Gene expression regulation during cellular differentiation. Dom Helmlinger CRBM 24/10/2017

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1 Gene expression regulation during cellular differentiation Dom Helmlinger CRBM 24/10/2017

2 Outline 1. Gene expression regulation: why and how? 2. Yeast as model systems. 3. Control of sexual differentiation in S. pombe. 4. Control of meiotic commitment in S. cerevisiae.

3 Defining gene expression regulation and why should you care?

4 Problem Genes alone can account for the extraordinary complexity of a living organism. Genes interact with each other and with their environment (medium, cells): House-keeping genes: always ON Regulated genes: ON or OFF Gene expression regulation

5 Difference prokaryotes / eukaryotes Prokaryotes: ON is the default state regulators = repressors Eukaryotes: OFF is the default state regulators = activators

6 Difference prokaryotes / eukaryotes Prokaryotes: ON is the default state regulators = repressors although: archeal histones Eukaryotes: OFF is the default state regulators = activators although: pervasive transcription

7 Importance of regulating gene expression Self-renewal or differentiation Adaptation to the environment Perturbations during oncogenesis

8 Structure of an eukaryotic gene Genomic DNA +1 exon intron exon Promoter ncrna: rrna trna snrna, snorna mirna, sirna RNA mrna Protein Terminator

9 Regulating the expression of a gene Genomic DNA +1 exon intron exon 1. TRANSCRIPTION Promoter 5. ncrna RNA Protein 2. PROCESSING 3. DEGRADATION 4. TRANSLATION, PROCESSING, PTMs, DEGRADATION Terminator

10 Regulating transcription +1 exon intron exon

11 Regulating transcription 1. Chromatin structure +1 exon intron Nucleosomes exon

12 Regulating transcription 1. Chromatin structure 2. Sequences (cis-regulatory elements) UAS TATA exon intron exon +1 Nucleosomes

13 Regulating transcription 1. Chromatin structure 2. Sequences (cis-regulatory elements) 3. DNA-binding factors (trans-acting factors) UAS Activator TATA +1 exon intron Nucleosomes exon

14 UAS Activator Regulating transcription 1. Structure de la chromatine 1. Chromatin structure 2. Séquences (cis) 2. Sequences (cis-regulatory elements) 3. Facteurs DNA-binding DNA-binding factors (trans-acting (trans) factors) Elongation Factors Elongation General Factors TATA Co-activator Transcription RNA Pol. II exon General Factors intron exon Transcription +1 General Termination Co-activator Factors Transcription Termination Factors Nucleosomes Factors Factors Co-activator 4. Plein and d autres many other facteurs! regulatory proteins!

15 Regulation of transcription initiation 1/2 cis-regulatory elements Specific transcription factors: DNA binding domain: binds to specific motifs, 6-10 bp, within promoters / enhancers Trans-activation domain: recruits co-activators and general transcription factors General transcription factors: DNA binding: TATA box-binding protein (TBP) Initiations (vs. elongators + terminators)

16 Regulation of transcription initiation 2/2 Nucleosomes positioning: ATP-dependent chromatin remodeling complexes Histone modifications ( histone code ): histone-modifying complexes = transcription co-activators Additional layer: non coding RNAs, DNA methylation = Epigenetic regulation of transcription

17 Overall objective To understand the mechanisms of transcription initiation regulation Activator UAS General TATA Co-activator Transcription exon General Factors intron Transcription +1 General Co-activato Factors Transcription Nucleosomes Factors Co-activator RNA Pol. II exon Transcription Elongators Transcription Elongators Transcription Transcription Terminators Terminators

18 Classical view EXTERNAL SIGNAL Activator UAS General TATA Co-activator Transcription exon General Factors intron Transcription +1 General Co-activator Factors Transcription Nucleosomes Factors Co-activator RNA Pol. II exon Transcription Elongators Transcription Elongators Transcription Transcription Terminators Terminators

19 Recent discoveries EXTERNAL SIGNAL Activator UAS General TATA Co-activator Transcription exon General Factors intron Transcription +1 General Co-activator Factors Transcription Nucleosomes Factors Co-activator RNA Pol. II exon Transcription Elongators Transcription Elongators Transcription Transcription Terminators Terminators

20 Open questions 1/2 How are these transcriptional regulators controlled and coordinated? Mechanisms by which they regulate transcription initiation?

21 These regulators are typically large macro-molecular complexes Activator UAS 0,5-5 MDa components Multiple activities General TATA Co-activator Transcription exon General Factors intron Transcription +1 General Co-activator Factors Transcription Nucleosomes Factors Co-activator RNA Pol. II exon Transcription Elongators Transcription Elongators Transcription Transcription Terminators Terminators

22 Open questions 2/2 How are these activities synchronized, is their coupling important? How are these large complexes assembled?

23 Which model systems are available to address these questions?

24 Yeast as model systems Unicellular Eukaryotes Version 1.0 of higher Eukaryotes Conservation of many factors / mechanisms Budding yeast Saccharomyces cerevisiae Fission yeast Schizosaccharomyces pombe cerevisiae = beer in Latin pombe = beer in Swahili

25 The tree of all Eukaryotes Yeast Animals

26 Their importance Bread, beer & wine (agriculture) Unicellular - very easy to manipulate Biomedical research: Enzyme (1897) Cell cycle CDKs and cyclins (2001 Nobel) Telomeres (2009 Nobel) Trafficking (2013 Nobel) Autophagy (2016 Nobel) Epigenetics: histone-code, RNAi Role of aneuploidy / genome instability in cancer Diagnosis tool in breast cancer (BRCA1) Gene therapy concept

27 Some numbers Cell size: 4-12 mm Genome size: Mb Gene numbers: Evolutionary distance: Timing: Doubling time: few hours Getting 10L culture: 24 hrs Creating a novel mutant: 2 weeks

28 Experimental advantages Easy to manipulate, store, maintain Unlimited biochemistry, genetic approaches Tools: Genomes very well annotated Classical and system wide genetics feasible Knock-out, fluorescent tag, purification tag collections Best biological characterization of an eukaryotic organism (epigenome, transcriptome, proteome, interactome, metabolome, phenome) Novel techniques first developed in yeast

29 Gene expression regulation in yeast?

30 Normalized Int ensity (log scale) Stress response MergeDC-GR-JM coloured by Venn Diagram Meiosis Mitosis

31 Normalized Int ensity (log scale) MergeDC-GR-JM coloured by Venn Diagram Drugs Sex Rock n Roll

32 Cell fate determination in yeast Mating-type switching Proliferation vs quiescence vs sexual differentiation = conjugation meiosis sporulation Dimorphic switch = yeast-to-hyphae, controls virulence in many pathogens, eg. C. albicans These events are controlled by external cues, from the yeast s environment, typically nutrient quality.

33 Overall goal of my lab To understand how cells sense nutrient availability to coordinately regulate gene expression and control cell fate. High nutrients Proliferation Low nutrients Differentiation, Quiescence External cues Regulatory pathways Gene expression Cell fate decisions

34

35 Regulation of transcription during sexual differentiation in S. pombe: A model to study how signaling pathways directly control an epigenetic regulator

36 S. pombe life cycle High nutrients PROLIFERATION M G1 G2 S

37 S. pombe life cycle High nutrients PROLIFERATION Low nutrients DIFFERENTIATION M G1 G0 G2 S

38 S. pombe life cycle High nutrients PROLIFERATION Low nutrients DIFFERENTIATION M G1 G0 conjugation (diploid zygote) G2 S zygote meiosis sporulation (haploid) germination

39 The power of yeast genetics Conjugation of haploid yeast cells diploid zygote Diploids enter meiosis 4 haploid spores, physically kept within one bag (ascus) Each ascus has the 4 products of ONE meiosis (Mendel laws live!) Mutants are easy to create, propagate, and analyze This the power of yeast genetics!

40 Regulators of the switch proliferation vs. differentiation TORC1 (Raptor) Protein Synthesis HIGH NUTRIENTS LOW NUTRIENTS camp PKA p38 MAPK AMPK TORC2 (Rictor) CELL GROWTH, PROLIFERATION DIFFERENTIATION, QUIESCENCE Ste11 Bähler, Hiraoka, Hoffmann, Jones, Kawamukai, Moreno, Murakami, Nielsen, Okayama, Petersen, Russell, Toda, Uritani, Weisman, Yamamoto, Yanagida labs

41 Regulators of the switch proliferation vs. differentiation TORC1 (Raptor) Protein Synthesis HIGH NUTRIENTS camp PKA CELL GROWTH, PROLIFERATION LOW NUTRIENTS p38 MAPK TORC2 (Rictor)? DIFFERENTIATION, QUIESCENCE Ste11 AMPK Bähler, Hiraoka, Hoffmann, Jones, Kawamukai, Moreno, Murakami, Nielsen, Okayama, Petersen, Russell, Toda, Uritani, Weisman, Yamamoto, Yanagida labs

42 Regulators of the switch proliferation vs. differentiation TORC1 (Raptor) Protein Synthesis HIGH NUTRIENTS LOW NUTRIENTS camp PKA p38 MAPK Tra1/ TRRAP Gcn5 CELL GROWTH, PROLIFERATION DIFFERENTIATION, QUIESCENCE AMPK TORC2 (Rictor)? SAGA Spt7 Spt8 Ste11

43 SAGA: a double agent Opposing roles of the SAGA complex in the transcriptional regulation of the switch proliferation vs. differentiation

44 The SAGA complex 1/2 Transcription co-activator Multi-functional (19 subunits, 2 MDa) Conserved: yeast, plants, insects, mammals Functions: development, oncogenesis Act. UAS GTFs Coactivator TBP TATA +1 Nucleosome RNA Pol. II gene

45 The SAGA complex 2/2 Multiple, distinct regulatory activities: Scaffold: TAFs, Spt7, Ada1 Histone modifications: Gcn5 (HAT), Ubp8 (DUB) TBP recruitment : Spt8, Spt3 Promoter binding: Tra1 Structural organization = functional organization Spt3 DUB Spt8 Spt7 HAT Ada1 TAFs Tra1

46 Studying SAGA in S. pombe Never characterized, 2 nd yeast model S. pombe is highly divergent from S. cerevisiae: Some processes uniquely conserved Similarities: general rule within Eukaryotes Differences: novel insights 3 initial approaches: Purifications Mutants (KO) Transcriptome and phenotypic analyses

47 The S. pombe SAGA complex 1/3 SAGA purification from S. pombe: 1 2 Lane 1: no tag Lane 2: ada1-tap Its subunit composition is identical between S. pombe, S. cerevisiae and mammals.

48 The S. pombe SAGA complex 2/3 Growth phenotypes of viable SAGA deletion mutants WT tra1d spt3d spt8d gcn5d ada2d ada3d sgf29d WT spt7d ada1d spt20d sgf73d ubp8d sgf11d sus1d

49 The S. pombe SAGA complex 3/3 Transcriptome of SAGA KO mutants tra1d spt7d ada1d ada2d ada3d gcn5d sgf11d spt20d spt8d sus1d sgf29d sgf73d ubp8d

50 Transcriptome profile of gcn5d The transcriptome of gcn5d mutants = that of cells undergoing differentiation (nutrient starved). p = 3.6 x 10-37

51 De-repression of Ste11 target genes gcn5d gcn5d ste11d Ste11 targets

52 Expression of differentiation genes in gcn5d mutants

53 Differentiation phenotype h 90 gcn5 + h 90 gcn5d DAPI + DIC % mating 0% 24% Gcn5 HAT activity is required of the repression of differentiation in high nutrient conditions

54 Differentiation phenotype of other SAGA mutants Time upon starvation (hrs) genotype wild-type gcn5d spt8d ste11d Opposing regulatory roles of SAGA subunits in differentiation

55 Expression of differentiation genes in spt8d mutants rich starved Spt8 is required for the induction of expression of differentiation genes upon nutrient starvation

56 Subunit composition depending on nutrient availability Lane 1: no tag Lane 2: ada1-tap Lane 3: ada1-tap Lane 4: no tag Rich Starved SAGA subunit composition is identical between rich and starved conditions.

57 Are these roles direct? Chromatin ImmunoPrecipitation (ChIP): is SAGA bound to promoters of differentiation genes?

58 relative Gcn5 levels Are these roles direct? rich starved ste11 + mei2 + SAGA binds to promoters of differentiation genes, irrespective of nutrient levels

59 relative Gcn5 levels SAGA recruitment rich starved ste11 + mei2 + SAGA is recruited by a TF to promoters of differentiation genes, irrespective of nutrients

60 Thus, so far: With nutrients Without nutrients Spt8 Spt8 Gcn5 SAGA Gcn5 SAGA Rst2 ste11 + Rst2 ste11 + WHAT S GOING ON?

61 Which factors act directly? Epistasis analysis (double mutants) + nutriments: spt8d suppress gcn5d - nutriments: gcn5d suppress spt8d

62 Working model Spt8 SAGA Gcn5 Nutritional starvation Spt8 SAGA Gcn5 Act. ste11 + Act. ste11 + Vegetative growth Sexual differentiation

63 Conclusions so far SAGA directly regulates the expression of differentiation genes and is a crucial factor for cells to decide to proliferate or differentiate. SAGA switches from a repressor to an activator, depending on nutrients. Open questions: 1. Gcn5 repression? Not through histone acetylation! 2. How does SAGA sense nutrient availability to switch from a repressor to an activator of transcription?

64 Conclusions so far SAGA directly regulates the expression of differentiation genes and is a crucial factor for cells to decide to proliferate or differentiate. SAGA switches from a repressor to an activator, depending on nutrients. Open questions: 1. Gcn5 repression? Not through histone acetylation! 2. How does SAGA sense nutrient availability to switch from a repressor to an activator of transcription?

65 Regulators of the switch proliferation vs. differentiation TORC1 (Raptor) Protein Synthesis HIGH NUTRIENTS LOW NUTRIENTS camp PKA p38 MAPK AMPK TORC2 (Rictor)? Tra1/ TRRAP? SAGA Spt7 Gcn5 Spt8 CELL GROWTH, PROLIFERATION DIFFERENTIATION, QUIESCENCE Ste11

66 Direct regulation of the SAGA co-activator by nutrient availability?

67 Regulation of SAGA by nutrients Combine genetic and biochemical approaches: 1. Which nutrient-sensing signaling pathway controls SAGA? 2. Which SAGA subunit is targeted and how does it controls its activities? 3. What is the mechanism of this functional switch?

68 Differentiation gene expression Gcn5 functions downstream of TORC1 to repress differentiation High nutrients AKT Tsc1 Tsc2 Rhb1 TORC1 Differentiation High nutrients Starved WT tsc2d gcn5d gcn5d tsc2d

69 Gcn5 functions downstream of TORC1 to repress differentiation High nutrients AKT Tsc1 Tsc2 Rhb1 TORC1 Differentiation WT gcn5d tsc2d gcn5d tsc2d High nutrients Starvation

70 Functional interactions between TORC1, TORC2, and Gcn5 TORC1 (Raptor) Protein Synthesis HIGH NUTRIENTS STARVED? Tra1/ TRRAP Gcn5 CELL GROWTH, PROLIFERATION DIFFERENTIATION, QUIESCENCE TORC2 (Rictor) AKT SAGA Spt7? Spt8 differentiation genes ste11 +, mei2 +,

71 Functional interactions between TORC1, TORC2, and Gcn5 TORC1 (Raptor) Protein Synthesis HIGH NUTRIENTS STARVED Mechanism?? Tra1/ TRRAP Gcn5 CELL GROWTH, PROLIFERATION DIFFERENTIATION, QUIESCENCE TORC2 (Rictor) AKT SAGA Spt7? Spt8 differentiation genes ste11 +, mei2 +,

72 Increased phosphorylation of SAGA in response to nutrient starvation High Starved High Starved

73 Differential phosphorylation of the SAGA component Taf12, in response to nutrient levels Starved / High:

74 High Differential phosphorylation of the SAGA component Taf12, in response to nutrient levels Starved

75 High Differential phosphorylation of the SAGA component Taf12, in response to nutrient levels Starved

76 Discrepancy between genetics and biochemistry: Taf12 is not phosphorylated when TORC1 is active (high nutrients) Genetic analyses Similar to Gcn5, the PP2A-B55 phosphatase represses differentiation in rich conditions, downstream of TORC1. Nutrients TORC1 PP2A-Pab1 SAGA Differentiation

77 PP2A-B55 de-phosphorylates Taf12 in rich, proliferation conditions WT pab1d R S R S Phosphatase assay

78 TORC2-AKT catalyzes Taf12 phosphorylation upon nutrient starvation / differentiation Rich Starved Kinase assay

79 Taf12 phosphorylation inhibits differentiation upon nutrient starvation

80 Increased TORC2 activity negatively regulates differentiation, through Taf12 phosphorylation Both loss of Tor1 AND higher Tor1 activity reduces differentiation. (Halova D et al., J. Cell Biol., 2013) Starved Genotype Differentiating cells WT 39 ± 1% tor1-t1972a 25 ± 2% taf12-5a 50 ± 4% tor1-t1972a taf12-5a 55 ± 2%

81 Conclusions 1. SAGA functions downstream of both the TORC1 and TORC2 signaling pathways to regulate differentiation. 2. Taf12 phosphorylation is tightly and rapidly controlled by the opposing activities of TORC1-PP2A and TORC2-Gad8 AKT. High nutrients Starved TORC1 TORC2 Gwl Igo1 Gad8 PP2A Pab1 Psk1 P Taf12 Gcn5 SAGA Spt8 Proliferation Differentiation diff.

82 Conclusions 1. SAGA functions downstream of both the TORC1 and TORC2 signaling pathways to regulate differentiation. 2. Taf12 phosphorylation is tightly and rapidly controlled by the opposing activities of TORC1-PP2A and TORC2-AKT. 3. Suggest thats TORC2 both activates and inhibits differentiation, reminiscent of an INCOHERENT feed-forward loop. Starvation TORC2 P-SAGA AND Differentiation

83

84 Regulation of meiotic commitment in S. cerevisiae Role of a long non-coding RNA in regulating transcription

85 S. cerevisiae life cycle With nutrients PROLIFERATION Without nutrients DIFFERENCIATION a haploid MITOSE a haploid CONJUGAISON a/a diploid MEIOSE

86 Regulation of meiotic commitment Induced only in diploids (MAT a/a) Controlled by the IME genes The study of IME4 uncovered a novel mechanism of regulation:

87 Northern blot analysis of IME4 expression 1 = MAT a/a diploid 2 = MAT a haploid 3 = MAT a haploid

88 Sense and anti-sense expression are negatively correlated

89 Regulation of IME4 anti-sense transcript: ChIP & genetics

90 cis-regulation of IME4 sense transcript 1 = MAT a/a diploid 2 = MAT a haploid 3 = MAT a/a diploid IME4-Bam 4 = IME4-1-URA3-IME4-2- Bam/ime4D

91 Functions of the IME4 antisense transcript

92 Conclusion (Even) in a yeast, there are multiple mechanisms to regulate transcription and control cell fate decisions.

93 Further readings Gene expression & differentiation : Regulation of meiotic commitment by a lncrna in S. cerevisiae: Signaling to chromatin and cell fate decisions in response to nutrients in S. pombe:

94 Contact info Dom Helmlinger