Genotype-dependent binding and regulation of gene expression
by PU.1 at IL10 risk loci for Systemic Lupus Erythematosus
Mehak Chawla; Quinton Smith; Daniel Miller; Carmy Forney; Arthur Lynch; Amber Sauder; Dr. Leah Kottyan; Dr. Matthew Weirauch
Center for Autoimmune Genomics and Etiology, Cincinnati Children’s Hospital Medical Center
Background & Significance
Systemic Lupus Erythematosus (SLE) is an
autoantibody mediated disease
• B cells release complement-fixing autoantibodies
leading to the formation of immune complex deposits
and tissue damage.
• The immune complexes have multi-organ involvement
that leads to systemic inflammation.
• Women of color tend to develop SLE at a younger age,
experience more serious complications, and have higher
mortality rates.
Why PU.1
•Bioinformatics predicts that the immunological
transcription factor PU.1 binds at 33 of the 83 SLE risk loci
in B cells, and we chose to focus on the IL10 locus.
Figure 1 | Expression Quantitative Trait Loci (eQTL) of SNP rs3024490.
SNP rs3024490 is in complete linkage disequilibrium with our SNP of interest
rs3024505. The eQTL shows that there is differential expression between the
risk and non-risk variants of rs3024505. Gene expression is higher in SLE risk
(alternative) than non-risk (reference) (GTEx project data).
Non-coding genetic risk variants around the gene IL10
are a focus of our laboratory
• IL10 is a potent anti-inflammatory cytokine produced by
B cells in a genotype-dependent manner and has been
shown to proliferate the lifespan of B cells
• Genetic variants near the IL10 gene are highly and
robustly associated with SLE.
• Cells with the SLE risk variants near the IL10 gene
express more IL10 mRNA and protein.
Hypothesis
PU.1 binds to a SLE genetic risk variant at the IL10
locus in a genotype-dependent manner and results in
differential IL10 expression.
Our goal: Demonstrate that a SLE risk variant at the IL10
locus binds PU.1 and regulates the expression of IL10 in a
genotype-dependent manner.
Methods
Step 1. Identify which of the genetic variants at the IL10
locus is most significantly associated with SLE
Figure 2| Fine mapping the SLE genetic risk variants around IL10. We
calculated the allele frequencies in people with and without SLE for variants
around IL10 gene and performed regression analysis to determine which
variants are highly associated with SLE, determined by the lowest p-value.
rs3024505 was the most significant.
Step 2. Predict differential binding of PU.1 to the risk
and non-risk versions of rs3024505
Electrophoretic Mobility Shift Assay (EMSA)
• Extract nuclear lysate from cells  expose lysate to
the risk and non risk oligos of the genetic variant
rs3024505  add antibody specific to PU.1 to
identify PU.1 from the mixture of transcription
factors in the lysate.
• If genotype dependent binding is present, the allele
(risk or non-risk) that binds stronger to PU.1 will
show as a darker band on the gel.
Step 3. Perform luciferase reporter assays to
determine genotype-dependent expression of IL10
Insert the IL10 enhancer
with the risk or non-risk
variants of rs3024505
into a luciferase reporter
vector
Transfect these constructs
into separate actively
dividing LCLs
Measure the amount of
luciferase produced to
quantify enhancer activity
Results
Optimization EMSAs for rs3024505
• Ran an EMSA with previously determined DNA-
stabilizing additives that optimized clarity of bands
Figure 3| EMSA with optimized conditions with both risk and non-risk
variants. An EMSA was performed with the optimized conditions
determined in a previous assay. There does not appear to be any significant
difference in bands between the risk and non-risk variants.
• Ran an EMSA with PU.1 recombinant protein to see
if there is any differential binding between the risk
and non-risk variants.
Figure 4| Optimization EMSA with the PU.1 recombinant protein to
determine if there is any genotype-dependent binding of the rs3024505
variant. The EMSA showed that there is no significant difference in the
bands between the risk and non-risk variants with the addition of the PU.1
recombinant protein.
Transfection of constructs into B cells
• Successfully cloned the IL10 enhancer with the risk
and non-risk rs3024505 variants into a luciferase
reporter construct with a minimal promoter.
• Transfection of risk and non-risk constructs into
GM12878 B cells.
Figure 4| Luciferase analysis for the transfection of the constructs into B
cells. Empty vector expressed higher levels luciferase than both plasmid
constructs in GM12878, indicating that there may be an issue with the
construct or that this region is a repressor. Red indicates the risk variant.
Discussion
• Since B-cell lysate does not appear to show genotype-
specific binding between the risk and non-risk variants, a
supershift with anti-PU.1 was not tested.
• However, since numerous TFs bind to DNA around our
SNP of interest, PU.1 can be binding as part of a
transcriptional complex.
• While the EMSA with the PU.1 recombinant protein did
not show differential binding between the risk and non-
risk variants, it does let us know that the PU.1
recombinant protein does bind to DNA which gives us
the ability to test other SLE risk loci for genotype-
dependent binding.
• The preliminary luciferase data did not show increased
luciferase expression of both constructs compared to the
control vector. This may be caused by an improperly
constructed enhancer region or that this region is a
repressor rather than an enhancer.
Future Studies
• Re-visit the genome browser reevaluate the boundaries
of the IL10 enhancer.
• Clone IL10 region in front of a maximal promoter
instead of a minimal promoter to test repressor activity.
• Alternatively, clone IL10 region containing rs3024505
variants in front of a working IL10 endogenous promoter
• Use bioinformatics and ChIP data to determine other
immunological transcription factors that prefer the risk
over the non-risk allele of rs3024505.
References
Lagerfeld et al. Transancestral mapping and genetic load in systemic lupus
erythematosus. Nature Communications. Nature Communications. 2017 Jul
17;8:16021. PMID: 28714469.
Miller et al. Strategy for non-coding variant analysis. Journal of Visualized
Experimentation. Journal of Visualized Experimentation. 2016 Aug; (11).
PMID: 27585267.
Sakurai et al. Preferential binding to Elk-1 by SLE associated IL10 risk allele
upregulates IL10 expression. PLoS Genetics. 2013;9*10):e1003870. PMID:
24130510.
Weirauch et al. Determination and inference of eukaryotic transcription factor
sequence specificity. Cell. 2014 Sep 11; 158(6):1431-1443. PMID: 25215497.