Functional Genomics Research Stream Lecture: May 5, 2009 The Road to Publication
Final Lab Issues Lab Hours this Week: Wednesday 10 AM - 6 PM Thursday 10 AM - 6 PM One Hour Required (sign in, sign out) Dispose of ALL Resources (plates, reagents) Cleaning & Organization Assignments
Research Stream Party Monday, May 11-7:00 PM Wahoo s Fish Tacos (509 Rio Grande St.)
663/Lake Austin 101 1L/1M
The Research Plan
How Do Transitions Occur?
How Do Transitions Occur? general answer regulation
How Do Transitions Occur? general answer regulation protein modification
How Do Transitions Occur? general answer regulation protein modification histone occupancy
How Do Transitions Occur? general answer regulation protein modification histone occupancy histone modification
How Do Transitions Occur? general answer regulation protein modification histone occupancy histone modification transcriptional regulation
How Do Transitions Occur? general answer regulation protein modification histone occupancy histone modification transcriptional regulation gene activation
How Do Transitions Occur? general answer regulation protein modification histone occupancy histone modification transcriptional regulation gene activation gene repression
How Do Transitions Occur? general answer regulation protein modification histone occupancy histone modification transcriptional regulation gene activation gene repression Which cellular molecules mediate transcriptional regulation?
Transcription Factors Protein that binds specific DNA sequences controlling the activation or repression of transcription. transcription start transcription stop cis-regulatory sequence
Transcription Factors Protein that binds specific DNA sequences controlling the activation or repression of transcription. transcription start transcription stop cis-regulatory sequence
Transcription Factors Protein that binds specific DNA sequences controlling the activation or repression of transcription. transcription start transcription stop cis-regulatory sequence mrna produced AAAAAAAAAAAA
One Gene transcription start transcription stop cis-regulatory sequence mrna produced AAAAAAAAAAAA
Whole Genome (Saccharomyces cerevisiae has 16 chromosomes + mitochondrial DNA) 1000 200K transcription start transcription stop cis-regulatory sequence mrna produced AAAAAAAAAAAA
Whole Genome (Saccharomyces cerevisiae has 16 chromosomes + mitochondrial DNA) 1000 200,000 I II III IV V VI VII VIII I I II III IV V VI M
Whole Genome (Saccharomyces cerevisiae has 16 chromosomes + mitochondrial DNA) 1000 200,000 I II III IV V VI VII VIII I I II III IV V VI M
The Experiment (ChIP-seq)
(1) cross-link proteins to genomic DNA
(1) cross-link proteins to genomic DNA (2) shear genomic DNA
(1) cross-link proteins to genomic DNA (2) shear genomic DNA (3) affinity purify transcription factors
(1) cross-link proteins to genomic DNA (2) shear genomic DNA (3) affinity purify transcription factors (4) reverse the cross-links
(1) cross-link proteins to genomic DNA (2) shear genomic DNA (3) affinity purify transcription factors (4) reverse the cross-links (5) purify DNA fragments
(1) cross-link proteins to genomic DNA (2) shear genomic DNA (3) affinity purify transcription factors (4) reverse the cross-links (5) purify DNA fragments CGATTACGCGCGATTTTACGGACACACCCC TTACGAAAACTGCATATAGCCAGTCACCCT (5) sequence DNA fragments
CGATTACGCGCGATTTTACGGACACACCCC TTACGAAAACTGCATATAGCCAGTCACCCT (5) sequence DNA fragments
CGATTACGCGCGATTTTACGGACACACCCC TTACGAAAACTGCATATAGCCAGTCACCCT (5) sequence DNA fragments (6) map DNA sequences to genome CCGGTTCGATTACGCGCGATTTTACGGACA CCGGTTCGATTACGCGCGATTTTACGGACA GGTTCGATTACGCGCGATTTTACGGACACA GGTTCGATTACGCGCGATTTTACGGACACA TTCGATTACGCGCGATTTTACGGACACACC TTCGATTACGCGCGATTTTACGGACACACC CGATTACGCGCGATTTTACGGACACACCCC CGATTACGCGCGATTTTACGGACACACCCC GACATTTACGAAAACTGCATATAGCCAGTC GACATTTACGAAAACTGCATATAGCCAGTC ATTTACGAAAACTGCATATAGCCAGTCACC ATTTACGAAAACTGCATATAGCCAGTCACC TTACGAAAACTGCATATAGCCAGTCACCCT TTACGAAAACTGCATATAGCCAGTCACCCT CGAAAACTGCATATAGCCAGTCACCCTAAA CGAAAACTGCATATAGCCAGTCACCCTAAA
CGATTACGCGCGATTTTACGGACACACCCC TTACGAAAACTGCATATAGCCAGTCACCCT (5) sequence DNA fragments (6) map DNA sequences to genome CCGGTTCGATTACGCGCGATTTTACGGACA CCGGTTCGATTACGCGCGATTTTACGGACA GGTTCGATTACGCGCGATTTTACGGACACA GGTTCGATTACGCGCGATTTTACGGACACA TTCGATTACGCGCGATTTTACGGACACACC TTCGATTACGCGCGATTTTACGGACACACC CGATTACGCGCGATTTTACGGACACACCCC CGATTACGCGCGATTTTACGGACACACCCC GACATTTACGAAAACTGCATATAGCCAGTC GACATTTACGAAAACTGCATATAGCCAGTC ATTTACGAAAACTGCATATAGCCAGTCACC ATTTACGAAAACTGCATATAGCCAGTCACC TTACGAAAACTGCATATAGCCAGTCACCCT TTACGAAAACTGCATATAGCCAGTCACCCT CGAAAACTGCATATAGCCAGTCACCCTAAA CGAAAACTGCATATAGCCAGTCACCCTAAA (7) establish consensus sequences CCGGTTCGATTACGCGCGATTTTACGGACA CCGGTTCGATTACGCGCGATTTTACGGACA GGTTCGATTACGCGCGATTTTACGGACACA GGTTCGATTACGCGCGATTTTACGGACACA TTCGATTACGCGCGATTTTACGGACACACC TTCGATTACGCGCGATTTTACGGACACACC CGATTACGCGCGATTTTACGGACACACCCC CGATTACGCGCGATTTTACGGACACACCCC GACATTTACGAAAACTGCATATAGCCAGTC GACATTTACGAAAACTGCATATAGCCAGTC ATTTACGAAAACTGCATATAGCCAGTCACC ATTTACGAAAACTGCATATAGCCAGTCACC TTACGAAAACTGCATATAGCCAGTCACCCT TTACGAAAACTGCATATAGCCAGTCACCCT CGAAAACTGCATATAGCCAGTCACCCTAAA CGAAAACTGCATATAGCCAGTCACCCTAAA
CGATTACGCGCGATTTTACGGACACACCCC TTACGAAAACTGCATATAGCCAGTCACCCT (5) sequence DNA fragments (6) map DNA sequences to genome CCGGTTCGATTACGCGCGATTTTACGGACA CCGGTTCGATTACGCGCGATTTTACGGACA GGTTCGATTACGCGCGATTTTACGGACACA GGTTCGATTACGCGCGATTTTACGGACACA TTCGATTACGCGCGATTTTACGGACACACC TTCGATTACGCGCGATTTTACGGACACACC CGATTACGCGCGATTTTACGGACACACCCC CGATTACGCGCGATTTTACGGACACACCCC GACATTTACGAAAACTGCATATAGCCAGTC GACATTTACGAAAACTGCATATAGCCAGTC ATTTACGAAAACTGCATATAGCCAGTCACC ATTTACGAAAACTGCATATAGCCAGTCACC TTACGAAAACTGCATATAGCCAGTCACCCT TTACGAAAACTGCATATAGCCAGTCACCCT CGAAAACTGCATATAGCCAGTCACCCTAAA CGAAAACTGCATATAGCCAGTCACCCTAAA (7) establish consensus sequences CCGGTTCGATTACGCGCGATTTTACGGACA CCGGTTCGATTACGCGCGATTTTACGGACA GGTTCGATTACGCGCGATTTTACGGACACA GGTTCGATTACGCGCGATTTTACGGACACA TTCGATTACGCGCGATTTTACGGACACACC TTCGATTACGCGCGATTTTACGGACACACC CGATTACGCGCGATTTTACGGACACACCCC CGATTACGCGCGATTTTACGGACACACCCC GACATTTACGAAAACTGCATATAGCCAGTC GACATTTACGAAAACTGCATATAGCCAGTC ATTTACGAAAACTGCATATAGCCAGTCACC ATTTACGAAAACTGCATATAGCCAGTCACC TTACGAAAACTGCATATAGCCAGTCACCCT TTACGAAAACTGCATATAGCCAGTCACCCT CGAAAACTGCATATAGCCAGTCACCCTAAA CGAAAACTGCATATAGCCAGTCACCCTAAA (8) elucidate peaks
Whole Genome (Saccharomyces cerevisiae has 16 chromosomes + mitochondrial DNA) 1000 200,000 I II III IV V VI VII VIII I I II III IV V VI M
Whole Genome (Saccharomyces cerevisiae has 16 chromosomes + mitochondrial DNA) 1000 200,000 I II III IV V VI VII VIII I I II III IV V VI M
Whole Genome (Saccharomyces cerevisiae has 16 chromosomes + mitochondrial DNA) 1000 200,000 I II III IV V VI VII VIII I I II III IV V VI M
Which Transcription Factors? Characterized Cell Cycle Regulators MBP1 MCM1 ACE2 SWI4 FKH1 FKH2 transcription start transcription stop cis-regulatory sequence
Which Transcription Factors? Characterized Cell Cycle Regulators MBP1 MCM1 ACE2 SWI4 FKH1 FKH2 transcription start transcription stop cis-regulatory sequence
How Did I Choose These Factors? Cell (2001) vol. 106 (6) pp. 697-708
Cell (2001) vol. 106 (6) pp. 697-708
sample ~100 ml at each time point
1 2 9 sample ~100 ml at each time point 3 8 4 7 5 6
Possible Cell States
Synchronicity
sample ~100 ml at each time point
1 2 sample ~100 ml at each time point 9 3 8 4 7 5 6
What is the end goal?
1 2 Experimental Data Collected 1 2 3 4 5 6 7 8 9 9 3 MBP1 MCM1 8 4 ACE2 FKH2 7 5 SWI4 FKH1 6 RNA
research article High resolution genome-wide characterization of cell cycle transcriptional regulation and response Sophia Ali 1,2, Amanda Cady 1,2, John Cuenca 1,2, Lauren Fairchild 1,2, Andrew Hertsenberg 1,2, Michael Gonzales 1,2, Elizabeth Joslin 1,2, Bradley Koshy 1,2, Elizabeth Lessels 1,2, Huadan Liu 1,2, Robert McKenzie 1,2, Kristy Moryan 1,2, Andrew Mueller 1,2, Monica Noori 1,2, Christine Nguyen 1,2, Thomas Nguyen 1,2, Caroline Pham 1,2, Kayla Purcell 1,2, Lauren Rego 1,2, Ronak Shah 1,2, Alissa Shelton 1,2, Abel Silva 1,2, Jacquelyne Spear 1,2, Emeline Sukamtoh 1,2, Matthew Tien 1,2, Madeline Waggoner 1,2, Sebastien Weyn 1,2, Morgan Williams 1,2, Lauren Wolfe 1,2, Vishwanath R Iyer 2,3, Patrick J Killion 2,3,4 1. Contributed equally to this work. 2. College of Natural Sciences, Freshman Research Initiative, Functional Genomics Research Stream, University of Texas at Austin, 1 University Station A4800, Austin, Texas 78712 3. Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas at Austin, 1 University Station A4800, Austin, Texas 78712 4. Corresponding Author Global analysis of transcription factor-mediated regulation of cell cycle progression and transcriptional response is characterized in unprecedented resolution using high-throughput next-generation (Solexa) sequencing. In this study we have utilized affinity tagged strains of Saccharomyces cerevisiae to characterize genome-wide transcription factor binding (ChIP-seq) of several key cell cycle regulators (ACE2, FKH1, FKH2, MBP1, MCM1, SWI4). Synchronized cultures were harvested at time-points coinciding with cell cycle stages and transitions in order to capture both steady state patterns of binding as well as regulator events that facilitate the transition of cell cycle stages in proliferating cells. Simultaneously, quantitative RNA profiling (RNA-seq) has been performed at each time point in order to capture a high-resolution picture of absolute gene expression. These data-sets provide new clarity in the temporal and mechanistic bases of cellular growth and proliferation.
Publication Pitfalls Contamination Issues Time Course Adherence & Consistency ChIP Specificity DNA Purification & Processing RNA Sequencing Process Next Generation Sequencing Overall Ownership & Productivity
Publication Potential Unprecedented resolution of regulatory binding events. Unprecedented correlation of RNA expression during binding event measurement. First time-course application of ChIP-seq in Saccharomyces cerevisiae. First next-generation sequence-based measurement of RNA expression (RNA-seq). Of immediate value to genomics community (see Hu & Killion 2007, Harbison 2004, Spellman 1999).
Questions?