1. Identification of regulatory elements

2. Transcriptional Regulation Strongest regulation happens during transcription Best place to regulate: No energy wasted making intermediate products However, slow response time After 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

3. Transcription Factors Binding to DNA Transcription regulation: Certain transcription factors bind DNA Binding recognizes DNA substrings: Regulatory motifs

4. RNA Polymerase TBP Promoter and Enhancers Promoter necessary to start transcription Enhancers can affect transcription from afar Enhancer 1 TATA box Gene X DNA binding sites Transcription factors

5. 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 TATASP1 CCAAT AP2 HSE AP2CCAAT SP1 promoter of heat shock hsp70 0 --158 GENE Motifs:

6. The Cell as a Regulatory Network AB Make D C If C then D If B then NOT D If A and B then D D Make B D If D then B C gene D gene B B Promoter D Promoter B

7. Cluster Co-regulation (DeRisi et al, 1997)

8. Cluster of co-expressed genes, pattern discovery in regulatory regions 600 basepairs Expression profiles Upstream regions Retrieve Pattern over-represented in cluster

9. Some Discovered Patterns Pattern Probability ClusterNo.Total ACGCG 6.41E-3996751088 ACGCGT 5.23E-389452 387 CCTCGACTAA 5.43E-382718 23 GACGCG 7.89E-318640 284 TTTCGAAACTTACAAAAAT 2.08E-292614 18 TTCTTGTCAAAAAGC 2.08E-292614 18 ACATACTATTGTTAAT 3.81E-282213 18 GATGAGATG 5.60E-286824 83 TGTTTATATTGATGGA 1.90E-272413 18 GATGGATTTCTTGTCAAAA 5.04E-271812 18 TATAAATAGAGC 1.51E-262713 18 GATTTCTTGTCAAA 3.40E-262012 18 GATGGATTTCTTG 3.40E-262012 18 GGTGGCAA 4.18E-264020 96 TTCTTGTCAAAAAGCA 5.10E-262913 18 Vilo et al. 2001

10. The " GGTGGCAA " Cluster

11. Pattern discovery strategies Sequence based: Suffix tree scanning Alignment based: –MEME (Expectation Maximization) –GibbsMotif (Gibbs Sampler) –CisModule (Gibbs Sampler for combinations of several modules)

12. Circadian rhythm Follow the daily cycle (Circa diem: about a day) Many biological systems follow circadian rhythms

13. Circadian rhythm

14. Microarray analysis of circadian rhythm Grow plants in 12 hours light and 12 hours dark Switch off light, and start collecting plants every 4 th hour. light dark Start experiment Collect RNA every 4 th hour

15. 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

16. Clustering and promoter elements Harmer et al. 2000 Science 290:2110-2113

17. Bayesian Networks Analysis Friedman et al. 2000 J. Comp. Biol., 7:601-620

18. - Can only represent acyclic relations.

19. From Gifford 2001 Science 293:2049-2050 34 genes, 140 experiments

20. Chromatin IP Chip (ChIP-chip) Iver et al. 2000

21. Chromatin Immuno Precipitation - Chip

23. 10 6 295 188

24. 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

27. 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

29. DDM1 Decrease in DNA methylation 1 Encodes a chromatin remodelling ATPase Pleiotrophic phenotype –Late flowering

30. siRNA Changes in DNA methylation

32. 1.5 Mb hk4S Gene island

33. 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:

36. Gene traps