Comprehensive Analysis of Retrotransposon Insertions within the Survival Motor Neuron Genes Involved in Spinal Muscular Atrophy

Transposable elements (TEs) are interspersed repetitive DNA sequences with the ability to mobilize in the genome. The recent development of improved tools for evaluating TE-derived sequences in genomic studies has enabled an increasing attention to the contribution of TEs to human development and disease. Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease that is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. SMN2 gene is a nearly perfect duplication of SMN1. Both genes (collectively known as SMN1/SMN2) are highly enriched in TEs. A comprehensive analysis of TEs insertions in the SMN1/2 loci of SMA carriers, patients and healthy/control individuals was completed to perceive TE dynamics in SMN1/2 and try to establish a link between these elements and SMA.We found an Alu insertion in the promoter region and one L1 element in the 3’UTR that likely play an important role as an alternative promoter and as an alternative terminator to the gene, respectively. Additionally, the several Alu repeats inserted in the genes’ introns influence splicing, giving rise to alternative splicing events that cause RNA circularization and the birth of new alternative exons. These Alu repeats present throughout the genes are also prone to recombination events that can lead to SMN1 exons deletions, that ultimately lead to SMA. The many good and bad implications associated with the presence of TEs inside SMN1/2 make this genomic region ideal for understanding the implications of TEs on genomic evolution as well as on human genomic disease.

Alu-mediated weak CEBPA binding and slow B cell transdifferentiation in human

Many developmental and differentiation processes take substantially longer in human than in mouse. To investigate the molecular mechanisms underlying this phenomenon, here we have specifically focused on the transdifferentiation from B cells to macrophages. The process is triggered by exactly the same molecular mechanism -- the induction by the transcription factor (TF) CEBPA -- but takes three days in mouse and seven in human. In mouse, the speed of this process is known to be associated with Myc expression. We found that in this species, CEBPA binds strongly to the Myc promoter, efficiently down-regulating Myc. In human, in contrast, CEBPA does not bind this promoter, and MYC is indirectly and more slowly down-regulated. Attenuation of CEBPA binding is not specific to the MYC promoter, but a general trait of the human genome across multiple biological conditions. We traced back weak CEBPA binding to the primate-specific Alu repeat expansion. Many Alu repeats carry strong CEBPA binding motifs, which sequester CEBPA, and attenuate CEBPA binding genome-wide. We observed similar CEBPA and MYC dynamics in natural processes regulated by CEBPA, suggesting that CEBPA attenuation could underlie the longer duration in human processes controlled by this factor. Our work highlights the highly complex mode in which biological information is encoded in genome sequences, evolutionarily connecting, in an unexpected way, lineage-specific transposable element expansions to species-specific changes in developmental tempos.

PPAR-Responsive Elements Enriched with Alu Repeats May Contribute to Distinctive PPARγ–DNMT1 Interactions in the Genome

Background: PPARγ (peroxisome proliferator-activated receptor gamma) is involved in the pathology of numerous diseases, including UM and other types of cancer. Emerging evidence suggests that an interaction between PPARγ and DNMTs (DNA methyltransferase) plays a role in cancer that is yet to be defined. Methods: The configuration of the repeating elements was performed with CAP3 and MAFFT, and the structural modelling was conducted with HDOCK. An evolutionary action scores algorithm was used to identify oncogenic variants. A systematic bioinformatic appraisal of PPARγ and DNMT1 was performed across 29 tumor types and UM available in The Cancer Genome Atlas (TCGA). Results: PPAR-responsive elements (PPREs) enriched with Alu repeats are associated with different genomic regions, particularly the promotor region of DNMT1. PPARγ–DNMT1 co-expression is significantly associated with several cancers. C-terminals of PPARγ and DNMT1 appear to be the potential protein–protein interaction sites where disease-specific mutations may directly impair the respective protein functions. Furthermore, PPARγ expression could be identified as an additional prognostic marker for UM. Conclusions: We hypothesize that the function of PPARγ requires an additional contribution of Alu repeats which may directly influence the DNMT1 network. Regarding UM, PPARγ appears to be an additional discriminatory prognostic marker, in particular in disomy 3 tumors.

Elevated Alu retroelement copy number among workers exposed to diesel engine exhaust

BackgroundMillions of workers worldwide are exposed to diesel engine exhaust (DEE), a known genotoxic carcinogen. Alu retroelements are repetitive DNA sequences that can multiply and compromise genomic stability. There is some evidence linking altered Alu repeats to cancer and elevated mortality risks. However, whether Alu repeats are influenced by environmental pollutants is unexplored. In an occupational setting with high DEE exposure levels, we investigated associations with Alu repeat copy number.MethodsA cross-sectional study of 54 male DEE-exposed workers from an engine testing facility and a comparison group of 55 male unexposed controls was conducted in China. Personal air samples were assessed for elemental carbon, a DEE surrogate, using NIOSH Method 5040. Quantitative PCR (qPCR) was used to measure Alu repeat copy number relative to albumin (Alb) single-gene copy number in leucocyte DNA. The unitless Alu/Alb ratio reflects the average quantity of Alu repeats per cell. Linear regression models adjusted for age and smoking status were used to estimate relations between DEE-exposed workers versus unexposed controls, DEE tertiles (6.1–39.0, 39.1–54.5 and 54.6–107.7 µg/m3) and Alu/Alb ratio.ResultsDEE-exposed workers had a higher average Alu/Alb ratio than the unexposed controls (p=0.03). Further, we found a positive exposure–response relationship (p=0.02). The Alu/Alb ratio was highest among workers exposed to the top tertile of DEE versus the unexposed controls (1.12±0.08 SD vs 1.06±0.07 SD, p=0.01).ConclusionOur findings suggest that DEE exposure may contribute to genomic instability. Further investigations of environmental pollutants, Alu copy number and carcinogenesis are warranted.

Homotypic clustering of L1 and B1/Alu repeats compartmentalizes the 3D genome

Organization of the genome into euchromatin and heterochromatin appears to be evolutionarily conserved and relatively stable during lineage differentiation. In an effort to unravel the basic principle underlying genome folding, here we focus on the genome itself and report a fundamental role for L1 (LINE1 or LINE-1) and B1/Alu retrotransposons, the most abundant subclasses of repetitive sequences, in chromatin compartmentalization. We find that homotypic clustering of L1 and B1/Alu demarcates the genome into grossly exclusive domains, and characterizes and predicts Hi-C compartments. Spatial segregation of L1-rich sequences in the nuclear and nucleolar peripheries and B1/Alu-rich sequences in the nuclear interior is conserved in mouse and human cells and occurs dynamically during the cell cycle. In addition, de novo establishment of L1 and B1 nuclear segregation is coincident with the formation of higher-order chromatin structures during early embryogenesis and appears to be critically regulated by L1 and B1 transcripts. Importantly, depletion of L1 transcripts in embryonic stem cells drastically weakens homotypic repeat contacts and compartmental strength, and disrupts the nuclear segregation of L1- or B1-rich chromosomal sequences at genome-wide and individual sites. Mechanistically, nuclear co-localization and liquid droplet formation of L1 repeat DNA and RNA with heterochromatin protein HP1α suggest a phase-separation mechanism by which L1 promotes heterochromatin compartmentalization. Taken together, we propose a genetically encoded model in which L1 and B1/Alu repeats blueprint chromatin macrostructure. Our model explains the robustness of genome folding into a common conserved core, on which dynamic gene regulation is overlaid across cells.

Droplet Digital PCR for the Quantification of Alu Methylation Status in Hematological Malignancies

Introduction. Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. They are located in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu repeats methylation has been associated with tumor aggressiveness and investigated in some solid tumors, but the global Alu methylation level has not yet been investigated in hematological malignancies. Moreover, today, some of the techniques designed to measure global DNA methylation are focused on the methylation level of specific genomic compartments, including repeat elements. In this work we propose a new method for investigating Alu differential methylation, employing droplet digital PCR (ddPCR) technology, applied in patients affected by chronic lymphocytic leukemia (CLL), myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). Methods. The study included a total of 46 patients: 30 CLL patients, 7 patients with MDS at intermediate/high risk, and 9 CMML patients. The study also involved acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with azacytidine (AZA) 0.75 µM or decytabine (DEC) 0.75 µM. Four healthy donors (HD) were also included as controls. For each DNA sample two aliquots of 250ng of gDNA were simultaneously digested (with 1 unit of Alu-in/sensitive isoschizomers either MspI or HpaII) and ligated (to a previously prepared synthetic adaptor) in parallel in two separate tubes. Considering that the genomic DNA amount in a human diploid cell is about 6 pg/cell, for each sample we calculated the percentage of methylated consensus Alu sequences as the ratio between the sum of positive droplets obtained from the three wells of both HpaII (MH) and MspI (MM) final dilutions, according to the following formula: [1-(sumMH/sumMM)]x100. The significance level was set at p<0.05 for all analyses. Results. Using our ddPCR assay, we observed a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, and a minor decrease with AZA (p=0.058). Moreover, comparing the global Alu methylation levels at diagnosis and after AZA treatment in MDS patients, we observed a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis. We also extended the assessment of our assay in CLL patients at diagnosis. We observed a significant decrease of the Alu methylation level in CLL patients compared to HD. CLL patients were also classified in the following three cytogenetic risk groups according to the karyotypic alterations identified by Fluorescent In Situ Hybridization (FISH): low (with isolated 13q deletion), intermediate (without 11q, 13q and 17p deletions or with trisomy 12), and high risk (with 11q or, 17p deletions, or more than two chromosomal aberrations). Alu methylation status of the low and high-risk groups was more significantly reduced compared to HD, whereas considering intermediate-risk patients the difference was less evident. Finally, for CMML patients, a significant decrease of Alu sequences methylation was observed in patients harboring the main SRSF2 gene hotspot. However, these preliminary results should be confirmed by extending the analysis to other CMML patients. Conclusions. In our work, we propose a new method to investigate Alu differential methylation based on ddPCR technology. This assay represents an alternative to conventional quantitative-PCR (qPCR), introducing ddPCR as a more sensitive and immediate technique for Alu methylation analysis. Moreover, compared to qPCR, our ddPCR Alu assay may be carried out using very small amounts of digested gDNA (about 6 pg), and does not require a reference gene for the analysis of ddPCR data. To date, this is the first application of ddPCR to study global DNA methylation by inspecting DNA repeats. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose. Disclosures No relevant conflicts of interest to declare.