Transcription regulation Cis-element Promoter Cis-element (“in the same DNA molecule”) is a specific nucleotide sequence which can regulate the promoter The binding of the transcription factors to their targets is the key Trans-factor mechanism of regulation Trans-factor (a molecule that can be transferred freely) is usually a specific transcription factor, i.e. a DNA-binding protein that has a high specific affinity to the cis-element Lac operon of E. coli Operator CAP-site Promoter Operator 3 structural genes CAP (catabolite activator protein) high glucose level – low cAMP concentration Lac operon of E. coli: positive regulation cAMP The chemical signal of low glucose level Lac operon of E. coli: positive regulation cAMP binding, change in CAP structure Lac operon of E. coli: positive regulation RNAP Effective transcription initiation Lac operon of E. coli: negative regulation Repressor Low lactose level Catabolite activator protein lac Repressor Lac operon of E. coli: negative regulation Repressor Low lactose level Lac operon of E. coli: positive regulation RNAP High lactose level Allolactose binding to lac-repressor, the structural change is accompanied by the loss of the affinity Attenuation in Trp operon of E. coli Leader sequence Promoter Start codon Operator Leader Structural genes 1 2 3 4 U8 Two Trp codons Stop codon High Trp concentration Low Trp High Trp concentration concentration mRNAs Ribosome stopped Low Trp concentration 2 3 U8 Ribosome 3 4 arrested Termination signal Antitermination Transcription factor RNAP ter RNAP ter Attenuation in Trp operon of E. coli Low Trp High Trp concentration concentration Ribosome stopped 2 3 U8 Ribosome 3 4 arrested Termination signal Riboswitch Examples of riboswitches N cI cro cIII cII Гени P Гени рекомбінації RM реплікації PRE PL PR ~50 kb PR' Q Гени Гени лізису головки Гени хвоста Стадії експресії генів фага λ Передранні тільки гени N і cro гени реплікації та Ранні рекомбінації Пізні лізогенія лізіс cI і int, після інтеграції гени головки, хвоста активний тільки cI та лізісу N cI cro cIII cII Гени P Гени рекомбінації RM реплікації PRE PL PR ~50 kb PR' Q Гени Гени лізису головки Гени хвоста 3 2 1 OR 17 bp cI PRM PR cro repressor C Cro N 17 bp 17 bp N cro Репресія N Cro сІ та N Гени Гени рекомбінації реплікації cIII cII Q CІІІ CІІ Q Гени лізису та оболонки cI інтеграція λ-репресор Репресія сro та N N cro N cro Гени рекомбінації cIII cII Q Гени реплікації cI cI Гени лізису та оболонки Transcription regulation in eukaryotic cells: Transcription factors Regulatory RNAs Chromatin structure Hormone receptors DNA binding Activation Hormone binding domain domain domain Receptor is inactive Receptor is active Steroid hormone DNA binding domain of hormone receptor Hormone receptors Target gene Nucleus Cytoplasm Hormone receptor Steroid hormone Signal transduction Target gene Nucleus Cytoplasm Transcription factor Enzyme Phosphate Receptor Protein hormone Signal transduction Transcription factors Combinatorial principle Activation domain DNA binding domain There are about 700 sequence motifs for cis-elements in human genome, ~3 million elements in total AP1 NFAT Weak NFAT Weak AP1 Cooperative binding binding site binding site A multiprotein complex (enhanceosome) in regulatory regions Cofactors Transcription factors Cis-elements TSS Proteins DNA Architectural proteins: High Mobility Group (HMG) HMG-A: an intrinsically disordered protein, recognizes short AT-rich sites, serves as a platform for multiprotein complexes Other proteins HMG-A DNA HMGA Pro-Arg-Gly-Arg-Pro Enhanceosome AT-hook in HMG-A proteins HMG-box proteins (HMG-B) DNA HMG-B Transcription factors HMG-box binds to DNA for a short time through the minor groove, the binding induces a sharp bend of the double helix HMG-N provides chromatin decompactization H3 N-tail - -- HMG-N The positively charged part of the HMG-N binds to the nucleosome DNA while the negatively charged part interacts with the H3 N-terminal tail thus reducing the chromatin compaction A multiprotein complex (enhanceosome) in regulatory regions Cofactors Transcription factors Cis-elements TSS Basal promoter Enhancer Promoter proximal region Coding region AD Pol II TFIIA TFIIB AD AD + TFIID TFIIF AD TFIIE AD Mediator Co-activators TFIIH General transcription factors Activators Loop Enhancer Promoter proximal region RNA interference (RNAi) mRNA RdRP dsRNA Dicer siRNA, 19-21 bp Micro RNAs (miRNAs) miRNAs in the regulation of gene expression miRNA gene target gene RNAP II pri-miRNA pre-miRNA RISC Dicer RISC miRNA miRNAs are integrated in the regulatory networks Elementary motifs of the network Regulatory network in Drosopila cells Transcription factor gene miRNA gene Target gene Regulatory network in human cells Circular RNAs: miRNAs miRNA sponges miRNA targets 5' 3' 5' 3' CircRNA Splicing Large intergenic non-coding RNAs (lincRNAs) Loop Enhancer Transcription factors Promoter proximal region miRNA and other RNAs In human genome, on average, there are 3.9 distal regulatory regions (enhancers or silencers) per one TSS and 2.5 TSSs per one distal region Direct enhancer-promoter interaction Enhancer is a promoter: indirect interaction Enhancer is a promoter: direct interaction Transcription factories Transcription factories Enhancer RNAP Promoter Transcription factory Enhancer blocking insulators Enhancer Insulator Insulator proteins CTCF Cohesin CTCF insulator contributes to the loop domain formation Chromatin loop domains Transcription driven supercoiling in the loops DNA RNA pol Negative supercoiling Positive supercoiling Negative supercoiling induces different structural transitions DNA supercoiling in gene regulation DNA should be accessible for transcription factors Transcription factors Chromatin structure is very important for transcription regulation miRNA and other RNAs Repressed Inactive Potentially active Active Histone code: chemical marks are recognized by transcription factors and cofactors Histone acetylation is a mark of transcriptionally active state HAT bromodomain Marks of activation: Histone acetylation Methylation of H3Lys4 + + + + + Marks of repression: + Histone deacetylation Methylation of H3Lys9 DNA methylation DNA accessibility depends on nucleosome positioning Strong nucleosome position Statistically positioned nucleosomes Genome-wide nucleosome mapping Nucleosome positioning with respect to TSS –1 +1 5'-NFR 3'-NFR -800 -400 0 400 800 1000 bp Two types of promoters Activator Open (constitutive) –1 +1 Regulatory cis-element TATA box Covered (regulated) Nucleosome positions and density can change in regulated promoters Heat-shock- induced Heat-shock- repressed Nucleosome dynamics upon activation of PHO5 promoter 0.32 0.18 0.60 N-3 N-2 N-1 TATA Cis-elements Remodeling complexes Multiprotein ATP-dependent machines, which can change nucleosome positions and transiently remove the nucleosomes ATPase subunit Families of remodeling complexes Helicase HSA Bromo SWI/SNF SANT SLIDE ISWI Chromo CHD HSA INO80 ISWI: the two complexes SWI/SNF: a nucleosome bound bound to a nucleosome inside the cavity of the complex nucleosome Chromatin remodeling Intermediate nucleosome structure with destabilized histone- Homogeneous spacing DNA interactions of nucleosomes Nucleosome re-positioning Nucleosome ejection Mechanism of remodeling ATPase DNA binding subunit domain ATP ATP Helicase (translocase) Helicase binds to nucleosome DNA near the center, DNA binding domain binds at the exit Helicase translocates, but since the complex is immobilized on the nucleosome the translocation of DNA occurs, i.e. a loop is created The loop migrates thus changing the position of the nucleosome ISWI Dimer is bound The activity of a monomer is higher for a longer linker SWI/SNF ATP Nucleosome ATPase Histone complexes Histone SWI/SNF acceptors ATP Nucleosome repositioning Transient removal of nucleosomes Replacement of histone complexes Enzymes Transcription factors Nucleosome Chemical modifications Remodeling complexes RNA polymerase Main components of the transcription regulation system Nucleosome Transcription positions / remodeling factors Regulatory RNAs Chemical modifications •The vast majority (80.4%) of the human genome participates in at least one biochemical RNA- and/or chromatin-associated event in at least one cell type. ~60% of the genome is involved in transcription. •Much of the genome lies close to a regulatory event: 95% of the genome lies within 8 kilobases (kb) of a DNA–protein interaction, and 99% is within 1.7 kb of at least one of the biochemical events measured by ENCODE. •Classifying the genome into seven chromatin states indicates an initial set of 399,124 regions with enhancer-like features and 70,292 regions with promoter-like features, as well as hundreds of thousands of silent regions. Many genes have more than one TSS Transcripts gene 1 gene 2 gene 4 5' 3' 3' 5' gene 3 Many promoters are bidirectional Antisense cryptic unstable transcript (CUT) Transcription elongation through nucleosome RNA pol Nucleosome transfer in the region behind the polymerase is possible when the ionic strength is increased Transcription elongation through nucleosome no NTPs NTPs in vivo: Nucleosomes are destabilized by the positive supercoiling wave in front of RNA pol Transient removal of the histone complexes is facilitated by histone acceptors (which “mimic” an increase in ionic strength) – FACT, spt 4-6 etc. Usually nucleosomes are present in active genes during transcription Hyperactive transcription may remove nucleosomes completely 87A Heat Schock Locus Insulator