Analyses of Regulatory Regions of Human TNFAIP3 Gene with Humanized Mouse Models

Mark LiberHospital for Special Surgery

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

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

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-kappaBand cell death responses in A20-deficient mice. Science 289, 2350–2354

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

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

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-/-)

GWAS SNPs

1. RP11-10J5

2. CTD-2657E11 3. RP11-953L22

CD14+

Unstim.

TNF 24h

TT to

A

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

Simplified figure of Our BACs

3’TNFAIP3

Δ-DOWN BAC

Δ-UP BAC

1. Δ-Down BAC: missing regulatory elements downstream of the TNFAPI3 gene2. WT-BAC: is centered around the TNFAIP3 gene3. Δ-Up BAC: missing regulatory elements upstream of the TNFAIP3 gene

WT-BAC

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

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

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

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. RP11-953L22ΔUP-BAC

WT-BAC

A20 gene

Conclusion

2. CTD-2657E11ΔDOWN-BAC

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

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