goal of our research is to dissect the molecular mechanisms that allow mammalian cells to distinguish chromosome ends from sites of DNA damage.

Suppression of the DNA damage response at chromosome ends.
Telomeres consist of long TTAGGG repeats bound by a specific protein complex termed shelterin, which acts to protect chromosome ends. How this protection is achieved remains poorly understood. Using mouse genetics we recently found that within the shelterin complex TRF2 and POT1 specifically and independently suppress the two main mammalian DNA damage response pathways: ATM and ATR respectively. Current work in the lab is aimed towards the identification of the critical molecular properties of TRF2 and POT1 involved in the suppression of the DNA damage response. In addition we are taking advantage of the unique properties of telomeres to identify novel components of the DNA damage and DNA repair pathways by high-throughput screenings. Telomeres represent a unique system to study the DNA damage response in mammalian cells since individual branches of the DNA damage response can be activated at chromosome by genetic manipulation. Moreover the unique sequence of the telomeric DNA allows us to follow the DNA damage response events at the site of damage.

Consequences of telomere dysfunction
Telomere erosion occurs constantly during cellular proliferation and eventually results in critically short telomeres that cannot protect any longer chromosome ends. Dysfunctional telomeres arise frequently during tumorigenesis as well as in aging organisms. Using mouse genetics we are studying the influence of cellular compartments to the outcome of telomere dysfunction. Surprisingly we found that complete loss of telomere protection in the quiescent liver has no apparent advert effects on cellular and organismal viability despite the induction of frequent end-to-end chromosomal fusions. Current work in the lab is focused on the impact of telomere dysfunction on differentiated or pluripotent cell types to reveal the contribution of telomere dysfunction to aging and cancer development.
  
PUBLICATIONS 
Lazzerini Denchi, E. (2009). Maintenance of telomeres in cancer. In: Cell Cycle Deregulation in Cancer. Springer Science, in press.

Lazzerini Denchi, E. (2009). Give me a break: How telomeres suppress the DNA damage response. DNA damage and Repair., in press.

Lazzerini Denchi, E. L., T. de Lange (2007). Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1. Nature, 448(7157): 1068-71.
 
INDEX/TERMS/KEY WORDS
Cancer, Aging, Genomic Stability, DNA damage, Genetics, Cell Biology, Molecular Biology
 
Eros Lazzerini Denchi, Ph.D., Member of the Advanced Discovery Institute of the Scripps Research Institute.

Franc Lab Scientific Report 2009

Genetic Dissection of the Molecular Mechanisms of Phagocytosis of Apoptotic Cells
 
N.C. Franc, Principal Investigator;
J.W. Liu, Research Technician.
 
Phagocytosis is an essential function of innate immune cells (such as macrophages) that leads to the rapid recognition and silent removal of cells that are dying by apoptosis during development. It also leads to the recognition and inflammatory removal of cellular debris and microbes at wound sites in innate immune responses. Failure to clear bacteria can lead to sepsis, often a fatal condition, and failure to clear apoptotic cells can lead to autoimmune diseases, such as Systemic Lupus Erythematosus (SLE), as well as neurodegenerative diseases.

Phagocytosis was first described over a century ago. However, to date little is known still about the molecular mechanisms regulating this cellular immune process. We use Drosophila as a model system to genetically dissect the molecular mechanisms of apoptotic cell clearance. We recently developed a number of in vivo and in vitro phagocytosis assays and performed both forward and reverse genetic screens that allowed us to characterize novel molecules involved in phagocytosis of apoptotic cells.
In particular, we recently identified Undertaker (UTA), a molecule with membrane occupational recognition nexus (MORN) repeats. We found that UTA appears to be related to Junctophilins (JPs), a family of molecules involved in linking plasma membrane depolarization to endoplasmic reticulum (ER) calcium release and store-operated calcium entry (SOCE). We placed UTA downstream of the receptor Draper, (fly homologue of C. elegans CED-1) and its adaptor drCed-6 (fly homologue of CED-6), and found that the Ryanodine receptor Rya-r44F on the ER, the ER Ca2+ sensor dSTIM, and the Ca2+-release-activated Ca2+ (CRAC) channel dOrai act in the same pathway downstream of UTA to promote calcium homeostasis and phagocytosis. Thus, we linked Draper-mediated phagocytosis to Ca2+ homeostasis, highlighting a previously uncharacterized role for the CED1/6/7 pathway.

We anticipate that our studies will have a major impact on our understanding of the molecular mechanisms underlying phagocytosis of apoptotic cell, as well as of bacteria, as we have accumulated evidence that DRPR and UTA are also involved in bacterial phagocytosis.  Remarkably, in collaboration with Dr. K. Ravichandran, we found that calcium fluxes are also essential for phagocytosis of apoptotic cells in Caenorhabditis elegans and in mammalian systems, indicating some conservation in the molecular mechanism that drive these calcium events from worm to man. We therefore anticipate that homologues of the novel molecules, which we will continue to identify in the fly, will represent good potential targets for therapeutic exploitation in promoting phagocytosis in man.
 
PUBLICATIONS 
Silva, E.A. , Burden, J., Franc, N.C. Chapter three: In Vivo and In Vitro Methods for Studying Apoptotic Cell Engulfment in Drosophila. In: Methods in Enzymology. Khosravi-Far, R., Zakeri, Z., Lockshin, R.A., Piacentini, M. (Eds). Elsevier Inc. Academic Press, 2008, Vol. 446, p. 39.

Cuttell, L., Vaughan, A., Silva, E., Escaron, C.J., Lavine, M., Van Goethem, E., Eid, J-P., Quirin, M., Franc, N.C. Undertaker, a Drosophila Junctophilin, Links Draper-Mediated Phagocytosis and Calcium Homeostasis. Cell, 135(3):524, 2008.

Gronski, M.A., Kinchen, J.M., Juncadella, I.J., Franc, N.C, Ravichandran, K.S. An essential role for calcium flux in phagocytes for apoptotic cell engulfment and the anti-inflammatory response. Cell Death & Diff. In Press, 2009.
  
INDEX/TERMS/KEY WORDS
Genetics
Cellular immunity
Phagocytosis
Apoptosis
Macrophages
Drosophila melanogaster
Calcium signaling
Systemic Lupus  Erythematosus
Neurodegenerative diseases
 
N.C. Franc, Ph.D., Member of the Advanced Discovery Institute of the Scripps Research Institute.

Beutler Lab Scientific Report 2008

Probing Normal Function by Disrupting It: The Genetic Approach to Understanding Disease 
 
B. Beutler, C. Arnold, M. Barnes, M. Berger, A. Blasius, K. Brandl, K. Crozat, C. Domingo, X. Du, C. Eidenschenk, N. Gnauck, E. Hanley K. Hoebe, M. Kastner, K. Khovananth, P. Krebs, B. Layton, E. Moresco, N. Nelson, B. Ortiz, X. Li, S. Sovath, O. Siggs, N. Smart, K. Whitley, Y. Xia, N. Xiao
 
Genetics is the science of exceptions. It begins with a "phenotype," 1 of 2 or more alternative states of a phenomenon of interest. To understand why most animals are lean, we might find an exceptional animal that is obese; to understand why most animals resist a particular infection, we might find an animal that cannot. With current technology, the genetic cause of such an exception can be determined in short order. Geneticists can thus elucidate the fundamental molecular requirements for a given phenomenon. Without ever making hypotheses, they can sometimes draw profound conclusions about the functions of specific genes and the proteins the genes encode.

Phenotypes are the raw material for genetic inquiry, and much effort goes into making them. We have developed a prolific mutagenesis effort and have used forward genetics primarily to study immunologic phenomena: how humans resist infection by specific microbes, and why inflammatory or autoimmune diseases sometimes develop.

Beyond the immune system, we have also identified mutations that cause neurobehavioral, metabolic, developmental, hematologic, neoplastic, ocular, auditory, and pigmentation disorders. In so doing, we have broken new ground in understanding a wide variety of human diseases, including deafness, blindness, arthritis, obesity, cancer, schizophrenia, nutritional deficiency disorders, immunodeficiency, and autoimmunity. Indeed, we have discovered fundamentally new diseases that afflict humans but were not previously recognized as heritable defects. To date, we have created and preserved more than 230 phenotypes, mapped 132 of them to chromosomes, and identified the responsible mutation in 119 instances (nearly 60 of these during the past year; see http://mutagenetix.scripps.edu for a detailed description). The following is a brief description of a few such phenotypes.

The mutation mask, which has a visible phenotype marked by truncal (but not facial) hair loss and iron deficiency anemia, revealed how mammals sense iron deficiency and respond to it. The mask mutation indicated that a cell-surface protease, TMPRSS6, is necessary for the absorption of adequate amounts of iron from dietary sources. In the absence of TMPRSS6, which is expressed chiefly in the liver, a protein called hepcidin is produced in excess. Hepcidin is known as the master regulator of intestinal iron absorption. Through the identification of the mask phenotype and positional cloning of the mutation, patients with a comparable mutation responsible for refractory iron deficiency anemia have been identified. Moreover, mask has enlightened us about an important physiologic process. The mutant gene sphinx provides a model of liver tumors as well as a disturbance of function of natural killer cells and CD8 cells. The mutation spin offers a model of autoimmunity promoted by microbes and signaling by myeloid differentiation factor 88, specifically signaling via the receptor for IL-1. And the mutation add has a phenotype in which hyperkinesis, deafness, and vestibular disease have led to the identification of a completely new gene that encodes a protein required for maintenance of the hair cells of the inner ear.

Among the mutations created so far, 48 compromise the ability of the host to cope with mouse cytomegalovirus, a common pathogen of mice with a human equivalent that causes severe disease in immunodeficient patients and newborn infants. Of the 48 mutations, 26 have been identified. Among them was a mutation (3d) that foretold the existence of an equivalent human herpesvirus-susceptibility phenotype, which we have continued to investigate and understand mechanistically. Another mutation (warmflash) has pointed to a potential means of treating human herpesvirus infections. We have also collected a total of 28 mutations that impair awareness of infection mediated by the Toll-like receptors, key sensors of microbes in mammals. A total of 18 of these mutations have been identified at the molecular level, and all have contributed to our understanding of how the immune system becomes activated during infection (Fig. 1).

Bruce Beulter, Ph.D., Scientific Report 2008, figure 1

Fig. 1. Mutagenesis can be used to analyze biochemical pathways by which infection is sensed. Each red X depicts a mutation. The names of phenotypes are shown in yellow; protein names (where identified) are shown in pink. These mutations were identified by screening macrophages from mutant mice that did not produce TNF in response to stimulation with a Toll-like receptor.
  
One of the most recent success stories in immunology is the Elektra mutation. Identified because mice homozygous for the mutation died when infected with a normally harmless inoculum of mouse cytomegalovirus, Elektra seems to affect a protein that responds to viral infection within various immune cells, sending a signal to activate the T-cell response. The Elektra protein is also required for the development of a class of immune cells called natural killer T cells, which have resistance functions that are still poorly understood. Interestingly, Elektra points to a member of a family of proteins, and most likely the other members of the family have related functions.

The pace of identifying mutations has gathered speed with the development of special tools for the automated exploration of critical regions by DNA sequencing. As methods for sequencing become more advanced, most likely strange phenotypes will be found even more quickly, within days rather than months or years. In anticipation of this situation, we are striving to devise methods to find aberrant phenotypes faster than ever before and to make the resulting mutational models available to the scientific community. In years to come, we think that 50 to 100 phenovariants will typically be "solved" annually. Through interactions with other members of the Scripps Research community, in particular, members of the expanding Department of Genetics, we hope to study the proteins that are found and to understand their functions in mechanistic detail.