SP140L, an Evolutionarily Recent Member of the SP100 Family, Is an Autoantigen in Primary Biliary Cirrhosis

Abstract

The SP100 family members comprise a set of closely related genes on chromosome 2q37.1. The widely expressed SP100 and the leukocyte-specific proteins SP110 and SP140 have been associated with transcriptional regulation and various human diseases. Here, we have characterized the SP100 family member SP140L. The genome sequence analysis showed the formation of SP140L gene through rearrangements of the two neighboring genes, SP100 and SP140, during the evolution of higher primates. The SP140L expression is interferon-inducible with high transcript levels in B cells and other peripheral blood mononuclear cells. Subcellularly, SP140L colocalizes with SP100 and SP140 in nuclear structures that are devoid of SP110, PML, or p300 proteins. Similarly to SP100 and SP140 protein, we detected serum autoantibodies to SP140L in patients with primary biliary cirrhosis using luciferase immunoprecipitation system and immunoblotting assays. In conclusion, our results show that SP140L is phylogenetically recent member of SP100 proteins and acts as an autoantigen in primary biliary cirrhosis patients.

Figure 1

Sequence analysis of the SP100 gene family. (a) Genomic region of human chromosome 2 comprising the SP100 gene family. (b) Genomic structure of the SP140L gene. Exons are marked with solid boxes and numbered. Exon 2 (red) is not found in our cDNAs. Exon 11 and a fragment of exon 16 (purple) were spliced in some of our transcripts. (c) Structure of the SP140L protein and its isoforms and comparison to other SP100 family proteins. HSR/CARD (homogenously stained region/caspase recruitment domain), SAND (SP100, AIRE, NucP41/P75, and DEAF), PHD (plant homeodomain type zinc finger), Bromo (bromodomain), and PxVxL (HP1 binding motif). (d) Schematic representation of the putative genomic rearrangements creating the SP140L gene.

Figure 2

Phylogenetic clustering of SP140L protein domains. (a) HSR/CARD, (b) SAND, (c) PHD, and (d) bromodomain of human, chimpanzee, gorilla, orangutan, and macaque SP140L proteins.

Figure 3

Expression analysis of SP140L, SP140, SP100, and SP110 in PBMCs and cell lines. (a) The mRNA expression of the SP100 family members in the subpopulations of PBMCs: pDC (plasmacytoid dendritic cells), MO (monocytes), NK (natural killers), B (B cells), and CD4 (CD4+ T cells). (b) The mRNA expression of the SP100 family members in HeLa, HL60, U937, and THP-1 cell lines. (c) The activation of mRNA expression after interferon stimulation in U937 and HL60 cell lines. The mRNA expressions were detected using RT-qPCR. One representative experiment is shown and the error bars show the standard error of the mean (SEM) of the technical replicates. (d) Ectopic expression of SP140L and SP140 proteins in HEK293. The proteins were detected using anti-Myc and anti-FLAG antibodies, respectively.

Figure 4

Nuclear localization of the SP140L, SP140, SP110, and SP100A proteins in HeLa cells. SP140L (a–c) and SP140 (d–f) have a highly similar localization pattern in HeLa cell nuclei, which is distinctly different from either SP110 (g–i) or SP100A (j–l). Monoclonal anti-Myc antibody was used to detect SP140L and monoclonal anti-FLAG antibody was used to detect SP140, SP110, and SP100A. Scale bar: 5 μm.

Figure 5

Colocalization of SP140L with other SP100 family members. (a–c) SP140L and SP140. (d–f) SP140L and SP110. (g–i) SP140L and SP100A. (j) Cytofluorograms together with Pearson's correlation coefficients for the overlap of red and green pixel intensities correspond to the images on their left. Polyclonal anti-Myc antibody was used to detect SP140L and monoclonal anti-FLAG antibody was used to detect SP140, SP110, and SP100A. Scale bar: 5 μm.

Figure 6

Colocalization of SP140L, SP140, SP110, and SP100A with PML and p300. The nuclear body protein PML does not colocalize with SP140L (a–c), SP140 (d–f), or SP110 (g–i) in HeLa cells. PML colocalizes with its known interaction partner SP100A (j–l). Similarly, the transcriptional coactivator p300 does not colocalize with SP140L (m–o), SP140 (p–r), or SP110 (s–u). Cytofluorograms together with Pearson's correlation coefficients for the overlap of red and green pixel intensities correspond to the images on their left (v). SP140L was detected with monoclonal anti-Myc antibody and anti-FLAG antibody was used to detect SP140, SP110, and SP100A. Polyclonal anti-HA antibody was used to stain HA-PML and HA-p300. Scale bar: 5 μm.

Figure 7

Colocalization of SP140L with endogenous SP100 and PML in HeLa cells. (a–c) Ectopically expressed SP140L does not colocalize with endogenous PML. (d–f) Ectopically expressed SP140L colocalizes with endogenous SP100. (g) Cytofluorograms together with Pearson's correlation coefficients for the overlap of red and green pixel intensities correspond to the images on their left. The proteins were detected with polyclonal anti-Myc (SP140L) and monoclonal anti-SP100 and anti-PML antibodies. Scale bar: 5 μm.

Figure 8

Luciferase reporter gene activation assays with ectopically expressed SP140L, SP140, and SP110 in HEK293 and COS-1. (a) SP140L, SP140, and SP110 fused to GAL4 DNA-binding domain were cotransfected with the luciferase reporter plasmid containing three GAL4 response elements in the promoter. The results represent the mean ± SEM of 3 independent experiments. (b) SP140L, SP140, and SP110 expression plasmids were cotransfected with glucocorticoid receptor (GR) and MMTV-Luc reporter plasmid, and the cells were treated with dexamethasone (Dex). The results represent the mean ± SEM of 3 independent experiments. NC, negative control.

Figure 9

The presence of SP140L-specific autoantibodies in sera from patients with PBC. The LIPS assay detected autoantibodies against SP100A (a), SP140 (b), and SP140L (c) in ten, four, and three patient sera, respectively. Dashed line, 2 standard deviations from the average signal of the control group; LU, light units.