Biochemie III Chromatin Structure 2024 PDF

Biochemie III Chromatin structure Sofia Nasif [email protected] DCBP Creator: Beata Edyta Mierzwa UNIVERSITÄT BERN beatascienceart.com  Module Date Topic...

Biochemie III Chromatin structure Sofia Nasif [email protected] DCBP Creator: Beata Edyta Mierzwa UNIVERSITÄT BERN beatascienceart.com  Module Date Topic Lecturer 16.09.24 The role of proteins in DNA and RNA metabolism. DNA Structure Evan Karousis 23.09.24 DNA damage, mutations. DNA Repair Evan Karousis Genes and 30.09.24 Chromatin Structure Sofia Nasif chromosomes 07.10.24 DNA Replication Sofia Nasif 14.10.24 DNA Recombination Evan Karousis RNA Polymerase: Structure and Function 21.10.24 Sofia Nasif Prokaryotic and eukaryotic transcription, including control sequences Transcription: Transcriptional Regulation in Prokaryotes from DNA to RNA 28.10.24 Sofia Nasif Transcriptional Regulation in Eukaryotes (Part 1) 04.11.24 Transcriptional regulation in eukaryotes (Part 2) Sofia Nasif 11.11.24 Eukaryotic mRNA splicing, capping, polyadenylation (Part 1) Oliver Mühlemann Post- Eukaryotic mRNA splicing, capping polyadenylation (Part 2) 18.11.24 Oliver Mühlemann transcriptional RNA Editing RNA processing Elements of RNA degradation: mRNA degradation (general, small RNAs) 25.11.24 Oliver Mühlemann rRNA and tRNA processing Translation I: Elements of protein synthesis 02.12.24 Evan Karousis Structure of tRNAs and ribosomes Translation II: Initiation, Elongation and Termination Protein synthesis 09.12.24 Translation Regulation Evan Karousis Translation-dependent mRNA degradation 16.12.24 Gene expression regulation, examples from health and disease Evan Karousis Final exam 13.01.25  30.09.2024 21.10.2024 07.10.2024 28.10.2024 04.11.2024 A cautionary note… Look for common principles and related processes Biorender  Learning outcomes 1. DNA Supercoiling ❖ Define types of DNA supercoiling and explain their biological significance. ❖ Describe how Topoisomerases affect DNA supercoiling. 2. Chromatin structure ❖ Comprehensively describe the structure of chromatin, from its basic unit until higher-order structures. ❖ Assess the roles of histone variants and modifications in chromatin structure and gene expression. Chromosomes are much longer than the cellular or viral packages that contain them Electron micrograph of a lysed E.coli cell Genome “Compartment” Ratio length length (Virus head) Bacteriophage T2 50 µm ~500 100 nm (Cell) E. coli 1.7 mm ~850 2 µm Human (haploid) (Cell nucleus) 1m ~10^5 cells 5-10 µm Electron micrograph of a lysed bacteriophage T2 particle  How is this extreme DNA compaction achieved? DNA molecules are extremely compacted inside cells, still the information in the DNA has to be accessible during replication and transcription High degree of structural organization Keys to DNA compaction: neutralizing the negative charges of the phosphoryl groups in the DNA backbone with cations and polyamines a DNA structural alteration known as supercoiling  DNA supercoiling “Supercoiling” means the coiling of a coil When there is no net coiling of the DNA axis upon itself Supercoiled DNA is generally a manifestation of structural strain  Positive and Negative Supercoiling This torsional stress will induce the DNA molecule to coil on itself, creating supercoils Biorender (ahead of the replication and transcription machinery)  Linking number (Lk) The degree of supercoiling can be described by the linking number: the number of total times one strand winds around the other. Lk = twist + writhe Twist: total number of helical turns in the DNA (for a relaxed molecule = # bp/~10.5) Writhe: number of times the double helix crosses itself. The crossovers (nodes) are given positive or negative values depending on the 2100 bp Lk = Tw + Wr orientation of the DNA axis. Lk = 200 + 0 = 200 Underwinding (right-handed superhelix): negative Overwinding (left-handed superhelix): positive As long as the ends of DNA are fixed in space, Lk is an invariant value that cannot be changed without opening the system (i.e., breaking the DNA chain). Lk = Tw + Wr Lk = 200 + (-2) = 198 Lk = Tw + Wr DNAs that differ in their Lk are called topoisomers. Lk = 200 + 2 = 202  Most cellular DNA is underwound Plasmid DNA „relaxed“ (left) and with increasing degree of Supercoiling“ (to the right) Because supercoiled molecules are more compact than relaxed ones, they migrate more rapidly during electrophoresis.  Most cellular DNA is underwound ❑ Chromosomes in all cells are maintained in a state of torsional stress. DNA is underwound relative to the stable B-form structure (~6%), facilitating both the packaging of DNA and access to the genetic information contained within it. DNA is underwound with the aid of enzymes. DNA replication and transcription To maintain the underwound state the DNA must be: also affect supercoiling ▪ a closed circle ▪bound and stabilized by proteins  The underwinding and relaxation of DNA are catalyzed by DNA Topoisomerases Topoisomerases = enzymes that increase or decrease the extent of DNA underwinding by transiently breaking the DNA molecule, changing the spatial relationship between the strands, and re-ligating it. They change the Lk. – They are essential for bacteria and eukaryotes, because they play an important role in replication and DNA packaging Type I topoisomerases = act by transiently breaking one of the two DNA strands, passing the unbroken strand through the break, and rejoining the broken ends. Type II topoisomerases = break both DNA strands. Bacterial topoisomerase I Eukaryotic type IIα topoisomerase Covalent enzyme-DNA linkage Conserves the bond energy (no ATP required) Doesn’t allow the cleaved DNA to dissociate from the enzyme (genome integrity) Which type of topoisomerase is required to separate the newly formed chromosomes after bacterial DNA replication? Diversity in DNA Topoisomerases Relaxed DNA (the one and only) DNA Gyrase (Bact) underwinding overwinding How is eukaryotic DNA underwound  Eukaryotic DNA is underwound: Chromatin Assembly a) Relaxed, closed circular DNA. b) Binding of a histone core to form a nucleosome induces a negative supercoil. Without strand breakage, a positive supercoil forms nearby. c) Relaxation of this positive supercoil by topoisomerases causes the negative supercoil to become fixed (through the binding to the nucleosome’s histone core). Topoisomerases as cellular toxins  The structure of eukaryotic chromosomes Chromosomes are the nucleic acid molecules that are repositories of the genetic information Changes in chromosome structure during the eukaryotic cell cycle: Non-dividing cells (in G0 phase) and cells in interphase (G1, S, G2): amorphous chromatin Mitosis: Prophase: DNA condensation Metaphase: condensed chromosomes align in pairs Anaphase: separation of sister chromatids A DNA molecule of ~105 µm is packed into a cell nucleus with a diameter of 5-10 µm A 10’000-fold compaction of the DNA molecule HOW?  The structure of eukaryotic chromosomes Chromosomes are made of chromatin, which are filaments made of DNA and proteins (50:50, by mass), along with associated RNA molecules. Histones (70-80%) Non-histone proteins: ▪ Structural maintenance of chromosomes (SMC) ▪ Topoisomerases ▪ Transcription factors (TFs) Progressive levels of organization (in the form of coils upon coils upon coils) generate the higher- order structures that could provide the observed degree of DNA compaction in eukaryotic chromosomes.  Histones package and order DNA into nucleosomes Histones = proteins that are tightly associated with chromatin and function to package and order the DNA FIGURE 24-23 Nucleosomes Nucleosomes = the fundamental structural unit of chromatin – composed of core histone proteins bound to DNA Roger Kornberg (*1947) The eukaryotic DNA is organized into regular units: the evidence Treatment of chromatin with a nonspecific nuclease*, followed by: FIGURE 10-3b Nucleosomes as beads on a string Protein content DNA size analysis analysis by by agarose gel SDS-PAGE electrophoresis Unfolding of chromosomes: At low ion concentrations (< 5 mM) *Nonspecific nucleases, like chromatin unfolds into a "beads-on-a- micrococcal nuclease (MNase), string" structure in which the cut DNA where it is not nucleosomes are connected by DNA associated with proteins FIGURE 10-2 Histone FIGURE 10-1 Evidence of DNA representation packaging into repeating units In nucleosomes. obtained from an experiment using a nuclease.  Nucleosomes are the fundamental organizational units of chromatin Chromosomes are arranged as “beads-on-a- string” The beads are complexes of histones and DNA – nucleosome contains eight histone molecules: two copies each of H2A, H2B, H3, and H4 (core histones) – contains ~200 bp of DNA, of which 146 bp are bound tightly around the histone core in a left-handed solenoidal supercoil The nucleosome is the bead plus the connecting DNA that leads to the next bead (linker DNA) Histone H1 binds to the linker DNA (linker histone)  Histones are small, basic proteins Highly conserved (almost identical amino acid sequence in all eukaryotes) Structure of the histones ➤ unstructured N- and C-termini ➤ „Histone fold“ (approx. 70 amino acids in the middle): long α-helix, flanked on both sides by a loop and a short α-helix  Structure of the core nucleosome The interactions between histones and DNA are mainly between the conserved histone fold and the sugar-phosphate backbone and the minor groove of the DNA. ➤ 75% of the DNA surface remains accessible for interactions with other DNA-binding proteins. Voet, Fig 24-42: X-ray structure of the nucleosome core particle. Entire core particle viewed along its superhelical axis (left) and rotated 90° about the vertical axis (middle). In both views, the pseudo-twofold symmetry axis is vertical and passes through the DNA center at the top. (Right) The top half of the nucleosome core particle as viewed in the left. ❑ Histone binding is not random. ❑ Binding of the histone core seems to The interaction of DNA with histones is depend on a local abundance of A=T probably partly sequence-specific. base pairs. ❑ Staggering AA, AT, or TT dinucleotides at 10 bp intervals facilitates DNA binding around the histone core.  Stepwise assembly of the histone octamer (in vitro) Without DNA: no histone octamer assembly With DNA: nucleosome assembly (In vivo) histone chaperones are required: N-terminal tails of the bind histones and regulate histones protrude from nucleosome assembly after DNA the nucleosome replication and other chromatin Figure 4-26 Molecular Biology of remodeling events the Cell (© Garland Science 2008) Histone tails The N-terminal tails of one nucleosome protrude from the particle and interact with adjacent nucleosomes, helping to define higher-order DNA packaging (30 nm fiber). Are the target of chemical modifications (methylation, acetylation, ADP- ribosylation, phosphorylation, glycosylation, SUMOylation, ubiquitination...) that affect the net electrical charge, shape, and other properties of histones. Affect the structural and functional properties of chromatin. Histone tails are at the heart of the dynamic regulation of chromatin structure Canonical histones, variant histones, and their post- translational modifications Stabilize open chromatin Kinetochore attachment DNA repair Stabilize open chromatin Histone-fold domain X chromosome inactivation Histone variants confer special properties on the chromatin structure Histone variants are linked to specific cellular processes  Chromatin structure Transcription Structurally and functionally different types of chromatin: Heterochromatin condensed, transcriptionally inactive/repressed. (e.g. telomeres and centromeres). Euchromatin Decondensed, transcriptionally active Typical for active gene regions EM Image of Interphase Nucleus ➤ Different chromatin types are characterized by the presence of histone variants and of post-translational modifications of histones. ➤ The regulation of histone modifications and the incorporation of certain histone variants into nucleosomes has an important influence on the control of gene activity (see lecture transcriptional regulation).  Higher-order chromosome structure Fold compaction 6 to 7-fold 40-fold 10’000-fold  Histone H1 binds the nucleosome H1 = “Linker histone“, binds ~20 bp of the linker DNA + H1 (1 unit per histone octamer) Binding alters DNA entry and exit angles, facilitating packaging into higher-level chromatin structure - H1 ➤ 6-7-fold additional condensation ➤ H1 helps nucleosomes condense into a higher level of packaging, the 30 nm filament How does H1 impact gene expression?  The 30 nm fiber ✓ Nt tails of core histones are absolutely required, histone H1 helps Models of the organization of nucleosomes in the 30 nm fiber (exact arrangement still unclear) ▪ Solenoid model: consecutive nucleosomes are located next to each other in the fiber, folding into a simple one-start helix ▪ Zigzag model: nucleosomes are arranged in a zigzag manner, such that a nucleosome in the fiber is bound to the second neighbor, but not the first https://www.mbi.nus.edu.sg/mbinfo/what-are-nucleosomes/ Maeshima et al. Current Opinion Cell Biology, 2010. Interphase chromosomes are also organized Interphase nuclei of human breast epithelial cells. Bottom: two copies of chromosome 11 are green. Condensed chromosomes at the mitotic anaphase in cells of the bluebell (Endymion sp.) Misteli 2020 Higher-order chromatin structure Misteli, 2020 Higher-order chromatin structure: loops Chromatin loops: ➤ Chromatin fiber interacts with itself to form loops ➤ Length of loops: kbs-Mbs Function: ➤ Interaction of regulatory elements (enhancer - promoter); 10-100s kbs ➤ 3D condensation of the genome; regulation of gene clusters in precise temporal and spatial sequence (upstream elements - target genes); up to Mbs  Loops of DNA are attached to a chromosomal scaffold Extraction of histones leaves a proteinaceous chromosomal scaffold surrounded by naked DNA Scaffold Major components of The DNA is organized in the chromosomal loops attached at their scaffold: SMC proteins base to the scaffold (Structural Maintenance of Chromosomes)  The SMC proteins SMC = Structural maintenance of chromosomes → Found in all organisms, from bacteria to eukaryotes. In eukaryotes: Next to histones and topoisomerases, the third major class of chromatin proteins. ➤ integral roles in DNA condensation and chromosome segregation during mitosis, as well as in DNA repair. (DNA repair) Eukaryotes have two types of SMCs: Cohesins: link sister chromatids immediately after replication and hold them together when chromosomes condense. Condensins: important for condensing chromosomes as they enter mitosis. The roles of cohesins and condensins during the cell cycle  The SMC proteins "loop extrusion mechanism": formation of chromatin loops by cohesin complex (ATP-dependent molecular motor activity) ➤ SMC complex composition determines function Potential functions of loop extrusion preventing promoter- facilitating promoter- enhancer interations enhancer interations SMC complexes actively fold DNA using their ATPase activity Davidson & Peters, Nat Rev Mol Cell Biol 22, 445-464 (2021)  Higher-order chromatin structure: TADs Chromatin domains: ➤ TADs: Topologically associating domains: genome regions that preferentially interact with each other (rather than with surrounding sequences). ➤ several hundred kbs in length ➤ arise by "loop extrusion mechanism"; loop then folds with itself to form a domain whose boundaries are defined by the architectural chromatin protein CTCF CTCF (CCCTC binding factor): DNA-binding protein Function: ➤ Restriction of regulatory elements to genes within a domain (regulatory units) ➤ Structural framework for the regulation of genes ➤ facilitate chromatin-chromatin interactions FIGURE 24-28  Higher-order chromatin structure: chromatin compartments Chromatin compartments : ➤ higher ordered structure of TADs FIGURE 24-28 Chromosomal organization in the eukaryotic nucleus. Higher-order chromatin structure: chromatin compartments Chromatin compartments : ➤ higher ordered structure of TADs ➤ spatial separation of heterochromatin and euchromatin Structurally and functionally different types of chromatin: Heterochromatin condensed, transcriptionally inactive/repressed. (e.g. telomeres and centromeres). Euchromatin Decondensed, transcriptionally active Typical for active gene regions EM Image of Interphase Nucleus  Higher-order chromatin structure: chromosome territories Chromosomes: ➤ main unit of genome organization ➤ exist in the nucleus in the form of chromosome territories Chromosome territories: area within a nucleus occupied by a chromosome in interphase In interphase: compact, spatially limited territory in the nucleus (not scattered in the nucleus); approx. 2-3 µm in diameter. ➤ The interchromatin compartments are enriched in transcriptional machinery and have abundant actively transcribed genes. Genome function drives structure  DNA molecules are much longer than their cellular packages Example E. coli: 4,641,652 bp double-stranded circular DNA ➤ 1.7 mm length ➤ 850 x length of an E. coli cell (2 µm) Plasmid FIGURE 24-4 DNA lysed from an E. coli cell. Large circular DNA chromosome (nucleoid) Small, circular DNA (free in cytosol) ➤ Plasmids  Organization of bacterial DNA Bacteria have no nucleosomes, but bacterial DNA is also compacted by genome organization Nucleoid = DNA-containing area in the prokaryotic cell. Bacteria also have a scaffold-like structure (SMC proteins) that arranges the circular chromosome into about 500 loops of about 10 kbp. The domains are not static and shift along the DNA during replication. FIGURE 24-35 Looped domains in the E. coli chromosome. Looped DNA domains in the bacterial chromosome are topologically constrained → if the DNA is cleaved in one domain, only the DNA in that domain becomes relaxed Literature ➤ Nelson & Cox. Lehninger «Principles of Biochemistry», 8th edition. W.H. Freeman, chapter 24 ➤ Cox et al. «Molecular Biology. Principle and Practice», 2nd edition: chapter 9+10 Reviews: ➤ Misteli T (2020) The Self-Organizing Genome: Principles of Genome Architecture and Function. Cell, 183:28-45