Control of gene expression Transcriptional Post-transcriptional Epigenetics and long range control

Control of gene expression Transcriptional Initiation of transcription Transcription factors Tissue specific transcription factors Binding of hormones, growth factors etc. to response elements Use of alternative promoters in a single gene

Control of gene expression Initiation of transcription (RNA pol I) Confined to the nucleolus Transcribes rRNAs Two transcription factors required to bind to a core promoter and the upstream control element UBF(x2) binds first, recruiting SL1. UBF and SL1 recruit RNA pol I

Control of gene expression Initiation of transcription (RNA pol III) tRNA and 5s RNA promoter elements A, B, and C boxes located within the coding sequence A, B form a bipartite promoter, C is a single independent promoter for 5s RNAs Several transcription factors bind, recruiting RNA pol III

Control of gene expression RNA pol II Trancription of polypeptide and snRNA genes Conserved locations of promoter elements in eukaryotes

Control of gene expression Tissue-specific transcription factors The DNA complement in all cells of an organism is essentially identical. cis-acting factors aid in regulating tissue-specific gene expression. Enhancers and silencers

Control of gene expression Insulin gene promoter organization NRE negative regulatory element CRE cAMP response element

Control of gene expression HS-40 alpha-globin regulatory site Tissue specific regulation (many sites)

Control of gene expression Structural domains in transcription factors Alpha-helices – amino-acid cylinders constructed via hydrogen bonding HTH HLH Leucine zipper - amphipathic alpha-helices Zn Finger

Control of gene expression Steroid receptors and response elements Gene expression can be altered by external factors Response elements – DNA elements that are not constitutive but are bound in response to an activator transcription factor Steroid receptors (GR, ER,PR, RAR,TR,VDR) bind to specific response elements

Control of gene expression Transcription regulation by glucocorticoids Glucocorticoid receptor is normally inactive due to binding by Hsp90 When glucocorticoids are present, Hsp90 is released and the receptors bind to any of several glucocorticoid response elements, activating gene expression

Control of gene expression Target gene expression via signal transduction Hydrophilic molecules cannot diffuse through the plasma membrane The “water-line” approach Kinases and phosphatases induce conformational changes via phosphorylation/dephosphorylation Protein kinase: hormonal signaling through cAMP-protein kinase A pathway Cytoplasmic transcription factor NF kappa B and translocation to the nucleus

Regulatory subunits Catalytic subunits

Control of gene expression Major Classes of Cell Surface Receptor G protein Inactive – trimer bound to GDP Activation - GDP replaced by GTP Active – monomer + GTP, dimer Serine-Threonine kinase - phosphorylate serine or threonine in the affected polypeptide Tyrosine kinase Tyrosine kinase associated JAK (janus protein kinase) activity in JAK-STAT signaling Ion channel-linked – channels can be opened to allow signal transduction

Control of gene expression

Control of gene expression Secondary Messengers in Cell Signaling Cyclic AMP (cAMP) Cyclic GMP (cGMP) Phospholipids/Ca

Control of gene expression Alternative transcription Genes with multiple promoters Dystrophin has at least 7 promoters

Control of gene expression rRNA synthesis The major human rRNAs are transcribed from a common 13 kb transcription unit. Arrows indicate cleavage sites

Control of gene expression Post-transcriptional control Alternative splicing

Control of gene expression Differential RNA splicing Wt1 Wilm’s tumor (four splice forms) Calcitonin gene (tissue specific products)

Control of gene expression Post-transcriptional control Alternative splicing Alternative polyadenylation Tissue specific RNA editing - rare Translational control mechanisms

Control of gene expression Tissue specific RNA editing Apolipoprotein B gene (rare)

Control of gene expression Translational control Changing the fate of already existing transcripts The IRE binding protein and iron-response elements (IREs)

Control of gene expression Epigenetics Epigenetics – inheritable but not caused by a change in DNA sequence Methylation and cell memory

Control of gene expression Methylation and gene expression Largely confined to CpG dinucleotides CpG islands Methylation patterns change during development Sex-specific regulation

Control of gene expression Methylation CpG islands – associated with human genes Regions of ‘normal’ GC content

Control of gene expression Methylation Changes in methylation throughout development

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Control of gene expression Methylation Sex-specific regulation of the Dnmt1 methyl transferase gene through specific promoters 1so somatic 1sp spermatocytes 1oo oocytes

Control of gene expression Transcriptional repression by histone deacetylation The presence of acetyl groups at the N-terminus of histones promotes an open conformation, transcriptional activity. Mediated by methylation Methylated CpG’s bound by MeCP2 repressor which, in turn, recruites HDAC.

Control of gene expression Gene clusters Coordinated switching within a locus control region (LCR) LCR = a cluster of hypersensitive sites - Globin genes

Control of gene expression Chromatin structure Allelic exclusion (rearrangements, imprinting, X inactivation) Long range control by chromatin structure (position effects Pax6 in aniridia) Cell position-dependent, short range signaling

Control of gene expression Imprinting DNA methylation key component Selective expression of genes Nonequivalence of maternal and paternal genomes

Control of gene expression Monoallelic expression from biallelic genes Allelic exclusion according to parent of origin Genomic imprinting Allelic exclusion independent of parent of origin X-chromosome inactivation and dosage compensation – female mosaics Programmed DNA rearrangement Unknown mechanism

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Control of gene expression Programmed rearrangement Ig and TCR loci in B and T lymphocytes The adaptive immune system is required to recognize and respond to millions of different antigens

Control of gene expression Programmed rearrangement Ig heavy chain locus on 14q32 86 variable (V) sequences 30 diversity (D) sequences 9 joining (J) segments 11 constant (C) sequences

Control of gene expression Programmed rearrangement Light chain synthesis Somatic recombination V to J RNA splicing VJ to C Heavy chain synthesis Two sequential somatic recombination events yield DJ and VDJ RNA splicing VDJ to C Somatic recombination VDJ to different C (switching)

Control of gene expression Inversion or deletion based splicing Ig kappa light chain V-J splicing