1. Analyses of Regulatory Regions of
Human TNFAIP3 Gene with
Humanized Mouse Models
Mark Liber
Hospital for Special Surgery

2. Some Background on A20
• A20’s function is cell-type dependent
• A20 exerts its activities via multiple mechanisms,
including ubiquitin editing, primarily to negatively
regulate proinflammatory signaling
• A20 expression and activity is regulated
transcriptionally
Catrysse,L, et al.A20 in inflammation and autoimmunity. Trends in Immunology, January 2014, Vol. 35, No. 1

3. Some Background on A20
• Human genetic studies (GWAS) have identified A20
as a disease susceptibility gene
– Various SNPs have been associated with
autoimmune disease
• Gene targeting studies confirm the important role of
A20 in controlling tissue homeostasis
Catrysse,L, et al.A20 in inflammation and autoimmunity. Trends in Immunology, January 2014, Vol. 35, No. 1

4. Biological Consequences of A20 Deficiency
• A20-deficient mice die prematurely as a result of
cachexia and severe inflammation and tissue
damage in multiple organs.
• A20-deficient mouse embryonic fibroblasts (MEFs)
and myeloid cells fail to terminate TNF-and LPS-
induced NF-κB activation and show high levels of
proinflammatory cytokines
Lee, E.G. et al. (2000) Failure to regulate TNF-induced NF-kappaB
and cell death responses in A20-deficient mice. Science 289, 2350–
2354

5. A20 acts through various pathways within the cell…
Catrysse,L, et al.A20 in inflammation and autoimmunity. Trends in Immunology, January
2014, Vol. 35, No. 1

6. Catrysse,L, et al.A20 in inflammation and autoimmunity. Trends
in Immunology, January 2014, Vol. 35, No. 1
…which leads to tissue abormalities throughout
the body and autoimmune disease

7. Our Project
• Three different BACs containing different
regulatory regions of the human TNFAIP3
gene were injected into pronuclei of fertilized
mouse eggs of C57BL/6 background
• These 3 groups of humanized mice were born,
raised in our facility, and bred
– Mice were bred to be deficient in mouse A20, but
positive for their specific human A20 (hA20)
transgene (TG+/- A20-/-)

8. GWAS SNPs
1. RP11-10J5
2. CTD-2657E11
3. RP11-953L22
CD14+
Unstim.
TNF 24h
TTtoA
H3K27ac
Our BACs & the their location with respect to the
A20 (TNFAIP3) locus
ΔDOWN-BAC
ΔUP-BAC
WT-BAC
WT-BAC : 5’: 76 kb bp; 3’: 83 kb
D-DOWN-BAC1: 5’: 174 kb ; 3’: 36.7 kb
D-UP-BAC: 5’: 25.6 kb ; 3’: 190 kb
TT>A: 41578 bp downstream of TSS

9. Simplified figure of Our BACs
3’
TNFAIP3
Δ-
DOWN
BAC
Δ-UP
BAC
1. Δ-Down BAC: missing regulatory elements downstream of the TNFAPI3 gene
2. WT-BAC: is centered around the TNFAIP3 gene
3. Δ-Up BAC: missing regulatory elements upstream of the TNFAIP3 gene
WT-BAC

10. Aims
• To identify any phenotypic differences
between the mice of various human BACs and
determine which regulatory regions around
the TNFAIP3 gene are most important for A20
expression and regulation of inflammation

11. Comparing the BACs involved…
• Harvesting and studying primary cells from these
mice
– Measured hA20 RNA and protein expression in bone
marrow-derived macrophages, splenic T & B
lymphocytes, and fibroblast-like synoviocytes (FLS)
• Analyzing of serum autoimmune markers
• Challenging mice with antigens and comparing
their ability to tolerate infection and curtail
inflammation

12. Disease
-Autoantibody presence
-Susceptibility to
infection
A20 expression
-Cellular analyses
C57 Bl/6
WT BAC
Δ-Up BAC
Δ-Down BAC
A20 -/-
Our Findings Summarized
Healthier
Sicker

13. The decrease in mRNA and protein levels in Δ-Down BAC transgenic line
demonstrates that the enhancers located in the region between 36.7-83
kb downstream of TNFAIP3 TSS are required for inducible human A20
expression, and their absence may be at least partially responsible for
RA and SLE-like phenotypes observed
1. RP11-10J5
3. RP11-953L22
ΔUP-BAC
WT-BAC
A20 gene
Conclusion
2. CTD-2657E11
ΔDOWN-BAC

14. Clinical Implications
• Further study of the TNFAIP3 regulatory
elements downstream of the gene may
provide insight into the genetic causes of
phenotypes related to RA and SLE, and
overexpression of these regions may provide
therapeutically beneficial down the road

15. Skills Gained and Utilized
• Animal breeding
• Immunohistochemistry
• Fluorescence microscopy
• Data Analysis
• Scientific presentation
• Creation of mouse bone marrow chimeras
• Mouse intraperitoneal and intraorbital injections
• Utilization of the Quantstudio 3D Digital PCR system (Copy number determination)
• Cell culture with THP1 and IMR90 cell lines
• Primary cell culture (mouse and human monocytes, macrophages, B lymphocytes, T lymphocytes, and fibroblast-
like synoviocytes)
• PBMC purification from human blood
• Quantitative real-time PCR
• Southern blotting
• Western blotting
• Flow cytometry and cell sorting
• ELISA
• ChIP-sequencing
• Genomic DNA purification and amplification
• Restriction enzyme digest
• Mini-and maxipreparations of plasmid DNA for BAC construction