Eukaryotic transcription (II)
Transcription factors Prokaryote: Sigma factors they are similar to general transcription factor of eukaryotes, they help bring RNA pol to the promoter Transcription factors Such as laci and CAP, which bind to DNA to facilitate or inhibit transcription. Eukaryote: General transcription factors also called TFII factors, theyare required for transcription of all or most genes. TFII factors are required for basal transcription (relatively low level of transcription). Higher level of transcription are activated by transcription activators. Basal transcription can be suppressed by transcription repressors. Gene-specific transcription factors also simply referred to as transcription factors. They are transcription regulators that bind to DNA to activate (activator) or suppress (repressor) transcription of specific genes. Human genome encode about 2600 (~10% genome capacity) transcription factors, plants genome encode over 2200 transcription factors (~10% genome capacity).
1. Types of transcription factors Zinc finger Homeodomain Leucine zipper Helix-loop-helix Transcription factors are usually classified by DNA-binding domains, there are a few dozens different types, some major ones are listed here. Zinc finger: bind zinc ion by specific C or H residues (e.g. Sp1, Gal4, GR) Homeodomain: a special type of helix-turn-helix domain, found in transcription factors that regulate organ development (e.g. Ant, Ey, etc) bzip (basic Leucine Zipper): containing Leu-rich alpha helix DNA-binding domain and basic activation domain, usually act as dimer (e.g. GCN4, Fos, Jun) bhlh (basic helix-loop-helix): in bhlh, the basic region binds DNA, the HLH region acts as the DNA-binding and dimerization domain (e.g. Myc, MyoD). bhlh transcription factors often bind the E box DNA sequence (CANNTG).
2. Structure of transcription factors 1. Transcription factors have two major domains: a DNA-binding domain and an activation or suppression domain. The DNAbinding domain interacts with specific DNA sequences, the activation or suppression domain activates or suppresses transcription. 2. Examples of DNA-binding domains: homeodomain, zincfinger, leucine zipper, and helix-loop-helix. A DNA-binding domain contain the domain-specific amino acid sequence. Transcription factors are usually classified by the DNA-binding domain. 3. Examples of transcription regulatory (such as activation) domains: acidic domain, basic domain, and Q-rich domain. Activation domains allow transcription factor to interact with other proteins, such as TFII factors or various subunits of RNAPII to activate transcription.
A transcription activator contains a DNA binding domain and a transcription activation domain Different types of transcription activators have different DNAbinding domains and/or different transcription activation domains Different DNAbinding domains bind DNA by different mechanisms
(1) Zinc-finger proteins 1. Zinc-finger protein are transcription factors that bind zinc 2+ ions. There are many types of zinc finger proteins with different zinc finger domains. Zinc-finger domains contain regularly spaced cysteines and/or histidines. Zif268 (monomer) 2. One zinc finger protein may have one or more zinc fingers. One zinc finger is usually composed of a beta-strand and an alpha helix 3. Zinc finger proteins may bind DNA as monomers or dimers. GAL4 (dimer by coiledcoil domain)
cortisol Many mammalian steroid hormone receptors are Zinc Finger proteins Glucocorticoid receptor (GR) is one of the families of zinc finger transcription factors called nuclear receptors. GR is normally locked in the cytosol by its association to HSP s (heat shock proteins). Binding to hormone glucocorticoid (e.g. cortisol) disrupts the binding between GR and HSP, allowing GR to transport into the nucleus, bind to DNA, and activate or suppress transcription of ~15% of genes of the human genome.
(2) Homeodomain proteins 1. Homeodomain protein are homeodomain-containing transcription factors, which are particularly important for the developmental regulation, such as formation of body parts. 2. Homeodomain (HD) is a ~60- residue novel helix-turn-helix DNAbinding domain. The DNA sequence encoding the homeodomain is called Homeobox. Most HD has a relatively weak DNA-binding activity, so they need help of other proteins to bind to DNA. 3. Homeodic genes are genes by which a mutation can cause transformation of one body part to another (or missing or addition of body parts).
HD proteins are developmental regulators Fly Ey gene Ey encodes a homeodomain protein, it acts as the master control the expression of about 2500 genes required for eye development. Transgenic fly expressing the Ey gene in organs other than eye develop eyes in other body parts, such as legs. Ey Human Synpolydactyly (SPD) Human genome encodes many homeodomain proteins, including 38 so-called HOX proteins. Mutations in the HOXD13 gene cause SPD. Hoxd13 mutants
(3) Leucine-zipper proteins Leucine zipper proteins contain stretches of leucine-rich sequence that allow hydrophobic interaction between two leucine zippers. They usually bind DNA as dimers via a coiled-coil structure. Leucine zipper DNA-binding domain is often also rich in basic residues, so called bzip (e.g. Jun, Fos). The forceps structure of ZIP allows a very stable DNA-protein interaction. GCN4 Side view top view
e.g. The AP1 transcription activator AP1 is a transcription activator composed of two bzip proteins: Jun and Fos (c-fos and c-jun are proto-oncogenes). AP1 is important for many cellular functions and it is regulated by several different mechanisms: GCN4 (1) The protein kinase JNK phosphorylates Jun. Phosphorylated Jun binds to Fos to form active AP1 dimer that binds to DNA to activate transcription. (2) The co-factor A (CoA) can bind to phosphorylated Jun to stabilize the Jun-Fos dimer. (3) coa may also bind to nuclear receptor NR, which inhibits JNK activity to suppress Jun- Fos dimerization.
(4) bhlh (basic Helix-loop-helix) proteins Structurally related to the bacterial helix-turn-helix proteins, such as LacI. The basic region and one helix serve as the DNA-binding domain, the second helix severs as the dimerization domain. An eukaryotic genome often encodes hundreds of bhlh factors, most of them bind to the E box DNA sequence (CANNTG). C-Myc and Max (Myc-associated factor X) are among the first discovered bhlh factors. They are also called bhlh-zip factor, because they have ZIP domain in addition to bhlh domain. Myc is a oncogene.
How do we know the bhlh heterodimer bind to DNA? DNA-CIBs Free DNA E-box DNA (CANNTG) Control DNA (AAAAAA) Experiment (gel shift) Label E-box DNA (left) or the control DNA (right) with biotin. Mix labeled DNA probes with either one type of CIB proteins (e.g. CIB1, or CIB2, etc) or two types of bhlh proteins (e.g. CIB1+CIB2, or CIB1+CIB4, etc). Run the DNA-protein mixture in a native gel, and visualize the labeled probe (as in lecture 4) in an X-ray film. Results: none of the single CIB factors bind to the E-box DNA, but all CIBs bind to E-box DNA when two different CIBs were mixed (heterodimer).
Transcription factors and cancer Cancer is a group of diseases characterized by the spread of uncontrolled cell growth. Cancer is the #2 killer in US, causing ~1/4 death. In 2013, 1.6 million new cancer cases will be diagnosed, resulting in ~1,600 death/day (~0.5m/yr). US Life Expectancy Cancer incidence Mutations accumulate, so cancer is more likely to occur when we get older.
The bhlh factor myc is an oncogene Cancer results from accumulation of multiple mutations in two types of genes that control cell growth: proto-oncogenes and tumor suppressor genes. Mutations in both proto-oncogene and tumor suppressor genes lead to cancer development. To deal with multiple mutations is one reason why it is difficult to treat cancer. c-myc that encodes a bhlh transcription factor c-myc was among the first proto-oncogens discovered. The c-myc gene may be mutated to overexpress or become hyperactive. Those mutants are called Myc, which are oncogenes encoding myc oncoproteins. Mutations of multiple proto-oncogenes are often found in cancer patients. The mice experiment on the left showed a synergistic effect of two oncogenes, myc and ras D (a small GTPase).
Oncogenes and proto-oncogenes Oncogenes are mutant genes that cause cancer, they are usually the gain-of-function mutant of the normal genes called proto-oncogenes. Proto-oncogenes often encode proteins that stimulate cell division and growth. Transcription factor is an important group of protooncogenes (e.g. c-myc, c-max, c-jun, c-fos). Mutations that cause increased expression or increased activity of a protooncogene converts it to a oncogene (e.g. Myc, Max, Jun,Fos).
Tumor suppressor genes -Tumor suppressor genes are genes that the loss-of-function mutations cause cancer. Normal tumor suppressor gene products usually suppress cell proliferation. Many tumor suppressor genes also encode transcription factors. The best known tumor suppressor gene is p 53 (53kD, with zinc finger DNA-binding domain). Loss-of-function mutations of the P 53 gene contribute to many types of cancers. P 53 can act as either a transcriptional activator or repressor. P 53 affects genes transcribed by all three RNA polymerases
3. Mechanisms of transcription factors 1. Transcription activators may help recruit TFIID to the promoter, or to help recruit RNA polymerase to the promoter. They may also change the enzymatic activity of RNAP. 2. Different domains of a transcription factors may act independently, which means one domain can perform its normal function when physically separated from other domains of the protein. 3. Transcription activators usually bind to enhancer far away from the promoter, but relay its action by DNA looping 4. One gene is usually regulated simultaneously by multiple transcription activators and repressors to achieve optimal level of transcription in a specific cell and specific condition. This type of regulation is called combinatorial regulation. 5. The function of a transcription activator is often dependent on other proteins (e.g. mediators, insulators, co-activators). A transcription activator may interact with a co-activator to activate transcription, or it may interact with a co-repressor to inhibit transcription.
(1) Two modes of transcription activators Transcription activators may recruit GTF or RNAPII subunits to the promoter to activate transcription. activator
Activators help recruit RNAP to promoter Experiment 1: 1. Effector plasmid encodes the LexA-GAL11, which is a fusion protein of the DNA-binding domain of a bacterial transcription repressor LexA and a yeast TFII general transcription factor GAL11 (known to bind to yeast RNA PolII). 2. Reporter plasmid contains the LexA-LacZ reporter gene, which encodes β- gal (lacz) controlled by the promoter and the LexA-binding site (LexA operator). 3. Transform both plasmids to yeast cell to test lacz transcription by the β- gal activity. Result: LexA-GALl11 chimeric protein can activate lacz expression Reasoning: because the LexA-Gal11 chimeric protein bind to RNA Pol (by Gal11) and LexA-operator near the promoter (by LexA), it most likely recruit RNA pol to the promoter. Conclusion: a transcription factor can binds to DNA and help bring RNAP to the promoter to activate transcription.
(2) Different domains of a transcription factor can function independently GAL4 activation domain GAL4 DNAbinding domain (a) Native GAL4 transcription factor binding to the native UAS (upstream activation sequence) to activate the UAS-GAL1-lacZ reporter GAL4 activation domain LexA DNAbinding domain (b) Chimeric LexA-GAL4 transcription factor binding to LexA operator to activate the LexA-GAL1-lacZ reporter
Experiment 2 1. Effector plasmid encodes the chimeric transcription factor LexA-GAL4. An eukaryotic activation domain (e.g. GAL4) activates only eukaryotic RNA pol, but a DNA-binding domain of a prokaryotic transcription factor (e.g. lexa) can work in eukaryotic cells as long as the DNA sequence it recognizes is there. 2. Reporter plasmid contains the LexA-GAL1-lacZ reporter gene, which contains a LexA operator, GAL1 promoter, and lacz coding sequence. 3. Transform both effector and reporter plasmids to yeast cell, measure the -galactosidase activity for the transcriptional activity of the reporter gene. The β-gal enzyme is active (for the color reaction) in both prokaryotic and eukaryotic cells. Result: the DNA-binding domain of LexA-GAL4 can bind to the LexA operator, and the activation domain of the GAL4 activation domain activates transcription of the GAL1 promoter in yeast cells. This method is widely used in the study of transcription factors
(3) Activators bind to enhancers to bring together sequences located far away by DNA looping Possible mechanisms Coil (no evidence) Sliding Looping tracking
(4) Combinatorial regulation of transcription A protein may be needed in different cells at different concentration, and its level of expression may change in response to different conditions. The level of gene expression has to be regulated by multiple transcription activators (and repressors) that bind to the same or different DNA elements. This type of transcription regulation is called combinatorial regulation. The level of transcription is determined by the combined effects of all transcription factors. For example, a human metallothionine gene is regulated by at least 4 enhancer sequences that bind to different activators, including Sp1, Ap1 (Jun-Fos), MR (metal response regulators), and GR (glucocorticoid receptor). GR Ap1 MR Ap1 Sp1
Multiple transcription activators may loop around to form enhenceosome Enhanceosome is the DNA-protein complex of multiple transcription activators associated with enhancer. Different enhancers of a promoter may spread far away on the linear DNA. But a DNA-protein complex may loop together to become spacialy associated. Some transcription factors bend DNA to facilitates the formation of enhanceosome. Enhanceosome helps integrate combinatorial effects of different transcription factors.
(5) An activator can be converted to a repressor Some transcription activators may be converted to transcription repressors, depending on other proteins binding to the enhancer/repressor DNA. For example, a transcription activator binding to the A enhancer of the sea urchin Endo 16 gene converts to a repressor in two of the five developmental stages, when F/E or DC sites (but not F/E+DC) are occupied by co-repressors..
(6) Insulator regulation of activators/repressors The way transcription activators work raises two questions: (1) if enhancer of a gene is located far away, does it affect other genes? (2) What happens if a gene no longer needs to be activated or repressed? Both problems may be solved by another type of transcription regulator: insulator. Insulator is a protein that may suppress the activity of an enhancer by preventing DNA looping, or it may prevent transcription inhibition by stop spreading of chromatin condensation that usually inhibits transcription.
How does an insulator work? An insulator may bind to DNA between an enhancer and promoter to prevent an activator from sliding closer to the promoter An insulator may also loop out an activator-bound enhancer so that the activator/enhancer cannot form a loop with the promoter to activate it.
4. Regulation of transcription factors The level and activity of transcription activators themselves are tightly regulated by different mechanisms in the cell. 1. Change the level of co-activator or co-repressors. 2. Change the level of mrna encoding the transcription factor. 3. Change subcellular localization (e.g. GR) 4. Change protein level of the transcription factor by ubiquitination and degradation 5. Change activity of a protein by chemical modifications (1). Protein phosphorylation (add a phosphate) (2) Ubiquitination (add a short peptide ubiquitin) (3) Sumoylation (add a short peptide SUMO) (4) acetylation or methylation (add an acetyl or methyl group).
(1) Mediator: co-activator Mediator is a multiple-subunit protein complex that is not required for basal transcription, but required for activator-dependent stimulation of transcription. Experiment: test the effect of Mediator on the activity of a chimeric activator Gal4-VP16 (Gal4-DNA-binding-domain + VP16-activation-domain). Result: Gal4-VP16 does not activate transcription without Mediator. Increased amount of Mediator stimulates the activity of Gal4-VP16. Conclusion: mediator is a co-activator required for activator-dependent stimulation of transcription. + Gal4-VP16 - Gal4-VP16
(2) Protein phosphorylation e.g. RNA Pol II must be phosphorylated at CTD to become active in transcription initiation and elongation.
(3) UPS and protein degradation UPS: ubiquitination/26s proteasome system Ubiquitin (Ubq) is a highly conserved peptide of 76 amino acids. Addition of multiple Ubq to Lys residues of a protein tags it for degradation. UPS: Ubiquitin 26S proteasome system
Three enzymes of the UPS pathway Three enzymes are needed to ubiquitinate a protein. E1 (Ubq-activating enzyme) bring ubq to E2. E2 (Ubq-conjugating enzyme) transfers Ubq to the substrate. E3 (Ubq-ligase) recognizes and brings the substrate to E2 to facilitate the transfer of Ubq from E2 to the substrate. Ubiquitination occurs at lysine residues of the substrate. Cells have many different E3's, each recognizes a specific protein substrate. 26S proteasome is a large protein degradation complex. Ubiquitinated proteins are degraded by 26S proteasome.
e.g. an E3 ubiquitin ligase regulates the tumor suppressor transcription factor P 53 UPS degradation DNA damage 1. P53 normally binds to HDM2 ubq E3 ligase, HDM2 inhibits P53 from binding to DNA, and it also ubiquitinates P53 to trigger its degradation. 2. P53 is phosphorylated in response to DNA damage, the phosphorylated P53 binds to Pin1 transcription coactivator to disrupt the P53-HDM2 interaction. Without HDM2, P53 is not degraded and it binds DNA to activate transcription.
(4) Protein acetylation and methylation Acetylation: adding acetyl group (C 2 H 3 O) to a protein, at the N-terminal or at internal Lys. Acetylation of the N-terminal α-amine of proteins is a widespread modification in eukaryotes. 40-50% of yeast proteins, and 80-90% of human proteins are N- terminal acetylated, catalyzed by N-alphaacetyltransferases. In addition, some proteins (e.g. transcription activator, histones) can also be acetylated at the epsilon ( )-amine group of lysines, by protein acetyltransferases (e.g. CBP). Arginine is usually not acetylated. Methylation: adding a methyl (CH 3 ) group to, usually at Lys or Arg, of a protein. Both lysine and arginine of transcription factors may be methylated by protein methyltransferases (e.g. CARM1).
A comparison of three types of protein modifications Ubiquitination acetylation methylation Arg E3 HAT SAM HMT substrate E3: ubiquitin ligase HAT: histone acetyltransferase HMT: histone methyltransferase
5. Transcription and signal transduction 1. Signal transduction is a series cellular events in response to internal (e.g. levels of metabolites, gene expression, etc ) or external (e.g. hormones, nutrition, light, temperature, contact by other cells, etc) signals. 2. Complicated signal transduction cascades can (1) amplify the signal, and (2) provide interactions (also called crosstalk) of different signals 3. A signal may activate a signal transduction pathway, different signal transduction pathways may converge at one point or another to form signal transduction networks. 4. Signal transduction often activates or inhibits transcription of genes, by regulating the level and activity of transcription factors.
Histone acetylation affects transcription The following example illustrates this phenomenon. PKA (Protein kinase A) stays in the cytosol when camp is low, but it moves into nucleus when camp level is high. In the nucleus, PKA phosphorylates transcription activator CREB (camp response-element binding protein). Phosphorylated CREB binds to transcription co-activator CBP (CREB-binding protein, also called P300), and via CBP to contact basal (pre-initiation) complex to activate transcription. CBP is a histone acetyltransferase (HAT) that acetylates histone to activate transcription. Unphosphorylated CREB cannot bind CBP nor contact basal complex, so it cannot activate transcription at low camp.
e.g.2. RAS pathway - a phosphorylation cascade Phosphorylated Elk-1 is a transcription activator. Many genes of RAS pathway are proto-oncogenes
e.g.3.wnt pathway - a ubiquitination break APC (adenomatous polyposis coli,) recruits β- Catenin (a transcription co-activator) to UPS for degradation. β-catenin is a transcription co-activator and a proto-oncogene. APC is a tumor suppressor, associated with colon cancers.
How multiple pathways interact? RAS pathway Hormone pathway PKA pathway The co-activator CBP acts as the converge point of three signal transduction pathways that all lead to activation of transcription
Summary: regulation of transcription factors 1. The activity of transcription factors can be regulated by interacting with other proteins such as insulators, mediators, co-activators, and other proteins, or by chemical modifications, including phosphorylation, acetylation, methylation, ubiquitination, etc. 2. The protein level of transcription factors can be regulated by the ubiquitination/26s proteasome pathway. 3. Transcription is regulated by the chromatin structure. So the activity of transcription factors may be affected by histones. Recruitment of histones modifying enzymes (HAT, HMT, etc) to the promoter DNA is a major mechanism underlying the function of transcription factors. 4. Cellular signal transduction networks regulate the level and activity of transcription factors to eventually affect transcription of genes