1、BioSci 145A lecture 14 page 1 copyright Bruce Blumberg 2000. All rights reserved,BioSci 145A Lecture 14 - 2/21/2002 Transcription factors III,Finish up with identifying regulatory regions Major families of transcription factors and their functions zinc finger genes nuclear hormone receptors helix-tu
2、rn-helix homeobox genes helix-loop-helix myogenic genes bZIP proteinsAdditional reading Evans (1988) Science 240, 889-895 Blumberg and Evans (1998) Genes and Development 12, 3149-3155Last years final exam is now posted. I will post answers in a couple of weeks after you have had time to work through
3、 the questions,BioSci 145A lecture 14 page 2 copyright Bruce Blumberg 2000. All rights reserved,Identification of regulatory elements,Given a gene of interest, how does one go about studying its regulation? First step is to isolate cDNA and genomic clones. Map cDNA to genomic sequence identify intro
4、ns, exons locate approximate transcriptional start recognizing elements, e.g. TATA box 5 primer extension or nuclease mapping get as much 5 and 3 flanking sequence as is possible fuse largest chunk of putative promoter you can get to a suitable reporter gene. Test whether this sequence is necessary
5、and sufficient for correct regulation how much sequence is required for correct regulation? what is correct regulation? In cultured cells in animals? typical result is the more you look, the more you find. questions are usually asked specifically. That is, what part of the putative promoter is requi
6、red for activity in cultured liver cells? doesnt always hold in vivo.,BioSci 145A lecture 14 page 3 copyright Bruce Blumberg 2000. All rights reserved,Identification of regulatory elements (contd),Promoter mapping nuclease footprinting of promoter to identify regions that bind proteins make various
7、deletion constructs Previously made by ExoIII deletions or insertion of linkers (linker scanning) typical method today is to PCR parts of the promoter and clone into a promoterless reporter map activity of promoter related to deletions incremental changes in activity indicate regions important for a
8、ctivity test elements for activity,BioSci 145A lecture 14 page 4 copyright Bruce Blumberg 2000. All rights reserved,Identification of binding proteins,How to identify what factors bind to putative elements? examine the sequence does it contain known binding sites? if yes, do such proteins bind to th
9、e isolated element in gel-shift experiments? do the elements bind proteins from nuclear extracts? gel shift (EMSA) experiments clone the elements into reporters with minimal promoters. do these constructs recapitulate activity? Biochemical purification of binding proteins tedious, considerable bioch
10、emical skill required two basic approaches fractionate nuclear extracts chromatographically and test fractions for ability to bind the element in EMSA DNA-affinity chromatography multimerize the element and bind to a resin pass nuclear extracts across column and purify specific binding proteins prot
11、ein microsequencing predict DNA sequence from amino acid sequence look in GENBANK database prepare oligonucleotides and screen library,BioSci 145A lecture 14 page 5 copyright Bruce Blumberg 2000. All rights reserved,Identification of binding proteins (contd),Biochemical purification of binding prote
12、ins (contd) advantages gold standard if you can purify proteins, this will always work disadvantages slow, tedious need good protein sequencing facility biochemical expertise required expense of preparing preparative quantities of nuclear extracts Molecular biological approaches oligonucleotide scre
13、ening of expression libraries (Singh screening) multimerize oligonucleotide and label with 32P screen expression library to identify binding proteins advantages straightforward much less biochemical expertise required relatively fast disadvantages cant detect binding if multiple partners are require
14、d fair amount of “touch” required,BioSci 145A lecture 14 page 6 copyright Bruce Blumberg 2000. All rights reserved,Identification of binding proteins (contd),Molecular biological approaches (contd) yeast one-hybrid assay clone element of interest into a reporter construct (e.g. -gal) and make stable
15、 yeast strain transfect in aliquots of cDNA expression libraries that have fragments of DNA fused to yeast activator if the fusion protein binds to your element then the reporter gene will be activated advantages somewhat more of a functional approach eukaryotic milieu allows some protein modificati
16、on disadvantages slow, tedious purification of positives cant detect dimeric proteins sensitivity is not so great,BioSci 145A lecture 14 page 7 copyright Bruce Blumberg 2000. All rights reserved,Identification of binding proteins (contd),Molecular biological approaches (contd) expression cloning (si
17、b screening) clone element of interest (or promoter) into a suitable reporter construct (e.g. luciferase) transfect (or inject, or infect, etc) pools (10,000 cDNAs each) of cDNA expression libraries and assay for reporter gene retest positive pools in smaller aliquots (1000) repeat until a pure cDNA
18、 is found advantages functional approach presumably using the appropriate cell type so modifications occur possibility to detect dimers with endogenous proteins disadvantages VERY TEDIOUS very slow, much duplication in pools, extensive rescreening is required could be expensive,BioSci 145A lecture 1
19、4 page 8 copyright Bruce Blumberg 2000. All rights reserved,Identification of binding proteins (contd),in vitro expression cloning (IVEC) Make small pools of cDNAs (100) transcribe and translate cDNA libraries in vitro into protein pools EMSA to test protein pools for element binding unpool cDNAs an
20、d retest advantages functional approach smaller pools increase sensitivity disadvantages cant detect dimers very expensive (TNT lysate) considerable rescreening still required tedious, countless DNA minipreps required,BioSci 145A lecture 14 page 9 copyright Bruce Blumberg 2000. All rights reserved,I
21、dentification of binding proteins (contd),hybrid screening system 1 begin with cDNA libraries in 384-well plates, 1 cDNA per well pool cDNAs using robotic workstation prepare DNA with robotic workstation transcribe and translate protein in vitro test for ability to bind DNA element using sensitive,
22、high-throughput assay fluorescence radioactive assay retest components of positive pools advantages very fast, only two steps required, 2 weeks little work required disadvantages expense of robotics wont detect dimers (unless 1 partner known) expense of reagents (TNT, radionuclides, fluorescent labe
23、ls,BioSci 145A lecture 14 page 10 copyright Bruce Blumberg 2000. All rights reserved,Identification of binding proteins (contd),hybrid screening system 2 prepare reporter cell line with element or promoter driving reporter gene (e.g. luciferase) prepare cDNA pools as in system 1 use robotic workstat
24、ion to transfect cDNA libraries into reporter cells assay for reporter gene advantages very fast truly functional approach use of cells allows modifications can detect dimers if one partner is already present in cell disadvantages expense of equipment OK, you have your element and binding protein, n
25、ow what? functional analysis depends on type of protein you are dealing with goal will be to prove that this protein is necessary and sufficient to confer regulation onto the promoter, in vivo many just stop at works on the element,BioSci 145A lecture 14 page 11 copyright Bruce Blumberg 2000. All ri
26、ghts reserved,Transcription factors bind to regulatory elements,The response element binding proteins you have carefully identified are transcription factors. There are many types. The primary mode of classification is via the type of DNA-binding domains and intermolecular interactions Features of t
27、ranscription factors typically these proteins have multiple functional domains can frequently be rearranged or transferred DNA-binding domains these domains take many forms that will be discussed next time see also the list in TRANSFAC http:/transfac.gbf.de/TRANSFAC/ Activation domains these are pol
28、ypeptide sequences that activate transcription when fused to a DNA-binding domain these are diverse in sequence, 1% of random sequences fused to GAL4 can activate many activation domains are rich in acidic residues and assume an amphipathic -helix conformation when associated with coactivator protei
29、ns interact with histone acetylases that destabilize nucleosomes and open chromatin,BioSci 145A lecture 14 page 12 copyright Bruce Blumberg 2000. All rights reserved,Transcription factors bind to regulatory elements (contd),Features of transcription factors (contd) repression domains functional oppo
30、site of activation domains short and diverse in amino acid sequence some are rich in hydrophobic aa others are rich in basic aa some interact with proteins having histone deacetylase activity, stabilizes nucleosomes and condenses chromatin others compete with activators for the same sequence and con
31、tacts with the transcription machinery protein:protein interaction domains these are diverse in sequence but do contain structural motifs leucine zipper helix-loop-helix,BioSci 145A lecture 14 page 13 copyright Bruce Blumberg 2000. All rights reserved,Regulating transcription factor activity (contd)
32、,-catenin/ armadillo,BioSci 145A lecture 14 page 14 copyright Bruce Blumberg 2000. All rights reserved,Regulating transcription factor activity (contd),How can the activity of a transcription factor be restricted to a particular cell type or time? Factor is not generally present but synthesized only
33、 where it is needed some developmental regulators The factor is present but must be modified to be active heat shock factors - phosphorylated -catenin/armadillo - dephosphorylated A ligand is required for activity (or inactivity) nuclear hormone receptors The factor is localized to an inactive compa
34、rtment (e.g. cell membrane) and required cleavage for activity sterol response factors (primarily cholesterol) The factor may be bound to an inhibitory factor in the cytoplasm NF-B and I-B A dimeric factor can have multiple partners. Which partner is present determines activity some dimers are activ
35、e others are inactive eg bHLH and bZip proteins,BioSci 145A lecture 14 page 15 copyright Bruce Blumberg 2000. All rights reserved,Zinc finger genes,Zinc fingers are found in a variety of transcription factors two basic types Cys-His, consensus sequence is cys-X2-4-cys-X3-phe-X5-leu-X2-his-X3-his typ
36、ical gene has 3 or more fingers found in factors for Pol II and Pol III Cys-Cys, consensus sequence is cys-X2-cys-X13-cys-X2-cys typical gene has only 2 fingers found in steroid hormone receptor superfamily members may be involved in both DNA and RNA binding, presence of finger does not indicate whi
37、ch eg TFIIIA binds DNA and RNA product eIF2 recognizes translational initiation sites,BioSci 145A lecture 14 page 16 copyright Bruce Blumberg 2000. All rights reserved,Zinc finger genes (contd),purpose of fingers is to arrange residues such that zn ions can be coordinated fingers may form -helical s
38、tructures that fit into the major groove of the DNA helix multiple fingers may act cooperatively to bind nucleic acids,BioSci 145A lecture 14 page 17 copyright Bruce Blumberg 2000. All rights reserved,Zinc finger genes (contd),cys-cys fingers in nuclear receptors only 1st finger binds to DNA second
39、finger is responsible for protein:protein interactions spacing between fingers can vary quite a bit finger 1 contains a regions that determines target specificity - P-box CGSCKA - AGAACA CEGCKG - AGTTCA these can be swapped and change specificity of the receptor used in ecdysone-inducible system,Bio
40、Sci 145A lecture 14 page 18 copyright Bruce Blumberg 2000. All rights reserved,Hormonal signaling pathways,Hormones are chemical messengers that coordinate cellular activity Can act in different ways endocrine - on distant cells paracrine - on neighboring cells autocrine - on cells which secrete the
41、m Active at very low concentrations - typically less than 1 ppb (1 ppb = 3 nM) Involved in numerous biological processes - many hundreds of hormones reproduction - estrogen, testosterone, progesterone, FSH, LH, activin metabolic rate - thyroid hormone, TSH, GH stress - glucocorticoids, ACTH, CRF blo
42、od pressure - aldosterone, renin, angiotensin, vasopressin calcium homeostasis - vitamin D3, calcitonin, PH Some vitamins or vitamin derivatives are hormones Vitamin A all-trans-retinoic acid 9-cis-retinoic acid 14-OH-retroretinol Vitamin D3,BioSci 145A lecture 14 page 19 copyright Bruce Blumberg 20
43、00. All rights reserved,Nuclear hormone receptors,Domains are assortable and transferable DNA-binding domain (DBD) responsible for direct binding to DNA discriminates half site sequence determines spacing between half sites contains an important dimerization motif Ligand binding domain (LBD) respons
44、ible for ligand binding has a general dimerization motif contains an important transactivation domain may interact with amino terminus to modulate activation amino terminal region (A/B domain) contains an activation domain in many receptors may interact with other components of the transcriptional m
45、achinery many receptors have alternative splicing or promoter usage to yield different A/B domains linker region (D) may influence activation, repression, nuclear translocation or DNA-binding,A/B C D D F,BioSci 145A lecture 14 page 20 copyright Bruce Blumberg 2000. All rights reserved,Nuclear hormon
46、e receptors (contd),bind to specific target DNA sequences activate transcription of target genes upon ligand binding function at very low levels of ligand (10-9M or ppb) bind to small (300d) lipophilic molecules steroids retinoids thyroid hormone vitamin D3,BioSci 145A lecture 14 page 21 copyright B
47、ruce Blumberg 2000. All rights reserved,Nuclear hormone receptors (contd),Many receptor ligands are related to cholesterol steroids bile acids oxysterols Vitamin D3 ecdysone can move freely through tissues penetrate to a target diffuse from a source,BioSci 145A lecture 14 page 22 copyright Bruce Blu
48、mberg 2000. All rights reserved,Nuclear hormone receptors (contd),more orphan than known receptors why study orphan receptors (not particularly easy) novel signaling pathways new developmental hormones target gene networks potential teratogens roles in adult physiology and endocrinology cancer treat
49、ment,BioSci 145A lecture 14 page 23 copyright Bruce Blumberg 2000. All rights reserved,Nuclear hormone receptors (contd),Payoff from orphan receptor research so far (4 biotechs) LXR and FXR regulate cholesterol metabolism LXR diverts cholesterol into bile acid pathway FXR negatively regulates uptake of bile acids PPARs regulate fat metabolism PPAR is insulin sensitizer SXR and PXR regulate metabolism of steroids, xenobiotics and environmental compounds CAR also mediates drug breakdown,BioSci 145A lecture 14 page 24 copyright Bruce Blumberg 2000. All rights reserved,
