Metode za študij interakcij DNA-proteini

Proteomika transkripcijskih faktorjev

Funkcionalne študije promotorja

Metode za določanje količine RNA

(Tehnologija siRNA)

Metode za študije interakcij DNA-proteini:

- metoda premika na gelu (gel shift, EMSA),

- metoda odtisa, odtis z DNAzo (DNAse footprint),

- metilacijska interferenca,

- kromatinska imunoprecipitacija in metoda Chip-chip.

Metoda premika na gelu

+

-

Odtis z DNAzo

Odtis z DNAzo

LDL LDL SRE1 SRE1 SRE2 SRE2

Binding of recombinant SREBP proteins to CYP51-sterol responsive DNA elements (SREs)

LDL TTT GAA AAT CAC CCC ACT GCA LDL TTT GAA AAT CAC CCC ACT GCA

CYP51CYP51--SRE1 SRE1 --239 GGC CGA GAT CAC CTC AGG CGC T 239 GGC CGA GAT CAC CTC AGG CGC T

CYP51CYP51--SRE2 SRE2 --143 CGT GTC CCG GCA CCC CGC ACC CGG 143 CGT GTC CCG GCA CCC CGC ACC CGG

DNADNA--protein complexprotein complex

free DNAfree DNA

probeprobe

supershiftsupershift

+anti-SREBP1acompetition

Rozman D, Fink M, Fimia GM, Sassone-Corsi P, Waterman MR.Mol Endocrinol. 1999 Nov;13(11):1951-62.

http://universe-review.ca/I10-33-epigenetic.jpg

Metilacija DNA

DNA methylation is one of several epigenetic modifications. It involves the covalent addition of a methyl group to the 5'-carbon of cytosine in a CpG dinucleotide.

DNA methylation

Assays to Detect DNA Methylation

DNA methylation can be detected by the following assays currently used in scientific research:

•Bisulfite sequencing, which is based on a chemical reaction that converts unmethylated CpGsites to UpG, followed by traditional PCR to determine the (converted) sequence and thus deduce the (original) methylation status.

•The HELP assay, which is based on restriction enzymes' differential ability to recognize and cleave methylated and unmethylated CpG DNA sites.

•ChIP-on-chip assays, which is based on the ability of commercially prepared antibodies to bind to DNA-methylation associated proteins like MCP2.

•Restriction landmark genomic scanning, a complicated and now rarely-used assay based upon restriction enzymes' differential recognition of methylated and unmethylated CpGsites; the assay is similar in concept to the HELP assay.

•Methylated DNA immunoprecipitation (MeDIP), analogous to chromatin immunoprecipitation, immunoprecipitation is used to isolate methylated DNA fragments for input into DNA detection methods such as DNA microarrays (MeDIP-chip) or DNA sequencing (MeDIP-seq).

DNA metilacijska analiza(HELP) - HpaII-tiny fragment Enrichment by Ligation-mediated PCR

MspI – cepi metilirana in nemetiliranaCCGG zaporedja

HpaII – cepi le nemetilirane CCGG

Metilacijska interferenca-metilacija DNA

-premik na gelu

-demetilacija s piperidinom

Če metilacija na specifičnem G nimavpliva na vezavo proteina (primera A in C), bosta vezana in nevezanaoblika DNA vsebovali enaki količinimetiliranih G na tem mestu.

V primeru zaščite zaradi metilacije(začetni oligonukletid B), bo le nevezana DNA vsebovala metiliranG na tem mestu.

Metode in vivo

-odtis z DNAzo in vivo: permeabilizacija jeder z DNAzo; Tretiranje celic z DMS, ki modificira A in G in omogoči cepitev DNA. Po ligaciji specifičnih oligonukleotidov in PCR,fragmente tretiranih in netretiranih jeder detektiramo s southernovo analizo

-Imunoprecipitacija kromatina (X-chip): tretiranje celic sformaldehidom, ki kovalentno poveže na DNA vezane TF inv nativni kromatinski strukturi (protein-protein, DNA-protein). S protitelesi, specifičnimi za preiskovani TF, oborimo protein in z G-sefarozo izločimo molekule DNA, ki so vezale TF. Z izbiro primernih oligonukleotidnih začetnikov pomnožimo izločenedele DNA.

Zaščita z DNAzo (DNAse protection assay)

Kromatinska imunoprecipitacija (ChIP) –metoda za študije DNA-protein interakcij in vivo

1. Fiksiranje proteinov na DNA v celici.2. Sonifikacija.3. Imunoprecipitacija.4. Analiza imunoprecipitirane DNA.

Optimizacija!!

• X-ChIP - s formaldehidom fiksiran kromatin, fragmentiran s sonifikacijo. Za študije vseh DNA-vezavnih proteinov.

• N-ChIP – nativni, nefiksiran kromatin, razgrajen z DNA-zo.učinkovita imunoprecipitacija, ni potrebe po amplifikaciji imunoprecipitirane DNA. Za študije histonskih proteinov, ki so močno vezani na DNA.

Figure 1: General principle of Chromatin ImmunoPrecipitation assay

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowTOC&rid=inserm.TOC

ChIP-ChipA combination of ChIP and whole genome

microarrays (ChIP-chip) has been used successfully to create high-resolution genome-wide maps of in

vivo DNA-protein interactions.

Buck, M.J. and J.D. Lieb. 2004. ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments. Genomics 83:349-360.

Genomics. 2004 Mar;83(3):349-60.

ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments.

Buck MJ, Lieb JD.

Department of Biology and Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA.

Chromatin immunoprecipitation (ChIP) is a well-established procedure to investigate interactions between proteins and DNA. Coupled with whole-genome DNA microarrays, ChIPS allow one to determine the entire spectrum of in vivo DNA binding sites for any given protein. The design and analysis of ChIP-microarray (also called ChIP-chip) experiments differ significantly from the conventions used for locus ChIPapproaches and ChIP-chip experiments, and these differences require new methods of analysis. In this light, we review the design of DNA microarrays, the selection of controls, the level of repetition required, and other critical parameters for success in the design and analysis of ChIP-chip experiments, especially those conducted in the context of mammalian or other relatively large genomes.

Proteomika transkripcijskih faktorjev

KloniranjeČiščenje in karakterizacija

Določevanje domenInterakcije z drugimi proteini

Določanje neznanih proteinov

Kloniranje rekombinantnih transkripcijskih faktorjev z nalepkamiPriprava z His, tag, Flag, GST…nalepkami označenih rekombinantnih proteinov

en.wikipedia.org/wiki/His-tag

Nalepke (angl. Tags)

en.wikipedia.org/wiki/His-tag

Čiščenje z afinitetno kromatografijo

Čiščenje z afinitetno kromatografijo

Mapiranje domen transkripcijskih faktorjev

Metode za določanje potranslacijskih sprememb histonov

Metoda potega za iskanje proteinov, ki prepoznajo spremenjene histone

Methods, Vol. 40, 339-343 (2006)

Metode za določanje interakcij med proteini:

-ko-imunoprecipitacija (preiskujemo, če kompleks TF-protitelo v celičnem lizatu veže še kakšen protein)

-metoda potega (pull-down assay):v E. coli proizvedemo s repkom(GST, myc, etc.)-združen TF, ga izločimo z GST/myc-agarozo in izpostavimo proteinom, ki potencialno interagirajo. Detekcija z westernovo ali daljno westernovo analizo.

-daljna westernova analiza (far western analysis): z radioaktivno označenim TF delujemo na potencialne partnerske proteine, vezane na nitrocelulozni membrani.

-analiza dvojnih hibridov (two-hybrid assay): izrablja lastnost TF, da imajo ločeno DNA vezavno in transaktivacijsko domeno

Imunoprecipitacija proteinov

Protein-Protein Interactions

Approaches: experimental or computional

Experimental: The yeast two-hybrid systemTagged protein mass spectrometry

Computional:To infer protein interaction networks and predict the function of proteins

For example, once a function of a protein (Called A) is identified, it can be used to seek proteins whose peptide sequences are similar to A.This method based on similarity infererence extends the functional knowledge of protein to other protein.Larg-scale protein interaction pathway network can be accumulated by this inference with support of experimental result.

Analiza dvojnih hibridov

Prey – fuzijski protein z V16 transaktivacijsko domeno

Bait – fuzijski protein z GAL4 DNA vezavno domeno

Če pride do stabilne interakcije proteinov A in B je aktivirano prepisovanjesporočilnega gena

Trokomponentni sistem:

1. Poročevalec (lacZ) pod kontrolo promotorja z GAL4 DNA vezavnimi mesti

2. Ekspresijski vektor, ki usmerja izražanjeDNA vezavne domene GAL4, vezane na področje proteina, ki vstopa v protein-proteinInterakcije (bait)

3. Vektor, ki usmerja izražanje fuzijskegaproteina z močno transaktivacijsko domeno,(prey) povezanega s proteinsko domeno, za katerose predpostavlja protein-proteininterakcijas preiskovanim proteinom.

Preiskovanje cDNA knižnjic –funkcijsko kloniranje TF.

http://www.mcb.mcgill.ca/~hallett/GEP/PLecture6/2Hybrid_files/image002.jpg

Tagged protein mass spectrometry

http://www.sanger.ac.uk/Teams/Team17/gfx/mass-spec_600.gif

Mass spectrometer

http://www.ionsource.com/Card/phos/phos.h1.gifhttp://www.afcs.org/reports/v1/DA0008/DA0008Fig7.jpg

Določanje fosforiliranih proteinov

Funkcionalne študije promotorja

TGGTGGCATATTAATACCTGTCAGGTTGATTTTAGTTGGAGTGGGACGGGGCTGACCTC

CCGTCCTACCCTTCGGTATGAGGCCGGCGTCTTCACGACCTCGCTTTTCACAACCCCAG

CCCCGAGGGCTTATTTGGGGCTGTCGGAGTCCCCAAGACGTGGCCCCGGAGAAGGGTGA

GTGCCACAGTTCGAGGTGCCCCAGTACCCTGCGTCCGGACAGGGGGCGGTGCTCACGCC

AAGGCCCCGCTGACGCGATGTAGGCCGAGATCACCTCAGGCGCTCGCGGTGCAATCACA

AGCGCGCCGTCGCCGCTCGAGCTCGCCCTGAGATCCCTCCGCTCAGCGCCTTCAACGCC

TGTCCCGGCACCCCGCACCCGGGCACACAAAAAACGGGTCGCCCGCGATTCTCAGGGAT

GATCCGCCTCTTCAGGTAAGTTATCTTCCGGCCCCGTACCACTGTGCCACAGGCGCAGC

CGCTTCCTCAGGTGCCCTATCCCGCGCAAGAAGACCACGGCTTCACAGAGTGTTATTTA

+1

CYP51-SRE1CYP51-CRE2

GC-box

CYP51-CRE1

- 21

- 81

- 141

- 201

- 261

- 321

- 381

- 501

- 441

(CYP51-SRE2)

Kloniranje promotorskih regij v poročevalske (sporočilne) plazmide

Poročevalski (sporočilni) sistemi

Transfekcije v sesalske celice in merjenje poročevalske aktivnosti

Fig 4

+316

+316

A

B

CRE3SRE1CRE2GC-boxCRE1+1

SRE1GC-boxCRE1+1

SRE1GC-boxCRE1+1C10

-471

D7-334

+1CRE3

ΔD7 -121

CRE3SRE1CRE2GC-box+1

SRE1CRE2GC-box+1

SRE1GC-box +1+316

1, ΔD7

2, D7

3, C10

C

0

2

4

6

8

1 2 3 4

repo

rter

act

ivity basal

CREBCREM

D7 CRE2amut CRE3mut CRE2,3mut

0 2 4 6reporter activity

CREM

basal

Fig 4

+316

+316

A

B

CRE3SRE1CRE2GC-boxCRE1+1

SRE1GC-boxCRE1+1

SRE1GC-boxCRE1+1C10

-471

D7-334

+1CRE3

ΔD7 -121

CRE3SRE1CRE2GC-box+1

SRE1CRE2GC-box+1

SRE1GC-box +1

CRE3SRE1CRE2GC-boxCRE1+1

SRE1GC-boxCRE1+1

SRE1GC-boxCRE1+1C10

-471

D7-334

+1CRE3

ΔD7 -121

CRE3SRE1CRE2GC-box+1

SRE1CRE2GC-box+1

SRE1GC-box +1+316

1, ΔD7

2, D7

3, C10

C

0

2

4

6

8

1 2 3 4

repo

rter

act

ivity basal

CREBCREM

D7 CRE2amut CRE3mut CRE2,3mut

0 2 4 6reporter activity

CREM

basal

0 2 4 6reporter activity

CREM

basal

CAT poročevalec

Reporter-plasmid (CAT)

Control-plasmid (ß-Gal)

Cell

Cotransfection Experiment

Normalization = Reporter plasmidControl plasmid

Overexpression of transcription factor

β-galkontrolni plazmid

CoA + 3H-acetat 3H-Ac-CoA

3H-Ac-CoA + CHCl3 3H-Ac-CHCl3

Ac-CoA sintaza

CAT encim

CAT poročevalec

Luciferazni poročevalec

Cloned Luciferase Genes

Bacteria Luciferase (lux) Reporter Gene Assay

Photinus Luciferase (luc)Reporter Gene Assay

Renilla Luciferase (Rluc)Reporter Gene Assay

AequorinCa2+ Indicator

Vargula Luciferase Reporter Gene Assay

Dinoflagellate Luciferase no application

Photinus (Firefly) Bioluminescence

Luciferin+ ATP + O2

Luciferase Oxyluciferin + AMP+ PPi + CO2 +

Luciferase: Monomeric, 61,000 Daltons

Co-Substrat: ATP . Mg 2+

LIGHT

Renilla Bioluminescence

Coelenterazine+ O2

Luciferase Coelenteramid+ CO2 +

Luciferase: Monomeric, 36,000 DaltonsLIGHT

Luciferase Measurementsin Living Cells

External stimuli

ReceptorSignal

TransductionProtein-Protein

InteractionsTranscription

Factors

TranscriptionmRNA Processing

Ribozyme

Anti-sense RNATranslation

luc

Regulatory

Sequences

Protein Folding

LIGHT

Viral Infection

Cell-Lysate

LuciferaseAssay Reagent II (LAR)

Stop & GloTM

Reagent

(Photinus)

(Renilla)

Dual-Luciferase® Reporter Assay System

1. Rapid

2. Sensitive

3. Linear

4. Precise

30 s manual4 s automatic

or

LIGHT

LIGHT

2 measurements withina single system

Zeleni fluorescentni protein (GFP)

Metode za določanje količine RNA – ugotavljanjeravni izražanja genov ex vivo

-Kvantitativni PCR (PCR v realnem času)

- Mikromreže

Real-time PCR

is able to detect sequence-specific PCR products as they accumulate in "real-time" during the PCR amplification process.

As the PCR product of interest is produced, real-time PCR can detect their accumulation and quantify the number of substrates present in the initial PCR mixture before amplification began.

Molecular beacons are short segments of single-stranded DNA (Figure 1). The sequence of each molecular beacon must be customized to detect the PCR product of interest. This beacon is 33 nucleotides long with a reporter dye attached to the 5' end and a quencher attached to the 3' end. The nine 5' bases are able to form base pairs with the nine 3' bases which brings the reporter and quencher in very close proximity. Therefore, when the reporter is excited by the appropriate light, its emission is absorbed by the quencher and no fluorescence is detected. The pink lines represent nucleotides that can form base pairs with the PCR product under investigation.

www.bio.davidson.edu/.../ method/realtimepcr.html

Figure 5: Fluorogenic 5' nuclease chemistry. 1. Forward and reverse primers are extended with Taq polymerase as in a traditional PCR reaction. A probe with two fluorescent dyes attached, a reported (R) and a quencher (Q), anneals to the gene sequence between the two primers. 2. When both dyes are attached to the probe, reporter dye emission is quenched. As the polymerase extends the primer, the probe is displaced. 3. An inherent nuclease activity in the polymerase cleaves the reporter dye from the probe. 4. Once separated from the quencher, the reporter dye emits its characteristic fluorescence.

Light cycler

Figure 3. One of the views available after completion of the run is an amplification window.This window shows the amount of fluorescence obtained in each amplification cycle for each reaction. The threshold cycle (Ct) is shown by the darker horizontal line.

RNA quantification: Real time PCR

Figure 4: SYBR Green 1. At the beginning of amplification, the reaction mixture contains the denatured DNA, the primers, and the dye. The unbound dye molecules weakly fluoresce, producing a minimal background fluorescence signal which is subtracted during computer analysis. 2. After annealing of the primers, a few dye molecules can bind to the double strand. DNA binding results in a dramatic increase of the SYBR Green I molecules to emit light upon excitation. 3. During elongation, more and more dye molecules bind to the newly synthesized DNA. If the reaction is monitored continuously, an increase in fluorescence is viewed in real-time. Upon denaturation of the DNA for the next heating cycle, the dye molecules are released and the fluorescence signal falls.

A DNA microarray (also commonly known as gene chip, DNA chip, or biochip) is a collection of microscopic DNA spots attached to a solid surface, such as glass, plastic or silicon chip forming an array for the purpose of monitoring of expression levels for thousands of genes simultaneously.

The affixed DNA segments are known as probes (although some sources will use different nomenclature), thousands of which can be used in a single DNA microarray.

Microarray technology evolved from Southern blotting, where fragmented DNA is attached to a substrate and then probed with a known gene or fragment.

Measuring gene expression using microarrays is relevant to many areas of biology and medicine, such as studying treatments, disease, and developmental stages. For example, microarrays can be used to identify disease genes by comparing gene expression in disease and normal cells.

A DNA microarray

Principle of expression profilingVrste DNA mikromrežglede na izbiro genov

• DNA mikromreže celotnih genomov

•DNA mikromreže posameznih kromosomov

•DNA mikromreže za iskanje mutacij in polimorfizmov

•Tematske DNA mikromreže

•Itd. ....................

Problem kvalitete mikormrež in standardizacije

High-density tiled microarrays for transcript mapping.

Genome-wide, exon-level analysis on a single array —a survey of alternative splicing and gene expression.

Robust, simple representation focusing on the 3' ends.

Different parts of the gene as probes on the array

http://www.affymetrix.com/products/arrays/index.affx

DNA chip/microarray:

-Surface-bound microscopic groups of thousands of DNA molecules with known nucleotide sequence

-Each gene is represented by at least one group of identical molecules (probe)

-Organized array of DNA groups represents a matrix that is used later (after hybridization, etc.) for definition of differences between the sample and the control

Hybridization: Watson-Crick base pairing; AT(U), GC

Principle of expression profilingčitalec

označevanje

Obratno prepisovanje

Emisija

Prekrivanje

Računalniška analiza

Laser 1Test Referenca

Laser 2

ko-hibridizacija

Ekspresijsko profiliranje z DNA mikromrežami

Geni zastopani scDNA ali z oligonukleotidi

Robot-nanašalec(spotter)

Poznavanje interakcij DNA-proteini

Identifikacija transkripcijskih faktorjev

Identifikacija funkcionalnega promotorja

Vpliv transkripcijskega faktorja na endogeno izražanje tarčnega gena

Razumevanje mehanizmov, ki uravnavajo hitrostprepisovanja določenega gena