IMM1016: Recent Advances in Immunology October 15, 2012 Michele K. Anderson Sunnybrook Research Institute [email protected] The “Who Cares” Question Developmental processes result in the establishment of cellular identity Cell identity in a large part is dependent on the genes that are expressed and those that are poised to be expressed in response to external stimuli During development genes are turned on that start the process of establishing a certain identity, but other genes need to be turned off to allow the locking in of that identity Networks of interactions are required to keep the system stable even in the absence of the signals that set the whole process in motion Early stages of T cell development: setting the stage Specification versus Commitment in T Cell Development Specification: initiation of the T-cell gene expression program Commitment: loss of the ability to become other cell types These can be decoupled by genetic manipulation Key players: Notch1 TCF1 Bcl11b GATA3 HEB (E-proteins) Notch1 is critical for entry into the T-cell lineage Ligand-activated receptor that complexes with RBPj to initiate transcription Conditional deletion of Notch1: Mx-Cre, Lck Ectopic expression of Notch1 In vitro expression of DL Notch ligands OP9-DL1 or DL4 system (J.C. Zuniga-Pflucker) Widely used as a readout of T cell potential Can be used to generate large numbers of T cell precursors Used to test impact of transcription factor deficiency Developmental potential of early DN thymocytes cultured on OP9 bone marrow stromal cells in the presence (OP9-DL1) or absence (OP9-control) of Dll1 expression. Schmitt T M et al. J Exp Med 2004;200:469-479 © 2004 Rockefeller University Press Two waves of transcription factor upregulation in response to Notch signaling Bcl11b enforces T cell commitment Zinc-finger transcription factor that acts primarily as a repressor Conditional deletion interrogated using OP9-DL1 co-culture Deletion of Bcl11b allows development to DN3 stage OP9-DL1 co-culture Deletion of Bcl11b abolishes DN3 commitment OP9 Co-culture Deletion of Bcl11b favors NK and myeloid fates Bcl11b primarily inhibits alternative lineage regulators Distinct subcircuits drive specification vs. commitment HEBAlt and HEBCan belong to the E-protein family of TFs Network of interactions between T-cell specific transcription factors Student Presentation How does commitment work? How is the T cell program deployed? How do these processes intersect? Global examination of gene expression and chromatin status Strategy: 1) Isolate five different stages of T cell development DN1 DN2a DN2b DN3 DP Commitment as defined by OP9-DL1 co-culture assays 2) Use RNAseq to generate a complete catalogue of gene expression at each developmental stage Map onto the genome Example of RNA seq data: HEBAlt in TCRgamma locus activation Jg1 Cg1 Global changes in gene expression between each stage All genes differentially expressed between at least two populations by 2-fold •Two biological replicates of each population •Good agreement between replicates •Few changes between DN1 and DN2a •Large changes between DN2a and DN2b •FLDN2b and ThyDN3 very similar to each other Coordinated upregulation of pre-TCR genes at DN1 to DN2 •CD3gamma •CD3delta •CD3epsilon •CD3zeta •Pre-Talpha •Itk •LAT •Rag1 DN1 DN2a DN2b DN3 DP Two major changes in TF usage: DN2a to DN2b and DN3 to DP * Increased: Bcl11b, Lef1, SpiB, Tcf1, Hes1, GATA3, HEBAlt Decreased: PU.1, GATA2, C/EBPbeta, Bcl11a, LMO2, Hhex, Lyl1 Broad downregulation of regulators of alternative lineages 2) Use ChiPseq to generate a complete catalogue of chromatin marks on cis-regulatory elements at each stage Three histone modifications linked to activation status •Histone3K(9,14) acetylation (H3Ac): linked to activation at transcriptional start sites (TSSs) •Histone3K4me3: linked to silencing •Histone3K4me2: poised for activation or silencing (developmentally labile) Chromatin marks on expressed and silent genes at each stage •H3Ac and me2 marks are on most expressed genes whereas me3 marks are not •Most silent genes have no marks but those that do have me2 or me3 •Chromatin marks are good indicators of activation status of genes in these cells No drastic changes in overall chromatin status between stages Distal elements are more labile than promoter elements Me2=labile Me3=silenced Each distally marked region was assigned to the closest gene Some distal elements corresponded to known regulatory sites Patterns of gene expression over developmental time C C Upregulated C V Transient C C V C Downregulated V Timing of chromatin changes at TSSs relative to transcription H3Ac tightly correlated with RNA Me2 often present before and after RNA Me3 associated with repressed genes but most silent genes don’t acquire this mark Next….Zoom in on hematopoietic genes… Upregulation of T-cell specific genes in DN and DP stages Lineage-inappropriate genes downregulated and marked Lineage-inappropriate genes not expressed but marked Activation at DN2a to DN2b transition: CD3 versus Bcl11b CD3 enhancer available even in DN1 CD3 gradual acquisition and loss of HAc3 and me3 Bcl11b much more abrupt acquisition and loss of marks at DN2a to DN2b Histone marks on T-regulator GATA3 and B-regulator Pax5 Me3 (remember, not on all silenced genes) Alternative lineage regulators downregulated at DN2a to DN2b PU.1 in hematopoiesis and early T cell development PU.1 is expressed at high levels in myeloid cells, intermediate levels in B cells, and low levels in T cell precursors PU.1 is high in DN1, low in DN2, very low in DN3, and absent from DP, SP, and mature T cell precursors Overexpression of PU.1 in either B cell or T cell precursors redirects them to the myeloid fate in the presence of the myeloid growth factors Overexpression of PU.1 in T cell precursors in FTOC blocks T cell development at the DN stages progressively However, T cells cannot develop without PU.1. Is this because of an overall defect in pre-thymic precursors or is PU.1 playing a positive role in the context of Notch signaling? Examine binding of PU.1 in T cell precursors using ChIP-Seq PU.1 binding in T cell precursors versus B cells and macrophages Macrophages B cells DN1 PU.1 bound 34K sites in DN T cells, comparable to the number bound in B cells and macrophages However, these were primarily different sites than those bound in B cells or macrophages The tighter the plots, the more similar the samples are PU.1 binding decreases as DN stages progress but the binding sites remain largely the same DN1 by DN2a DN1 by Me2 DN2a by DN2b DN2b by me2 PU.1 binding is tightly correlated with H3K4me2 (poised) status throughout the early stages of T cell development PU.1 binding and chromatin mark status on Tal1 (SCL) locus during T cell development Me2 persists after mRNA halts Me3 increases and spreads as T cell development proceeds and mRNA PU.1 decreases but remains bound to the same sites as T cell development proceeds, PU.1 binding and chromatin mark status on IL7R during T cell development Me2 precedes mRNA expression Not many me3 marks PU.1 stays bound to the same site before and during T cell development PU.1 is associated with expressed genes during early T cell development prior to commitment GATA3 is essential for several stages of T cell development GATA3 is expressed throughout T cell development at fairly constant levels Lack of GATA3 inhibits even the earliest ETP stage of T cell development Overexpression of GATA3 in precursors, however, blocks T cell development at the DN2 stage Addition of IL3 and SCL and withdrawal of DL Notch ligands can however rescue cells into the mast cell lineage GATA3 plays different roles at different stages of T cell development: bselection, CD4 lineage, Th1 lineage Does GATA3 bind the same sites throughout T cell development? ChIPseq analysis GATA3 binds different sites at different stages of T cell development Lyl1 Ets2/Erg Itk E2A GATA3 sites on Ly1-1 (expressed early) and Ets2 (expressed in DP) Lyl1 Ets2/Erg GATA3 sites on Itk (expressed late) and Ets2 (expressed throughout) Itk E2A Network of interactions between T-cell specific transcription factors What are the target genes? How are they regulated? ChIP-seq + RNAseq Need better antibodies to ChIP seq more of these factors!