Regulation of gene expression in eukaryotes and cell signaling https://www.khanacademy.org/science/biology/gene-regulation/gene-regulation-in-eukaryotes/a/overview-of-eukaryotic-gene- regulation 5_regulace_2020FaF 1 Gene regulation is the process of controlling which genes in a cell will be expressed. • Different cells of a multicellular organism may express very different sets of genes, even if they contain the same DNA. • A set of genes expressed in a cell identifies the set of proteins and functional RNAs it contains, giving it its unique properties. • In eukaryotes, such as humans, gene expression involves many steps, and gene regulation may occur in any of these steps. However, many genes are regulated primarily at the transcriptional level. Relationship between cell expression and signaling. Regulation of gene expression - in general https://www.khanacademy.org/science/biology/gene-regulation/gene-regulation-in-eukaryotes/a/overview-of-eukaryotic-gene- regulation 5_regulace_2020FaF 2 Products of all genome genes are not necessary at every point in a cell's life conditions and variability of the environment play a significant role the complexity of the gene expression process is mostly energetic cell variability during the cell cycle - in unicellular: reactions to environmental changes (temperature, osmotic pressure, nutrient availability, etc.) - - in multicellular: reactions to changes in the environment + communication between cells of the same organism + developmental processes within the organism Constitutive and regulatable genes • there are different types of genes (constitutive, regulated) • constitutive genes are expressed in most cells • ensure stable (continuous) expression of genes that encode the components of cells necessary to maintain normal - operational - functions ("housekeeping functions") • eg expression of genes for rRNA, tRNA, ribosome proteins, RNA polymerases, proteins involved in proteosynthesis, enzymes catalyzing operational functions • the expression of regulatable (inducible / repressible) genes increases or decreases as needed • refers to (inducible / repressible) genes whose products • they are only needed under certain conditions • the synthesis of these genes is under the control of special regulatory systems • constitutive expression of these genes would mean an unnecessary energy load on the cell, • evolutionary advantages - regulation • common for both prokaryotes and eukaryotes 5_regulace_2020FaF 3 Regulace exprese u eukaryot: ➢komplikovaný proces mnoha faktorů působících v závislosti na čase a místě ➢produkty téhož genu mají v různých tkaních jinou funkci ➢v různých fázích ontogenetického vývoje jsou exprimovány různé geny kódující podobné produkty 5_regulace_2020FaF 4 Naše úžasné tělo obsahuje stovky různých typů buněk, od imunitních buněk přes kožní buňky až po neurony. Téměř všechny vaše buňky obsahují stejnou sadu pokynů pro DNA - tak proč vypadají tak odlišně a dělají tak odlišnou práci? Odpověď: odlišná regulace genů! Regulace u eukaryot složitější než u prokaryot vysoký počet genů, které jsou různě exprimovány v různých tkaních - nepřístupnost DNA heterochromatinu transkripci - exprese genu vyžaduje přítomnost několika aktivátorů - problém: DNA v jádře, transkripční faktory a proteinové regulátory vznikají v cytoplazmě • pozitivní: gen může byt exprimován, pokud dostane určitý pozitivní signál-aktivátor • negativní: exprese genu je tlumena represorem a může byt zahájena jen po jeho odstraněni – závislém na přijeti signálu • pozitivni i negativni regulace je tak zavisla na male molekule – induktoru, který se váze k regulačnímu proteinu • signálů ovlivňujících expresi určitého genu může byt větší počet • společné pro prokaryota i eukaryota 5_regulace_2020FaF 5 - pozitivní a negativní regulace Regulatory levels 5_regulace_2020FaF 6 ➢DNA and chromosome levels ➢ level of transcription ➢level of transcript editing ➢translation initiation level ➢Proteins ➢Transcription factors https://employees.csbsju.edu/hjakubowski/classes/ch331/bind/olbindtransciption.html 5_regulace_2020FaF 7 TEST 1. úroveň DNA a chromozomů 2. úroveň transkripce 3. úroveň úpravy transkriptů 4. úroveň iniciace translace ➢Regulatory levels: http://www.discoveryandinnovation.com/BIOL202/notes/lecture19.html 5_regulace_2020FaF 8 •there are many changes in the chromatin structure at the transcription site •positive mechanisms regulate transcription much more often than negative ones. •transcription and translation occur at spatially and temporally different places and times. 9 Regulation of gene transcription availability - chromatin Chromatine in nucleus: • Condensed (heterochromatin) genes are inactive • Diffuse (euchromatin) genes produce mRNA In cells of differentiated tissues, only those genes that play a role in a given cell are manifested During development, there are changes in the activity of genes, chromatin changes from the condensed form to the diffuse and vice versa. 10 Influencing gene expression at the level of chromosome structure ➢DNA accessibility in chromatin ➢ histone acetylation regulators ➢chromatin remodeling complexes ➢ DNA methylation ➢ gene DNA rearrangement ➢ gene amplification ➢ gene deletion Epigenetic modifications they regulate genome function by altering the local structure of chromatin - primarily by regulating availability and compactness 11 •densely composed DNA in chromatin cannot be transcribed; RNA polymerase does not have access to the promoter • histone acetylation affects chromatin compactness: non-acetylated histones form highly condensed chromatin, acetylated histones - less condensed chromatin • the degree of acetylation is determined by the enzymes: histone acetlytransferases (HAT) and histone deacetylases (HDAC) a change in chromatin status that leads to the activation of transcription •release of the nucleosome from the chromatin • Development of a section of DNA from a nucleosome using ATP cleavage • Covalent modification of histone ends by acetylation (acetylation of the -amino group in the lysine side chain at the N-termini of histones H2A, H2B, H3 and H4). DNA accessibility in chromatin, chromatin remodeling Regulators chnges acetylation of histones • coactivators of transcription are HAT (histone acetyltransferases) eg CBP / p300 proteins • transcription corepressors are HDAC (histone deacetylase) • coactivators and corepressors do not bind to DNA, but interact with transcription factors • - binding of the transcription factor to DNA • - HAT binding to transcription factor • - HAT acetylates surrounding histones and releases them • binding of nucleosomes to DNA • - chromatin remodeling complexes change • nucleosome organization - accessibility • DNA is elevated • - binding of other transcription factors • - RNA polymerase binding to DNA Complexes for chromatin remodeling they complement nucleosome changes resulting from histone acetylation they move nucleosomes across Activation of eukaryotic transcription gene - sequence of events ATP-dependent chromatin remodeling complexes multiprotein complexes that alter the conformation of histones and DNA (in promoter regions) use energy from ATP hydrolysis • the substrate is not a mononucleosome, but rather a chain of nucleosomes - it changes the position of nucleosomes on DNA and forms "nucleosome-free" regions • SWI / SNF, RSC, NURF, CHRAC, ACF, FACT • classification according to ATPase subunit: SWI2 / SNF2 ISWI Mi-2 (+ deacetylase subunit) (CHD complexes) • their cooperation necessary for activators and repressors 14 Gene expression in eukaryotes is affected by DNA methylation - methylation of cytosine residues in DNA 5- methylcytosine - catalyzed by methyltransferase recognition sequences are short: GC in animals and GNC in plants (GC islands) -Methylation of DNA weakens gene expression - constitutive operational genes do not have GC methylated islands - Tissue-specific genes do not have GC islands methylated only if their products are needed in that tissue -methyl groups protrude into a large groove of DNA and thus prevent proper binding of transcription factors Ex .: genes for globin are methylated in non-erythroid cells (hemoglobin synthesis does not take place here), in erythroblasts and reticulocytes (erythrocyte precursors) these genes are not methylated covalent modification of cytosine at position 5´ in the CpG dinucleotide • cytosine methylation takes place by transfer of a methyl group from the donor: S-adenosylmethionine with the participation of DNA methyltransferases • DNMT1 - "maintenance" - 10x higher affinity for semimethylated DNA, much more active than DNMT3a and 3b • DNMT3a and DNMT3b - de novo - methylate unmethylated DNA • Deletion of DNMT in mice is embryonally lethal • The pattern of DNA methylation is relatively stable in adult cells, significant changes are described in connection with aging . gene silencing The DNA methylation landscape of vertebrates is very particular compared to other organisms. In mammals, around 75% of CpG dinucleotides are methylated in somatic cells,[15] and DNA methylation appears as a default state that has to be specifically excluded from defined locations.[12][16] By contrast, the genome of most plants, invertebrates, fungi, or protists show “mosaic” methylation patterns, where only specific genomic elements are targeted, and they are characterized by the alternation of methylated and unmethylated domains.[17][18] Methylace DNA Methylation patterns can account for gene silencing (in which one gene in a pair of identical chromosomes is not expressed) and inactivation of one entire X chromosome in a female (who has 2 X chromosomes). In general, transcription from genes that are methylated is inhibited. https://employees.csbsju.edu/hjakubowski/classes/ch331/bind/ olbindtransciption.html CpG DNA methylation is associated with repression of transcription - with maintaining a more stable state of chromatin methylated DNA regions are recognized by the MeCP2 protein (its methyl-DNA binding domain) and specifically interact (transcriptional repressor domain) with the Sin3 corepressor, which brings HDACs to the methylated regions Metylace DNA a deacetylace histonů Přechodná represe stabilní represe Prof. Šmardová – přednáška epigenetika Other histone modifications: -phosphorylation of histones is functionally linked to acetylation - methylation of histones (lysines and arginines can be methylated; H2B, H3 (lys 4, 9, 27, 36) and H4 (lys 20)) - monoubiquitination, at the C-terminus of histones: lysine 119 histone H2A and lysine 123 histone H2B - associated with a transcriptionally active or, conversely, repressed (H2A K119) chromatin state - Histone polyubiquitination is also an integral part of DNA repair 17 Gene amplification In gene amplification, a region of a chromosome undergoes repeated cycles of DNA replication Newly synthesized DNA is excised to form small, unstable chromosomes (double minutes) These integrate into other chromosomes and the corresponding gene is thus amplified Normally, amplification is caused by errors in DNA replication and cell division - under certain circumstances, they may be encoded in the genome. Ex .: Patients treated with methotrexate (a dihydrofolate reductase inhibitor) developed drug resistance (the drug ceased to be effective). The reason is an increase in the number of dihydrofolate reductase genes due to amplification. Gene rearrangement DNA segments can rearrange and associate with other genes within the genome Ex .: Gene rearrangement in antibody-producing cells (immunoglobulins) 18 II. Regulation at the transcriptional leve Basic regulation of transcription (common to all genes) Regulation by components of the "basal transcription complex" (RNA polymerase binding to the TATA box, TATA binding proteins and other "basal" transcription factors binding to the RNA polymerase or in the promoter region) Genes regulated only in this way: Constitutively expressed genes Specific effects on gene expression: Through regulatory sequences in DNA and specific transcription factors. Promoter in eukaryotes Binding of basal transcription factors