Research interests

Genetics of Blood Cell Development and Disease

Our research focuses on the formation and function of blood cells. We study mechanisms that drive developmental assembly of the red blood cell (RBC) membrane skeleton, a complex multi-protein structure critical to RBC stability; the development of intracellular organelles critical to platelet function; and genetic interactions that influence red cell formation and baseline white blood cell (WBC) count. These studies are highly relevant to human disease. Hemolytic anemia due to membrane skeleton defects is one of the most common inherited diseases in Northern Europeans (1:2000 to 1:3000). Defects in organelle biogenesis cause platelet storage pool disease (SPD), the second most common cause of inherited bleeding in humans. Baseline WBC count is a significant risk factor for disease severity and early mortality in sickle cell anemia, a disease affecting ~1/300 people of African descent.

The RBC Membrane Skeleton

An underlying spectrin-based membrane skeleton supports the plasma membrane of most cells. In RBCs, the major component of the membrane skeleton is spectrin, which is present as tetramers of α- and β-subunits. Spectrin tetramers are cross-linked into a two-dimensional array (Figure 1) by short actin filaments at junctional complexes, which also include the protein adducin. The spectrin array is attached to the overlying plasma membrane by ankyrin, which binds spectrin to the cytoplasmic domain of the integral membrane protein termed band 3, and by protein 4.1-glycophorin C interactions at the junctional complexes.

During RBC development, membrane skeleton components are synthesized asynchronously. According to the "sequential assembly" hypothesis, pre-assembled spectrin and ankyrin shift from the cytoplasm to the membrane only upon induction of band 3 synthesis and its insertion into the RBC lipid bilayer. Studies from our laboratory, however, dispute this hypothesis, as a membrane skeleton is assembled normally in band 3-null RBCs. Hence, some other mechanism must exist to direct skeleton assembly.

Adducin shows characteristics in vitro that make it an attractive candidate to direct membrane skeleton assembly. Three genes encoding α- (Add1), β- (Add2), and γ- (Add3) adducin have been identified. Hence a full assessment of the role of adducin in RBCs requires multiple targetings with interbreeding to produce double and triple homozygotes. We have successfully generated β-adducin null mice and a germline conditional α-adducin strain. We are presently targeting γ-adducin. Hence, we will soon be able to assess the role of the adducins in RBC development in vivo. Interestingly, in addition to its potential role in membrane skeleton assembly, adducin has been implicated in the control of systemic blood pressure, although this is highly controversial. Similarly, adducin is present in blood platelets, but its role is unknown. The conditional adducin knockout strains will allow us to assess its role in platelet function, and determine the impact on systemic blood pressure as well.

Platelet Storage Pool Disease (Hermansky-Pudlak Syndrome)

Platelet SPD causes excessive bleeding due to a lack of platelet-specific organelles termed dense bodies, which are required for formation of the platelet plug at the site of injury. In one of the most severe forms of SPD, Hermansky-Pudlak syndrome (HPS), defects in developmentally related organelles, melanosomes and lysosomes result in albinism and lysosomal storage disease, respectively, in addition to the bleeding diathesis. HPS is a defect in organelle biogenesis; as a result of mis-sorting of proteins or failure of proper vesicle fusion and docking, the lysosome-related organelles (LROs) do not develop properly.

In both humans and mice, HPS is genetically heterogeneous. We have recently cloned the genes responsible for two mouse HPS mutations, cappuccino (cno) and reduced pigmentation (rp), and shown that both genes encode novel proteins. Notably, we have shown biochemically that CNO and RP are both components of a complex termed BLOC-1 (biogenesis of lysosome-related organelle complex-1), which includes the protein products of the mouse mutations pallid, muted, and sandy. As these mutations all cause severe HPS, it is clear that BLOC-1 is critical to the biogenesis and/or function of the LROs. Our future goals include the analysis of genetic modifiers of HPS using a mutagenesis strategy, and the assessment of the role of conserved non-coding sequences flanking the cno and rp loci to determine if they function as cis-regulatory elements.

In addition, we have cloned lrod (lysosome-related organelle defects), a mouse mutation caused by a retroviral insertion in a novel gene that shows homology to TRAPP proteins, which are involved in endoplasmic reticulum-Golgi apparatus trafficking. The lrod mutation causes overt variegated pigment dilution. We have demonstrated that rp interacts genetically with lrod to produce an exacerbated pigment phenotype and prolonged bleeding, suggesting that LROD is a component of a pathway that is distinct from but interacts with BLOC-1.

The lrod phenotype and its genetic interactions with rp suggest that lrod defects may underlie HPS, but the highly mosaic nature of the lrod phenotype precludes a definitive analysis. Hence, we generated an lrod knockout. Unexpectedly, lrod deficient mice die in utero from heart defects. Hence, to assess its role in platelet and melanocyte function, we are generating a conditional lord allele.

Genetic Modifiers: Quantitative Trait Loci (QTLs) Studies

In sickle cell disease, baseline WBC count is a strong predictor of disease severity, including development of acute chest syndrome, stroke, and early mortality. These findings reflect the inflammatory aspects of sickle cell disease. Our laboratory is part of a multicenter sickle cell project headed by Dr. Orah Platt (Children's Hospital, Harvard Medical School, Boston, Mass.) to dissect the complex sickle cell phenotype. We are working to identify genetic modifiers of baseline WBC count in mice as predictors of modifying genes in humans. To date, two significant QTLs have been identified on mouse Chromosome 1 using two F2 crosses (NZW/LacJ x SM/J and C57BLKS/J x SM/J) and haplotype analyses. We are currently analyzing candidate genes within each critical interval.

New Mouse Models

Phenotype-driven approaches such as the analysis of spontaneous or chemically induced mutations are a powerful method to assign function to genes. Therefore, we continue to analyze spontaneous and N-ethyl-N-nitrosourea (ENU)-induced and spontaneous mouse mutations showing anemia or platelet-dysfunction phenotypes. Two potential new HPS mutants and two novel hemolytic anemia mutants are currently in various stages of development, including heritability testing and phenotypic characterization.

Publications

• Rabenstein RL, Addy NA, Caldarone BJ, Asaka Y, Gruenbaum LM, Peters LL, Gilligan DM, Fitzsimonds RM, Picciotto MR. 2005. Impaired synaptic plasticity and learning in mice lacking beta-adducin, an actin-regulating protein. J Neurosci. 25:2138-2145.
• Yang SH, Shrivastav A, Kosinski C, Sharma RK, Chen MH, Berthiaume LG, Peters LL, Chuang PT, Young SG, Bergo MO. 2005. N-myristoyltransferase 1 is essential in early mouse development. J Biol Chem. 280:18990-18995.
• De Franceschi L, Rivera A, Fleming MD, Honczarenko M, Peters LL, Gascard P, Mohandas N, Brugnara C. 2005. Evidence for a protective role of the Gardos channel against hemolysis in murine spherocytosis. Blood. 106:1454-1459.
• Peters LL, Zhang W, Lambert AJ, Brugnara C, Churchill GA, Platt OS. 2005. Quantitative trait loci for baseline white blood cell count, platelet count, and mean platelet volume. Mamm Genome. 16:749-763.
• Inoue Y, Peters LL, Yim SH, Inoue J, Gonzalez FJ. 2005. Role of Hepatocyte nuclear factor 4alpha in control of blood coagulation factor gene expression. J Mol. Med. Epub ahead of Print.
• Peters LL, Swearingen RA, Andersen SG, Gwynn B, Lambert AJ, Li R, Lux SE, Churchill GA. 2004. Identification of quantitative trait loci that modify the severity of hereditary spherocytosis in wan, a new mouse model of band-3 deficiency. Blood 103:3233-3240.
• Peterson KR, Fedosyuk H, Zelenchuk L, Nakamoto B, Yannaki E, Stamatoyannopoulos G, Ciciotte S, Petters LL, Scott LM, Papayannopoulou T. 2004. Transgenic Cre expression mice for generation of erythroid-specific gene alterations. Genesis 39:1-9.
• Gwynn B, Martina JA, Bonifacino JS, Sviderskaya EV, Lamoreux ML, Bennett DC, Moriyama K, Huizing M, Helip-Wooley A, Gahl WA, Webb LS, Lambert AJ, Peters LL. 2004. Reduced pigmentation (rp), a mouse model of Hermansky-Pudlak Syndrome, encodes a novel component of the BLOC-1 complex. Blood 104:3181-3189.
• Clark AT, Goldowitz D, Takahashi JS, Vitaterna MH, Siepka SM, Peters LL, Frankel WN, Carlson GA, Rossant J, Nadeau JH, Justice MJ. 2004. Implementing large-scale ENU mutagenesis screens in North America. Genetica122:51-64.
• Bruce LJ, Beckmann R, Ribeiro ML, Peters LL, Chasis JA, Delaunay J, Mohandas N, Anstee DJ, Tanner MJA. 2003. A band 3-based macrocomplex of integral and peripheral proteins in the red cell membrane. Blood 101:4180-4188.
• Da Costa L, Narla G, Willig T-N, Peters LL, Parra MK, Fixler J, Tchernia G, Mohandas N. 2003. Ribosomal protein S19 (RPS19) expression during erythroid differentiation. Blood 101:318-324.
• Gridley DS, Nelson GA, Peters LL, Kostenuik PJ Bateman TA, Morony S, Stodieck LS, Lacey Dl, Simske SJ, Pecaut MJ. 2003. Effects of spaceflight in the C57BL/6 mouse II: Activation, cytokines, erythrocytes, and platelets. J App Physiol 94:2095-2103.
• Lee G, Spring FA, Parsons SF, Mankelow TJ, Peters LL, Koury MJ, Mohandas N, Anstee DJ, Chasis JA. 2003. Novel secreted isoform of adhesion molecule ICAM-4: Potential regulator of membrane-associated ICAM-4 interactions. Blood 101:1790-1797.
• Mouro-Chanteloup I, Delaunay J, Gane P, Nicolas V, Johansen M, Brown EJ, Peters LL, Kim C, Cartron JP, Colin Y. 2003. Evidence that the red cell skeleton protein 4.2 interacts with the Rh membrane complex member CD47. Blood 101:338-344.
• Pecaut MJ, Nelson GA, Peters LL, Kostenuik PJ. Bateman TA, Morony S, Stodieck LS, Lacey DL, Simske SJ, Gridley DS. 2003. Genetic models in applied physiology: selected contribution: effects of spaceflight on immunity in the C57BL/6 mouse I: Immune population distributions. J App Physiol94:2085-2094.
• Ciciotte SL, Gwynn B, Moriyama K, Huizing M, Gahl WA, Bonifacino JS, Peters LL. 2003. Cappuccino, a mouse model of Hermansky-Pudlak syndrome, encodes a novel protein that is part of the pallidin-muted complext (BLOC-1). Blood 101:4402-4407.
• Svenson KL, Bogue MA, Peters LL. 2003. Identifying new mouse models of cardiovascular disease: A review of high-throughput screens of mutagenized and inbred strains. J App Physiol 94:1650-1659.
• Paw BH, Davidson AJ, Zhou Y, Li R, Pratt SJ, Lee C, Trede NS, Brownlie A, Donovan A, Liao EC, Ziai JM, Drejer AH, Guo W, Kim CH, Gwynn B, Peters LL, Chernova MN, Alper SL, Zapata A, Wickramasinghe SN, Lee MJ, Lux SE, Fritz A, Postlethwait JH, Zon LI. 2003. Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band-3 deficiency. Nat Genet 34:59-64.
• Johnson KR, Gagnon LH, Webb LS, Peters LL, Hawes NL, Chang B, Zheng QY. 2003. Mouse models of USH1C and DFNB18: phenotypic and molecular analyses of two new spontaneous mutations of the USH1C gene. Hum Mol Genet 12:3075-3086.