Oct 14 Lecture

Gene Expression Analysis II 徐国良 中科院生化细胞所 [email protected] 5492 1332

To start a new experiment •

Find somebody who has done it and get a protocol.

•

Read the protocol and the relevant chapter in «Molecular Cloning»; Try to understand principles.

•

Talk to the person who has experience. Understand critical steps.

•

Prepare reagents you cannot get from other.

•

Set up checkpoints in the course of lengthy experimentation.

Protein expression is regulated at multiple levels DNA

Transcription

Gene copy number Chromosome structure Methylation Promoter activity Inducible transcription factor

RNA

Alternative splicing mRNA transport and stability Ribosome binding site Codon usage Termination

Translation

Protein

Protein folding and targeting Protein stability Phosphorylation Glycosylation Ubiquitination Sumonylation Palmolylation …

Post-translational modification

Biological Activity

Cellular process

Wang C.

Techniques covered in this session

Northern hybridization RNase protection assay Gel shift (or EMSA) Footprinting Hypersensitive site mapping Chromatin IP DNA methylation analysis

Northern Hybridization To measure the amount (abundance) and size (length) of specific RNA. Based on base pairing between labeled DNA strand (probe) and denatured target RNA fixed on a membrane. Major steps: • • • • • • •

Isolation of intact mRNA or total RNA (TRIZOL: acid phenol-guanidinium thiocyanate) Separation of RNA in a denaturing agarose gel with formaldehyde; Transfer the RNA to a membrane in 10xSSC; Fixation of the RNA to the membrane （ vacuum bake, 80ºC, 30 min). Hybridization of probes to immobilized RNA Wash in SSC/SDS solution (monitor with Geiger counter) Detection by autoradiography （用 X 光片） or phosphaimaging ( 用磷屏） .

分析 Specificity and sensitivity • Use two probes from different regions of cDNA • Use cloned DNA of known amounts (5, 15, 100 pg)

Preparation of Probe

25 ng (11 µl) denatured DNA 4 µl High Prime solution (from Roche etc.) 5 µl (50 µCi) [a32P]dCTP 37°C, 10 min.

High Prime is a mixture containing random oligonucleotides, Klenow pol., dATP, dGTP, dTTP and reaction buffer with 50% glycerol.

Specific activity: radioactive incorporation (cpm) per pmol of probe

Marker

F9

P19

ES

EG

Marker

F9

P19

ES

EG

Northern 实例

rRNA 28s

(4.6-5.3 kb)

Dnmt3L

18s

(1.8-2.0 kb)

RNA resolved on agarose gel 30 µg each lane

Autoradiogram

Northern Troubleshooting • Bad quality of RNA preparations • Inefficient transfer to the membrane • Lack of specificity of hybridization • No or weak signal • Multiple bands • High background (Molecular Cloning 7.45) Concept of Stringency

Ribonuclease Protection To measure the abundance of specific mRNA To map topological features Relies on the digestion of unhybridized single strand RNA probes. tenfold more sensitive than hybridization

Flow Chart of RNase Protection Assay

Radiolabeled RNA after digestion with RNase

Mol. Cloning 7.64

RNase Protection 实例

TSA is an inhibitor of histone deacetylase. 5Aza-dC is a demethylating reagent.

Tumor suppressor GPC3 is silenced in lung tumor cell lines by histone deacetylation and/or DNA methylation. Kim H et al., 2003

Techniques for Quantitating RNA Material amount Or sensitivity

Northern blotting

10-30 ug total RNA 1-2 ug mRNA

Susceptible to RNA Degradation

Size & abundance determination

Yes

Simultaneously

Ribonuclease protection

Very sensitive

less

abundance +end mapping

Quantitative RT-PCR

most sensitive

Less

abundance

Real-time PCR

most sensitive

Less

abundance (most quantitative)

The methods above measure the steady-state mRNA levels; Nuclear run-on assay allows the measurement Of promoter transcription activity at a given time.

Chromation Immunoprecipitation (ChIP) = IP+PCR; To ask if a specific region of DNA is bound by a protein directly or indirectly; Major steps: • • • • • • • • •

Fix cells with formaldehyde ( 甲醛 ). Isolate nuclei and lyse (this step left out sometimes). Sonicate chromatin to generate small DNA fragments (200-1000 bp); The sonicated DNA is immunoprecipitated using an antibody against a DNAbinding protein; IP with a specific antibody and protein A sepharose beads. Anti-crosslink at 65°C for >6 hours. Proteinase K and RNase A treatment. Extract DNA with Qiagen’s PCR purification kit PCR reactions are performed with multiple pairs of primers to determine which DNA region is “pulled down” in IP.

ChIP 原理与流程

Trouble Shooting Low specificity: 2. Precleaning with IgG and protein A beads; 3. IP with protein A beads only control; 4. IP with IgG and protein A beads control; 5. IP with related antibodies

ChIP Applications -Study leukemogenesis by MLL-AF10 fusion

Okada et al.,Cell 121, p167,2005

ChIP 实例 1a: Determine the histone modification state of specific genomic loci

Primer pair

Okada et al., Cell 121, p167,2005

ChIP 实例 1b: Identification of genomic regions where a DNA-binding protein binds

MLL-DOT1 融合蛋白在 Hox 基因上的结合位点分析

Okada et al., Cell 121, p167,2005

ChIP 实例 2a: Establish the Role of histone H3K27 methylation in Polycomb-group silencing

Ru et al Science 2002

Model depicting the relation between ESC-E(Z)-mediated H3K27 methylation and PcG silencing. PRE: polycomb response element; PRC1: polycomb repressive complex 1.

ChIP 实例 2b: ESC-E(Z ） HMTase complex is required for H3K27 methylation in Polycomb-group silencing

Ru et al Science 2002

Gel Retardation Assay ( eletrophoresis mobility assay, gel shift assay)

To identify proteins which bind to a specific DNA sequence To identify binding sequence of a DNA-binding protein

The assay exploits electrophoretic mobility differences between a rapidly migrating DNA And a more slowly migratig complex of protein bound to DNA.

Gel Shift Assay 实例 Anti-Cdx2 antibody Anti-HNF1α antibody 20 pmol of non-related DNA 20 pmol of cold probe 2 µg of Caco-2 nuclear extract 0.1 pmol of [32P]-labeled probe

+

+ +

+ + +

+ + +

+ + +

+ + +

+ + + + Supershift

Complexes

* Free probe 1

2

3

4

5

6

7

HNF1α [32P]-labeled probe

（李伯良组提供）

-912

-769

Cdx2-1

2

3

4

Footprinting •

Measures the ability of a protein to protect a radiolabeled DNA fragment against digestion by DNaseI or chemical cleavages.

•

The position of footprint reveals where on the DNA molecule the protein X has bound.

Footprinting

DNA fragment is radio-labeled at the left end

Footprinting 实例 -Binding of lactose repressor to its operator

32

p

The lac operator

Light blue arrows indicate purines protected by repressor against methylation; Dark blue arrows: Purines where methylation is enhanced by repressor; Brown arrows: thymines that can be crosslinked to repressor

Mol. Biol. Of the Gene, 4th edition, p470

Micrococcal Nuclease Digestion of Chromatin

To map “open” chromatin regions Appearance of hypersensitive sites is associated with transcriptional activation

Hypersensitive Sites & Chromatin Structure

Gene VII, Fig. 19.41

Mapping of Hypersensitive Sites in Chromatin

Gene VII, Fig. 19.39

Define Active Genes or Regions In Chromatin by DNase I Sensitivity Analysis

Gene VII, Fig. 19.42

Chromatin state determined by DNase I Sensitivity Analysis

实例

Gene VII, Fig. 19.43

Detection of DNA Methylation

• Restriction digestion with methylation-sensitive restriction enzyme followed by Southern hybridization; • Bisulfite sequencing

Chemistry of the Bisulphite Reaction

Bisulfite Conversion of a DNA Sequence

Data of Methylation Analysis by Bisufite Sequencing H19

Snrpn

MII oocytes

Sperm

Liver

TaqI (ACGT) H19 and Snrpn genes are paternally and maternally imprinted resp. Filled circles represent methylated CpG sites Li et al, Genomics 2004

Online Resources for Gene Expression Analysis

Promoter element prediction including CpG island Exon-intron prediction Tissue and developmental stage specific expression patterns

“Data mining means your data are mine and my data are mine” By Sidney Brenner It is the automated extraction of hidden predictive information from databases. Textbook

Analysis of Gene Expression • High throughput analysis of RNA expression. – ESTs • Virtual Northerns

– SAGE • Sequencing of SAGE tags

– Microarrays • cDNAs • Oligos • Gene chips (Affymetrix)

Expressed Sequence Tags • Single-pass sequencing of “random” cDNAs – 5’ or 3’ end – Relatively low quality sequence

• ESTs are cDNAs – Represent mRNAs – Correspond to transcribed sequences of the genome – “Normalization” of libraries

Individual clones sequenced

ESTs • Genome sequencing is great but… – In eukaryotes, most of the genome (>90%) does not contain protein coding genes. – Since ESTs focus on transcribed sequences, they represent only the coding portion of the genome. – This approach is sufficient for many types of genomic analyses.

EST and Gene Expression • ESTs collections can be used to estimate levels of gene expression in a particular tissue. – Random ESTs from a cDNA libraries that accurately represent the mRNA distribution in the source tissue. – The more often a particular cDNAs shows up, the more abundant that mRNA was in the cells. – Libraries are often not representative and are sometimes manipulated to remove highly abundant transcripts, making it easier to sequence rare cDNAs.

Importance of ESTs • ESTs provide molecular tags for genes. – The more ESTs sequenced, the deeper the representation of genes from a give source. – 5’ sequence gives maximum chance to identify sequence similarity. – 3’ sequence provides potential genes specific sequence. – Number of ESTs in dbEST – Typical EST entry

Gene expression analysis: EST Profile Viewer

Expression profile suggested by analysis of EST counts. Mm.178246- Chd3: Chromodomain helicase DNA binding protein 3 Breakdown by Tissue Mm.178246 Bone

0

0

/

38920

Bone Marrow

116

4

/

34476

Brain

345

168

/

486601

Colon

38

2

/

52041

Eye

181

26

/

143641

Heart

37

2

/

53270

Kidney

51

6

/

116877

Liver

9

1

/

104394

Lung

0

0

/

43593

Lymph Node

0

0

/

25598