Two gene regulatory networks for T cell development

1. Two gene regulatory networks for T cell development MBL GRN course Ellen Rothenberg Division of Biology, California Institute of Technology Oct. 18, 2011

2. The strategy of “adaptive immunity” and its developmental programming consequences • Every T or B cell has a “unique” antigen receptor recognition specificity via immune receptor (TCR or Ig) gene rearrangement and somatic mutation • Antigen receptor gene rearrangement process generates many dysfunctional products • Cells with bad receptor genes must be eliminated: >90% loss •  Large excess of precursors must be generated continuously

3. The strategy of “adaptive immunity” and its developmental programming consequences: T cell development population dynamics Est. cell numbers: 1E07 3 – 50? 2E04 ~5E04 1E06 ~1E06 2E08 3E07

4. T cell development requires distinct program phases • Precursor expansion phase • Cell cycle arrest, TCR gene rearrangement, application of selection checkpoint (DN3 –|| b-selection) • Selected cell expansion phase • Cell cycle arrest, TCR gene rearrangement, application of selection checkpoint (DP –|| positive selection) • Distinct positive selection differentiation programs (helper, killer)

5. T cell development depends on extrinsic signals: crucial role of Notch/Delta signaling in vitro & in vivo Notch1-DL4 interaction is the major inductive signal that precursors receive from the thymus environment in vivo B cells T cells In vitro system: T. Schmitt & J. C. Zuniga-Pflucker, 2002 Rel. expression (Taghon et al., 2005, Genes Dev) Days

6. Notch-DL4 signalling drives and governs early stages of T-cell development but has hit & run role NOTCH requirement

7. Lockdown mechanisms in commitment • Positive regulatory factors for cell type • Expressed stably • Can act in positive feedback loops • Regulatory factors for alternative cell types • Must be silenced in development • May be repressed by lineage-specific factors • Continuous repression needed??? • Repression may be stabilized by chromatin closing

8. Commitment in the DN2a to DN2b transition

9. Genes in play during T-cell lineage commitment: a genome-wide look by RNA-seq(J. Zhang, A. Mortazavi, B. Williams, B.Wold, EVR) FLDN1 FLDN2a FLDN2b ThyDN3 ThyDP

10. T cell identity genes: details of gene-specific regulation differ …but many are strongly activated in DN2a-DN2b stage Data from RNA-seq, J. Zhang, A. Mortazavi, B. Williams, B. Wold, E. Rothenberg

11. “Lineage identity” factors: prototypes from other hematopoietic lineages Erythroid fate: GATA-1 B cell fate: EBF1 + Pax5 Myeloid fates: PU.1 + C/EBPa • Direct positive drivers of lineage-specific genes • Direct antagonists of alternative-lineage regulators • Direct antagonists of stem/progenitor cell genes • Lineage “specific” expression

12. Tcf7 (encodes TCF-1 protein) is induced by Notch/Delta signaling … Upregulation by Notch Upregulation by Tcf7 vs. Notch (Weber, … & Bhandoola, 2011, Nature)

13. Tcf7 is induced by Notch …and activatedTCF-1can turn on T-cell genes withoutNotch/Delta signaling Upregulation by Notch Upregulation by Tcf7 vs. Notch (Weber, … & Bhandoola, 2011, Nature)

14. Feed-forward network circuits triggered by Notch pathway signaling activate T-cell transcription factor genes and marker genes Notch signal Notch signal • Crucial role of Notch signal to activate: direct inputs to TCF-1 (Tcf7), Bcl11b, and some differentiation genes • Direct input from TCF-1 to Bcl11b and from GATA-3 to TCRb (& Cd3d,e) shown by ChIP-seq (Tcf7) • Dose dependence: • Normally activated Notch/Su(H) & sub-maximal Tcf7 collaborate to start specification • But maximal Tcf7 can turn on a subset of these targets without Notch input • This high-dose positive role of TCF is not dependent on Wnt/ b-catenin • (Weber et al., Nature 2011; P. Liu et al., Science, 2010; Wei et al., Immunity, 2011; J. Zhang et al., submitted; network models: H. Y. Kueh & EVR, WIREs Sys Bio, 2011)

15. Feed-forward network circuits triggered by Notch pathway signaling activate T-cell transcription factor genes and marker genes Notch signal Notch signal (Tcf7) • Crucial role of Notch signal to activate: direct inputs to TCF-1 (Tcf7), Bcl11b, and some differentiation genes • Direct input from TCF-1 to Bcl11b and from GATA-3 to TCRb (& Cd3d,e) shown by ChIP-seq • (Weber et al., Nature 2011; P. Liu et al., Science, 2010; Wei et al., Immunity, 2011; J. Zhang et al., submitted; network models: H. Y. Kueh & EVR, WIREs Sys Bio, 2011)

16. An unbiased look at transcription factor and DNA binding factor gene expression changes genome-wide Exp higher in DN1 Exp higher in DN2b PU.1 Log2 ratio DN1 exp: DN2b exp Gata3 Tcf7 Bcl11b

17. Major stages in early intrathymic T-cell development Major pathvia “DP” cells Bcl11b level

18. Conditional knockout of Bcl11b to determine its function in T-lineage commitment A. Bcl11b locus 3’UTR Exon 4 Loxp Loxp Conditional KO mouse model from Dr. Mark Leid, Oregon State University Infected by NGFR-Cre retrovirus (48 hr) B. NGFR+ c-kit+ CD27+ cells were sorted OP9-DL1 Flt3L, IL7 Fetal liver HSC: Lin-c-kit+ SCF, Flt3L, IL7 Cells were cocultured with OP9-DL1 for ~9 days. 1-2 weeks DN2A and DN2B cells were sorted Analysis, cell sorting OP9-DL1 SCF, Flt3L, IL7 (Long Li, Mark Leid, & E.V.R., Science 2010)

19. Loss of Bcl11b: deficient cells “stall” at early DN2a stage DN2 DN1 Sorted DN2a cells from Cre-treated: Bcl11b conditionalControl B6 miceknockout mice a c-Kit b DN4 DN3 further culture& differentiation CD25 Normal pathway for early T cell development DN3, DN4& later cells more DN2a cells& some NK cells(non-T) Bcl11b is needed to terminate “self-renewal” in DN2 stage (Long Li, Mark Leid, & E.V.R., Science 2010)

20. DN2a cells with deleted Bcl11b: “stuck” in DN2 but enhanced self-renewal capability Bcl11b-/-DN2A Bcl11b-/- “DN2B” B6 DN2A B6 DN2B c-kit CD25 Production of more DN2a cells in T-cell conditions Cell Numbers B6 DN2a + + Bcl11bKO DN2a + + Input 2 5 2 5

21. Early roles of Bcl11b (Long Li, Mark Leid, & E.V.R., Science 2010)

22. Molecular Basis of the Function of Bcl11b in T-cell Lineage Commitment • T-cell program • Stem Cell program • NK program (IL7Rα) CD3ε (initial) CD3γ CD25 Rag1 Lck GATA3 E2A HEB Tcf7 (TCF-1) Ikaros SCL (Tal1) Lyl1 Cpa3 Bcl11a Flt3 Gfi1b GM-CSFRβ Hhex Kit Sfpi1 (PU.1) (IL7Rα) Id2 IL2Rβ T-bet Eomes Zbtb16 (PLZF) Nfil3 (E4bp4) Zfp105 The initiation of T-lineage specification is Bcl11b-independent. The repression of the stem cell regulatory program requires Bcl11b. The repression of the NK program requires Bcl11b.

23. Bcl11b is not required to start the T-cell differentiation program

24. Bcl11b is the developmental gatekeeper for irreversibility of commitment

26. Factors expressed before commitment have “rap sheets” • Alternative lineage factors: PU.1 (myeloid, dendritic cell); Gfi1b, SCL/Tal1 (erythroid); Bcl11a, Lyl1 (B) • Stem cell self-renewal genes: SCL/Tal1, Lmo2, Erg, Hhex, Gfi1b, Meis1 • T-lineage proto-oncogenes: SCL/Tal1, Hhex, Lyl1, Lmo2

27. How simple a regulatory process is T-lineage commitment? • Use epigenetic markings to detect whencis-regulatory elements become active • H3K4me2 marks at promoter, enhancers, other functional sites • H3K(9,14)Ac marks closely correlate with pol II recruitment • When & how are regulatory genes that compete with commitment silenced • H3K27me3 marks silencing by PRC2 (Ezh2, Suz12) Analysis by ChIP-seq: same samples as used for RNA-seqtranscriptome analysis (J. Zhang, A. Mortazavi, B. Williams, B. Wold, & EVR)

28. H3K4me2 H3K(9,14)Ac H3K27me3 RNA All genes during T cell specification:Histone modifications at the transcriptional start site vs. RNA expression DN1, DN2a, DN2b, DN3, DP stages, left  rightHeat map, log2 of integrated intensity

29. Lineage exclusion by repression of crucial regulators of other cell types • One or many different repression mechanisms required? • Temporally • Biochemically • When do these events occur – does this fit expectation for hierarchical order of changes in developmental potential? • Lose B cell potential earlier: EBF1, Pax5 • Lose DC, NK potential later: PU.1 (Sfpi1), Id2 • Stem cell self-renewal “potential”: SCL(Tal1), Lyl1, Erg, Gata2, Bcl11a, Hhex, Meis1 (Gfi1b)

30. Diverse repression mechanisms, not a single “master commitment factor”, silence regulators of different alternative fates: Pax5(B cell), Hhex (progenitor cell), Sfpi1/PU.1 (progenitors & myeloid cells) H3Ac H3K4me2 H3K27me3 RNA

31. Bcl11b knockout reveals a conditionally sustainable “Phase 1” state: what sustains it? E. V. Rothenberg, J. Zhang, L. Li 2010 Immunol Rev

32. An unbiased look at transcription factor and DNA binding factor gene expression changes genome-wide Exp higher in DN1 Exp higher in DN2b Three known roles: myeloid cells, B cells, multipotent progenitors PU.1 Log2 ratio DN1 exp: DN2b exp Gata3 Tcf7 Bcl11b

33. Impact on T cell development of removing PU.1 from DN1 stage: PU.1fl/fl FLPs OP9-DL1 co-culture OP9-DL1 co-culture 3 days 4 hrs Infection with Cre-carrying retroviruses e14.5 B6 and PU.1fl/fl FL cells 2 days Sort Cre+ DN1, DN2a and DN2b cells Conditional knockout mouse model from Dr. Stephen Nutt, WEHI DN progression

34. Accelerated T-cell differentiation in the absence of PU.1, but reduced cell recovery: tradeoff between phase 1 proliferation and phase 2 differentiation Ameya Champhekar

35. Two “phase 1” transcription factor genes with PU.1 binding sites are functional positive regulatory targets of PU.1 Upregulated in cells forced to express wildtype PU.1 (PU.1-WT) Downregulated in cells forced to express an artificial obligate repressor form of PU.1 (PU.1 Eng) (RNA expression rel. to b-actin, avg + SD, by qPCR) (Ameya Champhekar)

36. PU.1 has direct inputs into multiple phase 1 specific genes PU.1 binding targets Lmo1, Lmo2, (Tal1), Lyl1, Hhex, and Bcl11a are all proto-oncogenes PU.1 Sfpi1 Lyl1 Meis1 Flt3 Bcl11a Lmo2 Hhex PU.1 binding at promoters and specific distal elements defined by ChIP-seq; regulatory impact of inputs defined by gain/loss of PU.1 function effects (qPCR)

37. Phase 1 and phase 2 positive networks: what is their interaction? PU.1 Sfpi1 Lyl1 Meis1 Flt3 Bcl11a Lmo2 Hhex ? ? ? ? Notch signal Notch signal

38. PU.1 overexpression represses T-cell specific genes: a potential timing role for completion of commitment But does it do this directly or via activation of another phase 1 repressor? Strategy: use modified PU.1 gene with obligate repressor activity

39. PU.1-Engrailed fusion construct: test for PU.1 functional role by forced expression of obligate repressor PU.1 Transactivation DNA binding HDAC Repression of PU.1 target genes Gro/TLE

40. Control PU.1-WT PU.1-Eng Log10expression DN2b DN2b DN2b DN2a DN2a DN2a DN1 DN1 DN1 Stem cell and Alt. lineage genes PU.1 overexpression blocks or delays activation of specific T-cell genes in early T lineage cells T-cell genes

41. Control PU.1-WT PU.1-Eng DN2b DN2b DN2b DN2a DN2a DN2a DN1 DN1 DN1 Stem cell and Alt. lineage genes But…PU.1 repression of T-cell genes is probably indirect Form with obligate repressor biochemical action represses true PU.1 activation targets but activates T-cell genes T-cell genes

42. PU.1 overexpression represses T-cell genes through activating another negative regulator Positive regulators Positive regulators Upregulation by PU.1-Eng implies that endogenous PU.1 in vivo counterbalances action of positive drivers (e.g. Notch, GATA3, TCF-1, E proteins) that are already expressed before commitment

43. A regulated phase 1-phase 2 transition is required: “phase 1” factors as well as “phase 2” are part of T-cell program Thymus Stem cell Multipotent “Paucipotent” Committed Notchsignals ETPDN1 DN2a DN2b DN3a Grow! Restrain growth; make TCR ROLE IN STAGE: KEY FACTORS: “Stem/progenitor” PU.1, Lyl1, SCL, Hhex,Gfi1b, Erg, Bcl11a… T-cell factors…?

45. Transcription factor expression: multiple patterns from multipotent stage to T-cell lineage commitment Rising Falling DN3 peak Legacy  drop Genes from gene discovery project (David-Fung et al., Devel. Biol., 2009); levels measured by qPCR (log10 scale); patterns identified by supervised clustering (self-organizing matrices)

46. T cell development depends on extrinsic signals: crucial role of Notch/Delta signaling in vitro & in vivo B cells Tcf7=TCF-1 T cells Rel. expression (Taghon et al., 2005, Genes Dev) Days

47. T-cell development depends on transcription factors that are shared with other programs Implication: Not just presence of factor, but its network connections and stage of action determines T-lineage role