DIAMANTINA INSTITUTE for Cancer, Immunology and Metabolic Medicine

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1 DIAMANTINA INSTITUTE for Cancer, Immunology and Metabolic Medicine Defining MYB Transcriptional Network by Genome-wide Chromatin Occupancy Profiling (ChIP-Seq) 2010 E.Glazov, L. Zhao

2 Transcription Factors: Past & Present 1961 Discovery of LacZ operon in bacteria by Francois Jacob & Jacques Monod DNA binding factors can directly activate or repress genes

3 Transcription Factors: Past & Present 2001 The Human Genome More than 2000 proteins encoded in human genome have DNA-binding domain(s) To understand mechanisms of gene regulation we need to know: Transcription factors Where and when do they interact with DNA? How do they interact with each other?

4 Transcription Factors: Where & When Where? Utilize DNA-binding properties of transcription factors to isolate DNA regions that they bind When? Take a series of snapshots of different developmental stages and cell types

5 Transcription Factors: Where & When Where? Utilize DNA-binding properties of transcription factors to isolate DNA regions that they bind When? Take a series of snapshots of different developmental stages and cell types ChIP-Seq Chromatin ImmunoPrecipitation (ChIP) coupled with Next Generation Sequencing addresses these question on the genome-wide scale

6 ChIP ChIP-Seq Workflow Seq Analysis

7 Chromatin Immunoprecipitation (ChIP) Quality of the ChIP is critical for the success of any ChIP-Seq project: Specificity and affinity of antibodies Level of enrichment of immunoprecipitated chromatin compared to input DNA QC real-time PCR on known target genes

8 Illumina 2 nd Generation Sequencing (Seq) 1. Sample Preparation 2a. Flow Cell 2b. Cluster Generation 6. Publication 5. Data Analysis 4. Initial Image Analyses 3. Sequencing & Imaging

9 ChIP-Seq Data Analyses Workflow P. Park 2009 Nature Rev Genetics

10 Defining MYB Transcriptionall Network Using ChIP-Seq MYB is a transcription factor and an oncogene Key regulator of normal formation of blood cells Highly expressed in immature progenitor cells of all blood cell lineages Actively contributes to leukemias Can block differentiation and transform immature blood cells Activated in human leukaemias due to structural rearrangements (e.g. chromosomal translocation, duplication)

11 MYB is a Dual Action Transcription Factor MYB? Target gene Self-renewal Differentiation Survival

12 MYB Target Genes Direct vs Indirect MYB MYB Over 60 reported Myb target genes (e.g. Bcl2, Mpo) What are the critical direct target genes?

13 Inducible ER-MYB Cell Model + β-estradiol - β-estradiol for 3 days Mouse myeloid progenitor cell line transformed with an inducible version of activated Myb (ER-MYB) Committed to monocytic-macrophage differentiation when Myb is inactivated Partially reversible differentiation process

14 Experiment Design β-estradiol withdrawal re-addition ERMYB cells Myb activity on off off on Time (h) Cross-linked chromatin Total RNA ChIP-Seq Illumina bead array Genome-wide occupancy of Myb Myb regulated genes Identification of direct Myb target genes

15 Sequencing and Mapping Reads Each sample produces million short sequence reads. Sequence reads are mapped to the reference genome Distribution of mapped reads allows identification of enriched peak regions within the genome and binding sites within peaks Review of read aligners and peak callers: S. Pepke, Nature Methods v.6 (11) Oct 2009

16 Data Analysis Alignments and Peak Calling Sequence reads were aligned to mouse genome (UCSC, mm9) using bowtie (Langmead et al. Genome Biol. 2009) Ambiguously mapped reads were excluded Genome-wide peak identification was performed using MACS (Zhang et al. Genome Biol. 2008)

17 Quality Assessment of Sequencing Data Using Known MYB Target Genes IgG - β-e2 Myb off Sequence reads are highly enriched in MYB On replicate samples + β-e2 Myb on

18 Quality Assessment of Sequencing Data Using Known MYB Target Genes + β-e2 Myb on -β-e2 Myb off + β-e2 Myb on - β-e2 Myb off

19 Genome-wide Analysis of MYB Binding Sites P. Park 2009 Nature Rev Genetics Peak normalization and differential analyses were performed using EdgeR package (Robinson et al., 2009) Criteria used in MYB ChIP- Seq analysis: Replication in two samples Background cut-off 10 reads Enrichment ratio 2 fold P-value < 10-5

20 Genome-wide occupancy pattern of Myb We identified 7646 sites occupied by Myb in vivo These sites were associated with 4892 genes

21 Overrepresented sequence motifs in Myb binding regions (MBRs) Myb binding consensus identified by MEME (Tim Bailey, IMB) TRANSFAC MYB

22 Effect of Myb DNA Occupancy on Gene Expression Myb bound genes 4, Myb direct target genes Non-responsive target genes Co-activators and/or corepressors Timing Activated by Myb Repressed by Myb

23 Myb contributes to the LSC maintenance program Gene Enrichment analysis (GSEA, Tamayo, et al PNAS) Up-regulated gene set in LSC from MLL leukaemia Down-regulated gene set in LSC from MLL leukaemia NES: 2.62 FDR: NES: FDR: Somervaille, TC. et al. Cell Stem Cell 4:129 (2009)

24 Summary ChIP-Seq is powerful method for studying DNA-protein interactions BioInformatics - Integrated experiment design and analysis approach produces most informative outcome Future trends simultaneous profiling of multiple transcription factors, chromatin state, and RNA expression.

25 ACKNOWLEDGEMENTS AND CONTACTS Molecular Oncogenesis Group Liang Zhao Diwakar Pattabiraman Faisal Al-Owaidi Thomas Gonda Bioinformatics Ping Zhang Paul Leo Evgeny Glazov DI Musculoskeletal genetics group Phone e.glazov@uq.edu.au