09.9.9 1 CHAPTER 5, THE PARADIGM OF DIFFERENTIAL GENE EXPRESSION ZOO3603C 9/10/2009 Satomi Kohno Developmental history of the leopard frog, Rana pipiens  Without deferential gene expression, a fertilized egg could not differentiate into the different cells, tissues or organ with the genomic equivalence. An integrated account of gene expression I 1. Receptor activation 2. Transcription factor activation 3. Nuclear localization 4. Chromatin decompaction 5. Coupled initiation and 5’ capping 6. Coupled transcription and mRNA processing 7. Splicing 1 2 3 4 5 6 7 An integrated account of gene expression II 8 Cleavage and 3’ polyadenylation 9 mRNA packaging 10 mRNA export 11 Translation 12 Protein folding and release 13 Protein modification 8 9 10 11 12 13 An integrated account of gene expression I 1. Receptor activation 2. Transcription factor activation 3. Nuclear localization 4. Chromatin decompaction 5. Coupled initiation and 5’ capping 6. Coupled transcription and mRNA processing 7. Splicing 1 2 3 4 5 6 7 1. Receptor activation  Paracrine  Diffusion of inducers from cell to another.  Endocrine  Travel through the blood to exert their effects.  Autocrine  Secrete paracrine inducers also respond to them.  Juxtacrine  Direct contact between the inducing and responding cells. 1 09.9.9 2 An integrated account of gene expression I 1. Receptor activation 2. Transcription factor activation 3. Nuclear localization 4. Chromatin decompaction 5. Coupled initiation and 5’ capping 6. Coupled transcription and mRNA processing 7. Splicing 1 2 3 4 5 6 7 Chromatin decompaction 4 Mechanical and chemical modifications on the chromatin regulate gene expressions. • Dosage compensation • Methylation • Insulator Nucleosome and chromatin structure Chromatin: a complex of DNA and protein, histone Heterochromatin: highly coiled chromatin; inactive for transcription. Euchromatin: normal chromatin; active for transcription. Chromatin decompaction 4 Mechanical and chemical modifications on the chromatin regulate gene expressions. • Dosage compensation • Methylation • Insulator Inactivation of a single X chromosome in mammalian XX cells Barr Bodies XX female XXX female Heterocromatic X chromosome becomes a Barr Body. X chromosome contained a lacZ making β-galactosidase. If this X activated, the cells would become blue. X chromosome inactivation: imprinting Initiated by XIST? X chromosome inactivation in mammals X chromosome contain an orange allele for pigmentation gene, and the other X chromosome bear a black allele for that pigmentation gene. 09.9.9 3 X chromosome inactivation in mammals (Part 1) X chromosome inactivation in mammals Both Active Paternal inactive Paternal inactive Both Active Randomly Active No imprinting Chromatin decompaction 4 Mechanical and chemical modifications on the chromatin regulate gene expressions. • Dosage compensation • Methylation • Insulator DNA methylation Methylation stabilizes nucleosomes and prevents transcription factor from binding. Methylation of globin genes in human embryonic blood cells Methylation pattern changes during development. DNA methylations are on enhancer or promoter region: genomic imprinting Chromatin modification induced by DNA methylation  Histone H3  Inactive = Methylation ↔ Active = Acetylation  Methylation of histone H3 increases the nucleosome’s stability. 1. MeCP2 binds to methylated regions of DNA and histone deacetylase. 2. EZH2 binds to DNA methyltransferase which would methylate DNA and histone. 3. Histone H1 binds to methylated regions of DNA. 09.9.9 4 Chromatin decompaction 4 Mechanical and chemical modifications on the chromatin regulate gene expressions. • Dosage compensation • Methylation • Insulator The BEAF32 Insulators in Drosophilla 1. prevent the enhance form activating an adjacent promoter. 2. prevent the condensed chromatin of neighboring loci from repressing the actively transcribed gene. Yellow: overlap of green & red Green: anti-BEAF32 insulator antibody Red: propidium iodide, an intercalating agent for dsDNA. Insulators on folate/β-globin genes preventing the enhance Keeping the enhance between insulators preventing the inactivation Recruiting acetyltransferases An integrated account of gene expression I 1. Receptor activation 2. Transcription factor activation 3. Nuclear localization 4. Chromatin decompaction 5. Coupled initiation and 5’ capping 6. Coupled transcription and mRNA processing 7. Splicing 1 2 3 4 5 6 7 Basic anatomy of the gene Promoter region: responsible for the binding of RNA polymerase and the initiation of transcription. Initiation of Transcription Basic structures to regulate the transcription.  Promoter: DNA site to initiate transcription; immediately upstream  TATA box: part of promoter; about 30-bp upstream from the first base  Enhancer: controlling the efficiency of transcription; on the same chromosome at great distance  Silencer: one type of enhancer; negative enhancer; can repress the transcription.09.9.9 5 Formation of the active eukaryotic transcription initiation complex (Part 1) 1. The TFIID complex binds to the TATA box through its TBP subunit. 2. TFIID is stabilized by TFIIA. 3. TFIIB and TFIIH join the complex on the TATA box; TFIIE and TFIIF associate with RNA polymerase II. Formation of the active eukaryotic transcription initiation complex (Part 2) 4. RNA polymerase II is positioned by TFIIB, and its carboxy-terminal domain is bound by TFIID. 5. The CTD is phosphorylated by TFIIH and is released by TFIID; RNA polymerase II can now transcribe mRNA. Reporter gene assay  To analyze the regulation of gene expression.  Enhancer  Transcription factor  Reporter  β-galactosidase  Green fluorescent protein  Luciferase ? Upstream enhancer of Pax6 β-gal 5’ 3’ ? Enhance is for optic cup. Transcription factor  DNA-binding domain  recognize DNA of enhancer or promoter region  Trans-activating domain  Interact with RNA polymerase  Protein interaction domain  Modulate activity Summary of the steps involved in the production of β-globin and hemoglobin Nuclear RNA 5’-cap: methylated guanosine on 5’-end of RNA; protection from the exonucleases; need to bind