1. |
- Castillejo-Lopez, Casimiro, et al.
(author)
-
Genetic and physical interaction of the B-cell systemic lupus erythematosus-associated genes BANK1 and BLK
- 2012
-
In: Annals of the Rheumatic Diseases. - : BMJ. - 0003-4967 .- 1468-2060. ; 71:1, s. 136-142
-
Journal article (peer-reviewed)abstract
- ObjectivesAltered signalling in B cells is a predominant feature of systemic lupus erythematosus (SLE). The genes BANK1 and BLK were recently described as associated with SLE. BANK1 codes for a B-cell-specific cytoplasmic protein involved in B-cell receptor signalling and BLK codes for an Src tyrosine kinase with important roles in B-cell development. To characterise the role of BANK1 and BLK in SLE, a genetic interaction analysis was performed hypothesising that genetic interactions could reveal functional pathways relevant to disease pathogenesis.MethodsThe GPAT16 method was used to analyse the gene-gene interactions of BANK1 and BLK. Confocal microscopy was used to investigate co-localisation, and immunoprecipitation was used to verify the physical interaction of BANK1 and BLK.ResultsEpistatic interactions between BANK1 and BLK polymorphisms associated with SLE were observed in a discovery set of 279 patients and 515 controls from northern Europe. A meta-analysis with 4399 European individuals confirmed the genetic interactions between BANK1 and BLK. As BANK1 was identified as a binding partner of the Src tyrosine kinase LYN, the possibility that BANK1 and BLK could also show a protein-protein interaction was tested. The co-immunoprecipitation and co-localisation of BLK and BANK1 were demonstrated. In a Daudi cell line and primary naive B cells endogenous binding was enhanced upon B-cell receptor stimulation using anti-IgM antibodies.ConclusionsThis study shows a genetic interaction between BANK1 and BLK, and demonstrates that these molecules interact physically. The results have important consequences for the understanding of SLE and other autoimmune diseases and identify a potential new signalling pathway.
|
|
2. |
- Castillejo-López, Casimiro, et al.
(author)
-
A regulatory element associated to NAFLD in the promoter of DIO1 controls LDL-C, HDL-C and triglycerides in hepatic cells
- 2024
-
In: Lipids in Health and Disease. - : BioMed Central (BMC). - 1476-511X. ; 23
-
Journal article (peer-reviewed)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.
|
|
3. |
- Castillejo-Lopez, Casimiro, et al.
(author)
-
Detailed Functional Characterization of a Waist-Hip Ratio Locus in 7p15.2 Defines an Enhancer Controlling Adipocyte Differentiation
- 2019
-
In: iScience. - : Elsevier BV. - 2589-0042. ; 20, s. 42-59
-
Journal article (peer-reviewed)abstract
- We combined CAGE sequencing in human adipocytes during differentiation with data from genome-wide association studies to identify an enhancer in the SNX10 locus on chromosome 7, presumably involved in body fat distribution. Using reporter assays and CRISPR-Cas9 gene editing in human cell lines, we characterized the role of the enhancer in adipogenesis. The enhancer was active during adipogenesis and responded strongly to insulin and isoprenaline. The allele associated with increased waist-hip ratio in human genetic studies was associated with higher enhancer activity. Mutations of the enhancer resulted in less adipocyte differentiation. RNA sequencing of cells with disrupted enhancer showed reduced expression of established adipocyte markers, such as ADIPOQ and LPL, and identified CHI3L1 on chromosome 1 as a potential gene involved in adipocyte differentiation. In conclusion, we identified and characterized an enhancer in the SNX10 locus and outlined its plausible mechanisms of action and downstream targets.
|
|
4. |
- Castillejo-Lopez, Casimiro, et al.
(author)
-
Drosophila exoribonuclease nibbler is a tumor suppressor, acts within the RNA(i) machinery and is not enriched in the nuage during early oogenesis
- 2017
-
In: Hereditas. - : BIOMED CENTRAL LTD. - 0018-0661 .- 1601-5223. ; 155
-
Journal article (peer-reviewed)abstract
- Background: micro RNAs (miRNAs) are important regulators of many biological pathways. A plethora of steps are required to form, from a precursor, the mature miRNA that eventually acts on its target RNA to repress its expression or to inhibit translation. Recently, Drosophila nibbler (nbr) has been shown to be an important player in the maturation process of miRNA and piRNA. Nbr is an exoribonuclease which helps to shape the 3' end of miRNAs by trimming the 3' overhang to a final length. Results: In contrast to previous reports on the localization of Nbr, we report that 1) Nbr is expressed only during a short time of oogenesis and appears ubiquitously localized within oocytes, and that 2) Nbr was is not enriched in the nuage where it was shown to be involved in piwi-mediated mechanisms. To date, there is little information available on the function of nbr for cellular and developmental processes. Due to the fact that nbr mutants are viable with minor deleterious effects, we used the GAL4/UAS over-expression system to define novel functions of nbr. We disclose hitherto unknown functions of nbr 1) as a tumor suppressor and 2) as a suppressor of RNAi. Finally, we confirm that nbr is a suppressor of transposon activity. Conclusions: Our data suggest that nbr exerts much more widespread functions than previously reported from trimming 3' ends of miRNAs only.
|
|
5. |
|
|
6. |
- Castillejo-Lopez, Casimiro
(author)
-
Repetitive DNA in search of a function - a study of telomeric and centromeric sequences in Chironomus
- 1998
-
Doctoral thesis (other academic/artistic)abstract
- Repetitive DNA is quantitatively the main component of telomeres and centromeres, structures responsible for maintenance of the eukaryotic chromosome. The telomere is the specialized nucleoprotein complex that terminates linear chromosomes. In most species the DNA component consists of short repeats which are generated by the enzyme complex telomerase. However, there are important exceptions such as the Drosophila melanogaster telomeres which are elongated by retrotransposons and, as documented in the present thesis, a third telomeric system in Chironomus pallidivittatus in the form of arrays of 340 bp long complex tandem repeats which extend to the end of their chromosomes. Complex repeats are not elongated by telomerase and one aim of my work has therefore been to elucidate possible regeneration mechanisms for telomeres with such repeats. I have obtained evidence for DNA increase through DNA sequences of nontelomeric origin being inserted into the telomeric repeat array via gene conversion. Immunolocalization of reverse transcriptase related proteins in the telomeric puff of the related species C. thummi revealed, on the other hand, a possible link between the regeneration of Chironomus telomeric complex repeats and mechanisms used by other eukaryotes. Only seven of the eight pairs of chromosome termini in C. pallidivittatus have 340 bp repeats. The remaining telocentric end contains another repeat, the centromeric 155 bp unit, probably extending to the chromosome end. In the arrays of this repeat, a putative homologue of the mammalian centromeric CENP-B box has been found and characterized. Its interspersed distribution and its surrounding direct repeats suggest a mobile origin. It is present in different recombined forms, which could be related to a role in recombination as has been suggested for the human CENP-B box.
|
|
7. |
- Castillejo-Lopez, Casimiro, et al.
(author)
-
The fat-like gene of drosophila is the true orthologue of vertebrate fat cadherins and is involved in the formation of tubular organs.
- 2004
-
In: Journal of Biological Chemistry. - 1083-351X. ; 279:23, s. 24034-24043
-
Journal article (peer-reviewed)abstract
- Fat cadherins constitute a subclass of the large cadherin family characterized by the presence of 34 cadherin motifs. To date, three mammalian Fat cadherins have been described; however, only limited information is known about the function of these molecules. In this paper, we describe the second fat cadherin in Drosophila, fat-like (ftl). We show that ftl is the true orthologue of vertebrate fat-like genes, whereas the previously characterized tumor suppressor cadherin, fat, is more distantly related as compared with ftl. Ftl is a large molecule of 4705 amino acids. It is expressed apically in luminal tissues such as trachea, salivary glands, proventriculus, and hindgut. Silencing of ftl results in the collapse of tracheal epithelia giving rise to breaks, deletions, and sac-like structures. Other tubular organs such as proventriculus, salivary glands, and hindgut are also malformed or missing. These data suggest that Ftl is required for morphogenesis and maintenance of tubular structures of ectodermal origin and underline its similarity in function to a reported lethal mouse knock-out of fat1 where glomerular epithelial processes collapse. Based on our results, we propose a model where Ftl acts as a spacer to keep tubular epithelia apart rather than the previously described adhesive properties of the cadherin superfamily.
|
|
8. |
- Castillejo-Lopez, Casimiro, et al.
(author)
-
The serine protease Sp7 is expressed in blood cells and regulates the melanization reaction in Drosophila.
- 2005
-
In: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 338:2, s. 1075-1082
-
Journal article (peer-reviewed)abstract
- Serine proteases play a central role in defense against pathogens by regulating processes such as blood clotting, melanization of injured surfaces, and proteolytic activation of signaling pathways involved in innate immunity. Here, we present the functional characterization of the Drosophila serine protease Sp7 (CG3006) by inducible RNA interference. We show that Sp7 is constitutively expressed in blood cells during embryonic and larval stages. Silencing of the gene impairs the melanization reaction upon injury. Our data demonstrate that Sp7 is required for phenoloxidase activation and its activity is restricted to a subclass of blood cells, the crystal cells. Transcriptional up-regulation of Sp7 was observed after clean, septic injury and in flies expressing an activated form of Toll; however, mutations in the Toll or the IMD pathway did not abolish expression of Sp7, indicating the existence of other regulatory pathways and/or independent basal transcription.
|
|
9. |
- Cook, Naomi L., et al.
(author)
-
CRISPR-Cas9-mediated knockout of SPRY2 in human hepatocytes leads to increased glucose uptake and lipid droplet accumulation
- 2019
-
In: BMC Endocrine Disorders. - : Springer Science and Business Media LLC. - 1472-6823. ; 19
-
Journal article (peer-reviewed)abstract
- BackgroundThe prevalence of obesity and its comorbidities, including type 2 diabetes mellitus (T2DM), is dramatically increasing throughout the world; however, the underlying aetiology is incompletely understood. Genome-wide association studies (GWAS) have identified hundreds of genec susceptibility loci for obesity and T2DM, although the causal genes and mechanisms are largely unknown. SPRY2 is a candidate gene identified in GWAS of body fat percentage and T2DM, and has recently been linked to insulin production in pancreatic β-cells. In the present study, we aimed to further understand SPRY2 via functional characterisation in HepG2 cells, an in vitro model of human hepatocytes widely used to investigate T2DM and insulin resistance.MethodsCRISPR-Cas9 genome editing was used to target SPRY2 in HepG2 cells, and the functional consequences of SPRY2 knockout (KO) and overexpression subsequently assessed using glucose uptake and lipid droplet assays, measurement of protein kinase phosphorylation and RNA sequencing.ResultsThe major functional consequence of SPRY2 KO was a significant increase in glucose uptake, along with elevated lipid droplet accumulation. These changes were attenuated, but not reversed, in cells overexpressing SPRY2. Phosphorylation of protein kinases across key signalling pathways (including Akt and mitogen activated protein kinases) was not altered after SPRY2 KO. Transcriptome profiling in SPRY2 KO and mock (control) cells revealed a number of differentially expressed genes related to cholesterol biosynthesis, cell cycle regulation and cellular signalling pathways. Phospholipase A2 group IIA (PLA2G2A) mRNA level was subsequently validated as significantly upregulated following SPRY2 KO, highlighting this as a potential mediator downstream of SPRY2.ConclusionThese findings suggest a role for SPRY2 in glucose and lipid metabolism in hepatocytes and contribute to clarifying the function of this gene in the context of metabolic diseases.
|
|
10. |
- Delgado-Vega, Angélica M., et al.
(author)
-
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
- 2012
-
In: Annals of the Rheumatic Diseases. - : BMJ. - 0003-4967 .- 1468-2060. ; 71:7, s. 1219-1226
-
Journal article (peer-reviewed)abstract
- ObjectivesTo perform fine mapping of the autoimmunity susceptibility gene BLK and identify functional variants involved in systemic lupus erythematosus (SLE).MethodsGenotyping 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. B (NFkB) binding.ResultsFine mapping of BLK identified two independent genetic effects with functional consequences: one represented by two tightly linked associated haplotype blocks significantly enriched for NF kappa B-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.ConclusionsThese 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.
|
|