Chapter 9-II - Transcriptional Control of Gene Expression

Chapter 9-II - Transcriptional Control of Gene Expression

Transcriptional Control of Gene Expression 9.3 RNA Polymerase II Promoters and General Transcription Factors Three types of promoter sequences in eukary otic DNA: TATA box prevalent in highly transcribed genes Initiator promoters in some genes CpG islands, the promoters for about 70 percent of protein-coding genes in vertebrates, are chara cteristic of genes transcribed at a low rate. Sequential binding of general transcription fa ctors initiates transcription of protein-coding genes by Pol II.

Core promoter elements of non-cpg island promoters in metazoans RNA polymerase II promoter sequences: Several different types of gene DNA sequences TATA boxes, initiators, and CpG islands (not shown) Specify where polymerase initiates transcription of an RNA complementary to the template strand of the gene DNA. TATA box single base change decreases gene transcription Initiator sequences degenerate sequence CpG islands ( p represents the phosphate between the C and G nucleotides): control housekeeping genes expressed at low constant levels harder to bend around nucleosomes form nucleosome-free regions (Larger font most frequently observed bases in that position; A +1 is the base at which transcription starts; Y is a pyrimidine (C or T), N is any of the four bases.)

Analysis of elongating RNA polymerase II molecules in human fibroblasts. Sense transcripts: peak at ~+50 Pol II pauses in the +50 to +200 region before elongating further many transcribe longer than 1 kb Antisense transcripts: peak at ~-250 Pol II pauses at the other end of the nucleosomefree regions in CpG island promoters fewer transcripts longer than 1 kb Nuclei isolated from cultured fibroblasts incubated in a buffer with a non-ionic detergent that allows RNA polymerase II continuation of transcription but prevents initiation of new transcription What do you know with this data? 1. Transcription start site? 2. Elongation? Treated nuclei incubated with ATP, CTP, GTP, and Br-UTP for 5 minutes at 30 C Pol II incorporates ~100 nucleotides

The chromatin immunoprecipitation technique localizes where a protein of interest associates with the genome. (a) Experiment: Step 1: Live cultured cells or tissues incubated in 1 percent formaldehyde covalently cross-links proteins to DNA and proteins to proteins Step 2: Preparation sonicated solubilizes chromatin and shears it into 200 500 bp fragments of DNA Step 3: Anti-RNA polymerase II antibody precipitates RNA polymerase II and its covalently linked DNA Step 4: Reverse cross-linking and isolate DNA Sequence DNA by massively parallel DNA sequencing. (b) Results (in mouse embryonic stem cells): Data are plotted as the number of times a DNA sequence in a 50-bp interval was found per million base pairs sequenced. (left) RNA polymerase II transcribing gene in both directions (right) RNA polymerase II transcribing gene only in the sense direction

Think See the figure (b) and discuss 1. What can you expect the promoter types of left and right panels? Please explain the reasons 2. You can see the peaks in both panels. What can you explain this in sense of transcriptional elongation?

Model for the sequential assembly of an RNA polymerase II preinitiation complex. General transcription factors: Bind to promoters of genes transcribed by RNA polymerase II Position RNA polymerase II at start sites and assist in transcription initiation Mechanism: Preinitiation complex (PIC) general transcription factors and purified RNA polymerase II (Pol II) bind sequentially to TATA box DNA TFIIH helicase ATP hydrolysis provides energy for unwinding DNA at the transcription start site and pushing downstream DNA into the polymerase The PIC TFIID TBP subunit holds DNA in position by binding to the TATA box Strain on DNA structure assists the TFIIB N- terminal region and Pol II to melt the DNA at the transcription start site, forming the transcription bubble Pol II: initiates transcription in the resulting open complex continues transcription away from the promoter The TFIIH kinase domain phosphorylates the Pol II CTD. General transcription factors dissociate from the promoter.

Eukaryotic transcription https://www.youtube.com/watch?v=iczjgzozkb8

Cryoelectron microscopic structure of the yeast RNA polymerase II preinitiation complex. Yeast Pol II preinitiation complex (PIC): Composed of 33 polypeptides (~size of a ribosomal subunit). RNA polymerase II General transcription factors TFIIH: Ssl2 subunit: helicase that binds the DNA and helps open the DNA strands at the transcription start site pushes DNA that is bound upstream to TBP, TFIIB, and TFIIA creates torsional stress that contributes to transcription bubble melting Kinase module phosphorylates serine 5 of the Pol II CTD

Elongation

Elongation regulation NELF: negative elongation factor DSIF (DRB sensitivity-inducing factor) The inhibition of Pol II elongation that results from NELF binding is relieved when DSIF, NELF, and serine 2 of the Pol II CTD repeat (Tyr-Ser-Pro-Thr- Ser-Pro-Ser) are phosphorylated by a protein kinase with two subunits, CDK9-cyclin T, also called P- TEFb, which associates with the Pol II, NELF, DSIF complex

Model of antitermination complex composed of HIV Tat protein and several cellular proteins NELF: negative elongation factor DSIF (DRB sensitivity-inducing factor) HIV Tat: A sequence-specific RNA-binding protein binds to the RNA copy of TAR sequence to form a stemloop structure near the 5ʹ end of the HIV transcript Functions as an antitermination factor permits RNA polymerase II to read through a transcriptional block Activates cyclin T-CDK9 HIV transcript TAR element binds Tat and cellular cyclin T Cyclin T Activates kinase CDK9 Positions CDK9 near its cellular substrates CTD of RNA polymerase II, NELF, and DSIF Pol II CTD phosphorylation of heptad repeat serine 2 required for transcription elongation Cellular proteins DSIF and the NELF complex involved in regulating Pol II elongation

Transcriptional Control of Gene Expression 9.4 Regulatory Sequences in Protein-Coding Genes and the Proteins Through Which They Function Expression of eukaryotic protein-coding genes is regulate d through multiple protein-binding transcription-control r egions located at various distances from the transcription start site. Promoters direct binding of RNA polymerase II to DNA, de termine the site of transcription initiation, and influence t he frequency of transcription initiation. Promoter-proximal elements and enhancers are cell-typespecific. Transcription activators and repressors modular protein s containing a single DNA-binding domain and one or a fe w activation or repression domains

Linker scanning mutations identify transcription control elements Results: LS mutations 1, 4, 6, 7, and 9 little or no effect on expression of the reporter gene regions contain no control elements LS mutations 2, 3, 5, and 8 reporter-gene expression significantly reduced regions contain control elements

General organization of control elements that regulate gene expression in multicellular eukaryotes and yeast. Find the differences upstream activating sequence (UAS) Mammalian genes with a CpG island promoter: Transcription initiates at several sites in both the sense and antisense directions from the ends of the CpG-rich region

How to get the region where the transcription factors bind?

DNase I footprinting reveals the region of a DNA sequence where a transcription factor binds. (a) Experiment: A DNA fragment known to contain a transcription-control element labeled at one end with 32 P (red dot) Labeled DNA digested with DNase I in the presence and in the absence of protein samples containing a sequencespecific DNA-binding protein DNase I hydrolyzes the phosphodiester bonds of DNA between the 3ʹ oxygen on the deoxyribose of one nucleotide and the 5ʹ phosphate of the next nucleotide. a low concentration of DNase I is used so that, on average, each DNA molecule is cleaved just once. (left) No protein binding: DNA fragment is cleaved at multiple positions between the labeled and unlabeled ends of the original fragment. generates multiple fragments (right) Bound protein: protects digestion of DNA bound to proteins blocks generation of some fragments DNA separated from protein, denatured to separate the strands, and electrophoresed Autoradiography: detects only labeled strands reveals fragments extending from the labeled end to the site of cleavage by DNase I missing gel bands constitute footprint of regions where proteins were bound

DNase I footprinting reveals the region of a DNA sequence where a transcription factor binds. (b) Results: Footprints produced by increasing amounts of TBP (triangle) and of TFIID on the strong adenovirus major late promoter

Binding of transcription factors?? How to detect it??? Vs.

Binding of transcription factors?? How to detect it??? + DNA element from gene Some proteins or a protein

The electrophoretic mobility shift assay can be used to detect transcription factors during purification. Protein fractions separated by column chromatography assayed for binding to a radiolabeled DNA-fragment probe containing a known regulatory element

An in vivo transfection assay measures transcription activity to evaluate proteins believed to be transcription factors Experimental system two plasmids: TF plasmid contains gene encoding the putative transcription factor (Protein X) Reporter gene plasmid contains one or more binding sites for protein X and a reporter gene (e.g., luciferase) Both plasmids simultaneously introduced into cells that lack the gene encoding protein X Measure production of reporter-gene RNA transcripts or encoded protein (luciferase) activity Results: compare reporter-gene transcription in the presence and absence of the X-encoding plasmid : Greater with Protein X Protein X is activator Less with Protein X Protein X is repressor

Which protein domain has a critical role for the transcription?

Schematic diagrams illustrating the modular structure of eukaryotic transcription activators. Transcription factors may contain more than one activation domain but rarely contain more than one DNA-binding domain: Gal4 and Gcn4 yeast transcription activators Glucocorticoid receptor (GR) promotes transcription of target genes when glucocorticoid hormones bind to the C-terminal activation domain SP1 binds to CpG promoter elements in a large number of mammalian genes

Interaction of bacteriophage 434 repressor with DNA. DNA binding motifs Proteins binding to specific DNA sequences: Commonly involves noncovalent interactions between atoms in an DNAbinding domain α helix and atoms on the edges of the bases within the major groove in the DNA May involve ionic interactions between positively charged repressor residues arginine and lysine and negatively charged phosphates in the sugarphosphate backbone and with atoms in the DNA minor groove Bacteriophage 434 repressor: dimeric protein helix-turn helix motif interacts intimately with one side of the DNA molecule over a length of 1.5 turns a recognition or sequence-reading α helix from each monomer inserts into the major groove in the DNA helix

Eukaryotic DNA-binding domains that use an α helix to interact with the major groove of specific DNA sequences. (a) C 2 H 2 zinc finger most common DNA-binding motif encoded in the human and other mammalian genomes Zn 2+ -binding motif: 23 26 consensus sequence residues contains two conserved cysteine (C) and two conserved histidine (H) residues, whose side chains bind one Zn 2+ ion GL1 DNA-binding domain: monomeric contains five C 2 H 2 zinc fingers fingers 2 5 interact with DNA (b) C 4 zinc finger proteins: Motif found in 50 human transcription factors, including nuclear hormone receptors Four conserved cysteines bind Zn 2+ Binds DNA as homodimer one α helix in each monomer interacts with the DNA (c) Leucine-zipper proteins: (d) bhlh proteins:

Combinatorial possibilities due to formation of heterodimeric transcription factors (a) Heterodimeric transcription factors in which the activation domain of each monomer recognizes the same DNA sequence (b) Heterodimeric transcription factors in which the activation domain of each monomer recognizes different DNA sequences:

Cooperative binding of two unrelated transcription factors to neighboring sites in a composite control element (a) NFAT-AP1 regulation of IL-2 gene: Monomeric NFAT and heterodimeric AP1 transcription factors each alone has low affinity for their respective binding sites in the IL-2 promoterproximal region NFAT and AP1 interaction two proteins bind cooperatively to the composite site increases overall stability of the NFAT-AP1-DNA complex

Transcriptional Control of Gene Expression 9.5 Molecular Mechanisms of Transcription Repression and Activation Eukaryotic transcription activators/repressors affect ge ne expression by binding to multisubunit co-activators /co-repressors that modulate chromatin structure or i nteract with Pol II and general transcription factors. The highly cooperative assembly of preinitiation compl exes in vivo requires several activators. A cell produces the specific set of activators required for transcription of a particular gene to express that gene.

Mediator complex forms a molecular bridge between activator bound to cognate DNA site and Pol II Mediator (coactivator) is a multiprotein complex that functions as a transcriptional coactivator in all eukaryotes. It was discovered in the lab of Roger D. Kornberg, winner of the 2006 Nobel Prize in Chemistry. Mediator complexes interact with transcription factors and RNA polymerase II. The main (but not exclusive) function of mediator complexes is to transmit signals from the transcription factors to the polymerase.

How to transmit the signal? Signal Receptor Promoter Enhancer Pol II Activator Mediator DNA Nucleus

Model of several DNA-bound activators interacting with a single Mediator complex. Individual Mediator subunits bind to specific activation domains Multiple activators may influence transcription from a single promoter by interacting with a Mediator complex simultaneously or in rapid succession. Different Mediator subunits interacting with specific activation domains may contribute to integration of signals from several activators at a single promoter Points 1. Mediator 2. Two activators 3. Loop of DNA

Cis-acting element: enhancer Signal Receptor Promoter Enhancer Pol II Activator Mediator DNA Nucleus

Consensus sequences of DNA response elements that bind five nuclear receptors. palindromic sequence Cis-acting elements

Example> CREB-mediated transcription

Activator Signal Receptor Promoter Enhancer Pol II Activator Mediator DNA Nucleus

CREB camp-responsive transcription factor CREB (camp response element-binding protein) is a cellular transcription factor. It binds to certain DNA sequences called camp response elements (CRE), thereby increasing or decreasing the transcription of the downstream genes. e.g. SNAT2 gene

CREB CREB recruits other regulatory proteins.

CREB in camp signaling Interacting with outside environment.

Interacting with outside world. Signal Receptor Promoter Enhancer Pol II Activator Mediator DNA Nucleus

Decondensation of Chromatin Histone acetylation and deacetylation Epigenetics

Chromatin and histone How to bind the DNA? Charge?

Structure of Histone Which charge is necessary?

Histone All histones have a highly positively charged N-terminus with many lysine and arginine residues.

Q. How to detach the DNA from histone? Charge?

Acetylation and Deacetylation "histone acetyltransferase" (HAT) or "histone deacetylase" (HDAC) Acetylation removes the positive charge on the histones, thereby decreasing the interaction of the N termini of histones with the negatively charged phosphate groups of DNA.

Acetylation and Deacetylation https://www.youtube.com/watch?v=tze3xr4kcj4

Proposed mechanism of histone deacetylation and hyperacetylation in yeast transcriptional control. (a) Ume6 repressor-directed deacetylation of histone N-terminal tails (b) Gcn4 activator-directed hyperacetylation of histone N-terminal tails

CREB-CBP => Acetylation

Expression of fusion proteins demonstrates chromatin decondensation in response to an activation domain. Cultured hamster cell line engineered to contain multiple copies of a tandem array of E. coli lac operator sequences integrated into a chromosome in a region of heterochromatin (a) lac repressor (LacI) expression vector transfected into these cells cells express lac repressor: lac repressor antibody (red) shows lac repressor binding to lac operator sites in a region of condensed chromatin (DNA stained with DAPI [blue]) diagram condensed chromatin (b) LacI fused to an activation domain and transfected into cells Activation domain causes chromatin decondensation into a thinner chromatin fiber fills a much larger volume of the nucleus Diagram decondensed chromatin with bound LacI-VP16 activation domain (AD) fusions and associated chromatin remodeling and histone acetylase complexes

9-3 Histone Post-Translational Modifications Associated with Active and Repressed Genes

Association of a repressed transgene with heterochromatin. a transgene containing binding sites for an engineered repressor fusion protein DAPI (blue) stains all DNA heterochromatin stains brighter (DNA concentration is higher than in euchromatin) (left) ( ) hormone Recombinant repressor retained in the cytoplasm Transgene transcribed and associated with euchromatin (right) ( + ) hormone Recombinant repressor in the nucleus Transgene expression repressed and associated with heterochromatin

Maintenance of histone H3 lysine 9 methylation during chromosome replication H3 lysine 9 methylation is maintained following chromosome replication.

X inactivation: Controlled by a ~100-kb domain on the X chromosome X-inactivation center X-inactivation center encodes several lncrnas required for the random inactivation of one entire X chromosome early in the development of female mammals.

Discussion with friends 1. An electrophoretic mobility shift assay (EMSA) was performed using a radiolabeled DNA fragment from the sequence upstream of gene X. This DNA probe was incubated with (+) or without (-) nuclear extract isolated from tissues A (bone); B (lung); C (brain); and D (skin). The DNA;protein complexes were then fractionated on nondenaturing polyacrylamide gels. The gels were exposed to autoradiographic film; the results are presented in the figure. a. Which tissues contain a binding activity that recognizes the sequence upstream of gene X? Is the transcription factor the same in each tissue? b. If the binding activity was purified, what test could be done to verify that this factor is in fact a transcription factor? 2. Find the X chromosome inactivation and its mechanisms. 3. What are the differences between histone methylation and DNA methylation?