Monica Britton, Ph.D. Sr. Bioinformatics Analyst June 2016 Workshop Introduction to the ChIP-Seq/RNA-Seq Experiment Whose Data We’ll Be Using Monica Britton, Ph.D. Sr. Bioinformatics Analyst June 2016 Workshop

The following slides are adapted Heny O’Geen’s lecture at our 2015 Workshops ….. Global analysis of ZNF217 chromatin occupancy in the breast cancer cell genome reveals an association with ERalpha Frietze S, O'Geen H, Littlepage LE, Simion C, Sweeney CA, Farnham PJ, Krig SR. BMC Genomics. 2014 Jun 24;15:520.

The 20q13.2 locus is amplified in 20-30% of breast tumors The 20q13 locus is one of twenty known “hot spots” found in breast cancer. 20q13 is clinically associated with aggressive disease and poor prognosis. ZNF217: multiple zinc finger motifs, suggested a DNA-binding protein and putative oncogene Hypothesis: ZNF217 overexpression gives a selective advantage by interfering with regulation of cell growth, cell death, differentiation or DNA repair. Collins et al., PNAS 1998

Genome-wide Mapping of ZNF217 in MCF7 cells to elucidate function Distribution of ZNF217 binding sites ENCODE data Promoters Enhancers

ZNF217 binding (33%) at intronic enhancer regions

Motif analysis: ZNF217 binding localizes to GATA, FOXA1 and ER (endocrine response) motifs

Gene Ontology supports ZNF217 co-binding with ER and FOXA1 GREAT: Pathway Commons Category

Heatmap clustering of ENCODE ChIP-seq datasets ZNF217 binding sites cluster with transcription factor binding sites in MCF7 cells.

ZNF217 co-binding with ER, GATA3 and FOXA1

ER co-immunopreciptation identifies 108 ER-associated proteins Mohammed et al., Cell Rep 2013

ZNF and ER protein correlate in breast tumors Actin

Hypothesis: ZNF217 regulates chromatin looping to assist ER gene regulation CtBP TSS ZNF217 RNA Pol II FoxA1 Chromatin Looping >5 kb Model for chromatin looping. Change in the three dimensional structure between the distal ER-bound enhancer region and the proximal promoter allows protein interactions with RNA pol II at the TSS.

Gene expression changes in ZNF217 knock-down cells

Gene expression changes in ZNF217 knock-down cells

Gene Ontology on differentially expressed genes co-bound by ZNF217 and ER Gene Set Enrichment Analysis (GSEA) compares differences between two biological states

Clinical Conclusions ZNF217 overexpression is a biomarker for poorer prognosis in breast cancer patients Prognostic: Identify a subset of ZNF217-overexpressing tumors with worse prognosis in the ER+ Luminal A subtype Predictive: Identify tumors with de novo anti-hormone resistance or more likely to relapse with anti-hormone treatment Hypothesis: ZNF217 overexpression in ER+ breast cancer leads to aberrant ER gene regulation Knowledge of the ZNF217 role in ER biology will have clinical impact ZNF217 overexpression alters ER co-regulator activity at the chromatin level

Our Workshop Exercises Although we will be working with real ChIP-Seq and RNA-Seq data from this experiment, we need our exercises to run very quickly. These fastq files are small subsets of the full data (see paper for NCBI accession info). We will only use chr 20, a single chromosome in the human genome.