The first genome-wide association study in the Argentinian and Chilean populations identifies shared genetics with Europeans in Alzheimer's disease.

INTRODUCTION: Genome-wide association studies (GWAS) are fundamental for identifying loci associated with diseases. However, they require replication in other ethnicities. METHODS: We performed GWAS on sporadic Alzheimer's disease (AD) including 539 patients and 854 controls from Argentina and Chile. We combined our results with those from the European Alzheimer and Dementia Biobank (EADB) in a meta-analysis and tested their genetic risk score (GRS) performance in this admixed population. RESULTS: We detected apolipoprotein E ε4 as the single genome-wide significant signal (odds ratio  = 2.93 [2.37-3.63], P = 2.6 × 10-23 ). The meta-analysis with EADB summary statistics revealed four new loci reaching GWAS significance. Functional annotations of these loci implicated endosome/lysosomal function. Finally, the AD-GRS presented a similar performance in these populations, despite the score diminished when the Native American ancestry rose. DISCUSSION: We report the first GWAS on AD in a population from South America. It shows shared genetics modulating AD risk between the European and these admixed populations. HIGHLIGHTS: This is the first genome-wide association study on Alzheimer's disease (AD) in a population sample from Argentina and Chile. Trans-ethnic meta-analysis reveals four new loci involving lysosomal function in AD. This is the first independent replication for TREM2L, IGH-gene-cluster, and ADAM17 loci. A genetic risk score (GRS) developed in Europeans performed well in this population. The higher the Native American ancestry the lower the GRS values.

Chromatin loop dynamics during cellular differentiation are associated with changes to both anchor and internal regulatory features.

Three-dimensional (3D) chromatin structure has been shown to play a role in regulating gene transcription during biological transitions. Although our understanding of loop formation and maintenance is rapidly improving, much less is known about the mechanisms driving changes in looping and the impact of differential looping on gene transcription. One limitation has been a lack of well-powered differential looping data sets. To address this, we conducted a deeply sequenced Hi-C time course of megakaryocyte development comprising four biological replicates and 6 billion reads per time point. Statistical analysis revealed 1503 differential loops. Gained loop anchors were enriched for AP-1 occupancy and were characterized by large increases in histone H3K27ac (over 11-fold) but relatively small increases in CTCF and RAD21 binding (1.26- and 1.23-fold, respectively). Linear modeling revealed that changes in histone H3K27ac, chromatin accessibility, and JUN binding were better correlated with changes in looping than RAD21 and almost as well correlated as CTCF. Changes to epigenetic features between-rather than at-boundaries were highly predictive of changes in looping. Together these data suggest that although CTCF and RAD21 may be the core machinery dictating where loops form, other features (both at the anchors and within the loop boundaries) may play a larger role than previously anticipated in determining the relative loop strength across cell types and conditions.

Temporal analysis suggests a reciprocal relationship between 3D chromatin structure and transcription.

To infer potential causal relationships between 3D chromatin structure, enhancers, and gene transcription, we mapped each feature in a genome-wide fashion across eight narrowly spaced time points of macrophage activation. Enhancers and genes connected by loops exhibit stronger correlations between histone H3K27 acetylation and expression than can be explained by genomic distance or physical proximity alone. At these looped enhancer-promoter pairs, changes in acetylation at distal enhancers precede changes in gene expression. Changes in gene expression exhibit a directional bias at differential loop anchors; gained loops are associated with increased expression of genes oriented away from the center of the loop, and lost loops are often accompanied by high levels of transcription within the loop boundaries themselves. These results are consistent with a reciprocal relationship where loops can facilitate increased transcription by connecting promoters to distal enhancers, whereas high levels of transcription can impede loop formation.

Oncogenic fusion proteins and their role in three-dimensional chromatin structure, phase separation, and cancer.

Three-dimensional (3D) chromatin structure plays a critical role in development, gene regulation, and cellular identity. Alterations to this structure can have profound effects on cellular phenotypes and have been associated with a variety of diseases including multiple types of cancer. One of several forces that help shape 3D chromatin structure is liquid-liquid phase separation, a form of self-association between biomolecules that can sequester regions of chromatin into subnuclear droplets or even membraneless organelles like nucleoli. This review focuses on a class of oncogenic fusion proteins that appear to exert their oncogenic function via phase-separation-driven alterations to 3D chromatin structure. Here, we review what is known about the mechanisms by which these oncogenic fusion proteins phase separate in the nucleus and their role in shaping the 3D chromatin structure. We discuss the potential for this phenomenon to be a more widespread mechanism of oncogenesis.

Phase separation drives aberrant chromatin looping and cancer development.

The development of cancer is intimately associated with genetic abnormalities that target proteins with intrinsically disordered regions (IDRs). In human haematological malignancies, recurrent chromosomal translocation of nucleoporin (NUP98 or NUP214) generates an aberrant chimera that invariably retains the nucleoporin IDR-tandemly dispersed repeats of phenylalanine and glycine residues1,2. However, how unstructured IDRs contribute to oncogenesis remains unclear. Here we show that IDRs contained within NUP98-HOXA9, a homeodomain-containing transcription factor chimera recurrently detected in leukaemias1,2, are essential for establishing liquid-liquid phase separation (LLPS) puncta of chimera and for inducing leukaemic transformation. Notably, LLPS of NUP98-HOXA9 not only promotes chromatin occupancy of chimera transcription factors, but also is required for the formation of a broad 'super-enhancer'-like binding pattern typically seen at leukaemogenic genes, which potentiates transcriptional activation. An artificial HOX chimera, created by replacing the phenylalanine and glycine repeats of NUP98 with an unrelated LLPS-forming IDR of the FUS protein3,4, had similar enhancing effects on the genome-wide binding and target gene activation of the chimera. Deeply sequenced Hi-C revealed that phase-separated NUP98-HOXA9 induces CTCF-independent chromatin loops that are enriched at proto-oncogenes. Together, this report describes a proof-of-principle example in which cancer acquires mutation to establish oncogenic transcription factor condensates via phase separation, which simultaneously enhances their genomic targeting and induces organization of aberrant three-dimensional chromatin structure during tumourous transformation. As LLPS-competent molecules are frequently implicated in diseases1,2,4-7, this mechanism can potentially be generalized to many malignant and pathological settings.

Triacylglycerol synthesis directed by glycerol-3-phosphate acyltransferases -3 and -4 is required for lipid droplet formation and the modulation of the inflammatory response during macrophage to foam cell transition.

BACKGROUND AND AIMS: The transition of macrophage to foam cells is a major hallmark of early stage atherosclerotic lesions. This process is characterized by the accumulation of large cytoplasmic lipid droplets containing large quantities of cholesterol esters (CE), triacylglycerol (TAG) and phospholipid (PL). Although cholesterol and CE metabolism during foam cell formation has been broadly studied, little is known about the role of the glycerolipids (TAG and PL) in this context. Here we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). METHODS: We used RAW 264.7 cells and bone marrow derived macrophages (BMDM) treated with oxidized LDL (oxLDL). RESULTS: We showed that TAG synthesis is induced during the macrophage to foam cell transition. The expression and activity of GPAT3 and GPAT4 also increased during this process, and these two isoforms were required for the accumulation of cell TAG and PL. Compared to cells from wildtype mice after macrophage to foam cell transition, Gpat4-/- BMDM released more pro-inflammatory cytokines and chemokines, suggesting that the activity of GPAT4 could be associated with a decrease in the inflammatory response, probably by sequestering signaling precursors into lipid droplets. CONCLUSIONS: Our results provide evidence that TAG synthesis directed by GPAT3 and GPAT4 is required for lipid droplet formation and the modulation of the inflammatory response during the macrophage-foam cell transition.

Mapping Alzheimer's Disease Variants to Their Target Genes Using Computational Analysis of Chromatin Configuration.

Genome-wide association studies (GWAS) have successfully identified hundreds of genomic loci that are associated with human traits and disease. However, because the majority of the genome-wide significant (GWS) loci fall onto the non-coding genome, the functional impact of many remain unknown. Three-dimensional chromatin interactions identified by Hi-C or its derivatives can provide useful tools to annotate these loci by linking non-coding variants to their actionable genes. Here, we outline a protocol to map GWAS non-coding variants to their putative genes using Alzheimer's disease (AD) GWAS and Hi-C datasets from human adult brain tissue. Putative causal single-nucleotide polymorphisms (SNPs) are identified by application of fine-mapping algorithms. SNPs are then mapped to their putative target genes using enhancer-promoter interactions based on Hi-C. The resulting gene set represents AD risk genes, as they are potentially regulated by AD risk variants. To garner further biological insights into molecular mechanisms underlying AD, we characterize AD risk genes using developmental brain expression data and brain single-cell expression profiles. This protocol can be expanded to any GWAS and Hi-C datasets to identify putative target genes and molecular mechanisms underlying various human traits and diseases.

Glycerol-3-phosphate acyltransferases 3 and 4 direct glycerolipid synthesis and affect functionality in activated macrophages.

Macrophage classical M1 activation via TLR4 triggers a variety of responses to achieve the elimination of foreign pathogens. During this process, there is also an increase in lipid droplets which contain large quantities of triacylglycerol (TAG) and phospholipid (PL). The functional consequences of this increment in lipid mass are poorly understood. Here, we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). Using bone marrow-derived macrophages (BMDMs) treated with Kdo2-lipid A, we showed that glycerolipid synthesis is induced during macrophage activation. GPAT4 protein level and GPAT3/GPAT4 enzymatic activity increase during this process, and these two isoforms were required for the accumulation of cell TAG and PL. The phagocytic capacity of Gpat3-/- and Gpat4-/- BMDM was impaired. Additionally, inhibiting fatty acid ß-oxidation reduced phagocytosis only partially, suggesting that lipid accumulation is not necessary for the energy requirements for phagocytosis. Finally, Gpat4-/- BMDM expressed and released more pro-inflammatory cytokines and chemokines after macrophage activation, suggesting a role for GPAT4 in suppressing inflammatory responses. Together, these results provide evidence that glycerolipid synthesis directed by GPAT4 is important for the attenuation of the inflammatory response in activated macrophages.

Small non-coding RNA landscape is modified by GPAT2 silencing in MDA-MB-231 cells.

Glycerol-3-phosphate acyltransferase-2 is a member of "cancer-testis gene" family. Initially linked to lipid metabolism, this gene has been recently found involved also in PIWI-interacting RNAs biogenesis in germline stem cells. To investigate its role in piRNA metabolism in cancer, the gene was silenced in MDA-MB-231 breast cancer cells and small RNA sequencing was applied. PIWI-interacting RNAs and tRNA-derived fragments expression profiles showed changes following GPAT2 silencing. Interestingly, a marked shift in length distribution for both small RNAs was detected in GPAT2-silenced cells. Most downregulated PIWI-interacting RNAs are single copy in the genome, intragenic, hosted in snoRNAs and previously found to be upregulated in cancer cells. Putative targets of these PIWI-interacting RNAs are linked to lipid metabolism. Downregulated tRNA derived fragments derived from, so-called 'differentiation tRNAs', whereas upregulated ones derived from proliferation-linked tRNAs. miRNA amounts decrease after Glycerol-3-phosphate acyltransferase-2 silencing and functional enrichment analysis of deregulated miRNA putative targets point to mitochondrial biogenesis, IGF1R signaling and oxidative metabolism of lipids and lipoproteins. In addition, miRNAs known to be overexpressed in breast cancer tumors with poor prognosis where found downregulated in GPAT2-silenced cells. In conclusion, GPAT2 silencing quantitatively and qualitatively affects the population of PIWI-interacting RNAs, tRNA derived fragments and miRNAs which, in combination, result in a more differentiated cancer cell phenotype.

Glycerol-3-phosphate acyltranferase-2 behaves as a cancer testis gene and promotes growth and tumorigenicity of the breast cancer MDA-MB-231 cell line.

The de novo synthesis of glycerolipids in mammalian cells begins with the acylation of glycerol-3-phosphate, catalyzed by glycerol-3-phosphate acyltransferase (GPAT). GPAT2 is a mitochondrial isoform primarily expressed in testis under physiological conditions. Because it is aberrantly expressed in multiple myeloma, it has been proposed as a novel cancer testis gene. Using a bioinformatics approach, we found that GPAT2 is highly expressed in melanoma, lung, prostate and breast cancer, and we validated GPAT2 expression at the protein level in breast cancer by immunohistochemistry. In this case GPAT2 expression correlated with a higher histological grade. 5-Aza-2' deoxycytidine treatment of human cells lines induced GPAT2 expression suggesting epigenetic regulation of gene expression. In order to evaluate the contribution of GPAT2 to the tumor phenotype, we silenced its expression in MDA-MB-231 cells. GPAT2 knockdown diminished cell proliferation, anchorage independent growth, migration and tumorigenicity, and increased staurosporine-induced apoptosis. In contrast, GPAT2 over-expression increased cell proliferation rate and resistance to staurosporine-induced apoptosis. To understand the functional role of GPAT2, we performed a co-expression analysis in mouse and human testis and found a significant association with semantic terms involved in cell cycle, DNA integrity maintenance, piRNA biogenesis and epigenetic regulation. Overall, these results indicate the GPAT2 would be directly associated with the control of cell proliferation. In conclusion, we confirm GPAT2 as a cancer testis gene and that its expression contributes to the tumor phenotype of MDA-MB-231 cells.

Interstitial telomeric sequences are not preferentially involved in the chromosome damage induced by the methylating compound streptozotocin in Chinese hamster cells.

The effect of the methylating compound streptozotocin (STZ) on interstitial telomeric sequences (ITSs) was investigated in Chinese hamster ovary (CHO) cells by using peptide nucleic acid-fluorescence in situ hybridization with a pantelomeric probe. Cells were exposed to increasing concentrations of STZ, and chromosomal aberrations were analyzed at the first mitosis after treatment. The frequency of chromosomal aberrations directly involving ITSs increased in STZ-treated cells by a factor of 2.6 (2 mM) and 3.6 (4 mM) when compared with the frequency of these aberrations in control cells (P < 0.05). However, no significant differences were found between control and exposed cells in the percentage of aberrations directly involving ITSs, demonstrating that these repeat regions were not preferentially involved in the chromosome damage induced by STZ. In addition, STZ did not alter telomerase activity, suggesting that this enzyme may not be involved in the induction of chromosomal aberrations by this compound.

Telomere instability is present in the progeny of mammalian cells exposed to bleomycin.

We analyzed the chromosomal aberrations involving telomeres in the progeny of mammalian cells exposed to the radiomimetic compound bleomycin (BLM) in order to determine if this antineoplastic drug induces long-term telomere instability. To this end, rat cells (ADIPO-P2 cell line, derived from adipose cells from Sprague-Dawley rat) were treated with a single concentration of BLM (2.5 µg/ml), and chromosomal aberrations were analyzed 18 h and 10 days after treatment by using PNA-FISH with a pan-telomeric probe [(TTAGGG)n repeats]. Cytogenetic analysis revealed a higher frequency of aberrations at 18 h and 10 days after treatment in BLM-exposed cultures vs. untreated cultures, although the yield of BLM-induced aberrations 10 days after treatment decreased about 25% compared with the one at 18 h after treatment. Moreover, the level of telomerase activity in BLM-treated cells compared with that of untreated control cells was significantly higher at 10 days after treatment, but did not differ at 18 h after treatment. These data indicate that in terms of unstable aberrations, the in vitro clastogenic effect of BLM on ADIPO-P2 cells persists for at least 10 days after exposure. In addition, our data demonstrate, for the first time, that BLM-induced telomere instability in mammalian cells (cytogenetically detectable as incomplete chromosome elements and telomere FISH signal loss and duplication) persists for several generations after exposure. Moreover, the appearance of telomere fusions in BLM-exposed cells 10 days after treatment suggests that this compound can induce delayed telomere instability. The increase in telomerase activity in BLM-exposed cells 10 days after treatment is accompanied by the presence of aberrations directly related to telomere dysfunction. This fact suggests that telomerase is not directly involved in BLM-induced telomere instability.

Extracellular sunflower proteins: evidence on non-classical secretion of a jacalin-related lectin.

Extracellular proteins from sunflower seedlings were analyzed by electrophoresis followed by peptide mass fingerprinting. Tentative identification revealed novel proteins for this crop. A significant number of those proteins were not expected to be extracellular because they lacked the typical signal peptide responsible for secretion. In silico analysis showed that some members of this group presented the characteristic disordered structures of certain non-classical and leaderless mammalian secretory proteins. Among these proteins, a putative jacalin-related lectin (Helja) with a mannose binding domain was further isolated from extracellular fluids by mannose-affinity chromatography, thus validating its identification. Besides, immunolocalization assays confirmed its extracellular location. These results showed that a lectin, not predicted to be secreted in strict requirement of the N-terminal signal peptide, occurs in a sunflower extracellular compartment. The implications of this finding are discussed.