Transcript MHC II Function - Andrew Pierce -

Regulation of MHC II Gene
Transcription
Charlotte S. Kaetzel PhD
Dept. of Microbiology, Immunology &
Molecular Genetics
February 28, 2008
MHC II Function
MHC II
TCR
• Expressed by antigenpresenting cells (APCs)
• Heterodimer of a and b chains
• Binds small peptides
• “Presents” peptides to T cells
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
Organization of MHC II genes
Short arm of human chromosome 6
MHC II a chain genes
MHC II pseudogenes
MHC II b chain genes
unrelated genes
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
The MHC II Promoter
Nekrep et al., Immunity 18:453, 2003
Factors Recruited to MHC II Promoters
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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
CIITA is a member of the CATERPILLAR family
of intracellular pattern recognition receptors
Nucleotidebinding domain
Leucine-rich
repeats
Transactivation
domain
Ting et al., Nat. Rev. Immunol. 6:183, 2006
Model for regulation of subcellular
distribution of CIITA
NLS = nuclear localization signal
NES = nuclear export signal
Ravalet al., J. Immunol. 170:922, 2003
Expression of CIITA Molecules
Constitutive
“Professional” APCs
• B cells
• Dendritic cells
Inducible by IFN-g
Many cell types
• Macrophages
• Epithelial cells
• Fibroblasts
• Others
Transcriptional Integration by CIITA
Wright & Ting, Trends Immunol. 27:405, 2006
Structure of the CIITA gene locus
p1
pIII
pIV
Wright & Ting, Trends Immunol. 27:405, 2006
Chromatin Remodeling
Zika and Ting, Curr. Opin. Immunol. 17:58, 2005
Role of CIITA in Chromatin Remodeling
Zika and Ting, Curr. Opin. Immunol. 17:58, 2005
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
METHODS
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”)
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.
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 sequencespecific 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_tra
dpcr.pdf
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
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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
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.
Expression Vectors
Constitutive Promoter
Protein coding region
• 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
Transcription Reporter Plasmids
MHC II or CIITA Promoter
“Reporter” protein
• 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
CIITA Transactivation assay
Strong
Promoter
GAL4
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GAL4
GAL4-binding motif
GAL4
GAL4
GAL4
CIITA (WT or mutant)
Weak
Promoter
Luciferase
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 GLA4:CIITA expression plasmid and GAL4:luciferase reporter plasmid are cotransfected 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