MOLECULAR CHARACTERIZATION OF PERFORIN GENE REGULATION

Project: Research project

Description

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells lyse
virus-infected cells and possibly emerging tumor cells but they also
contribute to transplant rejection and perhaps to autoimmune tissue
destruction. One of the lytic molecules of CTL and NK cells is a
pore-forming protein known as perforin. Perforin gene expression is
restricted to CTL and NK cells. While NK cells express perforin
constitutively, CTL express perforin upon their activation. Our
objective is to characterize relevant molecular mechanisms 1) that
determine the CTL and NK-cell restricted expression of perforin; 2) that
determine the induction of perforin in CTL upon their activation; and we
propose 3) to identify the transcription factors involved. Within the
first objective, we will characterize a cell-type specific cis-acting
element in the 5' flanking region of the perforin gene (specific aim 1.
1), determine the role of the proximal perforin promoter (specific aim
1.2) and analyze and determine the role of the chromatin structure of the
perforin gene locus (specific aim 1.3). To achieve these aims we will
analyze fragments of the perforin 5' flanking and promoter region for
their control over the "CAT" reporter gene after transient transfection
into different cell types and will compare DNase I hypersensitivity sites
of the perforin gene locus between different cell types. Within the
second objective, we will determine whether perforin mRNA induction in
CTL occurs primarily at the transcriptional level or the
posttranscriptional level (specific aim 2.1). We anticipate that the
regulation event takes place at the transcriptional level based on our
preliminary data and therefore propose to identify regulatory elements
involved (specific aim 2.2). To achieve these aims we will analyze, by
Northern blot analysis and nuclear run-off assay, perforin gene induction
or upregulation in cloned CTL in response to IL-2, T-cell receptor
signals or second-messenger pathway activating agents. We will identify
the cis-acting responsive regions by reporter gene analysis as outlined
above. Within the third objective, we propose to identify putative
transcription factors acting on relevant regulatory elements defined in
the previous aims (specific aim 3). We will apply in vitro DNase I
footprinting and will analyze a particular DNA binding site by gel shift
analysis.
StatusFinished
Effective start/end date9/1/923/31/04

Funding

  • National Institutes of Health
  • National Institutes of Health: $106,511.00
  • National Institutes of Health: $201,230.00
  • National Institutes of Health: $112,317.00
  • National Institutes of Health: $219,645.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $213,250.00
  • National Institutes of Health: $104,097.00
  • National Institutes of Health

Fingerprint

Perforin
Cytotoxic T-Lymphocytes
Genes
Natural Killer Cells
Reporter Genes
Deoxyribonuclease I
5' Flanking Region
Tumor Cell Line
Transcription Factors
Immune System
Hypersensitivity
Tumor Virus Infections
Gene Expression

ASJC

  • Medicine(all)