Transcription regulation BS222 – Genome Science Lecture 4 Transcription regulation Dr. Vladimir Teif

Module structure Genomes, sequencing projects and genomic databases (VT) (Oct 9, 2018) Sequencing technologies (VT) (Oct 11, 2018) Genome architecture I: protein coding genes (VT) (Oct 16, 2018) Genome architecture II: transcription regulation (VT) (Oct 18, 2018) Genome architecture III: 3D chromatin organisation (VT) (Oct 23, 2018) Epigenetics overview (PVW) (Oct 25, 2018) DNA methylation and other DNA modifications (VT) (Oct 30, 2018) NGS applications I: Experiments and basic analysis (VT) (Nov 1, 2018) NGS applications II: Data integration (VT) (Nov 8, 2018). Comparative genomics (JP, guest lecture) (Nov 13, 2018) SNPs, CNVs, population genomics (LS, guest lecture) (Nov 15, 2018) Histone modifications (PVW) (Nov 20, 2018) Non-coding RNAs (PVW) (Nov 22, 2018) Genome Stability (PVW) ) (Nov 27, 2018) Transcriptomics (PVW) (Nov 29, 2018) Year's best paper (PVW) (Dec 6, 2018) Revision lecture (all lecturers; spring term)

What is oversimplified in this video? Watch this video: https://www.youtube.com/watch?v=D3fOXt4MrOM What is oversimplified in this video?

Gene regulation involves many steps Signal transduction from the cell membrane to the cytoplasm to the nucleus (next year, BS327) Chromatin organization defines accessibility of regulatory regions (next lecture) Transcription regulation: Transcription initiation (this lecture) Transcription elongation Transcription termination Processing of RNA (BS221, next week) Translation (mRNA, microRNAs) Post-translational regulation (later, this module)

Example of cell signalling

Signal molecules enter the nucleus, then the signal has to reach the DNA https://micro.magnet.fsu.edu/cells/nucleus/images/chromatinstructurefigure1.jpg

TF-centric view of gene regulation Transcription factor (TF) concentrations Protein assembly at regulatory regions Transcription start site Proteins produced (including TFs) Teif et al. (2013), Methods. 62, 26-38

TF-centric view of gene regulation Transcription factor (TF) concentrations Enhancer RNA polymerase: enzyme which makes RNA Promoter Proteins produced (including TFs) Teif et al. (2013), Methods. 62, 26-38

Prokaryotic vs. eukaryotic transcription http://www.discoveryandinnovation.com/BIOL202/notes/lecture18.html

TF-DNA binding in gene regulation http://biomserv.univ-lyon1.fr/baobab/images/reg.jpg

Protein-DNA binding is very complex Repair protein cradles broken DNA (Goldberg et al. 2001) Lac repressor-DNA complex (Lu et al. 1987) CAP-DNA complex (Steitz et al. 1984)

Stud-and-tube coupling …Let’s make it simpler Stud-and-tube coupling ?

…And even simpler! h n j m(g) But this simple system of short DNA composed of 15 base pairs (bp) (blue) and proteins which cover 3 bp upon binding to DNA (yellow), can still be in 415 = 1,073,741,824 states! h protein DNA n m(g) j Strength of binding of this protein to this site Binding probability Sum of probabilities of all possible configurations of bound proteins to this DNA sequence

Transcription factor binding sites Any TF can bind to any location on the DNA (if it is free), but different TFs bind different DNA sequence motifs with different affinities (different energies). Why can they bind everywhere? What does it mean “free”? Why all locations have different affinities? Most TFs can be characterised by a single motif that they “like”. It is called the consensus motif. Genomic locations which contain sequences similar to the consensus motifs form TF binding sites

Learning TF-binding motifs in vitro Geertz and Merkl (2010) Brief. Func. Genom., 9, 362-373

Combinatorial TF binding Sequence logo DNA Proteins TF binding site Gene DNA Regulatory region Adapted from http://cs273a.stanford.edu [Bejerano Fall09/10]

TF-DNA binding concepts Sequence-specificity Different protein types Competitive binding Nucleosome positioning

TF-DNA binding concepts Cooperative binding Multilayer assembly Protein-induced DNA loops

Example: Bacteriophage  switch Infection Phage  Lysis Lysogeny UV

Ptaschne M. (2011) Nature Chemical Biology 7, 484–487

Bacterial promoters Source: http://www.mun.ca/biology/scarr/MGA2_03-09.html

CI Cro (RNAP) Teif V.B. (2010) Biophys J., 98, 1247-56

For this relatively simple bacterial system one can measure all binding energies in a test tube (in vitro) Teif V.B. (2010) Biophys J., 98, 1247-56

For each set of TF concentrations we can calculate TF binding maps …calculate binding maps For each set of TF concentrations we can calculate TF binding maps Teif V.B. (2010) Biophys J., 98, 1247-56

For any concentrations of proteins CI and Cro we can predict the activity of genes PR and PRM Wild-type phage  Phage  mutant Teif V.B. (2010) Biophys J., 98, 1247-56

Thus, gene activity is a function of TF concentrations. Which function? Figure from Mayo et al., 2006 Teif V.B. (2010) Biophys J., 98, 1247-56

Can we “digitise” gene regulation functions for all genes?

Gene networks Blais and Dynlacht (2005) Genes Dev., 19, 1499-1511

http://www.discoveryandinnovation.com/BIOL202/notes/lecture18.html

Eukaryotic transcription factors General transcription factors are involved in the formation of a transcription pre-initiation complex. Examples: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Specific transcription factors bind at promoters, enhancers and other regulatory regions. They can be distinguished by structure. Pioneering transcription factors are able to bind to packed chromatin and clear the way for other weaker transcription factors.

Eukaryotic promoters do not have a fixed structure (unlike prokaryotes). They may have different modules:

Example: herpes virus promoter http://darwin.bio.uci.edu/~faculty/wagner/hsv6f.html

Eukaryotic promoter classification TATA-containing (~24%) - usually with many additional elements. TATA-less – many of them are “housekeeping” genes which are always expressed INR-containing (~46%) CpG island containing (~72%) – these are regulated by DNA methylation Bidirectional (~10%) – transcription of overlapping genes in both direction

Gene enhancers Enhancer is a DNA region that can be bound by transcription factors to increase the likelihood that transcription of a particular gene will occur. Chromatin conformation loops and long-range gene regulation. (a) Linear representation of a gene (green box) regulatory landscape showing the position of the gene promoter (brown oval), the immediately proximal regulatory elements (light blue box), and the distal regulatory enhancer (dark blue box), which may be located hundreds or thousands of kilobases from the gene promoter. (b–d) Models of chromatin conformation that may underlie enhancer-promoter communication. In panel b, factors (transcription factors, coactivators, and chromatin remodeling complexes) bound to the enhancer and promoter reorganize the intervening chromatin into a loop that juxtaposes the distal enhancer and gene promoter. In panel c, enhancer-promoter interactions occur by the formation of chromatin miniloops; this model may be particularly applicable to complex regulatory landscapes composed of multiple dispersed enhancer elements. In panel d, the regulatory elements are in a chromatin conformation that keeps them relatively close to the gene promoter but does not necessitate direct enhancer-promoter contact. Factors can diffuse locally from the enhancer to the promoter.

Enhanceosome: higher-order protein complex assembled at the enhancer ß-interferon enhancer

Enhanceosome: higher-order protein complex assembled at the enhancer http://pdb101.rcsb.org/motm/122 ß-interferon enhancer

Promoters & enhancers have common features Kim and Shiekhattar (2015), Cell, 162, 948-959

How enhancers help promoters regulating gene expression? Li et al. (2016) Nature Reviews Genetics, 17, 207-223

How enhancers help promoters regulating gene expression? Li et al. (2016) Nature Reviews Genetics, 17, 207-223

How enhancers help promoters regulating gene expression? Li et al. (2016) Nature Reviews Genetics, 17, 207-223

How enhancers help promoters regulating gene expression? Li et al. (2016) Nature Reviews Genetics, 17, 207-223

Insulators can disrupt promoter/enhancer interactions Eeckhoute et al., J Cell Science (2009) 122, 4027-4034

Levels of cis-regulation: 1) Protein-binding DNA sequence motifs: TATA box, etc TF binding sites (TFBS) 2) Cis-regulatory modules composed of several motifs: Promoters Enhancers Insulators 3) Integration over several cis-regulatory modules: Gene regulatory networks (GRN) Chromatin domains (will discuss in next lectures)

Take home message Cooperative, combinatorial TF binding TF binding determines when and where to start transcription by RNA polymerase MUST KNOW: PROMOTER, ENHANCER, INSULATOR CIS-REGULATORY REGION, TRANSCRIPTION FACTOR (TF), Cooperative, combinatorial TF binding RNA POLYMERASE (RNAP, Pol II) PROMOTER-ENHANCER INTERACTION Transcription initiation in prokaryotes vs. eukaryotes TF binding site (TFBS), consensus motif