1. 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

2. 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 Jun 24;15:520.

3. 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

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

6. ZNF217 binding (33%) at intronic enhancer regions

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

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

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

10. ZNF217 co-binding with ER, GATA3 and FOXA1

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

12. ZNF and ER protein correlate in breast tumors
Actin

13. 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.

14. Gene expression changes in ZNF217 knock-down cells

15. Gene expression changes in ZNF217 knock-down cells

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

17. 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

18. 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.