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. 2018 Mar 12;19(1):194.
doi: 10.1186/s12864-018-4573-5.

Identification of a novel Dlg2 isoform differentially expressed in IFNβ-producing plasmacytoid dendritic cells

Shafaqat Ali  1   2 , Alexander Hoven  1 , Regine J Dress  1   3 , Heiner Schaal  4   5 , Judith Alferink  2   6 , Stefanie Scheu  7
Affiliations

Identification of a novel Dlg2 isoform differentially expressed in IFNβ-producing plasmacytoid dendritic cells

Shafaqat Ali et al. BMC Genomics. .

Abstract

Background: The murine discs large homolog 2 (DLG2; post synaptic density 93 (PSD-93); Chapsyn-110) is a member of the membrane-associated guanylate kinase (MAGUK) protein family involved in receptor assembly and associated with signaling enzymes on cell membranes. In neurons, DLG2 protein isoforms derived from alternatively spliced transcripts have been described to bind to NMDA (N-methyl-aspartate) receptors and K channels and to mediate clustering of these channels in the postsynaptic membrane. In myeloid cells of the immune system, such as dendritic cells (DCs), a lack of data exists on the expression or function of DLG2. In cDNA microarray transcriptome analyses, we found Dlg2 highly expressed in a subpopulation of plasmacytoid DCs (pDCs) stimulated to produce type I interferons (IFNs) such as IFNβ.

Results: Using RACE- and RT-PCR as well as immunoprecipitation followed by Western blotting we characterised the differential expression of the Dlg2 splice variants in IFNβ-producing pDCs. Besides Dlg2ɣ this cell population expressed a novel short Dlg2η transcript we termed Dlg2η3. Our expression data were integrated into information from genome databases to obtain a novel and comprehensive overview of the mouse Dlg2 gene architecture. To elucidate the intracellular localisation pattern of protein isoforms, ectopical expression analysis of fluorescently tagged DLG2 splice variants was performed. Here we found an enrichment of the larger isoform DLG2α1 at the plasma membrane while the newly identified shorter (DLG2η) isoform as well as DLG2ɣ were equally distributed throughout the cytoplasm. Additionally, DLG2η was also found in the nucleus. Analysis of Dlg2-knockout mice previously generated by deleting exon 9 surprisingly revealed that the protein for the novel DLG2η isoform was still expressed in the brain and in bone marrow-derived pDCs from mice carrying the homozygous deletion (Dlg2 ΔE9/ΔE9 ).

Conclusion: We describe a novel splice variant of the mouse Dlg2 gene termed Dlg2η and define the differential expression pattern of DLG2 isoforms in IFNβ-producing pDCs. The presence of DLG2η protein in the CNS of Dlg2 ΔE9/ΔE9 mice might influence the phenotype of these mice and has to be taken into account in the interpretation of results regarding the functional role of DLG2 in neuronal postsynaptic membranes.

Keywords: Dlg2; IFNβ; Isoforms; PSD-93; Plasmacytoid dendritic cells.

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Conflict of interest statement

Ethics approval and consent to participate

This study was carried out in strict accordance with the German act for animal welfare (Tierschutzgesetz) §8. The protocol was approved by the local ethics committee, the board for nature, environment and consumer protection (Landesamt für Natur-, Umwelt-, und Verbraucherschutz – LANUV) of the regional government of Düsseldorf (North Rhine-Westphalia, Germany); Permit number 84–02.05.30.13.034. All efforts were made to minimize suffering.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
High expression of DLG2 in IFNβ-producing pDCs. (a) Analysis of Dlg2 expression in splenic pDCs. IFNβ/YFP+ and IFNβ/YFP— splenic pDCs were ex vivo FACS sorted from IFNβmob/mob mice 6 h after i.v. injection with CpG. Dlg2 (white bars) and Ifnb (black bars) mRNA expression was analysed by quantitative RT-PCR. (b) Dlg2 expression in in vitro BM-derived pDCs. BM-derived DCs were generated from IFNβmob/mob mice in Flt3L cultures. IFNβ/YFP+ and IFNβ/YFP— BM-derived pDCs were FACS sorted 6 h after stimulation with CpG. Dlg2 (white bars) and Ifnb (black bars) mRNA expression was analysed by quantitative RT-PCR. Data shown in (a) and (b) is fold mRNA quantity in IFNβ-producing pDCs relative to IFNβ non-producers. Data shown is mean ± standard error of mean (SEM) from four independent experiments (each sample is pooled from 12 (a) or 6 (b) mice. Statistical differences between IFNβ/YFP+ and IFNβ/YFP— were analysed by two tailed, unpaired t test. ns: P > 0.05, *: P < 0.05, **: P < 0.01, ***: P < 0.001. (c) Immunofluorescence images of DLG2 expression in BM-derived Flt3L cultured pDCs left untreated (naïve; upper panel) and 24 h after CpG (CpG; lower panel) stimulation. Cells were stained with antibodies against DLG2 and mPDCA-1 and a YFP-crossreacting antibody against GFP. Data shown is one representative experiment out of three independent experiments with comparable results
Fig. 2
Fig. 2
Analysis of Dlg2 isoforms expressed in IFNβ/YFP+-producing pDCs. (a) Gene architecture and alternatively spliced transcripts of the Dlg2 gene. (a, upper graph) Genomic position of Dlg2 gene is presented. Exons are shown as vertical bars and introns as thin horizontal lines. Introns and exons are drawn to scale. Smaller exons (less than 1 point line space) are not to scale. (a, lower graph) Exons are shown as boxes and are drawn to scale. Exons are named or numbered as indicated. Alternative exon-exon-junctions are indicated with connecting lines. Grey boxes show protein coding regions whereas empty boxes represent untranslated mRNA regions. (b) RT-PCR of Dlg2 N-terminal isoforms in IFNβ/YFP+ and IFNβ/YFP— BM-derived pDCs (upper and middle panel). Flt3L cultures from six pooled IFNβmob/mob mice were stimulated with CpG for 6 h and FACS-sorted for YFP+ (pDC+) and YFP— pDCs (pDC—). Naïve brain from C57BL/6 N mice was used as positive and not reversely transcribed RNA from YFP+ pDCs as negative controls (Control). Lower panel shows Gapdh and Ifnb expression in the respective cDNA samples indicating successful stimulation and sorting of pDCs as well as equal template amounts. (c) SH3-GUK linker isoforms of Dlg2 in RT-PCR. SH3-GUK region was amplified using cDNA samples as described above in (b). (d) Restriction analysis of Dlg2 clones generated after 5´-RACE PCR. Empty vector or selected 5´-RACE clones were digested either with EcoRI (left), HindIII (middle), or with both restriction enzymes (right). Lower panel shows the plasmid maps for the clones shown in the upper panel. (e) Exon-intron structure of the Dlg2 isoforms expressed in pDCs. Exons are shown as boxes and are drawn to scale as shown in A (lower part)
Fig. 3
Fig. 3
Protein expression of a novel η-isoform of DLG2 in pDCs. (a) Detection of DLG2 isoforms at protein level in pDCs. DLG2 was immunoprecipitated from BM-derived Flt3L cultured DCs and brain lysates (left panel) from WT or Dlg2ΔE9/ΔE9 mice. The cells were left untreated or stimulated with CpG for 6 h. Immunoprecipitated DLG2 isoforms were analysed by DLG2 specific immunoblotting (upper panel); Iso: Isotype control antibody. In parallel, recombinant murine DLG2, isoforms (indicated) were expressed in HEK293 cells after transient transfection (right panel). DLG2 isoforms were analysed by immunoblotting as described above. (b) Protein domain organization of the DLG2 α-, γ-, and η-isoform expressed in pDCs. Indicated are the double cysteine-motif of the palmitoylation site within the α-isoform, and the PSD-95/Dlg/ZO-1 (PDZ), Src-homology-3 (SH3), and Guanylate Kinase (GK) domains. Protein length and relative positions of the domains are drawn to scale
Fig. 4
Fig. 4
Localisation of murine DLG2 isoforms ectopically expressed in NIH3T3 cells. EGFP (a) or murine DLG2 isoforms DLG2α (b), DLG2γ (c) or DLG2η (d) fused to EGFP were expressed in NIH3T3 cells. A day after transient transfection the cells were fixed and stained for Na/K ATPase as a cell membrane marker and nuclei (DAPI). Stained cells were analysed by confocal microscopy. Single channel views (left and middle panels) or merged views are displayed. Signal intensities of different markers are shown (right panel; profile) for the area under the arrow (merge) as analysed by ZEN software. Data shown is one representative picture out of a series of pictures analysed in at least three interdependent experiments
Fig. 5
Fig. 5
The effect of ectopic expression of Dlg2 isoforms on Ifnb expression. (a). Effect of IRF7 activation on Ifnb promoter activity. IRF7 or TBK1 were expressed alone or in combination in the presence of Ifnb reporter plasmid. Control (Cont.) shows the basal activity of Ifnb promoter in NIH3T3 cells. (b). Effect of expression of Dlg2 isoforms on Ifnb promoter activity. Increasing amounts of Dlg2 isoforms or TPI gene were transiently transfected along with Irf7, Tbk1 encoding plasmids and an Ifnb reporter vector in NIH3T3 cells. Ifnb promoter activity was measured from cell lysates one day after transfection. Data shown are ratios of relative light unites of Firefly luciferase (under the control of the Ifnb promoter) / Renila luciferase (under the control of the CMV promoter) from one representative experiment performed in quadruplicates out of three independent experiments. Data shown are the means ±SEM. Significance was analyzed by either One-way ANOVA followed by Bonferroni’s Multiple Comparison Test (a) or Two-way ANOVA followed by Bonferroni posttests (b). ns: P > 0.05, *: P < 0.05, **: P < 0.01, ***: P < 0.001
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References

    1. Feng W, Zhang M. Organization and dynamics of PDZ-domain-related supramodules in the postsynaptic density. Nat Rev Neurosci. 2009;10:87–99. doi: 10.1038/nrn2540. - DOI - PubMed
    1. Funke L, Dakoji S, Bredt DS. Membrane-associated guanylate kinases regulate adhesion and plasticity at cell junctions. Annu Rev Biochem. 2005;74:219–245. doi: 10.1146/annurev.biochem.74.082803.133339. - DOI - PubMed
    1. Grant SG. A general basis for cognition in the evolution of synapse signaling complexes. Cold Spring Harb Symp Quant Biol. 2009;74:249–257. doi: 10.1101/sqb.2009.74.033. - DOI - PubMed
    1. Won S, Levy JM, Nicoll RA, Roche KW. MAGUKs: multifaceted synaptic organizers. Curr Opin Neurobiol. 2017;43:94–101. doi: 10.1016/j.conb.2017.01.006. - DOI - PMC - PubMed
    1. Zhu J, Shang Y, Zhang M. Mechanistic basis of MAGUK-organized complexes in synaptic development and signalling. Nat Rev Neurosci. 2016;17:209–223. doi: 10.1038/nrn.2016.18. - DOI - PubMed

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