2024 Master Lecture - Molecular Cell Biology PDF

Master - Lecture „Molecular Cell Biology“ 1 Cellular regulation 1 Regulation of gene expression B. Jungnickel Molecular Cell Biology 2 Cellular regulation 2 Regulation of protein function B. Jungnickel 3...

Master - Lecture „Molecular Cell Biology“ 1 Cellular regulation 1 Regulation of gene expression B. Jungnickel Molecular Cell Biology 2 Cellular regulation 2 Regulation of protein function B. Jungnickel 3 The cell nucleus Structure and function P. Hemmerich 4 Cellular homeostasis 1 Cellular Ca2+ homeostasis R. Schönherr 5 Cellular homeostasis 2 Cellular redox homeostasis R. Schönherr 6 Cellular shape 1 Movement of and within cells M. Kessels 7 Cellular shape 2 Cell polarity and attachment B. Jungnickel 8 Immunobiology 1 Cellular Basics B. Jungnickel Molecular Biomedicine 9 Immunobiology 2 Cellular Networks B. Jungnickel 10 Neurobiology 1 Cellular Basics B. Jungnickel 11 Neurobiology 2 Cellular Networks B. Jungnickel 12 Cellular Plasticity 1 Cell Biology of Cancer B. Jungnickel 13 Cellular Plasticity 2 Stem cells and aging B. Jungnickel Intro: Epigenetics and cellular plasticity During the Cambrian Radiation (500 Million years ago, often called „Cambrian explosion“), the complex building plans of most multicellular / vertebrate organisms developed within only 50 million years. However, genome size of these organisms did not change considerably during that time. Therefore, they must have acquired the ability to „make more of their genes“ by allowing changes to gene expression, mRNA splicing and processing etc. by establishment and implementation of epigenetic changes. Epigenetics is also responsible for the enormous variability and plasticity of cells within multicellular organisms and in particular in vertebrates. Content of this lecture Epigenetic states and chromatin Transcription regulatory networks mRNA processing, export and decay Regulation of translation (initiation) Mechanisms of miRNA control  Mechanisms of epigenetic memory Epigenetics Mitotically/meiotically inheritable changes in gene function, not via changes in DNA sequence Trans epigenetic states Self-maintaining transcription factor network (already existing in bacteria) Cis epigenetic states On the gene (DNA / histone modification) (emerged with the appearance of multicellular organisms / particularly vertebrates) Criteria for epigenetic change Applicability for cis states 1) Mechanism for propagation - DNA methylation: 1, 2 and 3 applicable 2) Evidence of transmission - Histone modification: case less clear, 3) Effect on gene expression to be decided for each mark individually? DNA methylation CpG islands close to promotors of genes, mostly hypomethylated. Repetitive elements / intergenic / intronic regions etc. are CpG poor but usually methylated De novo methylation: during embryonic development and cellular differentiation / cell changes DNA methyltransferase 3A/B (DNMT3A/B) Maintenance methylation (DNMT1): during cellular proliferation to maintain methylation marks on both strands  Functional differences of DNA methyltransferases DNA Demethylation: In plants, enzymes may directly remove the methyl group. In vertebrates, mechanisms of demethylation are much more complex.  DNA demethylation in vertebrates Passive DNA Demethylation: Since unmethylated cytosines are inserted during DNA replication, the mark may disappear indirectly with successive cell divisions Active DNA Demethylation: Deamination of the methyl-cytidine with or without previous hydroxylation leads to thymidine or 5-hydroxymethyl-uracil, which are recognized by cellular DNA repair pathways and processed back to cytidine Cyt: Cytidine, 5mC: 5-methyl-cytidine, 5hmC: 5-Hydroxymethyl-cytidine, Thy: Thymidine, 5caC: 5-Carboxycytidine, 5hmU: 5-hydroxymethyl-uracil, AID: Activation-induced cytidine deaminase, AB1,3: Apobec1/3, Tet: ten eleven translocation proteins, TDG: Thymidine-deglycosylase, SMUG: single strand selective Detection of DNA methylation Bisulfite conversion: all cytidines are converted to uracils, but methylated cytidines are protected. PCR, cloning and sequencing reveal modified nucleotides. But: Hydroxymethylcytidine is not detected!  Detection of DNA methylation MeDIP: Methylated DNA immunoprecipitation. Methylated DNA is bound by antibodies and the relative frequency is determined by microarrays or sequencing. Chromatin immunoprecipitation (ChIP) Sample preparation: crosslinking, lysis and chromatin fragmentation, immunoprecipitation (antibodies for DNA-bound proteins or modifications), crosslink reversal „simple“ ChIP (Quantitative) PCR detection of bound sequences relative to input ChIP on chip: Analysis via array containing e.g. promotor sequences ChIP-seq: Analysis by second generation sequencing of bound DNA sequences  Histone modifications Modification Inserted by Removed by Methylation Histone methyltransferases (HMT) Histone demethylases Acetylation Histone acetyltransferases (HAT) Histone deacetylases (HDACs) Phosphorylation (different kinases) (different phosphatases) Examples: Promotors: H3K9me3 Repressed: H3K27me3 (mark of Polycomb-repressors) Heterochromatin: H3K9me3 Enhancer: p300 (protein) Insulator: CTCF (protein): blocking of enhancer activity  Reinforcement, spreading and transmission Establishment / Erasure of histone modifications Enzyme recruitment by transcription factor (e.g. polycomb: repression, trithorax: activation) or recruitment of enzyme by the process of transcription, or via non-coding RNA Reinforcement and spreading Histone modification binders may recruit respective histone modifiers (e.g. H3K9me: Suv39H1 and HP1 for heterochromatin). Transmission Clear for methylation: during replication Not so clear for histone modifications: maybe also involving binder/modifier pairs or alternatively via (unknown) 2nd signal  Interplay of epigenetic marks Epigenetic marks on histones often affect DNA methylation and vice versa e.g. establishment of heterochromatin Histone deacetylases (HDACs) deacetylate nucleosomes. This allows for H3K9me transfer by SUV39H1 (a histone lysine methyltransferase). This provides a binding site for Heterochromatin protein 1 (HP1). DNA-methyltransferases are then recruited and methylate cytidines (filled circles).  Chromatin remodeling ATP – dependent chromatin Different classes of chromatin remodeling factors move, eject or remodelling complexes are known, restructure nucleosomes to allow such as the SWI/SNF family. access for transcription factors.  Transcription factors (TFs) Binding and action: Bind to promotor and enhancer sequences, often in cooperation or competition with other TFs. May be aided by coactivators. One gene often contains several transcription regulatory regions. TFs contain DNA binding domain and may contain transcription activation / repression domain. Families / Redundance: Several TFs of one family may cooperate / compete / replace each other. They may or may not include transactivation domain or regulatory sequences (e.g. NFkB family). Trancription factor binding sites Binding sites: With core sequence that may tolerate certain changes, effectiveness may be affected by adjacent sequences Cis regulatory module (CRM): TF binding sites clustered in CRMs determining final TF effect on gene expression Regulation: Genes regulated by CRM form proteins (TFs) regulating other genes etc. Transcription regulatory networks a) Network motifs: Autoregulation Feedforward loop Single input motif Multiple input motif Red circle: TF blue box: target b) Network modules: Contains nodes / highly interconnected hubs c) Network: e.g. yeast, containing 17873 known relationships Red transcription regulators, black: regulated genes  Transcription initiation and pausing RNA polymerase II requires specific phosphorylations of its highly repetitive C-terminal domain (CTD) to move from initiation to pausing to elongation during transcription. Transcriptional pausing may be used by cells to regulate important genes, such as c-myc.  mRNA processing during transcription The Pol II CTD and its phosphorylation may regulate mRNA processing reactions. It is speculated that epigenetic marks on the chromatin may be converted to marks on the Pol II CTD and hence affect mRNA processing to „make more of our genes“ PIC. Preinitiation complex, names of other factors in this slide are not relevant for the main message J mRNA processing and export mRNAs are subject to capping / splicing / polyadenylation in the nucleus Exosome : Consists of several exoribonucleases, can degrade rRNA, shRNA, snoRNA, mRNA, even during transcription, may act in mRNA surveillance in nucleus / cytoplasm Quality control check at nuclear pore : Formation of export-competent mRNP may fail due to errors in transcript etc., these are retained in nucleus (e.g. to complete splicing), process involves MLP / others Further processes in cytoplasm check mRNA for integrity / modulate stability mRNA surveillance in the cytoplasm Detection and destruction of faulty transcripts, to maintain translational fidelity Nonsense-mediated decay (NMD): Destruction of mRNAs with premature stop codon (not in last exon) – next slide Non-stop decay: Elimination of mRNAs without stop (e.g. due to premature polyadenylation), via two pathways and XRN1/exosome No-go decay: Detects stalled ribosomes and recruits endonuclease, then XRN1 / exosome Nonsense-mediated mRNA decay: upstream marker model Major model for NMD, others are discussed Exon junction complex: is deposited during splicing at junction 1st round of translation: EJC is removed / remodeled by ribosome, translation proceeds Premature stop: Ribosome stalls prior to EJC, this recruits factors triggering degradation  General mRNA decay / turnover Responsible for regulation of up to 50% of all mRNAs (see also: trypanosoma). Most frequent: Deadenylation (CCR4/NOT) /Decapping cause degradation by XRN1/ exosome. mRNA degradation occurs in specialized cytoplasmatic P (processing) bodies.  Levels of regulation of mRNA translation Initiation / Elongation / Termination - Most regulation occurs at level of translation initiation. CAP-dependent translation initiation: - for most mRNAs, involves initiation factors, CAP / PolyA – mRNA, Met-tRNA etc. - scanning of mRNA for first AUG IRES – dependent translation initiation: - IRES: internal ribosome entry site - discovered in viral mRNAs but also active for cellular mRNAs Regulation by miRNAs: May involve regulation at level of mRNA stability translation initiation other steps of translation IRES – mediated translation initiation Mechanism: Structurally different IRES use different mechanisms of recognition, Major subtypes = 1 and 2 IRES: Recruits critical eIFs directly, then 43S complex assembles. Some eIFs are not required. IRES-mediated translation initiation may thus be resistant to some forms of cellular regulation targeting canonical CAP-dependent initiation! IRES – mediated translation initiation: Cells and viruses IRES - mediated translation initiation does NOT require several factors required for CAP-dependent translation Viruses have evolved strategies to shut down host protein synthesis while boosting their own miRNA control: miRNA biogenesis Pri-miRNA Precursor transcribed by PolII Drosha Nuclear processing to pre-miRNA Exportin - 5 Export of pre-miRNA Dicer Processing of cytoplasmic miRNA RISC Binding of miRNA and effector function, contains Argonaute (Ago) proteins, GW182 and other proteins  miRNA features Seed sequence : Mismatch sensitive 5‘ region of miRNA, 3‘ region is less sensitive, matching may determine mechanism of miRNA action  miRNA alternatives siRNAs: Endogenous, exogenous mostly lead to mRNA cleavage miRNAs: Endogenous, repression of translation, mRNA degradation piRNAs: piwi-interacting RNAs (piwi = Ago subfamily), 25-30 nt long, Silence repetitive elements in germ cells siRNA/miRNA mediated mRNA cleavage RISC binding brings Ago (Argonaute) proteins to the mRNA / siRNA complex. These contain PAZ and Piwi domains that position and cleave the RNA. miRNA mediated translation inhibition RISC binding: Mostly at 3‘ UTR Ago/GW182/PABP complex Initiation block: Interference with CAP recognition or ribosome assembly Postinitiation block? No mechanistic insight yet. Elongation block? Ribosome disassembly? Proteolysis of protein?  miRNA mediated mRNA decay miRISC interacts with CCR4 / NOT = deadenylase to facilitate deadenylation of mRNA and subsequent decay. Decapping may follow deadenylation and promote mRNA degradation.  Control questions 1) Define the following terms: nucleosome, euchromatin, heterochromatin, histone code, gene regulatory element, promotor, enhancer, insulator, silencer, capping, polyadenylation, 5‘UTR, 3‘UTR, miRNA, siRNA, translation initiation / elongation / termination! How do 5‘ Cap, 5‘UTR, 3‘UTR, polyA, miRNAs affect stability or translation of an mRNA? 2) What links epigenetics and epigenetic states to the Cambrian Radiation? 3) What are trans- and cis- epigenetic states and the 2 major forms of the latter? 4) Which enzymes mediate DNA methylation in which context and where in the genome? 5) Explain 2 methods for detection of DNA methylation! How may DNA demethylation occur? 6) Explain the common features and differences of ChIP, Chip-on-chip and ChIP-seq! 7) Name three enzymes mediating histone modifications and briefly explain their function! 8) Discuss the quality of inheritance / transmission of the two major cis epigenetic states! 9) What is the role of chromatin remodeling complexes in regulation of gene expression? 10) Where in the genome can transcription factors bind and how do they usually bind? 11) What are feedback/-forward loops, modules, hubs in transcription regulatory networks? 12) Which features/functions of the PolII CTD allow regulation of transcription / processing? 13) How are mRNAs processed / controlled for their quality before nuclear export? 14) Explain differences between the 3 main pathways of cytoplasmic mRNA surveillance! 15) Explain the major mode of NMD! How may it affect mRNA levels of mutated genes? 16) Which factors mediate deadenylation – dependent mRNA decay, how and where? 17) How do viruses exploit the main difference between CAP- and IRES-mediated translation? 18) Explain the biogenesis of miRNAs, the importance of the seed sequence and alternative small regulatory RNAs! How may these affect stability or translation of target mRNAs?