Molecular Mechanisms of Lupus Susceptibility Allele PBX1D

Abstract

Pre-B cell leukemia homeobox 1 (PBX1) controls chromatin accessibility to a large number of genes in various cell types. Its dominant negative splice isoform, PBX1D, which lacks the DNA and Hox-binding domains, is expressed more frequently in the CD4+ T cells from lupus-prone mice and patients with systemic lupus erythematosus than healthy control subjects. PBX1D overexpression in CD4+ T cells impaired regulatory T cell homeostasis and expanded inflammatory CD4+ T cells. In this study, we showed that PBX1 message expression is downregulated by activation in CD4+ T cells as well as in B cells. PBX1D protein was less stable than the normal isoform, PBX1B, and it is degraded through the ubiquitin-proteasome-dependent pathway. The DNA binding domain lacking in PBX1D has two putative ubiquitin binding sites, K292 and K293, that are predicted to be in direct contact with DNA. Mutation of K292-293 reduced PBX1B stability to a level similar to PBX1D and abrogated DNA binding. In addition, contrary to PBX1B, PBX1D is retained in the cytoplasm without the help of the cofactors MEIS or PREP1, indicating a different requirement for nuclear translocation. Overall, these findings suggest that multiple post-transcriptional mechanisms are responsible for PBX1D loss of function and induction of CD4+ T cell inflammatory phenotypes in systemic lupus erythematosus.

FIGURE 1.. Activation decreased Pbx1 expression in CD4⁺ T cells and B cells.

A. Pbx1 expression normalized to Ppia in naïve CD4⁺ T cells isolated from B6 or CD4-Pbx1-d (Tg) mice and activated with anti-CD3 and anti-CD28 antibodies for 24 h. Comparison with paired t tests between naïve (n) and activate (act) cells for each strain. N = 6. B. Pbx1 expression normalized to Ppia in B6 B cells either unstimulated, or stimulated with anti-IgM, anti-CD40 and IL-4, or LPS. Comparison with Dunn’s multiple comparison tests. Mean ± SEM, **: P < 0.01, ***: P < 0.001.

FIGURE 2.. PBX1D protein is less stable than PBX1B.

A. HEK293T were transfected either pcDNA, pcDNA-PBX1B, or pcDNA-PBX1D plasmid for 48 h. PBX1 expression normalized to β-Actin was analyzed by Western blot analysis of 5 and 10 μg of total protein. J-PBX1B or J-PBX1D cells were treated with either DMSO or CHX for indicated time points. PBX1 expression normalized to β-Actin was analyzed by Western blot analysis. Representative Western blot (B) and quantification normalized to β-Actin relative to the starting amount (C) from three independent experiments. mean± SEM, N = 3, 2-way ANOVA, ***: P < 0.001.

FIGURE 3.. PBX1D accelerated degradation is mediated by the Ub-proteasome pathway.

(A – B) PBX1 expression was analyzed by Western blot in J-PBX1B and J-PBX1D cells treated with CHX in the presence or absence of proteasome inhibitor MG132 for 6 and 12 h. (A) Representative Western blot images. (B) Quantification of PBX1 expression normalized to β-Actin relative to the starting amount from three independent experiments. Mean ± SEM (N = 3 J-PBX1D and 4 J-PBX1B). The J-PBX1D samples were compared with 2-way ANOVA. The J-PBX1B samples at 12 h were compared with a t test. (C) The blot shown in (A) was re-probed with an anti-Ub antibody. (D) PBX1 expression was analyzed by Western blot in J-PBX1B and J-PBX1D cells treated with CHX in the presence or absence of USP9X inhibitor WP1130 for 6, 12 and 24 h. The blot was re-probed with anti-USP9X and anti-Ub antibodies.

FIGURE 4.. Putative lysine ubiquitination sites and nuclear localization sequences in PBX1B

(A) Amino acid sequence alignment between PBX1B (top lane) and PBX1D (bottom lane). Residues in red are proposed ubiquitination target lysine sites. Two nuclear localization signal sequences (NLS) are identified by the blue boxes. (B) Crystal structure of the PBX1 homeodomain bound to DNA (PDB code 1PUF). PBX1 is shown as a cartoon with a transparent surface: cyan for carbon, blue for nitrogen and red for oxygen. Lysine residues at positions 287, 292 and 293 and shown in red. DNA is shown with the backbone in gold.

FIGURE 5.. K292–293R mutations reduced PBX1B stability and DNA binding activity

The K292 and K293 lysine residues in PBX1B were substituted with arginine, and the PBX1B (K292–293R) mutant was overexpressed in Jurkat cells. (A – B) PBX1 protein stability was analyzed by Western blot in J-EV, J-PBX1B and J-PBX1B(K292–293) cells treated with CHX for 6 h. The blot was re-probed with anti-Ub antibody (N = 3 – 4) (A) Representative Western blot images. (B) Quantification of PBX1 expression normalized to β-Actin relative to the starting amount from three independent experiments. Mean ± SEM (N = 3 J-PBX1D and 4 J-PBX1B(K292–293) compared with a t test. (C) After immunoprecipitation with anti-PBX1 antibody or isotype control IgG, the PBX1 binding site on the RTKN2 promoter was amplified by qPCR. Results are presented as fold change (FC) relative to the IgG negative control (N = 3 – 4) compared with Šídák’s multiple comparisons tests. Mean ± SEM, *: P < 0.05, **: P < 0.01.

FIGURE 6.. PBX1D requires co-factors for nuclear translocation

(A) Representative images of HEK 293T cells transfected with PBX1A-HA, PBX1B-HA or PBX1D-HA. PBX1 isoforms were stained with anti-HA antibody (red). Scale bar = 5 μm. (B) Cytosol to nucleus ratio of mean intensity of each PBX1 isoforms compared with a t test ****: P < 0.001. (C) Representative images of HEK 293T cells co-transfected with PBX1D-HA and individual cofactors: MEIS2A-GFP, MEIS2B-GFP, MEIX2C-GFP, MEIX2D-GFP, PREP1-Flag as indicated. PBX1 and PREP1 were stained with anti-HA and anti-Flag antibody, respectively. Scale bar = 5 μm. (D) Cytosol to nucleus ratio of mean intensity of PBX1 staining. Statistical significance is shown for a one-way ANOVA. Each point represents a cell, mean ± SEM.