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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 • • • • • • • 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 • • 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 • • • • • • 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