10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation1 10/19/05 Gene Regulation (formerly Gene Prediction - 2)

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation2 Gene Prediction & Regulation Mon- Overview & Gene structure review: Eukaryotes vs prokaryotes Wed - Regulatory regions: Promoters & enhancers - Predicting genes Fri - Predicting genes - Predicting regulatory regions Next week: Predicting RNA structure (miRNAs, too)

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation3 Reading Assignment (for Wed) Mount Bioinformatics Chp 9 Gene Prediction & Regulation pp Predicting Promoters Ck Errata: * Brown Genomes 2 (NCBI textbooks online)NCBI textbooks online) Sect 9 Overview: Assembly of Transcription Initiation Complex Sect DNA binding proteins, Transcription initiation * NOTE: Don’t worry about the details!!

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation4 Optional Reading Reviews: 1)Zhang MQ (2002) Computational prediction of eukaryotic protein- coding genes. Nat Rev Genet 3: )Wasserman WW & Sandelin (2004) Applied bioinformatics for the identification of regulatory elements. Nat Rev Genet 5:

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation5 Review last lecture: Genes & Genomes (formerly Gene Prediction - 1) Eukaryotes vs prokaryotes Cells Genome organization Gene structure

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation6 Eukaryotes vs Prokaryotes Eukaryotic cells are characterized by membrane-bound compartments, which are absent in prokaryotes. “Typical” human & bacterial cells drawn to scale. BIOS Scientific Publishers Ltd, 1999 Brown Fig 2.1

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation7 Comparison of Gene Structures BIOS Scientific Publishers Ltd, 1999 Brown Fig 2.2

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation8 Summary: Genes & Genomes (formerly Gene Prediction - 1) Genes in eukaryotes vs prokaryotes Have different structures and regulatory signals Eukaryotic genomes Are packaged in chromatin and sequestered in a nucleus Are larger and have multiple chromosomes Contain mostly non-protein coding DNA (98-99%)

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation9 Eukaryotic genes Are larger and more complex * Contain introns that are “spliced” to generate mature mRNA * Undergo alternative splicing, giving rise to multiple RNAs Are transcribed by 3 different RNA polymerases * In biology, statements such as this include an implicit “usually” or “often” Summary: Genes & Genomes (formerly Gene Prediction - 1)

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation10 Gene regulation in eukaryotes vs prokaryotes Primary level of control? Prokaryotes: Transcription Eukaryotes: Transcription is important, but Expression is regulated at multiple levels e.g., RNA processing, transport, stability, protein processing, post-translational modification, localization, stability Recent discoveries: small RNAs (miRNA, siRNA) may play very important regulatory roles, often at post-transcriptional levels Summary: Genes & Genomes (formerly Gene Prediction - 1)

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation11 Gene prediction? Prokaryotes: relatively “easy” Eukaryotes: harder Genomic organization and gene structures differ in different organisms Best results obtained with “customized” software for a particular species In general: Methods are “good” at locating genes Have trouble with “details” Summary: Genes & Genomes (formerly Gene Prediction - 1)

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation12 DNA Interactive: "Genomes" A tutorial on genomic sequencing, gene structure, genes prediction Howard Hughes Medical Institute (HHMI) Cold Spring Harbor Laboratory (CSHL)

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation13 But first: a few more words about cDNA & ESTs Promoters & enhancers Gene prediction programs (?) Today: Gene Regulation (formerly Gene Prediction - 2)

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation14 Thanks to Jonathan Pevsner for following Figs & Slides Slightly modified from: "Introduction to Bioinformatics" based on Chp 6 in Pevsner's text: Bioinformatics & Functional Genomics J. Pevsner

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation15 Pevsner p161 exon 1exon 2exon 3intron Transcription RNA splicing (remove introns) Capping & polyadenylation Export to cytoplasm AAAAA 3’5’ 3’ 5’3’ 7Me G

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation16 DNARNA cDNA Phenotypeprotein [1] Transcription [2] RNA processing (splicing) [3] RNA export [4] RNA surveillance Pevsner p160

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation17 Relationship of mRNA to genomic DNA (for RBP4) Pevsner p162

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation18 Analysis of gene expression in cDNA libraries A fundamental approach to studying gene expression is through cDNA libraries Isolate RNA (always from a specific organism, region, and time point) Convert RNA to complementary DNA (with reverse transcriptase) Subclone into a vector Sequence the cDNA inserts These are ESTs or Expressed Sequence Tags vector insert Pevsner p

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation19 UniGene: unique genes via ESTs Find UniGene at NCBI: UniGene clusters contain many ESTs UniGene data come from many cDNA libraries. Thus, when you look up a gene in UniGene you get information on its abundance and its regional distribution Pevsner p164

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation20 Cluster sizes in UniGene This is a gene with 1 EST associated; the cluster size is 1 Pevsner p164

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation21 Cluster sizes in UniGene This is a gene with 10 ESTs associated; the cluster size is 10 Pevsner p164

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation22 Cluster sizes in UniGene - (in 2002) Cluster sizeNumber of clusters 134, , , , , , ,0003 >16,0001 Pevsner p164

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation23 Other Resources Current Protocols in Bioinformatics Finding Genes 4.1 An Overview of Gene Identification: Approaches, Strategies, and Considerations 4.2 Using MZEF To Find Internal Coding Exons 4.3 Using GENEID to Identify Genes 4.4 Using GlimmerM to Find Genes in Eukaryotic Genomes 4.5 Prokaryotic Gene Prediction Using GeneMark and GeneMark.hmm 4.6 Eukaryotic Gene Prediction Using GeneMark.hmm 4.7 Application of FirstEF to Find Promoters and First Exons in the Human Genome 4.8 Using TWINSCAN to Predict Gene Structures in Genomic DNA Sequences 4.9 GrailEXP and Genome Analysis Pipeline for Genome Annotation 4.10 Using RepeatMasker to Identify Repetitive Elements in Genomic Sequences

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation24 Gene Regulation Promoters & enhancers What does an RNA polymerase "see"? Eukaryotes vs prokaryotes Regulatory regions Prokaryotic operons & promoters Eukaryotic promoters & enhancers Eukaryotic transcription factors

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation25 What does an RNA polymerase (or a transcription factor) “see” ?

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation26 Promoters for prokaryotic RNA polymerases (e.g., bacterium, E. coli) BIOS Scientific Publishers Ltd, 1999 Brown Fig 9.17

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation27 Prokaryotic genes & operons Genes with related functions are often clustered in operons (e.g., lac operon) Operons are transcriptionally regulated as a single unit - one promoter controls several proteins mRNAs produced are “polycistronic” - one mRNA encodes several proteins; i.e., there are multiple ORFs, each with AUG (START) & STOP codons

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation28 Prokaryotic promoters RNA polymerase complex recognizes promoter sequences located very close to & on 5’ side (“upstream”) of initiation site RNA polymerase complex binds directly to these. with no requirement for “transcription factors” Prokaryotic promoter sequences are highly conserved -10 region -35 region

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation29 Eukaryotic genes Genes with related functions are not clustered, but share common regulatory regions (promoters, enhancers, etc.) Chromatin structure must be in “right” configuration for transcription

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation30 Eukaryotic genes have large & complex regulatory regions Cis-acting regulatory elements include: Promoters,enhancers, silencers Trans-acting regulatory factors include: Transcription factors, chromatin remodeling enzymes, small RNAs BIOS Scientific Publishers Ltd, 1999 Brown Fig 9.26

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation31 Eukaryotic genes are transcribed by 3 different RNA polymerases BIOS Scientific Publishers Ltd, 1999 Brown Fig 9.18

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation32 Eukaryotic promoters & enhancers Promoters located “relatively” close to initiation site (but can be located within gene, rather than upstream!) Enhancers also required for regulated transcription (these control expression in specific cell types, developmental stages, in response to environment) RNA polymerase complexes do not specifically recognize promoter sequences directly Transcription factors bind first and serve as “landmarks” for recognition by RNA polymerase complexes

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation33 Assembly of an initiation complex for eukaryotic RNA polymerase II

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation34 But, it’s actually more complicated: “Activator & Mediator protein” actually represent a large complex of transcription factors (connected via DNA-protein & protein-protein interactions) that are usually associated with clusters of TF binding sites BIOS Scientific Publishers Ltd, 1999 Brown Fig 9.27

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation35 Eukaryotic transcription factors Transcription factors (TFs) are DNA binding proteins that also interact with RNA polymerase complex to activate or repress transcription TFs contain characteristic “DNA binding motifs” TFs recognize specific short DNA sequence motifs “transcription factor binding sites” Several databases for these, e.g. TRANSFACTRANSFAC generegulation.com/cgibin/pub/databases/transfachttp://www. generegulation.com/cgibin/pub/databases/transfac

10/19/05 D Dobbs ISU - BCB 444/544X: Gene Regulation36 Zinc finger-containing transcription factors Common in eukaryotic proteins Estimated 1% of mammalian genes encode zinc-finger proteins In C. elegans, there are 500! Can be used as highly specific DNA binding modules Potentially valuable tools for directed genome modification (esp. in plants) & human gene therapy BIOS Scientific Publishers Ltd, 1999 Brown Fig 9.12

Building “Designer” Zinc Finger DNA-binding Proteins J Sander, Fengli Fu, J Townsend, R Winfrey D Wright, K Joung, D Dobbs, D Voytas (ISU)