Gene Expression Regulation Lesson 8 Transcription and regulation

“Gene Expression Regulation” Lesson 8

Transcription and regulation in eucaryotes Procaryotes: 1 main RNA polymerase Eucaryotes: 3 main RNA polymerases RNA pol I: transcribes (mainly in the nucleolus) mainly r. RNA genes. RNA pol II: transcribes (mainly in the nucleoplasm) the precursors of m. RNA and small non coding RNA (mi. RNA), lnc. RNAs, e sn. RNA) involved in posttranscriptional regulation. RNA pol III: trascribes (mainly in the nucleoplasm) genes that code for r. RNA 5 S, t. RNA and small RNA involved in the processing and production of mature m. RNA (sn. RNA, U 6 involved in the splicing, l’RNA 7 SL as part of SRP complex, that plays a role mainly in translation control. Two main problems: - DNA – histones complex (chromatin organization): epigenetic control - Requirement of transcription factors for the efficient recruitment of RNA pol

RNA pol procaryotes RNA pol eucaryotes Subunits shared by eucaryotes and procaryotes RNA pol Subunits shared by all eucaryotes RNA pol Specific subunits Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

RNA polymerases • RNA polymerase I synthesizes r. RNA in the nucleolus. • RNA polymerase II synthesizes m. RNA in the nucleoplasm. • RNA polymerase III synthesizes small RNAs in the nucleoplasm. • All eukaryotic RNA polymerases have ~12 subunits and are aggregates of >500 k. D. • Some subunits are common to all three RNA polymerases. • The largest subunit in RNA polymerase II has a CTD (carboxyterminal domain) consisting of multiple repeats of an eptamer.

CTD: C Terminal Domain RNA pol procaryotes RNA pol eucaryotes Subunits shared by eucaryotes and procaryotes RNA pol Subunits shared by all eucaryotes RNA pol Specific subunits Unstructured tail constituted by several repetitions of the following sequence: Tyr-Ser-Pro-Thr-Ser-Pro-Ser (26 in S. Cerevisiae, up to 52 in mammals) It plays a crucial role for the assembly of the transcription initiation complex and for the release of the enzyme from the promoter. Its function is thightly regulated by phosphorylation.

In eucaryotes RNA polymerase cannot bind to the promoter by itself and transcription factors are essential for its recruitment to DNA. PROMOTER: DNA region involved in transcription initiation as well as in its regulation

PROMOTERS bacteria Pol II yeast Pol II multicell Pol III upstream control element upstream activation element insulator distal regulatory elements t. RNA proximal promoter elements Core promoter TFIIB core initiator promoter 5 S eucaryotes Promoter elements downstream TSS eucaryotes Figure 11. 2 Comparison between bacterial promoter and promoters of the three eukaryotic polymerases. (A) Promoter of bacteria where there are the -10 and -35 sequences, recognized by the sigma factor. (B) Promoter of Pol I in eukaryotes. It consists of two regions: a section overlapping the site of the beginning of the transcription (TSS, indicated by the red arrow) constitutes the nucleus of the promoter (core); a more upstream element, called UPE or UCE element, which extends from nucleotide 107 to -180. (C) and (D) Promoters of Pol II in eukaryotes. In the case of unicellular eukaryotes (C) the promoter is typically constituted by a TATA sequence which is located at -26 from the TSS and from a UAS element (Upstream Activating Sequence) which is recognized by the specific activators for that transcript. In multicellular eukaryotes (D) the promoters are more extensive and complex than the yeast correspondents, and are also very heterogeneous. They contain recognition sequences, in the near (proximal) part of the TSS, usually consisting of the TATA box, in position -26, and a series of elements positioned both upstream (up to -200 pb) and downstream of the TSS. The TATA box is present in about 50% of the promoters. Baseline factors are usually linked to these elements near the transcription start site (proximal elements). There also distant elements, the enhancers, where the specific activators for the gene to be transcribed are linked, which can also be found at different kpb distances from the TSS. In addition to enhancer elements, there are elements defined as isolators (insulators) that regulate the action of enhancers, canceling their function. Not all genes transcribed by RNA polymerase II contain all these elements. (E) and (F) Promoters of genes for t. RNA (E) and for RNA 5 S (F) transcribed by Pol III in eukaryotes. The elements of the promoter are in this case located in the transcribed region of the gene, downstream of the TSS. In the case of t. RNA genes the promoters are made of a box A and a box B, while in the case of genes for the 5 S r. RNA there a box A and a box C. These elements are recognized by the starting factors that place the polymerase upstream of the TSS. Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

Focus on RNA Pol II promoters: complex situation Promoter regions are larger Requirement for much more factors compared to RNA pol I and RNA pol III promoters Gene promoter organization may be very different Basal or General Transcription Factors (GTF): required to recruit RNA Pol II and assembly the initiation complex. Basal apparatus: RNA Pol II + Basal factors Minimal Promoter: binds basal factors and allows transcriptional initiation. Factors responsible for the regulation of gene expression: activators and repressors Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

Core promoter: constituted by the minimal DNA region required to bind basal factors and allow transcription initiation B responsive element Upstream proximal and distal promoter elements YYANWYY Downstream Promoter Element TATA-less promoters Core promoter: constituted by the minimal DNA region required to bind basal factors and allow transcription initiation, Lnr contains the start site, TSS usually an A flanked by pyrimidines, TATA box at -26, often preceded by BRE and sometimes followed by one or more DCE. DPE is present in TATA-less promoters. Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

RNA II pol Pre-initiation complex PIC: 1) TFIID (TBP + several transcription activation factors) binds to TATA; 2) TFII A and TF II B binding; 3) POL II and TF IIF are recruited; TFIIE and TFIIH recruitment; 4) TFIIH opens DNA (helicase activity) and allows the open conformation, and is responsible for CTD phosphorylation that allows the elongation step. Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

TBP is a positioning factor shared by all RNA pol • TBP is required to select the right position for the RNA pol on the promoter • It is part of different complexes for the different RNA pol : TBP + TAFs (these factors are different depending on the complex and on the RNA pol) • TBP: TATA Binding Protein, it has been originally identified as able to bind to TATA sequence. This name is not fully correct as TBP does not always bind to TATA box • TBP is also part of the SL 1 complex for RNA pol I dependent transcription and of the TFIIIB complex for RNA pol III dependent transcription: these promoters do not have a TATA box. • TBP recognizes TATA box in the RNA pol II promoters that contain TATA box, although it also recognizes the TATA-less RNA pol II dependent promoters. It is part of the TFIID complex.

TBP in the TF IID complex • TBP interacts with the RNA pol. Moreover on the promoters where it recognizes the TATA box it binds to DNA in a very conserved way (from yeast to mammals). • TBP binding may occur only when the nucleosome has been removed. Therefore the presence of nucleosomes may impair transcription initiation. • TBP complexes with several TAFs II (some are tissue specific, therefore there are several TFIID complexes). Some of the TAFs II are very similar to histone proteins and indeed participate also to histone acetylation and chromatin remodelling.

• TBP binds to the minor groove and bends the DNA Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

TRANSCRIPTION • PRE-INITIATION COMPLEX ASSEMBLY • INITIATION / ELONGATION • TERMINATION

Required for open DNA and allow the Pol to move The Pol may move and complexes with Elongation factors Required to allow the Pol to move and start elongation P Required to to allow RNA processing and modification

• TERMINATION There are no stop sequence RNA pol II elongation Endonuclease cuts downstream poly A signal exonuclease RNA pol II moves further Exonuclease 5’ to 3’ degrades the tail of the transcript Transcription stops when exonuclease meets RNA pol II Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

TBP structure and interaction with TATA box in the RNA Pol II promoter. A) Cristallografic structure of TBP: a monomer with two simmetric domains. B) TBP binds as a saddle to DNA st the TATA, interacting with the minor groove. This binding cause a bending of the DNA of about 80°, TFIIB interacts also with regions that are upstream and downstream TATA and drives Pol II to the initiation site of transcription. Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

Model of the structure of the pre-initiation complex. A) Components. B) Components assembled on the DNA. From: Kornberg (2007), «The molecular basis of eukaryotic transcription» , Proc. Natl. Acad. Sci. USA, 104, pp. 12955 -12961. ] Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

Basal Proteins that participate to RNApol. II dependent transcription Factor N subunits MW (k. Da) Functions Stabilize TBP and TFIIB binding Selects the starting point and recruits pol II From 15 to 250 Interacts with regulation factors Selects the starting point and recruits pol II Subunit of TFIID, specifically binds to TATA box Recruits TFIIH From 38 to 98 Helicase activity, opens promoter, phosphorylates CTD From 10 to 220 Catalyse RNA synthesis using DNA as a template More than 1000

Mediator Complex: mediates functional interactions between proximal and distal elements Mediator (TRAP/ARC/PC 2) is a large (22 -28 subunit) protein complex that binds RNA polymerase II and controls transcription from class II genes. Structure of RNA POL II in complex with the mediator (oloenzyme). The mediator complex helps the polymerase to correctly bind to the DNA, by interacting with proximal components such as TFIID and distal components such as enhancer binding factors. Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

POL II structure and Elongation step of transcription Figure 11. 10 Structure of the active site of RNA polymerase II. (A) On the left, split of RNA polymerase II; on the right, enlargement of the active site. (B) Detail of the placement of the nucleotides in the active site: from the color code it is possible to recognize the nucleotides, the DNA mold, the RNA and the bridge helix that connects the two large subunits of the polymerase. [Source: Kornberg L. (2007), «The molecular basis of eukaryotic transcription» , Proc. Natl. Acad. Sci. USA, 104, pp. 12955 -12961. ] Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

Trigger Loop: riconosce il nucleotide corretto, promuove il legame fosfodiesterico e l’avanzamento della Pol Figure 11. 11 Trigger loop and its role in the transcription. The trigger loop (violet), through the interactions of some lateral residues such as histidine 1085, positions the NTP (orange) exactly in the active site. If the pairing is correct, the nucleotide is able to form stacking interactions with the RNA (red) chain, to approach the right distance to the 3'-OH RNA and to position itself correctly with respect to the DNA strand ( green). In addition, histidine promotes a flow of electrons that allow nucleophilic attack by the 3'OH and detachment of the pyrophosphate, indicated with black arrows. [Source: Kronberg, L. (2007), «The molecular basis of eukaryotic transcription» , Proc. Natl. Acad. Sci. USA, 104, pp. 12955 -12961. ] Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

Elongation step of transcription: phosphorylated CTD on RNA pol II allows the recruitment of several factors that may modulate m. RNA synthesis (processing, splicing, poly. A) Similarly to bacteria, Pol. II pauses, verifies and eventually corrects : TFIIS

REGULATION Mechanisms that modulate RNA pol II transcription Regulation factors may promote or repress basal gene expression Regulation factors may modulate chromatin accessibility

Eucaryote promoters are constituted by “box” regions -140 -120 -100 -80 -60 SV 40 (promotore precoce) Timidina kinasi Istone H 2 B Ottamero CAAT GC -40 -20 1 10

Costitutive activators Modulo Activator DNA bound Consensus Distribution CAAT box CTF/NF 1 22 bp GGCCAATCT ubiquitous GC box SP 1 20 bp GGGCGG ubiquitous Octamer Oct-1 20 bp ATTGCAT ubiquitous Octamer Oct-2 23 bp ATTGCAT lymphoid k. B NFk. B 10 bp GGGACTTTCC ubiquitous ATF 20 bp GTGACGT ubiquitous

Inducible activators Regulation factor Responsive elements Consensus Factor Dimensions (dalton) Heat shock HSE CNNGCCNNTCCNNG HSTF 93. 000 Glucocorticoids GRE TGGTACAAATGTTCT GR 94. 000 Cadmio MRE CGNCCCGGNCNC TPA TRE TGACTCA AP 1 39. 000 Serum SRE CCATATTAGG SRF 52. 000

PROMOTER OF metallothionein Structure of human metallothionein promoter (example of eucaryote promoters). Basal transcription is ensured by elements and factors circled in green. Inducible transcription is highlighted by elements and factors circled in red. Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

DISTAL ELEMENTD THAT MODULTE GENE EXPRESSION • Enhancer: sequences where transcription factors bind, mainly activators. • Enhanceosome: complex of enhancer + its binding factors • Located upstream, downstream or in intronic sequences. • May be bidirectional • Classically a sequence of about 500 bp lacated 7001000 bp from the starting point, that can bind about 10 -12 factors.

Enhancer Up to -10 Kbp Up to +10 Kbp Enhanceosome Mediator Enhanceosome complex Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

DISTANCE promoter Transcription unit DIRECTION POSITION promoter Transcription unit Figura 11. 15 Enhancer can work in different ways. The enhancers can work (A) remotely, (B) in both orientations and (C) in different positions, even within the transcription unit. Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

• Enhancer: have several sequences present also in proximal promoter regions • Transcriptional control comes from cooperation of promoter and enhancer regions • Cooperative binding of transcription factors to enhancer that comunicate via the mediator to the core promoter and its binding factors • Independent binding of enhancer and promoter: combined effect ensured by the mediator • Other regulatory elements: Silencer and Insulator

Silencer: sequences that can bind inhibitors that may mask the activation regions required to bind activators or may compete for the mediator binding. Insulator: sequences positioned so that they can modulate the action of the contiguous enhancers

ON promoter insulator ON promoter OFF insulator promoter ON insulator promoter Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

AN INSULATOR MAY BLOCK AN ENHANCER ACTIVATED A PROMOTER Promoter Transcription ON THE INSULATOR INTERFERES WITH THE ENHANCER ACTIVITY Insulator Promoter Transcription OFF Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p. A. Copyright © 2005

THE INSULATOR MAY BLOCK HETEROCHROMATIN PROPAGATION PROMOTER Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p. A. Copyright © 2005

Study the functionality of a promoter Reporter systems are used to study promoter regions or enhancers and their interaction with trans-regulators. The activity of a promoter and the functional role of the control elements located in thus region can be assayed upon cloning this region upstream a reporter gene, inside an expression plasmid. Recombinant plasmids can be introduced into ad hoc cell lines and the concentration of the reporter protein or its enzymatic activity can be measured and it is proportional to the activity of the promoter.

Search for regulatory sequences Control sequences promoter

Search for regulatory regions by deletions Silencer Promoter Expression Enhancer deletion Silencer deletion Lower Expression Higher Expression

Search for regulatory regions by deletion and fusion to reporter gene Control sequences Promoter Gene of interest Replace the gene of interest with a Reporter Gene Promoter Control sequences

Reporter genes generally code for an enzymaticn activity that has the following features: : • it is usually absent in the host cell or anyway easily distinguishable from endogeneous activity; • it should not interfere or compete with other endogenous enzymatic functions; • the assay should be easy, fast, simple and reproducible. For eucaryote cells the most used reporter genes are: CAT ( cloramphenicol acetyl transferase) β-galattosidase GUS (β-glucuronidase) LUC (Luciferase) GFP (green fluorescent protein)

Reporter systems to study gene expression X Regulatory factors Y Promotor A protein easy to quantify Z Reporter gene Reporter systems allow to study the functionality of cis and trans regulatory elements and their ability to modulate transcription. Moreover it is possible to screen for molecules that may interfere with transcription and reporter gene expression.

Reporter Assay ER ER Transfection ERE (Estrogen Responsive Element) Transient Expression Assay Reporter Gene Activity

REPORTER GENES GENE ADVANTAGES DISADVANTAGES Bacteria BGalattosidasi () Very wellcharacterized, stable easy to detect Endogenous activity in the cell Luciferase (firefly) High specific activity, no endogenous activity (very low background) Requires the addition of cofactor, O 2, ATP. Akcalyn phosphatase (human) This protein is secreted. The assay is very sensitive. High endogenous activity in some cell types. fluorescent proteins (GFP, varianti RFP, BFP) monomeric, no cofactors required, absent from mammalian cells, different variants with different fluorescence l low sensitivity because there is no amplification (it is not an enzymatic reaction)

Fluorescent proteins Green Fluorescent Protein (GFP) is a small protein (238 aa 26, 9 kd) produced by jellyfish Aequorea victoria that can be excited by UV radiation (l 395 nm), but also in the visible(l 475 nm, blue) and emits a green radiation (505 nm) Osamu Shimomura Nobel 2008

GFP fluorophore is a tripeptide cyclic Ser-deidro. Tyr-Gly : p-idrossibenzilideneimidazolidone In the tridimensional structure of GFP some basic residues are engaged in hydrogen bridge bonds with Oxygen atoms (His 148 with Tyr 66 and Gln 94 or Arg 96 with imidalozolone) stabilizing the fluorophore Light is emitted by an autocatalytic process in the absence of other factors. The reaction is termosensitive but once produced GFP is very stable.

The protein has been cristallized and its structure defined. GFP works like a dimer and the fluorophore is protected inside the two cylinders. This particular structure with beta sheets out and alpha helixes inside identifies a class of proteins named beta-can. This structure is very stable and difficult to be degraded

The knowledge of the structure allowed the production of several mutants; the first one is a point mutation (S 65 T) described by Roger Tsien in 1995 that improves the frequency of emission (509 nm) with a more intense and more stable fluorescence and the shift of the exciting radiation at 488 nm. In the following years, Tsien group developed a wide range of fluorescent proteins (YFP, EBFP 2, citrine, Venus, ecc) Roger Tsien, Nobel 2008

Identification of important regulatory elements in a promoter region: the promoter is cloned upstram a reporter gene whose expression can be easily detected. It is possibile to introduce specific mutations, indicated by the X, in sequences that are postulated to be important for gene expression and assay the ability of a panel of mutated constructs to trigger the expression of the reporter gene. Mutations that affect expression of the reporter gene identify important regulatory regions in the promoter. Modified from the following text book: Amaldi, Pesole, Benedetti, Plevani. Biologia Molecolare, Zanichlli Editore

Luciferases are a class of oxidative enzymes that act on a specific substrate causing the production of photons( firefy and its recombinant derivates, Luc 2, . . luciferin + ATP → luciferil adenylate + PPi luciferil adenylate + O 2 → ossiluciferin + AMP + photons

VECTORS WITH LUCIFERASE REPORTES - The choice of the host cell is important - Simple assay Luciferase p. GL 2 -Basic Eucaryote expression vector, Transfection in eucaryote cells Protein extract+ luciferin Light (luminometer)

Luciferase assay

Site directed mutagenesis to investigate The role of specific residues for DNA binding or for protein interactions