Pollards Notes 2021 Lecture 8: Transcription Factors and Chromatin PDF

Pollards Notes 2021 Lecture 8: Transcription Factors and Chromatin Carl Wu I. Transcription Regulation (Eukaryotic): Physical interactions between promoters (proximal to genes) and enhancers (distal from genes): Cis regulatory interactions: 1. Encouraged by cohesion ring looping (TADS) 2. L...

Pollards Notes 2021 Lecture 8: Transcription Factors and Chromatin Carl Wu I. Transcription Regulation (Eukaryotic): Physical interactions between promoters (proximal to genes) and enhancers (distal from genes): Cis regulatory interactions: 1. Encouraged by cohesion ring looping (TADS) 2. Linked by enhancer: sequence specific TF + coregulators : mediator : polymerase + general TFs : TATA box (via TBP) 3. Culminate in polymerase [initiation] complex w. \~56 polypeptides: general TFs (including all the TFIID TAFs) and mediator (which the Polymerase CTD may interact with) nearly all discovered via biochemical reconstitutions TFs: 1. Sequence specific: regulate specific genes, based off of sequence logos 2. Sequence logos recognized by different binding domains: Zinc-Finger (CTCF has 12 Zn fingers), Leucine zipper, Helix-turn-helix, 3. Master developmental regulators and reprogramming factors (Ex. Fibroblast into muscle vs. neuron) 4. Diversely bind nucleosomes, but usually destabilize the DNA 5. Pioneer: bind with high affinity, increasing nucleosome accessibility for other TFs Enhancers: 1. 1 Kb to 1 Mb away from gene 2. Upstream or downstream of gene 3. Orientation independent 4. Super-enhancers (clustered) regulate pluripotency genes/oncogenes 5. Identifiable by epigenetic signature (H3K27ac, lack of nucleosomes) Nucleosomes: 1. Organizational unit of chromatin: DNA nucleosome beads on a string 30 nm fiber (zigzag vs. ~~solenoid~~, ~~two-start twisted helix~~) 120 nm chromonema 300-700 nm chromatid 1400 nm chromosome) 2. Absent at active promoters and all enhancers (aka nucleosome free or nucleosome-depleted regions, NDRs) 3. Structure: a. 8 histones/nucleosome (octamer): 2 H2A + 2 H2B (in dimers) and 2 H3 + 2 H4 (in tetramers) b. Binds to DNA in a handshake motif, DNA wrap left-handed, forms nucleosome core particle (NCP) c. Histone tails are conserved, basic, unstructured ("intrinsically disordered") and transiently interact w. DNA PTMs can alter the affinity of DNA interaction (Phos and Ac can weaken DNA binding) Influences chromatin state via directed or spontaneous unwrapping/sliding A. Directed: i. Baseline extended tail inter-nucleosome contacts ii. PTM-induced compaction intra-nucleosome contacts iii. PTM-induced weakening CAP binding, enhanced remodeling, transcription B. Spontaneous: i. Unwrapping/sliding (measured with Cy3 DNA/Cy5 Histones) TF invasion d. 145 bp of DNA wrapped 1.7x around octamer. Minor groove "attached" to histones by +Arg side chains of H3, H4 e. H3, H2B tails emerge through minor grooves. H4, H2A tails emerge from face f. "Chomatosome": Linker DNA between nucleosomes and on-dyad DNA can bind H1 histone (very dynamic, globular + have major DNA binding function at C'), leading the structure to be wrapped vs. unwrapped (If unwrapped, chromatin elasticity and bendability is observed), vertebrates have 12 H1 subtypes, therefore often need triple-plus KO to study the function g. Overall, 11 nm x 5 nm h. Acidic/hydrophobic patch (contributed too by diff histones) on each side fits different interacting protein motifs i. Can be modeled with Molecular Dynamics BUT histone tails collapse j. NCP is fragile = incipient instability, facilitates TF invasions 4. Can be "remodeled" mechanism of transcription regulation 5. The histones are "modified" normally on tails mechanism of transcription regulation Logo, company name Description automatically generated Chromatin: 1. Two models: zigzag, two start helix model by Cryo-EM, disordered model by ChromEMT 2. "A structurally disordered, 5-25 nm-diameter granular chain with different particle arrangements, conformations, densities, and 3D motifs" 3. CVCs: % interphase (uncondensed) chromatin volume per 120 nm cube. Increases during mitosis, but max is \