We quantified genome-wide patterns of lysine H3K27 acetylation (H3K27ac) in entorhinal cortex samples from Alzheimer's disease (AD) cases and matched controls using chromatin immunoprecipitation and ...highly parallel sequencing. We observed widespread acetylomic variation associated with AD neuropathology, identifying 4,162 differential peaks (false discovery rate < 0.05) between AD cases and controls. Differentially acetylated peaks were enriched in disease-related biological pathways and included regions annotated to genes involved in the progression of amyloid-β and tau pathology (for example, APP, PSEN1, PSEN2, and MAPT), as well as regions containing variants associated with sporadic late-onset AD. Partitioned heritability analysis highlighted a highly significant enrichment of AD risk variants in entorhinal cortex H3K27ac peak regions. AD-associated variable H3K27ac was associated with transcriptional variation at proximal genes including CR1, GPR22, KMO, PIM3, PSEN1, and RGCC. In addition to identifying molecular pathways associated with AD neuropathology, we present a framework for genome-wide studies of histone modifications in complex disease.
Abstract
RNA can directly bind to purine-rich DNA via Hoogsteen base pairing, forming a DNA:RNA triple helical structure that anchors the RNA to specific sequences and allows guiding of transcription ...regulators to distinct genomic loci. To unravel the prevalence of DNA:RNA triplexes in living cells, we have established a fast and cost-effective method that allows genome-wide mapping of DNA:RNA triplex interactions. In contrast to previous approaches applied for the identification of chromatin-associated RNAs, this method uses protein-free nucleic acids isolated from chromatin. High-throughput sequencing and computational analysis of DNA-associated RNA revealed a large set of RNAs which originate from non-coding and coding loci, including super-enhancers and repeat elements. Combined analysis of DNA-associated RNA and RNA-associated DNA identified genomic DNA:RNA triplex structures. The results suggest that triplex formation is a general mechanism of RNA-mediated target-site recognition, which has major impact on biological functions.
To prevent autoimmunity, thymocytes expressing self-reactive T cell receptors (TCRs) are negatively selected, however, divergence into tolerogenic, agonist selected lineages represent an alternative ...fate. As thymocyte development, selection, and lineage choices are dependent on spatial context and cell-to-cell interactions, we have performed Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) and spatial transcriptomics on paediatric human thymus. Thymocytes expressing markers of strong TCR signalling diverged from the conventional developmental trajectory prior to CD4
or CD8
lineage commitment, while markers of different agonist selected T cell populations (CD8αα(I), CD8αα(II), T
, T
(diff), and T
) exhibited variable timing of induction. Expression profiles of chemokines and co-stimulatory molecules, together with spatial localisation, supported that dendritic cells, B cells, and stromal cells contribute to agonist selection, with different subsets influencing thymocytes at specific developmental stages within distinct spatial niches. Understanding factors influencing agonist T cells is needed to benefit from their immunoregulatory effects in clinical use.
Retroelements constitute a large part of the human genome. These sequences are mostly silenced in normal cells, but genome-wide DNA hypomethylation in cancers might lead to their re-expression. ...Whether this re-expression really occurs in human cancers is largely unkown. We therefore investigated expression and DNA methylation of several classes of retroelements in human prostate cancer tissues and cell lines by quantitative reverse transcription-polymerase chain reaction and pyrosequencing, respectively. The most striking finding was strong and generalized increased expression of the HERV-K_22q11.23 provirus in cancers, including de novo expression of a spliced accessory Np9 transcript in some tumors. In parallel, DNA methylation in the long terminal repeat (LTR) decreased. Conversely, HERVK17 expression was significantly diminished in cancer tissues, but this decrease was unrelated to LTR methylation. Expression of both proviruses was restricted to androgen-responsive prostate cancer cell lines and LTRs sequences containing steroid hormone-responsive elements bound the androgen receptor and conferred androgen responsiveness to reporter constructs. Expression of LINE-1 5′-untranslated region (UTR) and 3′-UTR sequences in prostate cancers rather decreased, despite significant hypomethylation of the internal LINE-1 promoter. Increased expression of the young AluYa5 and AluYb8 families was restricted to individual tumors. Our findings demonstrate a surprising specificity of changes in expression and DNA methylation of retroelements in prostate cancer. In particular, LINE-1 hypomethylation does not lead to generalized overexpression, but specific human endogenous retrovirus-K proviruses display conspicuous changes in their expression hinting at significant functions during prostate carcinogenesis.
•SARS-COV-2 Variant PCR based on the protocol by Vogels et al provides fast results, demands relatively few human and data resources, and delivers high throughput.•In 1,642 SARS-CoV-2 positive ...samples, no Variant of Concern was missed by Variant PCR, confirmed by whole genome sequencing.•All samples identified as “non-VoC lineages” by the Variant PCR, were confirmed by whole genome sequencing.•The Variant PCR based on the protocol by Vogels et al., is a good and effective method for rapid screening for variants of concern, applicable for most diagnostic high-volume laboratories.
The emerging SARS-CoV-2 variants of concern (VoC), B.1.1.7, B.1.351 and P.1, with increased transmission and/or immune evasion, emphasise the need for broad and rapid variant monitoring. Our high-volume laboratory introduced a PCR variant assay (Variant PCR) in January 2021 based on the protocol by Vogels et al.
To assess whether Variant PCR could be used for rapid B.1.1.7, B.1.351 and P.1 screening, all positive SARS-CoV-2 airway samples were prospectively tested in parallel using both the Variant PCR and whole genome sequencing (WGS).
In total 1,642 SARS-CoV-2 positive samples from individual patients were tested within a time span of 4 weeks. For all samples with valid results from both Variant PCR and WGS, no VoC was missed by Variant PCR (totalling 399 VoC detected). Conversely, all of the samples identified as “other lineages” (i.e., “non-VoC lineages”) by the Variant PCR, were confirmed by WGS.
The Variant PCR based on the protocol by Vogels et al., is an effective method for rapid screening for VoC, applicable for most diagnostic laboratories within a pandemic setting. WGS is still required to confirm the identity of certain variants and for continuous surveillance of emerging VoC.
DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing of the RNA in the major groove of the DNA duplex have been observed in vitro, but the extent to which these interactions occur in ...cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1α-AS1 was retrieved as a top hit. Endogenous HIF1α-AS1 reduces the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1α-AS1 is down-regulated in pulmonary hypertension and loss-of-function approaches not only result in gene de-repression but also enhance angiogenic capacity. As exemplified here with HIF1α-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control.
Novel commercial kits for whole genome library preparation for next-generation sequencing on Illumina platforms promise shorter workflows, lower inputs and cost savings. Time savings are achieved by ...employing enzymatic DNA fragmentation and by combining end-repair and tailing reactions. Fewer cleanup steps also allow greater DNA input flexibility (1 ng-1 μg), PCR-free options from 100 ng DNA, and lower price as compared to the well-established sonication and tagmentation-based DNA library preparation kits.
We compared the performance of four enzymatic fragmentation-based DNA library preparation kits (from New England Biolabs, Roche, Swift Biosciences and Quantabio) to a tagmentation-based kit (Illumina) using low input DNA amounts (10 ng) and PCR-free reactions with 100 ng DNA. With four technical replicates of each input amount and kit, we compared the kits' fragmentation sequence-bias as well as performance parameters such as sequence coverage and the clinically relevant detection of single nucleotide and indel variants. While all kits produced high quality sequence data and demonstrated similar performance, several enzymatic fragmentation methods produced library insert sizes which deviated from those intended. Libraries with longer insert lengths performed better in terms of coverage, SNV and indel detection. Lower performance of shorter-insert libraries could be explained by loss of sequence coverage to overlapping paired-end reads, exacerbated by the preferential sequencing of shorter fragments on Illumina sequencers. We also observed that libraries prepared with minimal or no PCR performed best with regard to indel detection.
The enzymatic fragmentation-based DNA library preparation kits from NEB, Roche, Swift and Quantabio are good alternatives to the tagmentation based Nextera DNA flex kit from Illumina, offering reproducible results using flexible DNA inputs, quick workflows and lower prices. Libraries with insert DNA fragments longer than the cumulative sum of both read lengths avoid read overlap, thus produce more informative data that leads to strongly improved genome coverage and consequently also increased sensitivity and precision of SNP and indel detection. In order to best utilize such enzymatic fragmentation reagents, researchers should be prepared to invest time to optimize fragmentation conditions for their particular samples.
Craniosynostosis (CS) is a common congenital anomaly defined by premature fusion of one or more cranial sutures. Syndromic CS involves additional organ anomalies or neurocognitive deficits and ...accounts for 25%-30% of the cases. In a recent population-based study by our group, 84% of the syndromic CS cases had a genetically verified diagnosis after targeted analyses. A number of different genetic causes were detected, confirming that syndromic CS is highly heterogeneous. In this study, we performed whole-exome sequencing of 10 children and parents from the same cohort where previous genetic results were negative. We detected pathogenic, or likely pathogenic, variants in four additional genes (NFIA, EXTL3, POLR2A, and FOXP2) associated with rare conditions. In two of these (POLR2A and FOXP2), CS has not previously been reported. We further detected a rare predicted damaging variant in SH3BP4, which has not previously been related to human disease. All findings were clustered in genes involved in the pathways of osteogenesis and suture patency. We conclude that whole-exome sequencing expands the list of genes associated with syndromic CS, and provides new candidate genes in osteogenic signaling pathways.
The two oppositely imprinted and expressed genes, DLK1 and MEG3, are located in the same gene cluster at 14q32. Previous studies in bladder cancer have suggested that tumor suppressor genes are ...located in this region, but these have not been identified.
We observed that both DLK1 and MEG3 are frequently silenced in urothelial cancer tissues and cell lines. The concomitant downregulation of the two genes is difficult to explain by known mechanisms for inactivating imprinted genes, namely deletion of active alleles or epitype switching. Indeed, quantitative PCR revealed more frequent copy number gains than losses in the gene cluster that were, moreover, consistent within each sample, excluding gene losses as the cause of downregulation. Instead, we observed distinctive epigenetic alterations at the three regions controlling DLK1 and MEG3 expression, namely the DLK1 promoter; the intergenic (IG) and MEG3 differentially methylated regions (DMRs). Bisulfite sequencing and pyrosequencing revealed novel patterns of DNA methylation in tumor cells, which were distinct from that of either paternal allele. Furthermore, chromatin immunoprecipitation demonstrated loss of active and gain of repressive histone modifications at all regulatory sequences.
Our data support the idea that the main cause of the prevalent downregulation of DLK1 and MEG3 in urothelial carcinoma is epigenetic silencing across the 14q32 imprinted gene cluster, resulting in the unusual concomitant inactivation of oppositely expressed and imprinted genes.
Epigenetic dysregulation comprising DNA hypermethylation and hypomethylation, enhancer of zeste homologue 2 (EZH2) overexpression and altered patterns of histone modifications is associated with the ...progression of prostate cancer. DNA methylation, EZH2 and histone modifications also ensure the parental-specific monoallelic expression of at least 62 imprinted genes. Although it is therefore tempting to speculate that epigenetic dysregulation may extend to imprinted genes, expression changes in cancerous prostates are only well documented for insulin-like growth factor 2 (IGF2). A literature and database survey on imprinted genes in prostate cancer suggests that the expression of most imprinted genes remains unchanged despite global disturbances in epigenetic mechanisms. Instead, selective genetic and epigenetic changes appear to lead to the inactivation of a sub-network of imprinted genes, which might function in the prostate to limit cell growth induced via the PI3K/Akt pathway, modulate androgen responses and regulate differentiation. Whereas dysregulation of IGF2 may constitute an early change in prostate carcinogenesis, inactivation of this imprinted gene network is rather associated with cancer progression.
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