Evaluating Vitamin D Receptor Binding Site

ChIP-Seq Data for Novel Motifs, Histone

Modifications, and Disease-Associated SNPs

Jane Shmushkis
GCB535 Spring 2017
Healthy vitamin D levels are essential to good overall health.

➔ Produced by the skin

➔ Converted to active form
(calcitriol) by liver & kidneys
➔ Low levels linked to:
◆ Multiple sclerosis
◆ Rheumatoid arthritis
◆ Type 1 diabetes
◆ Autoimmune disorders
➔ Vitamin D deficiency: 1 million
worldwide
How vitamin D alters gene expression is poorly understood.

Signaling via calcitriol binding to

vitamin D receptor (VDR)
↓
Dimerizes with retinoid X
receptors (RXR)
↓
Heterodimer binds to genome
sequences
↓
Alters transcription
How does vitamin D alter gene expression?

‘’

GSE27438 GSE22484
Cells: THP-1 human monocytic leukemia cells Cells: Lymphoblastoid cell lines GM10855
Treatment: 1α, 25-dihydroxyvitamin D3 Treatment: calcitriol
Mapped to: hg19 Mapped to: hg18
Method: ChIP-seq with VDR antibody Method: ChIP-seq with VDR antibody
How does vitamin D alter gene expression?

Aim 1 Aim 2
Identify ChIP-seq peaks common to Identify de novo motifs using locations
both datasets of common peaks

Aim 3 Aim 4
Identify whether common VDR sites Determine whether common peaks
overlap with chromatin marks overlap with disease-associated SNPs
Aim 1: Identify peaks common to both datasets

Workflow Results
Downloaded ChIP-seq peaks from 258 peaks in both treated datasets
Genome Expression Omnibus Out of 1820 THP-1 leukemia peaks
↓ & 2864 lymphoblastoid peaks
Converted to same assembly
100 peak in both control datasets
mapping with liftOver
Out of 1169 THP-1 leukemia peaks
↓
& 1952 lymphoblastoid peaks
$ bedtools intersect
Aim 2: Identify de novo motifs using common peaks

Workflow
HOMER motif analysis
$ findMotifsGenome.pl
[common_peaks.bed] hg18 MotifOutput/ -
size 200 -mask -preparsedDir
parsed_genome -len 8
↓
Identified top known &
de novo motifs
Aim 2: Identify de novo motifs using common peaks
HOMER Known motifs enrichment results for common control peaks
% of Target % of Background
Rank Motif Name P-value
sequences with Motif sequences with Motif

1 NFY(CCAAT)/Promoter/Ho 1e-11 32.29 7.87

mer

2 Elk4(ETS)/Hela-Elk4-ChIP- 1e-7 30.21 10.32

Seq(GSE31477)/Homer

3 CTCF(Zf)/CD4+-CTCF- 1e-6 10.42 1.35

ChIP-
Seq(Barski_et_al.)/Homer

4 Fli1(ETS)/CD8-FLI-ChIP- 1e-5 34.38 14.67

Seq(GSE20898)/Homer

5 ETS(ETS)/Promoter/Homer 1e-5 19.79 5.53

Aim 2: Identify de novo motifs using common peaks
HOMER Known motifs enrichment results for common treatment peaks
% of Target % of Background
Rank Motif Name P-value
sequences with Motif sequences with Motif

1 VDR(NR),DR3/GM10855- 1e-75 36.07 1.97

VDR+vitD-ChIP-
Seq(GSE22484)/Homer

2 Etv2(ETS)/ES-ER71-ChIP- 1e-13 26.94 9.07

Seq(GSE59402)/Homer(0.967)

3 ETS1(ETS)/Jurkat-ETS1-ChIP- 1e-13 28.77 10.34

Seq(GSE17954)/Homer

4 Fli1(ETS)/CD8-FLI-ChIP- 1e-13 31.05 11.87

Seq(GSE20898)/Homer

5 PU.1-IRF(ETS:IRF)/Bcell- 1e-10 29.22 12.23

PU.1-ChIP-
Seq(GSE21512)/Homer
Aim 2: Identify de novo motifs using common peaks
HOMER de novo motifs results for common control peaks

HOMER de novo motifs results for common treatment peaks

Aim 3: Identify whether peaks overlap with chromatin marks

Workflow Results
Histone mark ChIP-seq from ENCODE Significant overlaps with H3K4me3
Homo sapiens sigmoid colon tissue female adult (53 y)
↓ 87/258 peaks
Downloaded bed narrowPeak format overlapped for treatment
H3K4me3, H3K9me3, H3K4me1 P-value: 1.8263e-93

↓ 96/100 peaks
$ bedtools fisher ... -g
overlapped for control
human.hg19.sorted.genome
P-value: 7.4975e-173
$ bedtools intersect
Aim 4: Identify overlaps with disease-associated SNPs

Workflow Results
GWAS Catalog for SNPs NOTHING!!!!
Celiac, Crohn’s, Multiple Sclerosis, Type 1 Diabetes None of the common peaks mapped to a
↓ disease-associated SNP
Adjusted SNP lists to only include ↓
chr# start_base end_base Checked for overlaps in each dataset
↓
Lymphoblastoid
Converted common peaks to hg19
Crohn’s, p-value: 0.00018326, 4/2860 peaks
assembly with liftOver Type 1 Diabetes, p-value 0.007524, 2/2862 peaks
↓
$ bedtools fisher ... -g THP-1 Leukemia
human.hg19.sorted.genome Multiple Sclerosis, p-value: 0.020834, 2/1818
$ bedtools intersect peaks
Future Directions

➔ Further investigation into mapped chromosome locations

➔ Compare more human ChIP-seq datasets with vitamin D treatment
➔ Investigate more disease-associated SNPs
◆ Rheumatoid arthritis
◆ Skin cancer
◆ Breast cancer
➔ Investigate more histone markers
References
1. Handel, Adam E., et al. "Vitamin D receptor ChIP-seq in primary CD4+ cells: relationship to serum 25-
hydroxyvitamin D levels and autoimmune disease." BMC medicine 11.1 (2013): 163.

1. Ramagopalan, Sreeram V., et al. "A ChIP-seq defined genome-wide map of vitamin D receptor
binding: associations with disease and evolution." Genome research 20.10 (2010): 1352-1360.

1. Heikkinen, Sami, et al. "Nuclear hormone 1α, 25-dihydroxyvitamin D3 elicits a genome-wide shift in
the locations of VDR chromatin occupancy." Nucleic acids research 39.21 (2011): 9181-9193.

2. Lee, Seong Min, et al. "1, 25-Dihydroxyvitamin D3 controls a cohort of vitamin D receptor target genes
in the proximal intestine that is enriched for calcium-regulating components." Journal of Biological
Chemistry 290.29 (2015): 18199-18215.