Research Activities

 
 

RESEARCH PROJECT:
CONTROL OF GENE EXPRESSION DURING B CELL DIFFERENTIATION

PROJECT STAFF
Santa Jeremy Ono, PhD  Principal Investigator
Gang Zhou, MS  Research Assistant 
Michael Radosevich, BS  Harvard Medical Student PhD Candidate 
Albert Tai, BSc (Hons.)  Research Assistant 

PROJECT SUMMARY
Over the past several years we have endeavored to understand B lymphocyte differentiation by studying molecular mechanisms controlling selective activation and repression of stage specific genes.  The target genes we have focused on are the class II genes of the human major histocompatibility complex (MHC), and recently the immunoglobulin heavy chain genes.  Class II MHC genes are expressed on mature and activated B cells, and transcription is silenced as B cells differentiate into immunoglobulin secreting plasma cells.  Immunoglobulin heavy chain genes are expressed throughout most of B cell differentiation, but the nature of the heavy chain exons utilized changes in response to cytokines produced by helper T cells (via a process termed isotype or class-switching).

Our studies on MHC class II gene regulation focus on mutant B cell lines which are defective in the expression of all, or a subset of the class II MHC genes.  We utilize both biochemical and molecular biology techniques to identify nuclear factors which regulate expression of these genes.  As examples, we are 1) characterizing mRNAs which are differentially expressed between wild-type and class II expression mutant B cells, and 2) we are probing CIITA-dependent and CIITA-independent transcription initiation mechanisms via transfection of reporter constructs, and by genomic footprinting.

Our investigation of immunoglobulin class-switching has just begun.  Our goal is to identify components of the elusive class-switch recombinase.  We have recently identified novel stimulus-dependent DNaseI hypersensitive sites in the Sa region, which only appear in B cells which are competent at switching to IgA.  These sites appear prior to gene rearrangement, but are not depndent on I region transcription.  We are now determining the boundaries of these hypersensitive sites, and will start to characterize nuclear factors which interact with this site.
 

RESEARCH PROJECT:
MOLECULAR IMMUNOLOGY OF ALLERGIC CONJUNCTIVITIS

PROJECT STAFF
Santa Jeremy Ono, PhD  Principal Investigator 
Andrea Keane-Myers, PhD  Postdoctoral Fellow 
Kam-Wa Cheung-Chau, BS  Research Assistant 
 
PROJECT SUMMARY
Conjunctivitis is the major atopic disease affecting the ocular surface. Indeed, it is one of the most commonly observed diseases in ophthalmic clinics.  Patients with the condition are hypersensitive to normally innocuous substances and suffer from symptoms ranging from  mild itching and burning, to chemosis, conjunctival hypermia, lid edema and at its worst, scarring of the ocular surface and permanent visual impairment. Previous data indicate that while the pathophysiology of atopic conjunctivitis resembles that of rhinitis and asthma, there are also important variables that determine whether allergic inflammation will occur in each, or only a subset of these disparate sites in a particular individual  We hypothesize that there are particular gene products that specifically target the allergic response to the eye in patients with conjunctivitis, just as there are thought to be specific asthma-predisposing genes.

We are investigating the pathogenesis of allergic conjunctivitis via two approaches: 1) we have developed a mouse model of the disease and are characterizing the nature of the immune response, and 2) in collaboration with Mark Abelson, we are initiating a sib-pair analysis of a large patient population with seasonal and/or vernal conjunctivitis.
 

RESEARCH PROJECT:
ROLE OF HMG I PROTEINS IN RETINAL FUNCTION AND RETINOBLASTOMA FORMATION

PROJECT STAFF
 
Santa Jeremy Ono, PhD  Principal Investigator
Guidalberto Manfioletti, PhD  Visiting Scientist 
Kai-Yin Chau, PhD  Postdoctoral Fellow 
Christian Parry, PhD Postdoctoral Fellow
Paola Arlotta, MS Research Assistant
Jason Kreisberg  MIT UROP student 

PROJECT SUMMARY
The high mobility group (HMG) I proteins are a set of three, small, nuclear proteins which are non-histone components of chromatin.  They are highly homologous and bind to DNA via three basic repeats which interact with the minor groove of DNA.  They are now thought to be architectural proteins which participate in gene activation and gene rearrangement by facilitating multiprotein complex formation at specific sites in the genome.  HMG I and HMG Y are encoded by a single gene and arise via alternative splicing.  HMG I-C is encoded by a distinct gene.

All three proteins are expressed primarily during embryogenesis.  HMG I-C transcription is completely silenced in normal adult tissues.   HMG I(Y) expression is also significantly repressed, and when expressed in adult tissue, it is only found at 1/200 the abundance as in embryonic tissue. During this past year, we have found that the HMG I(Y) proteins are paradoxically expressed at extremely high levels in the normal adult retina.  We hypothesize that  this may relate to the unusually high metabolic requirements placed on photoreceptor cells.  High levels of HMG I(Y) proteins may be required to promote transcription of photoreceptor specific genes.  In the upcoming year, we hope to probe the role of HMG I(Y) gene expression in retinal function.

We have also found that retinoblastoma cells and tumors (but not normal retina) express the HMG I-C gene.  Such derepression of HMG I-C gene expression has been seen in a variety of other tumors, but never in tumors of the eye.  We hope to uncover the molecular basis of derepression of I-C gene expression in retinoblastomas, and assess its contribution to the transformed phenotype.

Main Page

Selected References:

  1. Song, Z., Krishna, S. Thanos, D., Strominger, J.L., and Ono, S.J. A Novel Cysteine-Rich Sequence-Specific DNA-Binding Protein Interacts with the Conserved X-box Motif of the Human MHC Class II Genes via a Repeated Cys-His Domain and Functions as a Transcriptional Repressor. The Journal of Experimental Medicine. 180:1763, 1994.
  1. Ono, S.J., and Song, Z. Mapping of the binding site of the defective transcription factor in the class II MHC mutant cell line Clone-13 to the divergent X2-box motif. The Journal of Biological Chemistry. 270: 6396-6402, 1995.
  2. Stellato, C., Beck, L., Proud, D., Schall, T., Ono, S.J., Lichtenstein, L.M. and Schleimer, R.P. Expression of the chemokine RANTES by a human bronchial epithelial cell line: Modulation by cytokines and glucocorticoids. The Journal of Immunology. 155: 410-418, 1995.
  3. Abdulkadir, S.A., Krishna, S., Thanos, D., Maniatis, T., Strominger, J.L. and Ono, S.J. Functional Role of overlapping octamer and high mobility group protein I/Y binding sites in the HLA-DRA promoter I: Evidence for cooperativity. The Journal of Experimental Medicine. 182:487, 1995.
  4. Monos, D.S., Kappes, D., Ono, S.J., and Strominger, J.L. Transfectants expressing the HLA class II molecules implicated in the pathogenesis of human IDDM. Immunogenetics. 42: 172, 1995.
  5. Schwiebert, L.M., Schleimer, R.P., S. Radka and Ono, S.J. The effect of the glucocorticoid dexamethasone on human class II MHC gene expression: evidence for isotype-specificity. Cellular Immunology. 165: 12-19, 1995.
  6. Abdulkadir, S.A. and Ono, S.J. How are class II MHC genes turned on or off? The FASEB Journal. 9: 1429-1435, 1995.
  7. Ono, S.J. Transcriptional Regulation of the Genes of the Human Major Histocompatiblity Complex. Chicz, R. and Urban, R. (Eds): The Major Histocompatibility Complex. Austin, Texas: R.G. Landes Co. 1995.
  8. Casolaro, V., Georas, S., Song, Z., Zubkoff, I.D., Abdulkadir, S.A., Thanos, D., and Ono, S.J. Inhibition of NFATp-dependent transcription by NFkB: Implications for differential gene expression in T cell subsets. Proceedings of the National Academy of Sciences, USA. 92: 11623-11627, 1995.
  9. Song, Z., Casolaro, V., Monos, D. and Ono, S.J. Identification of polymorphic nucleotides within the human interleukin-4 gene promoter which mediate overexpression of the gene. The Journal of Immunology. 156:424-429, 1996.
  10. Ono, S.J. Biology and Genetics of Atopic Disease. H. Metzger. Ed. Current Opinion in Immunology. 8: 6, 1996.

Main Page

Research Facilities

Dr. Ono's laboratory is housed in the new Charles Building devoted to molecular and structural biology and immunology. The P.I.'s laboratory is part of a 5,000 square foot section of the building devoted to two new investigators and their shared equipment space. The P.I.'s lab has 1,500 square feet of space utilized by his graduate students/fellows, and there is an additional 1,000 square feet of space for tissue culture work and placement of heavy equipment. A dark room and warm room are shared by the two labs. The P.I.'s lab space has all of the required equipment to carry out most of the experiments underway in the lab. Ultra and high speed centrifuges, tissue culture centrifuges, microfuges, an X-ray film developer, phosphoimager and several tissue culture hoods and incubators are within the central space. In the Staniford Building, the institute has extensive support facilities for immunohistochemistry, flow cytometry, electron and fluorescence microscopy, confocal microscopy, and animal surgery and has extensive and state of the art animal facilities. Across the street, the Department of Molecular Biology of the Massachusetts General Hospital has extensive support facilities for molecular biological work including: DNA and peptide synthesizers, sequenators and facilities for transgenic and knockout mouse development. Other support facilities for photographic work and library are found in another building of the institute, the immediate MGH environment as well as the Harvard Medical School main quadrangles.

Main Page

Current and Past Pre- and Postdoctoral Trainees of the Laboratory of Molecular Immunology

Main Page