1Gene Regulation and ComputationVoichita D. Marinescu, PhDChildren’s Hospital Informatics Program6.872/HST.950 April 15th, 20042Topics for today• The transcriptional machinery and its regulation• The role of transcriptional regulation in development • Representations of transcriptional regulation components from a computational point of view• Popular tools and methods available• Examples3Definition• Gene regulation: a complex mechanism that allows the cell to vary the amount of mRNA produced for a gene• Consequences: – mediating the transition from genotype to phenotype– ensuring adaptation to environmental changes– making differentiation possible4The regulomeConsisting of:-transcription factors-DNA binding sites5The transcriptional mechanism(From Lewin, Genes VIII, 2004)6Stages and compartmentalization27Key players: transcription factors8Transcription factor binding sites and modules9Enhancers and Insulators10Coactivators11The complexity of gene regulationFrom Yuh et al., Science, 1998, 279(5358)12The importance of gene regulationTemporal and spatial gene expression patterns control:- developmental processes- morphogenesis- cell differentiation- tissue specificity- stress responsesFrom Yuh et al., Science, 1998, 279(5358)313The combinatorial control of eukaryotic transcriptionFrom Fickett and Wasserman, Curr.Opin.Biotechnol., 2000, 11(1)14The sea urchin model for a genomic regulatory networkDavidson EH, Science 2002, 295:166915The regulatory network for endomesoderm specificationDavidson EH, Science 2002, 295:166916Davidson EH, Science 2002, 295:1669Perturbations used in network analysis17How can we get there for other organisms?(From Qiu, Biochem. Biophys. Res. Commun, 2003, 495-501)18Functional genomics insights• Assumption: co-regulation = same cluster + common regulatory motifs• Cluster analysis of combined yeast data sets:• Can expression clusters be predicted?• Can clusters be used to predict structurally related genes?From Eisen et al., PNAS, 1998, 95(25)419The simple scenario• Perform genome-wide screening for identifying regulatory elements• Group the genes in classes sharing common motifs• Group the genes in clusters based on the expression profile• Look for the intersection of the two sets20Finding putative regulatory elements (e.g. TFBSs)• TFBS = a short sequence of DNA (6-20bp)• Conserved across the target genes• Usually identified experimentally by molecular biology techniques• Use this information to abstract a pattern21Deterministic TFBS representations– Exact string matching (e.g. “CAGTTCA”, “CATATGA”, “CAACTCA”, “CACGTGA”)– Regular expressions (e.g. “CA-X2-T-(C/G)-A”)22Probabilistic TFBS representations– Position weight matrices (PMWs)– oligonucleotide frequency analysis:• word counting algorithm• overrepresentation– dyad analysis:• many regulatory sites: XXX-(Y)n-XXX• count number of occurrences– HMMs23Position weight matricesBy far the most popular approachN – total number of sequencesni,j– number of times nucleotide i was observed in position jpi– a priori probability of letter ifi,j– frequency of letter i at position j 24PWMs – major disadvantages• Assumes that the positions are independent• Overemphasize a core matrix (3-4 bp in length)• Search methods based on them are prone to a very high number of false positives525Popular tools availableI. Databases: TRANSFAC• http://www.gene-regulation.com/index.html• Large and frequently updated database containing information on TF and their binding sites in target genes• Manually currated• Based on experimental data 26TRANSFAC data filesAC M00761ID V$P53_DECAMER_Q2 NA p53 decamer XX BF T00671; p53; Species: human, Homo sapiens. BF T01806; p53; Species: mouse, Mus musculus. BF T04997; p53; Species: rat, Rattus norvegicus. XX P0 A C G T 01 25 3 16 2 R 02 14 0 32 0 G 03 25 0 21 0 R 04 2 39 4 1 C 05 32 2 4 8 A 06 23 2 2 19 W 07 3 0 43 0 G 08 9 15 5 17 N 09 2 28 9 7 C 10 5 22 5 14 Y XX BA 46 compiled sequences XX BS R04373; Start: 1; Length: 10; Gaps:; Orientation: p. BS R04373; Start: 11; Length: 10; Gaps:; Orientation: p. -------------------------------------------------------- BS R11629; Start: 21; Length: 10; Gaps:; Orientation: n. BS R11629; Start: 31; Length: 10; Gaps:; Orientation: p. XX // MATRICESAC T00505 FA MEF-2 SY MEF-2; MEF-2A. OS mouse, Mus musculus ------------------------- XX MX M00006; V$MEF2_01. MX M00231; V$MEF2_02. MX M00232; V$MEF2_03. MX M00233; V$MEF2_04. MX M00405; V$MMEF2_Q6. MX M00405; V$MMEF2_Q6. XX BS R03583; AS$MEF2_01; Quality: 6. BS R03586; CHICK$CTNT_05; Quality: 6; cTNT, G000057; chick BS R09144; HS$DSMN_01; Quality: 6; DSMN, G001827; human BS R04212; HS$PGAMM_01; Quality: 4; PGAM-M, G000361; human BS R02201; MEF2$CONS_01; Quality: 6. BS R04530; MEF2$CONS_02; Quality: 6. BS R04804; MEF2$CONS_03; Quality: 6. BS R04805; MEF2$CONS_04; Quality: 6. BS R04806; MEF2$CONS_05; Quality: 6. BS R00244; MOUSE$MCK_05; Quality: 1; mck, G000557; mouse BS R03585; MOUSE$MCK_07; Quality: 6; mck, G000557; mouse BS R03587; RAT$AMHC_03; Quality: 6; MHC-A, G000694; rat BS R03588; RAT$AMHC_04; Quality: 6; MHC-A, G000694; rat BS R03589; RAT$MHC_02; Quality: 6; MHC, G000770; rat BS R03584; RAT$MLCC_01; Quality: 6; MLC2, G000771; rat XX --------------------------------- DR SwissProt: Q60929; MEFA_MOUSE XX RN [1] RX MEDLINE; 96104596. --------------------------------- XX // FACTORS27Databases (contd): JASPARhttp://jaspar.cgb.ki.se/28II. Search engines: Matchhttp://www.gene-regulation.com/pub/programs.html29Search engines (contd.): MatInspectorMatInspector output for sequence AF029081http://www.gene-regulation.com/pub/programs.html30TESS output for nucleotides 6000-8000 of sequence AF029081Search engines (contd.):TESShttp://www.cbil.upenn.edu/tess/631Search engines (contd.)http://bioprospector.stanford.edu/MDscan/32The next step: finding regulatory modulesModule representation:33III. Searching for cis-regulatory moduleshttp://trantor.bioc.columbia.edu/Target_Explorer/34Searching for modules (contd):Toucanhttp://www.esat.kuleuven.ac.be/~saerts/software/toucan.php35Searching for modules (contd.):