Deciphering the role of a SINE-VNTR-Alu retrotransposon polymorphism as a biomarker of Parkinson's disease progression.

SINE-VNTR-Alu (SVA) retrotransposons are transposable elements which represent a source of genetic variation. We previously demonstrated that the presence/absence of a human-specific SVA, termed SVA_67, correlated with the progression of Parkinson's disease (PD). In the present study, we demonstrate that SVA_67 acts as expression quantitative trait loci, thereby exhibiting a strong regulatory effect across the genome using whole genome and transcriptomic data from the Parkinson's progression markers initiative cohort. We further show that SVA_67 is polymorphic for its variable number tandem repeat domain which correlates with both regulatory properties in a luciferase reporter gene assay in vitro and differential expression of multiple genes in vivo. Additionally, this variation's utility as a biomarker is reflected in a correlation with a number of PD progression markers. These experiments highlight the plethora of transcriptomic and phenotypic changes associated with SVA_67 polymorphism which should be considered when investigating the missing heritability of neurodegenerative diseases.

A SINE-VNTR-Alu at the LRIG2 locus is associated with proximal and distal gene expression in CRISPR and population models.

SINE-VNTR-Alu (SVA) retrotransposons represent mobile regulatory elements that have the potential to influence the surrounding genome when they insert into a locus. Evolutionarily recent mobilisation has resulted in loci in the human genome where a given retrotransposon might be observed to be present or absent, termed a retrotransposon insertion polymorphism (RIP). We previously observed that an SVA RIP ~ 2 kb upstream of LRIG2 on chromosome 1, the 'LRIG2 SVA', was associated with differences in local gene expression and methylation, and that the two were correlated. Here, we have used CRISPR-mediated deletion of the LRIG2 SVA in a cell line model to validate that presence of the retrotransposon is directly affecting local expression and provide evidence that is suggestive of a modest role for the SVA in modulating nearby methylation. Additionally, in leveraging an available Hi-C dataset we observed that the LRIG2 SVA was also involved in long-range chromatin interactions with a cluster of genes ~ 300 kb away, and that expression of these genes was to varying degrees associated with dosage of the SVA in both CRISPR cell line and population models. Altogether, these data support a regulatory role for SVAs in the modulation of gene expression, with the latter potentially involving chromatin looping, consistent with the model that RIPs may contribute to interpersonal differences in transcriptional networks.

CRISPR deletion of a SINE-VNTR-Alu (SVA_67) retrotransposon demonstrates its ability to differentially modulate gene expression at the MAPT locus.

Background: SINE-VNTR-Alu (SVA) retrotransposons are hominid-specific elements which have been shown to play important roles in processes such as chromatin structure remodelling and regulation of gene expression demonstrating that these repetitive elements exert regulatory functions. We have previously shown that the presence or absence of a specific SVA element, termed SVA_67, was associated with differential expression of several genes at the MAPT locus, a locus associated with Parkinson's Disease (PD) and frontotemporal dementia. However, we were not able to demonstrate that causation of differential gene expression was directed by the SVA due to lack of functional validation. Methods: We performed CRISPR to delete SVA_67 in the HEK293 cell line. Quantification of target gene expression was performed using qPCR to assess the effects on expression in response to the deletion of SVA_67. Differences between CRISPR edit and control cell lines were analysed using two-tailed t-test with a minimum 95% confidence interval to determine statistical significance. Results: In this study, we provide data highlighting the SVA-specific effect on differential gene expression. We demonstrate that the hemizygous deletion of the endogenous SVA_67 in CRISPR edited cell lines was associated with differential expression of several genes at the MAPT locus associated with neurodegenerative diseases including KANSL1, MAPT and LRRC37A. Discussion: This data is consistent with our previous bioinformatic work of differential gene expression analysis using transcriptomic data from the Parkinson's Progression Markers Initiative (PPMI) cohort. As SVAs have regulatory influences on gene expression, and insertion polymorphisms contribute to interpersonal differences in expression patterns, these results highlight the potential contribution of these elements to complex diseases with potentially many genetic components, such as PD.

A polymorphic transcriptional regulatory domain in the amyotrophic lateral sclerosis risk gene CFAP410 correlates with differential isoform expression.

We describe the characterisation of a variable number tandem repeat (VNTR) domain within intron 1 of the amyotrophic lateral sclerosis (ALS) risk gene CFAP410 (Cilia and flagella associated protein 410) (previously known as C21orf2), providing insight into how this domain could support differential gene expression and thus be a modulator of ALS progression or risk. We demonstrated the VNTR was functional in a reporter gene assay in the HEK293 cell line, exhibiting both the properties of an activator domain and a transcriptional start site, and that the differential expression was directed by distinct repeat number in the VNTR. These properties embedded in the VNTR demonstrated the potential for this VNTR to modulate CFAP410 expression. We extrapolated these findings in silico by utilisation of tagging SNPs for the two most common VNTR alleles to establish a correlation with endogenous gene expression. Consistent with in vitro data, CFAP410 isoform expression was found to be variable in the brain. Furthermore, although the number of matched controls was low, there was evidence for one specific isoform being correlated with lower expression in those with ALS. To address if the genotype of the VNTR was associated with ALS risk, we characterised the variation of the CFAP410 VNTR in ALS cases and matched controls by PCR analysis of the VNTR length, defining eight alleles of the VNTR. No significant difference was observed between cases and controls, we noted, however, the cohort was unlikely to contain sufficient power to enable any firm conclusion to be drawn from this analysis. This data demonstrated that the VNTR domain has the potential to modulate CFAP410 expression as a regulatory element that could play a role in its tissue-specific and stimulus-inducible regulation that could impact the mechanism by which CFAP410 is involved in ALS.

Restricted differentiative capacity of Wt1-expressing peritoneal mesothelium in postnatal and adult mice.

Previously, genetic lineage tracing based on the mesothelial marker Wt1, appeared to show that peritoneal mesothelial cells have a range of differentiative capacities and are the direct progenitors of vascular smooth muscle in the intestine. However, it was not clear whether this was a temporally limited process or continued throughout postnatal life. Here, using a conditional Wt1-based genetic lineage tracing approach, we demonstrate that the postnatal and adult peritoneum covering intestine, mesentery and body wall only maintained itself and failed to contribute to other visceral tissues. Pulse-chase experiments of up to 6 months revealed that Wt1-expressing cells remained confined to the peritoneum and failed to differentiate into cellular components of blood vessels or other tissues underlying the peritoneum. Our data confirmed that the Wt1-lineage system also labelled submesothelial cells. Ablation of Wt1 in adult mice did not result in changes to the intestinal wall architecture. In the heart, we observed that Wt1-expressing cells maintained the epicardium and contributed to coronary vessels in newborn and adult mice. Our results demonstrate that Wt1-expressing cells in the peritoneum have limited differentiation capacities, and that contribution of Wt1-expressing cells to cardiac vasculature is based on organ-specific mechanisms.

Trypanosoma brucei colonizes the tsetse gut via an immature peritrophic matrix in the proventriculus.

The peritrophic matrix of blood-feeding insects is a chitinous structure that forms a protective barrier against oral pathogens and abrasive particles1. Tsetse flies transmit Trypanosoma brucei, which is the parasite that causes human sleeping sickness and is also partially responsible for animal trypanosomiasis in Sub-Saharan Africa. For this parasite to establish an infection in flies, it must first colonize the area between the peritrophic matrix and gut epithelium called the ectoperitrophic space. Although unproven, it is generally accepted that trypanosomes reach the ectoperitrophic space by penetrating the peritrophic matrix in the anterior midgut2-4. Here, we revisited this event using fluorescence- and electron-microscopy methodologies. We show that trypanosomes penetrate the ectoperitrophic space in which the newly made peritrophic matrix is synthesized by the proventriculus. Our model describes how these proventriculus-colonizing parasites can either migrate to the ectoperitrophic space or become trapped within peritrophic matrix layers to form cyst-like bodies that are passively pushed along the gut as the matrix gets remodelled. Furthermore, early proventricular colonization seems to be promoted by factors in trypanosome-infected blood that cause higher salivary gland infections and potentially increase parasite transmission.

The role of Ca2+ influx in endocytic vacuole formation in pancreatic acinar cells.

The inducers of acute pancreatitis trigger a prolonged increase in the cytosolic Ca(2+) concentration ([Ca(2+)]c), which is responsible for the damage to and eventual death of pancreatic acinar cells. Vacuolization is an important indicator of pancreatic acinar cell damage. Furthermore, activation of trypsinogen occurs in the endocytic vacuoles; therefore the vacuoles can be considered as 'initiating' organelles in the development of the cell injury. In the present study, we investigated the relationship between the formation of endocytic vacuoles and Ca(2+) influx developed in response to the inducers of acute pancreatitis [bile acid taurolithocholic acid 3-sulfate (TLC-S) and supramaximal concentration of cholecystokinin-8 (CCK)]. We found that the inhibitor of STIM (stromal interaction molecule)/Orai channels, GSK-7975A, effectively suppressed both the Ca(2+) influx (stimulated by inducers of pancreatitis) and the formation of endocytic vacuoles. Cell death induced by TLC-S or CCK was also inhibited by GSK-7975A. We documented the formation of endocytic vacuoles in response to store-operated Ca(2+) entry (SOCE) induced by thapsigargin [TG; inhibitor of sarcoplasmic/endoplasmic reticulum (ER) Ca(2+) pumps] and observed strong inhibition of TG-induced vacuole formation by GSK-7975A. Finally, we found that structurally-unrelated inhibitors of calpain suppress formation of endocytic vacuoles, suggesting that this Ca2+-dependent protease is a mediator between Ca(2+) elevation and endocytic vacuole formation.