Fine mapping and conditional analysis identify a new mutation in the autoimmunity susceptibility gene BLK that leads to reduced half-life of the BLK protein

OBJECTIVES: To perform fine mapping of the autoimmunity susceptibility gene BLK and identify functional variants involved in systemic lupus erythematosus (SLE). METHODS: Genotyping of 1163 European SLE patients and 1482 controls and imputation were performed covering the BLK gene with 158 single-nucleotide polymorphisms. Logistic regression analysis was done using PLINK and conditional analyses using GENABEL's test score. Transfections of BLK constructs on HEK293 cells containing the novel mutation or the wild type form were analysed for their effect on protein half-life using a protein stability assay, cycloheximide and western blot. CHiP-qPCR for detection of nuclear factor κ B (NFkB) binding. RESULTS: Fine mapping of BLK identified two independent genetic effects with functional consequences: one represented by two tightly linked associated haplotype blocks significantly enriched for NFκB-binding sites and numerous putative regulatory variants whose risk alleles correlated with low BLK mRNA levels. Binding of NFkBp50 and p65 to an associated 1.2 Kb haplotype segment was confirmed. A second independent genetic effect was represented by an Ala71Thr, low-frequency missense substitution with an OR=2.31 (95% CI 1.38 to 3.86). The 71Thr decreased BLK protein half-life. CONCLUSIONS: These results show that rare and common regulatory variants in BLK are involved in disease susceptibility and both, albeit independently, lead to reduced levels of BLK protein

A regulatory element associated to NAFLD in the promoter of DIO1 controls LDL-C, HDL-C and triglycerides in hepatic cells

Abstract Background Genome-wide association studies (GWAS) have identified genetic variants linked to fat metabolism and related traits, but rarely pinpoint causative variants. This limitation arises from GWAS not considering functional implications of noncoding variants that can affect transcription factor binding and potentially regulate gene expression. The aim of this study is to investigate a candidate noncoding functional variant within a genetic locus flagged by a GWAS SNP associated with non-alcoholic fatty liver disease (NAFLD), a condition characterized by liver fat accumulation in non-alcohol consumers. Methods CRISPR-Cas9 gene editing in HepG2 cells was used to modify the regulatory element containing the candidate functional variant linked to NAFLD. Global gene expression in mutant cells was assessed through RT-qPCR and targeted transcriptomics. A phenotypic assay measured lipid droplet accumulation in the CRISPR-Cas9 mutants. Results The candidate functional variant, rs2294510, closely linked to the NAFLD-associated GWAS SNP rs11206226, resided in a regulatory element within the DIO1 gene's promoter region. Altering this element resulted in changes in transcription factor binding sites and differential expression of candidate target genes like DIO1, TMEM59, DHCR24, and LDLRAD1, potentially influencing the NAFLD phenotype. Mutant HepG2 cells exhibited increased lipid accumulation, a hallmark of NAFLD, along with reduced LDL-C, HDL-C and elevated triglycerides. Conclusions This comprehensive approach, that combines genome editing, transcriptomics, and phenotypic assays identified the DIO1 promoter region as a potential enhancer. Its activity could regulate multiple genes involved in the NAFLD phenotype or contribute to defining a polygenic risk score for enhanced risk assessment in NAFLD patients

BANK1 and BLK Act through Phospholipase C Gamma 2 in B-Cell Signaling

<div><p>The B cell adaptor protein with ankyrin repeats (BANK1) and the B lymphoid tyrosine kinase (BLK) have been genetically associated with autoimmunity. The proteins of these genes interact physically and work in concert during B-cell signaling. Little is know about their interactions with other B-cell signaling molecules or their role in the process. Using yeast two hybrid (Y2H) we sought for factors that interact with BANK1. We found that the molecular switch PLCg2 interacts with BANK1 and that the interaction is promoted by B-cell receptor (BCR) stimulation. We found further that the kinase activity of BLK enhanced BANK1- PLCg2 binding and that the interaction was suppressed upon BLK depletion. Immunoprecipitation and mutational analysis demonstrated that the interaction between BANK1 and PLCg2 was dependent on specific tyrosine and proline residues on the adaptor protein. Our results provide new information important to understand the role of these two genes in basic B-cell physiology and immune-related diseases.</p> </div

Clones belonging to the phosphoinositide-specific phospholipase C and the family of Src kinases isolated in two yeast two-hybrid screens.

<p>(A) Representation of PLCg2 modular structure and the coding region of the clones identified in the Y2H. Clones named a- correspond to the first screen using as bait the full-length BANK1, clones starting with b- are the ones identified in the screen with the non autoactivating truncated protein BANK1 (331–785). The clone b-234 has a deletion of 25 aa between the cSH2 and SH3 domains. (B) Structure of the Src kinase FYN and the isolated clones belonging to this family of non- receptor kinase. PH: Pleckstrin homology domain, involved in the recruitment to membranes by binding to phosphatidylinositol containing lipids. X and Y are the two halves of the catalytic isomerase. SH2 (Src homology 2) conserved domain that typically binds to phosphorylated tyrosine residues. SH3 (Src homology 3) usually binds to proline-rich motifs. The C2 motif is present in many proteins that interact with membranes and are frequently involved in calcium dependent phospholipid binding and membrane targeting processes.</p

BANK1 and PLCG2 co-localize in cytoplasmic compartments.

<p>Confocal images of HEK293 cells co-expressing fluorescently tagged proteins. BANK1 is a cytoplasmic adaptor protein that shows a punctate and homogeneous pattern of distribution. PLCg2 shares the same sub-cellular compartments when co-expressed with BANK1. Upper row, co-expression of PLCg2 and BANK1 showing sub-cellular co-localization in punctate structures. Although a perfect co-localization between the two proteins is observed, there are dots (indicated by arrows, both in the merge image and in the amplification at the right upper corner) in which the ratio of the two proteins is reversed. Cross-talks between light channels were further ruled out by assessing the emission on cells expressing only one fluoresce protein and excitated sequencially with both lasers. Middle row, images showing co-localization between PLCg2 and BANK1 in a homogeneous cytoplasmic distribution. Lower row, images showing only partial co-localization between the cytoplasmic scavenger receptor, CD163 and BANK1. Scale bar: 10 µm.(-yfp; yellow fluorescence protein. –cfp; cyan fluorescence protein. –chr; cherry fluorescent protein. –gfp; green fluorescent protein).</p

Silencing of the BLK kinase leads to alteration of the association between BANK1 and PLCg2.

<p>(A) Immunoblot of extracts derived from human Daudi B-cells showing efficient silencing of endogenous BLK protein. The cell line shBLK was transduced with lentivirus coding for small hairpin RNAs targeting the BLK kinase while shControl lentivirus codes for unrelated sequences. Top, western blot analysis using antibody to BLK; bottom, western blot analysis using antibody to GAPDH as loading control. (B) The relative BLK mRNA is reduced to half in the silencing line (shBLK)(***P<0,001 based on Student´s test comparing mRNA expression between Daudi cell versus cBLK silencing cells and control silenced cells versus BLK silencing cells). (C) Immunoprecipitates (IPs) of stimulated silenced shBLK cells with anti-IgM, using anti-PLCg2 antibody and interrogated with anti-BANK1 to assay BANK1-PLCg2 association. Quantification of the immunoprecipitates normalized with BANK1 is displayed below the bands. Silencing of BLK leads to less association between BANK1-PLCg2. (D) Kinetics of the association between BANK1-PLCg2 assayed with <i>in situ</i> Proximity Ligation (PLA) in control and BLK-silenced cell lines. The association between BANK1-PLCg2 is reduced in silenced cells during resting conditions and after 15 minutes of IgM stimulation (***P≤0,001 based on Student´s test comparing control cells versus BLK silencing cells). The difference of BANK1-PLCg2 association between control and the silenced lines was not significant at 1 minute after stimulation.</p

The constitutive-active form of BLK enhances the binding between BANK1 and PLCG2.

<p>(A) Schematic representation of the constructs used to study the association between BANK1 and PLCg2 in transfected HEK293 cells. The constructs coding for wild-type forms of BLK, LYN, GFP and PLCg2 or the indicated mutated forms were fused to the epitope V5 at the C-termini. BANK1 was targeted with the Flag epitope at the N- terminus. The catalytic domains of BLK and PLCg2 are shown in red. The kinase dead form (BLK-KL-v5) has a substitution K (lysine) to L (leucine) at position 269 and the constitutively active form (BLK-YF-v5) has a Y501F substitution that prevents the phosphorylation of the inhibitory tyrosine. The lipidation in the amino terminal of BLK is indicated as back line. The myristoylation site was deleted by G2V substitution (glycine to valine) and the addition of an extra by palmitoylated site by L3C substitution (leucine to cysteine). The Src homology 3 domains (SH3) that bind to proline-rich motifs are drawn in orange and the SH2 domains in yellow. The Pleckstrin homology domain (PH) that binds to phosphatidylinositol lipids is shown in blue. In BANK1 are shown the Dof/BCAP/BANK (DBB) motif (amino acids 199–327), the double ankyrin repeat-like (ANK) motifs (amino acids 339-402) and the putative coiled coil (CC) region (amino acids 677–705). (B) HEK293 cells were transiently co-transfected with plasmids coding for the wild-type form of BLK, its functionally mutated forms (KL and YF), LYN or GFP in addition to plasmids expressing BANK1 and PLCg2. The lysates were immunoprecipitated using anti-PLCg2 antibody (above) and immunoblotted sequencially with anti-BANK1 antibody, anti-V5 to detect PLCg2, Srcs kinases and GFP and anti-phosphotyrosine antibody. (C). Mutation of lipidation sites of the kinases influence the formation of the BANK1-PLCg2 complex and the overall tyrosine phosphorylation on PLCg2. The blots were interrogated as in B.</p

Clones isolated in the yeast two-hybrid screen using the full-length BANK1.

<p>Clones isolated in the yeast two-hybrid screen using the full-length BANK1 (amino acids 1–785) as bait. The frame indicates if the coding sequences are in the same frame as Gal4-Activating Domain. In general polypeptides not having an in-frame (IF) position are not considered of biological interest. However, some of the proteins expressed from F1 or F2 can be translated in the correct frame, due to the existence of natural frame-shift events during translation in yeast. AA indicates the length of the clone in number of amino acids and (%) the percentage of frame corresponding to the annotation in GenBank.</p