CSHL : Systems Biology

1. CSHL : Systems Biology March 23-26, 2006

2. Schedule • Overture • Computational … cis-Reg Elements • Poster Session I • Poster Session II • Adv. Detection of TF/DNA Interactions • Trans. & Post-trans. Network Modelling • Comp. Genomics of Gene Regulation • Emerging Tech and Concepts

3. Overture - I • Aderem - keynote • wide array of data and techniques • time-course expression data • motif finding • kinetic model • microfluidics • Can manipulate single cell • RT-PCR and sensing with cell

4. Overture - II • Nir Friedman: Variety of regulatory relations between a pair of TFs in yeast • Mike Levine: Conserved kernel circuit, variable input • Rual: Human protein interaction network • Berman: Cross-species expression in yeast - relate back to motifs • Tenenbaum: Post-transcr. Elements, select RNA by RNPs

5. Computational Methods • Wasserman: PAZAR - bazaar for TF info • Benham: opening shifted by TF clamping • Weng: bi-d prom, different TF sharing • Segal: nucleosome positioning, Position of conserved sites relative to nuc • Bussemaker,Tanay: Using weaker ChIP-chip sites to get better model • Blanchette: same as UPenn • Andrews: gene toxicity w/ over expr; enriched for TF, cell cycle, and signalling • Williams: large db of mouse cross pheno, including expression and morphology

6. Posters • Sandelin - new RIKEN T-S TSS x106 • 4-5 CRM finders (inc. me) • Plant work

7. Identification and characterization of active promoters in the human genome. Bing Ren et al., Nature 436, 876-880 (11 August 2005)

8. Use of multiple promoters by human genes To verify experimentally our observations regarding multiple promoter use in IMR90 cells, we selected the WEE1 gene for further analysis. Two TFIID-binding sites were mapped within this gene, corresponding to the 5' ends of two distinct mRNAs, NM_003390 and AK122837 (Fig. 3a). Each mRNA encodes a distinct protein: one encodes a well-characterized full-length version of WEE1 protein, and the other only the kinase domain. We detected both transcripts in a steady-state, asynchronous population of IMR90 cells (Fig. 3b). The shorter transcript appears to be most abundant in the G0 phase, and the longer transcript is highly transcribed in both G0 and S phase (Fig. 3c), suggesting that the two promoters in the WEE1 gene might have distinct cell-cycle functions. Tissue Specific Promoters! Bing Ren et al., Nature 436, 876-880 (11 August 2005)

9. ChIP-PET The chromatin was fragmented by sonication. Immunoprecipitation using a specific antibody was used to capture the transcription factor bound to target sites (shown in red). The ChIP-enriched DNA was first cloned into a plasmid-based library, and we then used restriction enzymes to transform this original library into one that contained concatenated paired-end ditag (PET) sequences13. Each tag is 18 bp in length, and each ditag represents the 5'-most and 3'-most ends of the ChIP-enriched DNA fragments cloned into the original library. This second library increases the throughput of analysis, as each sequencing read identifies 10 to 15 PETs representative of 10 to 15 ChIP-enriched genomic fragments. We refer to this as the ChIP-PET methodology14. The concatenated PETs were sequenced and their locations were mapped to the mouse genome to demarcate the boundaries of transcription factor ChIP-enriched DNA. PET overlaps of four or more members were empirically determined to be high-confidence transcription factor binding sites. Random recovery of genomic DNA was observed in the form of PET singletons. To further establish the importance of the selective downstream targets of Oct4 and Nanog, we depleted the transcripts encoding these factors by RNAi and demonstrated their roles in maintaining ES cells in a nondifferentiated state. TFBS, transcription factor binding site. Nature Genetics 38, 431 - 440 (2006), Genome Institute of Singapore, Singapore

10. Description of SAGE Serial analysis of gene expression (SAGE) is a method for comprehensive analysis of gene expression patterns. Three principles underlie the SAGE methodology: A short sequence tag (10-14bp) contains sufficient information to uniquely identify a transcript provided that that the tag is obtained from a unique position within each transcript; Sequence tags can be linked together to from long serial molecules that can be cloned and sequenced; and Quantitation of the number of times a particular tag is observed provides the expression level of the corresponding transcript. www.sagenet.org

11. Identification of non-conserved transcriptional regulatory elements by histone acetylation (poster 122) Colocalization of acetylation islands with known regulatory elements. (A) CD4 locus. The upper panel shows the acetylation data, above which gene positions and known functional regulatory elements are indicated. The lower panel shows the VISTA human and mouse sequence comparison. (DE) Distal enhancer; (PE) proximal enhancer; (Pr) promoter; (Sil) silencer; (LCR) locus control region; (TE) thymocyte enhancer. The acetylation islands colocalized with known regulatory elements are highlighted in pink. The acetylation islands with no known functions are highlighted in green. (B) CD8 locus. The acetylation data and VISTA sequence analysis are shown as in A. The positions of the six clusters of DNase hypersensitive sites (HS) are indicated below the genes. Active chromatin domains are defined by acetylation islands revealed by genome-wide mapping. GENES & DEVELOPMENT 19:542-552, 2005

12. Optical Mapping it is a single molecule approach for the construction of ordered restriction maps. Optical Mapping dispenses with electrophoretic approaches, and uses light microscopy to directly image individual DNA molecules which are bound to specially derivatized surfaces and then cleaved by restriction enzymes. Importantly, cleaved fragments retain their original order, and cut sites are flagged by small, visible gaps. Optical Mapping solves the problem of determining fragment order, and in addition, works with only a handful of molecules. By determining the existence of these sequence-specific cut sites and the distances between them, we can create a landmark map of the DNA sequence. Such restriction enzyme maps also provide a useful scaffold for the alignment and verification of sequence data and are of particular value in assembling sequence from problematic DNA regions. Our laboratory collaborates extensively with genome centers to expedite the sequencing projects of both human and microbial genomes. Efforts in our laboratory, over the last two years have been to automate Optical Mapping, with the goal of creating high throughput systems for the analysis of a wide range of sample types. This work has entailed the fusion of advanced optical, and biochemical techniques with novel statistical and algorithmic developments. http://www.lmcg.wisc.edu/omm/omm.html

13. TF/DNA Interaction Detection • Farnham: chromatin mods over 200KB • Ren: C-C to identify histone modification code, patterns around TSS and enhancers • Myers: CpG methylation, pseudo-genes too • Ng: Oct4/nanog in ES, may reg 10k genes? • Buck: chromatin modifications with glucose • Van Steensal: hot-spot for C-C TF binding. • Walhout: Ce 650 TFs in yeast1 hybrids -> 283 PDIs

14. Post-transcriptional Networks • Keene: 3000 RBP, C-C-like to identify targets • Dikstein: core promoter element to identify novel motifs with post-t consequences, found my YY1 motif • Bailey-Serres: in plants, identifying number of ribos on mRNA as a function of stress, not all equally lower • Zaller: cMyc w/ C-PET… • Pe’er: SNPs explaining Scvs wild-type, chromatin mods • Rinn: Hs skin samples indentify 2-axis Hox code • Snyder: protein P+ testing on a chip, also network motifs • Le’Cuyer: mRNA localization in Dm hi-t high-resolution

15. Comparative Genomics • Mango: Ce gut development, time-gradient implies PHA-4 site strength controls stage of expression • Stubbs: Zn finger TFs • Kellis: oligo enum is proms and UTRs, tiss-spec, motifs evolution in fly. • Rubin: pufferfish/hs elements embryonic expression eval. - will validate predictions; gut-less worm symbiont sequencing • Ideker: protein networks query tool, Sc DNA damage, KO data does not correspond to transcriptional response • Gaul: Stubb in fly development, conservation - sites move • Lassig: combination of neutral drift and non-eq, fastest in prom + UTR • Sperling: clustering of tissue-expressed gene pairs

16. Emerging Tech & Concepts • Silver: synthetic biology to count cell division cycles. • Schwartz: optical mapping • Lim: miRNA targets, widely active, related to tissue-specificity • Keränen: beautiful digitized Dm embryo TF expression patterns • David: miRNA targets with functional and UTR-length correlates • Lee: CFP-TF cell sorting, proms are almost enough to explain expression

17. Take-Home Messages • ChIP-* • new techniques,lots of data, new analyses, conflicting results, ENCODE • miRNA • Methods of regulation • Post-transcriptional effects • Pre-transcriptional effects • Understanding Evolution