Regulation%20of%20MHC%20II%20Gene%20Transcription

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Regulation of MHC II Gene Transcription

• Charlotte S. Kaetzel PhD
• Dept. of Microbiology, Immunology Molecular
  Genetics
• February 28, 2008

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MHC II Function

• Expressed by antigen-presenting cells (APCs)
• Heterodimer of a and b chains
• Binds small peptides
• Presents peptides to T cells

MHC II
TCR
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Expression of MHC II Molecules

• Constitutive
• Professional APCs
• B cells
• Dendritic cells

• Inducible by IFN-g or LPS
• Many cell types
• Macrophages
• Epithelial cells
• Fibroblasts
• Others

Down-regulated in B cell ? plasma cell
differentiation
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Organization of MHC II genes
Short arm of human chromosome 6
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MHC II Gene Expression

• Heterodimers of a and b subunits
• Coordinate expression of multiple loci DR, DQ,
  DO, DM, DP
• Co-dominant expression of maternal and paternal
  alleles

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The MHC II Promoter
Nekrep et al., Immunity 18453, 2003
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Factors Recruited to MHC II Promoters

• Transcription factors that bind to conserved DNA
  elements
• RFX (trimer of RFXANK, RFX5 and RFXAP)
• CREB (cAMP response element binding protein)
• NF-Y (trimer of A, B and C subunits)
• OCAB (octamer binding protein)
• CIITA MHCII Transactivator acts as
  transcriptional integrator
• BRG1 Brahma-related gene 1 ATPase involved in
  remodeling nucleosome structure vertebrate
  homolog of yeast SWI/SNF
• CARM1 Histone methylase
• HAT histone acetyltransferase promotes open
  chromatin structure by acetylating core histones
  in nucleosomes
• Factors in pre-initiation complex (PIC) p-TEFb,
  TAFs, TBP, etc.
• RNAP II RNA polymerase II binds to Initiator
  element in MHC II promoter and catalyzes
  transcription elongation
• NOTE All of these proteins except CIITA are
  ubiquitously expressed

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CIITA is a member of the CATERPILLAR family of
intracellular pattern recognition receptors
Nucleotide-binding domain
Leucine-rich repeats
Transactivation domain
Ting et al., Nat. Rev. Immunol. 6183, 2006
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Model for regulation of subcellular distribution
of CIITA
NLS nuclear localization signal NES nuclear
export signal
Ravalet al., J. Immunol. 170922, 2003
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Expression of CIITA Molecules

• Constitutive
• Professional APCs
• B cells
• Dendritic cells

• Inducible by IFN-g
• Many cell types
• Macrophages
• Epithelial cells
• Fibroblasts
• Others

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Transcriptional Integration by CIITA
Wright Ting, Trends Immunol. 27405, 2006
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Structure of the CIITA gene locus
p1
pIII
pIV
Wright Ting, Trends Immunol. 27405, 2006
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Chromatin Remodeling
Zika and Ting, Curr. Opin. Immunol. 1758, 2005
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Role of CIITA in Chromatin Remodeling
Zika and Ting, Curr. Opin. Immunol. 1758, 2005
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Bare Lymphocyte Syndrome (BLS)

• Loss of constitutive and inducible expression of
  all MHC II genes
• Results in severe combined immunodeficiency
  because of loss of antigen recognition by T cells
• Mutations involve factors associated with MHC II
  transcription, NOT the MHC II genes themselves
• RFXANK
• RFX5
• RFXAP
• CIITA

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METHODS
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Electrophoretic Mobility Shift Assay (EMSA)

• In vitro assay of DNA-protein interactions
• Isolate protein extracts (nuclear or whole cell)
  from cultured cells or tissues following
  experimental treatment.
• Radiolabel short fragment of DNA or
  oligodeoxynucleotide containing a transcription
  factor binding site.
• Incubate labeled DNA with protein extract to
  allow protein-DNA binding.
• Separate protein-bound from unbound DNA by
  nondenaturating gel electrophoresis, and detect
  DNA by autoradiography. Protein-bound DNA will
  be shifted to a slower mobility than unbound
  DNA.
• Variation add an antibody against a specific
  transcription factor to the protein-DNA mix. The
  complex of antibody-protein-DNA will be shifted
  to a slower mobility than protein-DNA alone
  (supershift)

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Chromatin Immunoprecipitation (ChIP)

• Used to measure binding of proteins to DNA in
  native chromatin
• Add formaldehyde to living cells to form
  DNA/protein and protein/protein crosslinks
• Lyse cells and sonicate chromatin to break it
  into fragments with an average length of 500-1000
  bp
• Immunoprecipitate chromatin fragments with
  antibody to protein of interest
• Heat precipitated chromatin to reverse
  protein-DNA crosslinks and digest with RNAse A
  and proteinase K to purify DNA
• Amplify immunoprecipitated DNA fragments by PCR
  using primers for promoter of interest
• Variation use real-time PCR (see next slide) for
  a more quantitative measure of immunoprecipitated
  DNA.

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Reverse Transcriptase (RT)-PCR

• Used to measure steady-state levels of individual
  mRNAs
• Isolate total cellular RNA from cultured cells or
  tissues following experimental treatment
• Prepare complementary DNA (cDNA) by incubating
  RNA with random primers and reverse transcriptase
• Amplify transcript from gene of interest by PCR,
  using sequence-specific primers
• Real-time PCR uses fluorescent probes to
  analyze the level of amplified cDNA at each PCR
  cycle, and is more quantitative than end-point
  PCR, where the final amplified sample is analyzed
  by gel electrophoresis.
• For more information about real-time PCR, visit
  http//www.appliedbiosystems.com/support/tutorials
  /pdf/rtpcr_vs_tradpcr.pdf

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Fluorescence-Activated Cell Sorting (FACS)

• Used to measure protein expression in intact
  cells
• For example, expression of the MHC II protein
  HLA-DR on the cell surface
• Intact cells are incubated with
  fluorescent-labeled antibodies
• Can measure multiple proteins on the same cell if
  you use different colors of fluorescent labels
• Cells are sorted by machine and analyzed
  individually
• Data are expressed as histograms with number of
  cells on the Y-axis and fluorescence
  intensity on the X-axis

Immunofluorescence Microscopy

• Used to analyze intracellular localization of
  proteins
• Similar to FACS, but cells are fixed and stained
  on aslide, then imaged with a standard or
  confocal fluorescence microscope

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Immunoblot (Western Blot)

• Used to measure total protein expression in cells
  or tissues
• Cells or tissues are lysed in a denaturing
  buffer, and proteins are separated based on
  molecular weight by denaturing polyacrylamide gel
  electrophoresis (SDS-PAGE). Larger proteins will
  migrate more slowly in the gel.
• Separated proteins are transferred from the gel
  to a thin membrane of nylon or nitrocellulose
  (hence the term blot).
• Individual proteins bound to the membrane are
  visualized by specific antibodies labeled with an
  enzyme or fluorochrome. The intensity of the
  band is proportional to the level of the protein.
• Variation ectopically expressed proteins with an
  epitope tag (e.g., Flag or Myc) can be
  detected with an epitope-specific antibody.

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Expression Vectors

• Can be introduced into cells as plasmid or virus
  vectors
• Can be transcribed/translated in vitro or
  introduced into living cells by transfection
• Can encode wild-type or mutant form of protein
• Proteins can be tagged with extra sequences,
  such as a Flag or Myc epitope

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Transcription Reporter Plasmids

• Introduced into living cells by transfection
• Activity of reporter protein (e.g., luciferase,
  CAT) measured as an index of transcriptional
  activity
• Can be used to measure basal or inducible
  transcription
• Can encode wild-type or mutant form of promoter

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CIITA Transactivation assay

• A fusion protein is constructed by inserting a
  GAL4-binding motif at the 5 end of the CIITA
  coding sequence.
• A GAL4-dependent luciferase reporter plasmid is
  constructed by inserting 5 GAL4 sites upstream of
  a weak promoter driving luciferase transcription.
• The GLA4CIITA expression plasmid and
  GAL4luciferase reporter plasmid are
  co-transfected into living cells.
• Inside the nucleus, the GAL4-binding motif of the
  fusion protein binds to the GAL4 sites on the
  reporter plasmid.
• The CIITA portion of the fusion protein
  transactivates the weak promoter.
• Luciferase activity is measured as an index of
  transcriptional activity