C8545 Developmental Biology Lesson 10 Regulation of Gene Expression during Development Jan Hejátko Functional Genomics and Proteomics of Plants CEITEC and National Centre for the Biomolecular Research, Faculty of Science Masaryk University, Brno hejatko@sci.muni.cz, www.ceitec.eu 2 Literature  Fred H. Wilt and Sarah Hake, Principles of Developmental Biology (W.W. Norton & Company, New York, London, 2004)  Capron A, Chatfield S, Provart N, Berleth T 2009. Embryogenesis: Pattern Formation from a Single Cell. The Arabidopsis Book. Rockville, MD: American Society of Plant Biologists, doi: 10.1199/tab.0126, http://www.aspb.org/publications/arabidopsis/.  Selected original papers in scientific journals 3 Outline of Lesson 10 Regulation of Gene Expression during Development  Overview of levels of gene expression regulation  Transcriptional gene regulation  Modification of the chromatin structure and DNA methylation  Transcriptional activation  Post-transcriptional gene regulation  Splicing of hnRNA  Translation initiation  Localization of mRNA  Protein localization  RNA interference  Identification and mechanism of gene expression regulation via RNA interference  siRNA-mediated silencing  miRNA-mediated silencing 4 Outline of Lesson 10 Regulation of Gene Expression during Development  Overview of levels of gene expression regulation 5 6 Outline of Lesson 10 Regulation of Gene Expression during Development  Overview of levels of gene expression regulation  Transcriptional gene regulation  Modification of the chromatin structure and DNA methylation 7 Regulation by histone acetyl transferases or histone deacteylases 8 CpG or CpNpG CpNpNp CpG DNA methylation in animals vs. in plants methylation status methylation status Cell-specific methylation allows maintain of tissue-specific gene expression profiles Mechanism of transcriptional regulation by DNA methylation mostly unknown 9 Outline of Lesson 10 Regulation of Gene Expression during Development  Overview of levels of gene expression regulation  Transcriptional gene regulation  Modification of the chromatin structure and DNA methylation  Transcriptional activation 10 Formation of transcription initiation complex 11 12 Positive TFs Negative TFs Formation of transcription initiation complex 13 Mechanism of transcriptional regulation by TAFs Signal recognition Dimerization DNA binding and transcription activation every 7th aa 14 “Microprocessor-like” acting promoters ProENDO16:REPORTER Deletion mutagenesis Positive, interaction with TAFs Upregulation in the presence of A and B Developmental specificity Combinatorial control 15 “Microprocessor-like” acting promoters Regulation of β-globin type of hemoglobin chains expression Locus control region Development-dependent activation by LCR •Acetylation of H3? •Involvement of other genes? Cca 50 kbp 16 Outline of Lesson 10 Regulation of Gene Expression during Development  Overview of levels of gene expression regulation  Transcriptional gene regulation  Modification of the chromatin structure and DNA methylation  Transcriptional activation  Post-transcriptional gene regulation  Splicing of hnRNA 17 Splicing of hnRNA 18 Sex-specific splicing of DOUBLE SEX (DSX) hnRNA in Drosophila hnRNA Inhibition of male-type differentiation Inhibition of female-type differentiation 19 Outline of Lesson 10 Regulation of Gene Expression during Development  Overview of levels of gene expression regulation  Transcriptional gene regulation  Modification of the chromatin structure and DNA methylation  Transcriptional activation  Post-transcriptional gene regulation  Splicing of hnRNA  Translation initiation 20 Translation initiation after egg fertilization 21 Developmental Biology 8e Online (http://8e.devbio.com/)Puoti et al., EMBO Rep (2001) Inhibition of translation initiation via mRNA masking Cessation of sperm cells production TRA2 22 Outline of Lesson 10 Regulation of Gene Expression during Development  Overview of levels of gene expression regulation  Transcriptional gene regulation  Modification of the chromatin structure and DNA methylation  Transcriptional activation  Post-transcriptional gene regulation  Splicing of hnRNA  Translation initiation  Localization of mRNA 23 Regulation via mRNA localization Binding of STAUFEN Dimer structure of two BICOID mRNAs recognized by STAUFEN Developmental Biology 8e Online (http://8e.devbio.com/) 24 Regulation via mRNA localization 25 26 NANOS+PUMILO HUNCHBACK mRNA deacetylation and degradation 27 28 Regulation via mRNA localization NANOS+PUMILO 29 Outline of Lesson 10 Regulation of Gene Expression during Development  Overview of levels of gene expression regulation  Transcriptional gene regulation  Modification of the chromatin structure and DNA methylation  Transcriptional activation  Post-transcriptional gene regulation  Splicing of hnRNA  Translation initiation  Localization of mRNA  Protein localization 30 Leaving the Golgi apparatus Esterification and cholesterol addition Regulation via protein localization 31 Regulation via protein localization 32 Outline of Lesson 10 Regulation of Gene Expression during Development  Overview of levels of gene expression regulation  Transcriptional gene regulation  Modification of the chromatin structure and DNA methylation  Transcriptional activation  Post-transcriptional gene regulation  Splicing of hnRNA  Translation initiation  Localization of mRNA  Protein localization  RNA interference  Identification and mechanism of gene expression regulation via RNA interference 33 RNAi rnai Mello and Conte, Nature (2004) RNA interference as a natural mechanism of the gene expression 34 Mello, 2004 RNA-dependent RNA polymerase short hairpin RNA micro RNA Mello and Conte, Nature (2004) Mechanism of RNA interference + tasiRNAs 21-24 bp 35 From MacRae, I.J., Zhou, K., Li, F., Repic, A., Brooks, A.N., Cande, W.., Adams, P.D., and Doudna, J.A. (2006) Structural basis for double-stranded RNA processing by Dicer. Science 311: 195 -198. Reprinted with permission from AAAS. Photo credit: Heidi Dicer and Dicer-like proteins 36 Reprinted by permission from Macmillan Publishers Ltd: EMBO J. Bohmert, K., Camus, I., Bellini, C., Bouchez, D., Caboche, M., and Benning, C. (1998) AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO J. 17: 170–180. Copyright 1998; Reprinted from Song, J.-J., Smith, S.K., Hannon, G.J., and Joshua-Tor, L. (2004) Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305: 1434 – 1437. with permission of AAAS. Argonauta argo Argonaut pelagickýago1 Argonaute proteins 37 MIR gene RNA Pol AAAn AGO AAAn RNA Pol mRNA AGO AGO RNA Pol AGO AGO AAAn siRNA miRNA post-transcriptional gene silencingtranscriptional gene silencing transcriptional slicing translational repression binding to DNA binding to specific transcripts 38 The Nobel Prize in Physiology or Medicine 2006 Andrew Z. Fire Craig C. Mello USA USA Stanford University School of Medicine Stanford, CA, USA University of Massachusetts Medical School Worcester, MA, USA b. 1959 b. 1960 39 Outline of Lesson 10 Regulation of Gene Expression during Development  Overview of levels of gene expression regulation  Transcriptional gene regulation  Modification of the chromatin structure and DNA methylation  Transcriptional activation  Post-transcriptional gene regulation  Splicing of hnRNA  Translation initiation  Localization of mRNA  Protein localization  RNA interference  Identification and mechanism of gene expression regulation via RNA interference  siRNA-mediated silencing 40 Transcription DNA Histone proteins Silencing via covalent DNA modifications or histones Transcriptional gene silencing via covalent modifications of DNA Frequently associated with stable heterochromatin (centromeres) 41 O N NH2 N ~ O N N NH2 ~ CH3 cytosine 5-methylcytosine DNA methylation DNA methyltransferase Histone modification • molecular mechanism unknown • involvement of RNA polymerase IV a V Transcriptional gene silencing via DNA methylation 42 Complex Distribution Function RNA Polymerase I All eukaryotes Production of rRNA RNA Polymerase II All eukaryotes Production of mRNA, microRNA RNA Polymerase III All eukaryotes Production of tRNA, 5S rRNA RNA Polymerase IV Land plants Production of siRNA RNA Polymerase V Angiosperms Recruitment of AGO to DNA DNA RNA RNA Polymerase 43 From Herr, A.J., Jensen, M.B., Dalmay, T., and Baulcombe, D.C. (2005) RNA polymerase IV directs silencing of endogenous DNA. Science 308: 118–120. Reprinted with permission from AAAS. Arabidopsis with silenced GFP gene nrpd1a-1 Loss of function of an RNA Pol IV gene interferes with silencing 44 DNA methylation Histone modification DICER- mediated siRNA production Binding of siRNA to AGO Targeting of silencing machinery to the target via non-coding transcripts RNA Pol IV and V are necessary for transcriptional silencing 45 Kasschau, K.D., Fahlgren, N., Chapman, E.J., Sullivan, C.M., Cumbie, J.S., Givan, S.A., and Carrington, J.C. (2007) Genome-wide profiling and analysis of Arabidopsis siRNAs. PLoS Biol 5(3): e57. Abundance of small RNAs Abundance of transposon/ retrotransposons Chromosome Centromere Most siRNAs are produced from transposons and repetitive DNA 46 Pro35S: KAN Pro35S: HYG x Expected Results Selection on kanamycin only: 50% KanR Selection on hygromycin only: 50% HygR Selection on Kan + Hyg: 25% KanR and HygR Transcriptional gene silencing 47 Pro35S : HYG Observed Results Selection on kanamycin only: 50% KanR Selection on hygromycin only: 0% HygR Selection on Kan + Hyg: 0% KanR and HygR Pro35S : KAN Transcriptional gene silencing x 48 CaMV 35S pro : HYG CaMV 35S pro : HYG The promoter of the silenced gene become methylated, interfering thus with transcription. Pro35S : HYG DNA methylation Pro35S : KAN Transcriptional gene silencing 49  The siRNA pathway silences foreign DNA, transposons and repetitive elements.  In plants, siRNAs are produced by the action of Dicer-like proteins dicing dsRNA into 24 nt siRNAs  The siRNAs associate with AGO proteins and form silencing complexes  The silencing complexes can act post-transcriptionally on RNA targets, cleaving them or interfering with translation  The silencing complexes can also act on chromatin, silencing their targets by DNA methylation or histone modification siRNAs - summary 50 Outline of Lesson 10 Regulation of Gene Expression during Development  Overview of levels of gene expression regulation  Transcriptional gene regulation  Modification of the chromatin structure and DNA methylation  Transcriptional activation  Post-transcriptional gene regulation  Splicing of hnRNA  Translation initiation  Localization of mRNA  Protein localization  RNA interference  Identification and mechanism of gene expression regulation via RNA interference  siRNA-mediated silencing  miRNA-mediated silencing 51 AAAn RNA Pol II MIR gene RNA Pol II mRNA AGO AGO AAAn AAAn AGO Translational interference mRNA slicing Mechanisms of miRNAs action miRNAs in plants • small # of highly conserved miRNAs • hing # of non-conserved miRNAs • binding to 5’UTR and require almost complete complementarity • most of the plant miRNA induce slicing of target mRNAs 52 Reprinted from Margis, R., Fusaro, A.F., Smith, N.A., Curtin, S.J., Watson, J.M., Finnegan, E.J., and Waterhouse, P.M. (2006) The evolution and diversification of Dicers in plants FEBS Lett. 580: 2442-2450 with permission from Elsevier. AtDCL1 produces miRNA AtDCL2 - 4 produce siRNA miRNAs and siRNAs are processed by related but different DCL proteins 53 AGO1 AGO4 AGO1 preferentially slices its targets and associates with miRNAs but also some siRNAs AGO4 preferentially associates with siRNA and mediates methylation of source DNA. Reprinted from Vaucheret, H. (2008) Plant ARGONAUTES. Trends Plant Sci. 13: 350-358 with permission from Elsevier. miRNAs and siRNAs associate with several AGO proteins miRNAs in plants • small # of highly conserved miRNAs • hing # of non-conserved miRNAs 54 3' 5' miRNA miRNA* 3' 5' pri-miRNA miRNA MIR gene mRNA target MIR genes are transcribed into long RNAs that are processed to miRNAs Primary miRNA DCL1 processing and miRNA-miRNA* duplex formation Transport irom nucleus into the cytoplasm and miRNA* degrdation 55 Fahlgren, et al., PLoS ONE, 2007 •Non-conserved MIRNA families usually occur as single genes •Conserved ones have often duplicated to larger gene families Factors Some miRNAs are highly conserved and important gene regulators 56 Reprinted from Reinhart, B.J., Weinstein, E.G., Rhoades, M.W., Bartel, B., and Bartel, D.P. (2002) MicroRNAs in plants. Genes Dev. 16: 1616–1626. The MIR156 gene family is highly conserved Arabidopsis miR156 gene family miRNA* 57 miRNA gene family Target gene family Function 156 SPL transcription factors Developmental timing 160 ARF transcription factors Auxin response, development 165 HD-ZIPIII transcription factors Development, polarity 172 AP2 transcription factors Developmental timing, floral organ identity 390 TAS3 (tasiRNA) which acts on ARF transcription factors Auxin response, development 395 Sulfate transporter Sulfate uptake 399 Protein ubiquitination Phosphate uptake Adapted from Willmann, M.R., and Poethig, R.S. (2007) Conservation and evolution of miRNA regulatory programs in plant development. Curr. Opin. Plant Biol. 10: 503–511.. Targets of some conserved miRNAs 58 Reprinted from Willmann, M.R., and Poethig, R.S. (2007) Conservation and evolution of miRNA regulatory programs in plant development. Curr. Opin. Plant Biol. 10: 503–511 with permission from Elsevier. Gene duplication Plant miRNAs are thought to be distantly related to their targets 59 Germination zygote JUVENILE PHASE Vegetative phase change ADULT PHASE REPRODUCTIVE PHASE EMBRYONIC PHASE miRNAs and vegetative phase change 60 Photos courtesy of James Mauseth Adult Juvenile A J Vegetative phase change affects morphology and reproductive competence 61 Eucalyptus globulus Juvenile leaves: rounded, symmetrical, opposite phyllotaxy Adult leaves: elongated, asymmetrical, alternating phyllotaxy Phase change can affect leaf shape, phyllotaxy, and trichome patterns 62 Reprinted from Poethig, R.S. (2009) Small RNAs and developmental timing in plants. Curr. Opin. Genet. Devel. 19: 374-378, with permission from Elsevier. In Arabidopsis, phase change affects leaf shape and trichome patterning 63 Reprinted with permission from Bollman, K.M. Aukerman, M.J., Park, M.-Y., Hunter, C., Berardini, T.Z., and Poethig, R.S. (2003) HASTY, the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis. Development 130: 1493-1504. hasty Wild-type Phase change is specified by miRNAs miRNA export from nucleus 64 Reprinted from Hunter, C., Sun, H., and Poethig, R.S. (2003) The Arabidopsis heterochronic gene ZIPPY is an ARGONAUTE family member. Curr. Biol. 13: 1734–1739, with permission from Elsevier. Wild-type zippy Phase change is specified by miRNAs AGO7 65 Reprinted from Poethig, R.S. (2009) Small RNAs and developmental timing in plants. Curr. Opin. Genet. Devel. 19: 374-378, with permission from Elsevier. miR156 overexpression prolongs juvenile phase in Arabidopsis 66 miR156 binding site miR156 SPL ORF 3’ UTRPromoterSPL3 miR156 targets SQUAMOSA PROMOTER BINDING PROTEINLIKE (SPL) genes, promoters of phase change SBP-box TF 67 Reproduced with permission from Wu, G., and Poethig, R.S. (2006) Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3. Development 133: 3539–3547. ORF 3’ UTRPromoter ORFPromoter ORF 3’ UTRPromoter SPL3 SPL3Δ SPL3m miR156 binding site Wild- type miR156- resistant miR156 SPL miR156 SPL miR156-resistant SPL promotes precocious phase change 68 Reprinted from Poethig, R.S. (2009) Small RNAs and developmental timing in plants. Curr. Opin. Genet. Devel. 19: 374-378, with permission from Elsevier. Wild- type miR156- loss-of- function miR156 OE miR156 SPL SPL miR156 loss-of-function promotes precocious phase change 69 Aukerman, M.J., and Sakai, H. (2003) Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-Like target genes Plant Cell 15: 2730-2741. miR156 SPL miR172 glossy15 In Arabidopsis, SPL9 directly activates transcription of MIR172c Arabidopsis plants overexpressing miR172 flower early. Wild-type miR172-OX Phase change involves a temporal cascade of miRNAs and transcription factors 70 lin-14 mRNA lin-4 miRNA lin-14 gene 3’ untranslated region lin-4 binding sites Lee, R.C., Feinbaum, R.L., and Ambrose, V. (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75: 843–845. Wightman, B., Ha, I., and Ruvkun, G. (1993) Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75: 855–862. miRNAs regulate developmental timing in other organisms Proper larval development 71 Wild-type C. elegans lin-4 Loss-of-function lin-4 is a negative regulator of lin-14. In wild-type worms, lin-14 is expressed early and then shut off. lin-14 expressio n lin-4 loss-of- function causes lin-14 expression to remain high. Lee, R.C., Feinbaum, R.L., and Ambrose, V. (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75: 843–845. Wightman, B., Ha, I., and Ruvkun, G. (1993) Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75: 855–862. Downregulation of lin-14 by lin-4 is necessary for normal development 72 Vegetative phase change affects morphology and reproductive competence miRNAs contribute to the temporal control of gene expression and phase change miR156 promotes juvenile phase by preventing SPL gene accumulation SPL genes promote phase change and flowering In Arabidopsis, a SPL protein promotes transcription of miR172 mir172 triggers phase change by interfering with GLOSSY15 expression In the nematode C. elegans, lin-4 silencing of lin-14 is required for developmental progression miRNAs and phase change - summary 73 Key Concepts Regulation of Gene Expression during Development □ Regulation of gene expression occurs at different levels, from transcriptional till the posttranscriptional and posttranslational □ Basal promoters are co-regulated in a combinatorial way via spectrum of positive and negative factors □ mRNA and protein localizations belong to the most important posttranscriptional regulations of gene expression □ RNA interference is natural and powerful mechanism allowing regulation of gene expression at both transcriptional and posttranscriptional levels □ dsRNA is either trigger or intermediate in the RNAi-mediated regulation □ siRNA and miRNA are two major effector molecules regulating different and complementary spectrum of target genes 74 Discussion