Identification of regulatory elements

Transcriptional Regulation

• Strongest regulation happens during transcription

• Best place to regulate: No energy wasted making intermediate products

• However, slow response timeAfter a receptor notices a change:

1. Cascade message to nucleus

2. Open chromatin & bind transcription factors

3. Recruit RNA polymerase and transcribe

4. Splice mRNA and send to cytoplasm

5. Translate into protein

Transcription Factors Binding to DNA

Transcription regulation:

Certain transcription factors bind DNA

Binding recognizes DNA substrings:

Regulatory motifs

RNA Polymerase

TBP

Promoter and Enhancers

• Promoter necessary to start transcription

• Enhancers can affect transcription from afar

Enhancer 1 Enhancer 1 Enhancer 1

TATA box

Gene X

DNA binding sites

Transcription factors

Example: A Human heat shock protein

• TATA box: positioning transcription start

• TATA, CCAAT: constitutive transcription• GRE: glucocorticoid response• MRE: metal response• HSE: heat shock element

TATASP1CCAAT AP2HSEAP2CCAATSP1

promoter of heat shock hsp70

0--158

GENE

Motifs:

The Cell as a Regulatory Network

A B Make DC

If C then D

If B then NOT D

If A and B then D D

Make BD

If D then B

C

gene D

gene B

B

Promoter D

Promoter B

Cluster Co-regulation

(DeRisi et al, 1997)

Cluster of co-expressed genes, pattern discovery in regulatory regions

600 basepairs

Expression profiles

Upstream regions

Retrieve

Pattern over-represented in cluster

Some Discovered Patterns

Pattern Probability Cluster No. TotalACGCG 6.41E-39 96 75 1088ACGCGT 5.23E-38 94 52 387CCTCGACTAA 5.43E-38 27 18 23GACGCG 7.89E-31 86 40 284TTTCGAAACTTACAAAAAT 2.08E-29 26 14 18TTCTTGTCAAAAAGC 2.08E-29 26 14 18ACATACTATTGTTAAT 3.81E-28 22 13 18GATGAGATG 5.60E-28 68 24 83TGTTTATATTGATGGA 1.90E-27 24 13 18GATGGATTTCTTGTCAAAA 5.04E-27 18 12 18TATAAATAGAGC 1.51E-26 27 13 18GATTTCTTGTCAAA 3.40E-26 20 12 18GATGGATTTCTTG 3.40E-26 20 12 18GGTGGCAA 4.18E-26 40 20 96TTCTTGTCAAAAAGCA 5.10E-26 29 13 18

Vilo et al. 2001

The "GGTGGCAA" Cluster ORF Gene Description

YBL041W PRE7 20S proteasome subunit(beta6) YBR170C NPL4 nuclear protein localization factor and ER translocation component YDL126C CDC48 microsomal protein of CDC48/PAS1/SEC18 family of ATPases YDL100C similarity to E.coli arsenical pump-driving ATPase YDL097C RPN6 subunit of the regulatory particle of the proteasome YDR313C PIB phosphatidylinositol(3)-phosphate binding protein YDR330W similarity to hypothetical S. pombe protein YDR394W RPT3 26S proteasome regulatory subunit YDR427W RPN9 subunit of the regulatory particle of the proteasome YDR510W SMT3 ubiquitin-like protein YER012W PRE1 20S proteasome subunit C11(beta4) YFR004W RPN11 26S proteasome regulatory subunit YFR033C QCR6 ubiquinol--cytochrome-c reductase 17K protein YFR050C PRE4 20S proteasome subunit(beta7) YFR052W RPN12 26S proteasome regulatory subunit YGL048C RPT6 26S proteasome regulatory subunit YGL036W MTC2 Mtf1 Two hybrid Clone 2 YGL011C SCL1 20S proteasome subunit YC7ALPHA/Y8 (alpha1) YGR048W UFD1 ubiquitin fusion degradation protein YGR135W PRE9 20S proteasome subunit Y13 (alpha3) YGR253C PUP2 20S proteasome subunit(alpha5) YIL075C RPN2 26S proteasome regulatory subunit YJL102W MEF2 translation elongation factor, mitochondrial YJL053W PEP8 vacuolar protein sorting/targeting protein YJL036W weak similarity to Mvp1p YJL001W PRE3 20S proteasome subunit (beta1) YJR117W STE24 zinc metallo-protease YKL145W RPT1 26S proteasome regulatory subunit YKL117W SBA1 Hsp90 (Ninety) Associated Co-chaperone YLR387C similarity to YBR267w YMR314W PRE5 20S proteasome subunit(alpha6) YOL038W PRE6 20S proteasome subunit (alpha4) YOR117W RPT5 26S proteasome regulatory subunit YOR157C PUP1 20S proteasome subunit (beta2) YOR176W HEM15 ferrochelatase precursor YOR259C RPT4 26S proteasome regulatory subunit YOR317W FAA1 long-chain-fatty-acid--CoA ligase YOR362C PRE10 20S proteasome subunit C1 (alpha7) YPR103W PRE2 20S proteasome subunit (beta5) YPR108W RPN7 subunit of the regulatory particle of the proteasome

Pattern discovery strategies

• Sequence based: Suffix tree scanning• Alignment based:

– MEME (Expectation Maximization)– GibbsMotif (Gibbs Sampler)– CisModule (Gibbs Sampler for combinations of

several modules)

Circadian rhythm

• Follow the daily cycle (Circa diem: about a day)

• Many biological systems follow circadian rhythms

Circadian rhythm

Microarray analysis of circadian rhythm

• Grow plants in 12 hours light and 12 hours dark• Switch off light, and start collecting plants every

4th hour.

light

dark

Start experiment

Collect RNA every 4th hour

continued

• Extract RNA• Label and hybridize to microarray chip• Cluster by SOM• Identify cluster with circadian memory• Retrieve promoter of genes• Look for overrepresented words

Clustering and promoter elements

Harmer et al. 2000 Science 290:2110-2113

Bayesian Networks Analysis

Friedman et al. 2000 J. Comp. Biol., 7:601-620

P(X1,...,Xn ) P(Xi PaG(Xi)).i1

n

- Can only represent acyclic relations.

From Gifford 2001 Science 293:2049-205034 genes, 140 experiments

Chromatin IP Chip (ChIP-chip)

Iver et al. 2000

Chromatin Immuno Precipitation - Chip

10 6

295

188

Assembling motifs into networks

1. Define set of genes G that are bound by a set of regulators S at high significance

2. Find coexpressed subset of G

3. Establish core expression profile

4. Drop genes of G that do not match this profile

5. Include new genes that do, even if the binding by regulator is less significant

6. Repeat 4 and 5 until all combinations have been considered

Heterochromatin

• Dark staining regions, remain

condensed in interphase (centromeres

and telomeres)• Heterochromatic knobs

– Discovered in maize by McClintock (1951)

• In heterochromatin, there is heavy methylation of– Histones (H3mK9)– DNA (5mC)

• In euchromatin, DNA is not methylated, histones are not K9 methylated, but K4 instead (H3mK4) and acetylated

DDM1

• Decrease in DNA methylation 1• Encodes a chromatin remodelling ATPase• Pleiotrophic phenotype

– Late flowering

siRNA

Changes in DNA methylation

1.5 Mb hk4S Gene island

Chromatin IP – Chip (ChIP – Chip)

• Antibodies against– Histones (H3mK4)– Histones (H3mK9)– DNA (5mC)

are used to immunopreticipate fragmented DNA from DDM1 and WT.

Labeled red – green

Hybridized to chip with 1 kb PCR products spanning the hk4S region

• Tiling path:

Gene traps