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Van Nieuwerburgh, F.

Publications and source records attributed to Van Nieuwerburgh, F..

33 records · Page 2Linked to original sources

Transcriptional and translational dynamics underlying heat shock response in the thermophilic Crenarchaeon Sulfolobus acidocaldarius

High-temperature stress is critical for all organisms and induces a profound cellular response. For Crenarchaeota, little information is available on how heat shock affects cellular processes and on how this response is regulated. In this work, we set out to study heat shock response in the thermoacidophilic model crenarchaeon Sulfolobus acidocaldarius, which thrives in volcanic hot springs and has an optimal growth temperature of 75{degrees}C. Pulse-labeling experiments demonstrated that a temperature shift to 86{degrees}C induces a drastic reduction of the transcriptional and translational activity, but that RNA and protein neosynthesis still occurs. By combining RNA sequencing and TMT-labeled mass spectrometry, an integrated mapping of the transcriptome and proteome was performed. This revealed that heat shock causes an immediate change in the gene expression profile, with RNA levels of half of the genes being affected, followed by the more subtle reprogramming of the protein landscape. A limited correlation was observed in differential expression on the RNA and protein level, suggesting that there is a prevalence of post-transcriptional and post-translational regulation upon heat shock. Furthermore, based on the finding that promoter regions of heat shock regulon genes lack a conserved DNA-binding motif, we propose that heat-shock responsive transcription regulation is likely not to be accomplished by a classical transcription factor. Instead, in contrast to histone-harboring Euryarchaeota that have heat-shock transcription factors, it is hypothesized that Sulfolobales and other histone-lacking thermophilic archaea employ an evolutionary ancient mechanism relying on temperature-responsive changes in DNA organization and compaction, induced by the action of nucleoid-associated proteins.

microbiology↗

Single-cell profiling identifies a spectrum of human unconventional intraepithelial T lineage cells

In the human thymus, a CD10+ PD-1+ TCR{beta}+ differentiation pathway diverges from the conventional single positive T cell lineages at the early double positive stage. These cells are phenotypically and functionally similar to murine unconventional intraepithelial lymphocyte (uIEL) precursors. Here, the progeny of the human uIEL lineage was identified in antigen-inexperienced blood. The uIELs in thymus and peripheral blood share a transcriptomic profile, characterized by hallmark transcription factors (i.e. ZNF683 and IKZF2), and polyclonal TCR repertoire with autoreactive features, exhibiting a bias towards early TCR alpha chain rearrangements. Single-cell RNA sequencing confirmed a common developmental trajectory between the thymic and peripheral uIELs, and clearly delineated this unconventional lineage in peripheral blood. This population is phenotypically defined as CD3+ TCR{beta}+ CD4- CCR7- CD26-. It contains CD10+ recent thymic emigrants, Helios+ KIR+ CD8+ Tregs and CD8+ T cells. Thus, the uIEL lineage represents a well-defined but heterogeneous, unconventional TCR{beta}+ lineage mostly confined in human within the CD8 single positive T cells. SummaryBilliet et al. identify the postthymic progeny of the intraepithelial lymphocyte precursors in human based on shared characteristics of the T cell receptor repertoire and the transcriptome. This lineage represents a well-defined but heterogeneous, unconventional TCR{beta}+ lineage mostly confined within the CD8 single positive T cells.

immunology↗

The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

NK cells are innate lymphocytes that eliminate virus-infected and cancer cells by cytotoxicity and cytokine secretion. In addition to circulating NK cells, distinct tissue-resident NK subsets have been identified in various organs. Although transcription factors regulating NK cell development and function have been extensively studied in mice, the role of RUNX2 in these processes has not been investigated, neither in mice nor in human. Here, by manipulating RUNX2 expression with either knockdown or overexpression in human hematopoietic stem cell-based NK cell differentiation cultures, combined with transcriptomic and ChIP-sequencing analyses, we established that RUNX2 drives the generation of NK cells, possibly through induction of IL-2R{beta} expression in NK progenitor cells. Importantly, RUNX2 promotes tissue residency in human NK cells. Our findings have the potential to improve existing NK cell-based cancer therapies and can impact research fields beyond NK cell biology, since tissue-resident subsets have also been described in other lymphocyte subpopulations.

immunology↗

Cas9 targeted nanopore sequencing with enhanced variant calling improves CYP2D6-CYP2D7 hybrid allele genotyping

CYP2D6 is one of the most challenging pharmacogenes to genotype due to the high similarity with its neighboring pseudogenes and the frequent occurrence of CYP2D6-CYP2D7 hybrids. Unfortunately, most current genotyping methods are therefore not able to correctly determine the complete CYP2D6-CYP2D7 sequence. Therefore, we developed a genotyping assay to generate complete allele-specific consensus sequences of complex regions by optimizing the PCR-free nanopore Cas9-targeted sequencing (nCATS) method combined with adaptive sequencing, and developing a new comprehensive long read genotyping (CoLoRGen) pipeline. The CoLoRGen pipeline first generates consensus sequences of both alleles and subsequently determines both large structural and small variants to ultimately assign the correct star-alleles. In reference samples, our genotyping assay confirms the presence of CYP2D6-CYP2D7 large structural variants, single nucleotide variants (SNVs), and small insertions and deletions (INDELs) that go undetected by most current assays. Moreover, our results provide direct evidence that the CYP2D6 genotype of the NA12878 DNA should be updated to include the CYP2D6-CYP2D7 *68 hybrid and several additional single nucleotide variants compared to existing references. Ultimately, the nCATS-CoLoRGen genotyping assay additionally allows for more accurate gene function predictions by enabling the possibility to detect and phase de novo mutations in addition to known large structural and small variants. Author SummaryDuring the last decades, the usefulness of personalized medicine has become increasingly apparent. Directly linked to that is the need for accurate genotyping assays to determine the pharmacogenetic profile of patients. Continuing research has led to the development of genotyping assays that perform quite robustly. However, complex genes remain an issue when it comes to determining the complete sequence correctly. An example of such a complex but very important pharmacogene is CYP2D6. Therefore, we developed a genotyping assay in an attempt to generate complete allele-specific consensus sequences of CYP2D6, by optimizing a targeted amplification-free long-read sequencing method and developing a new analysis pipeline. In reference samples, we showed that our genotyping assay performed accurately and confirmed the presence of variants that go undetected by most current assays. However, the implementation of this assay in practice is still hampered as the selected enrichment strategies inherently lead to a low percentage of on-target reads, resulting in low on-target sequencing depths. Further optimization and validation of the assay is thus needed, but definitely worth considering for follow-up research as we already demonstrated the added value for generating more complete genotypes, which on its turn will result in more accurate gene function predictions.

genetics↗

Standing covariation between genomic and epigenomic patterns as source for natural selection in wild strawberry plants

Adaptive evolution is generally thought to be the result of natural selection predominantly acting upon pre-existing DNA sequence polymorphisms through gene-environment interactions. Epigenetic inheritance is, however, recently considered an additional molecular force driving adaptive evolution independent of DNA sequence variation. Through comparative analyses of genome-wide genetic (SNPs) and epigenetic (DMCs) variation of wild strawberry plants raised under distinct drought settings, we demonstrate intermediate levels of genome-wide covariation between SNPs and DMCs. Cases of high SNP-DMC covariation were significantly associated with (i) applied stress, (ii) non-adaptive SNPs, and (iii) solitary DMCs (as opposed to DMC islands). We also found that DMCs, drought-responsive DMCs in particular, typically co-vary with hundreds of SNPs, indicating high genomic redundancy as a basis for polygenic adaptation. Our findings suggest that stress-responsive DMCs initially co-vary with many associated SNPs under increased environmental stress (cfr. co-gradient plasticity), and that natural selection acting upon these SNPs subsequently reduce standing covariation with stress-responsive DMCs. In addition, the degree of covariation between SNPs and DMCs appears independent of their respective genomic distance, indicating that trans-acting associations between SNPs and DMCs are as likely as cis-acting associations. Our study is in favor of DNA methylation profiles representing complex quantitative traits rather than independent evolutionary forces, but further research is required to fully rule out SNP-independence of genome-wide DMCs. We provide a conceptual framework for polygenic regulation and adaptation shaping genome-wide methylation patterns.

evolutionary biology↗

MYCN-induced nucleolar stress drives an early senescence-like transcriptional program in hTERT-immortalized RPE cells

MYCN is an oncogenic driver in neural crest-derived neuroblastoma and medulloblastoma. To better understand the early effects of MYCN activation in a neural-crest lineage context, we profiled the transcriptome of immortalized human retina pigment epithelial cells with inducible MYCN activation. Gene signatures associated with elevated MYC/MYCN activity were induced after 24 h of MYCN activation, which attenuated but sustained at later time points. Unexpectedly, MYCN activation was accompanied by reduced cell growth. Gene set enrichment analysis revealed a senescence-like signature with strong induction of p53 and p21 but in the absence of canonical hallmarks of senescence such as beta-galactosidase positivity, suggesting incomplete cell fate commitment. When scrutinizing the putative drivers of this growth attenuation, differential gene expression analysis identified several regulators of nucleolar stress. This process was also reflected by phenotypic correlates such as cytoplasmic granule accrual and nucleolar coalescence. Hence, we propose that the induction of MYCN congests the translational machinery, causing nucleolar stress and driving cells into a transient pre-senescent state. Our findings shed new light on the early events induced by MYCN activation and may help unravelling which factors are required for cells to tolerate unscheduled MYCN overexpression during early malignant transformation. HighlightsO_LIActivation of MYCN attenuates proliferation in RPE1 cells C_LIO_LIGrowth arrest is associated with an early senescence-like transcriptional signature C_LIO_LITranscriptional and phenotypic evidence of nucleolar stress C_LIO_LICDKN1A upregulation in G2 phase primes cells for faltering in subsequent G1 C_LI

cancer biology↗

ADAR1 interaction with Z-RNA promotes editing of endogenous double-stranded RNA and prevents MDA5-dependent immune activation.

Loss-of-function of ADAR1 causes the severe autoinflammatory disease Aicardi-Goutieres Syndrome (AGS). ADAR1 converts adenosines into inosines within double-stranded (ds) RNA. This process called A-to-I editing masks self-dsRNA from detection by the antiviral dsRNA sensor MDA5. ADAR1 binds to dsRNA in both the canonical A-form and in the poorly defined Z-conformation (Z-RNA). Mutations in the Z-RNA binding Z-domain of ADAR1 are common in AGS patients. How loss of ADAR1/Z-RNA interaction contributes to disease development is unknown. Using ADAR1 Z-domain mutant human cells and knock-in mice, we demonstrate that abrogated binding of ADAR1 to Z-RNA leads to reduced A-to-I editing of dsRNA structures formed by pairing of inversely oriented SINEs. As a result, ADAR1 Z-domain mutant human cells and transgenic mice develop a spontaneous MDA5-dependent immune response. This shows that the interaction between ADAR1 and Z-RNA restricts sensing of self-dsRNA and prevents AGS development.

immunology↗

RRM2 is a target for synthetic lethal interactions with replication stress checkpoint addiction in high-risk neuroblastoma

Neuroblastoma is a pediatric tumor originating from the sympathetic nervous system responsible for 10-15 percent of all childhood cancer deaths. Half of all neuroblastoma patients present with high-risk disease at diagnosis. Despite intensive multi-modal therapies nearly 50 percent of high-risk cases relapse and die of their disease. In contrast to the overall paucity of mutations, high-risk neuroblastoma nearly invariably present with recurrent somatic segmental chromosome copy number variants. For several focal aberrations (e.g. MYCN and LIN28B amplification), the direct role in tumor formation has been established. However, for recurrent aberrations, such as chromosome 2p and 17q gains, the identification of genes contributing to tumor initiation or progression has been challenging due to the scarcity of small segmental gains or amplifications. In this study, we identified and functionally evaluated the ribonucleotide reductase regulatory subunit 2 (RRM2) as a top-ranked 2p putative co-driver and therapeutic target in high-risk neuroblastoma enforcing replicative stress resistance. In vitro knock down and pharmacological RRM2 inhibition highlight RRM2 dependency in neuroblastoma cells, further supported by the finding that co-overexpression of RRM2 in a d{beta}h-MYCN transgenic zebrafish line increased tumor penetrance with 80% and accelerated tumor formation. Given the critical role of RRM2 in replication fork progression and regulation of RRM2 through ATR/CHK1 signaling, we tested combined RRM2 and ATR/CHK1 small molecule inhibition with triapine and BAY1895344/prexasertib respectively, and observed strong synergism, in particular for combined RRM2 and CHK1 inhibition. Transcriptome analysis following combinatorial drugging revealed HEXIM1 as one of the strongest upregulated genes. Using programmable DNA binding of dCas9 with a promiscuous biotin ligase, RRM2 promotor bound proteins were identified including HEXIM1 and NurRD complex members, supporting a cooperative role for HEXIM1 upregulation together with CHK1 inhibition in further attenuating RRM2 expression levels. We evaluated the impact of combined RRM2/CHK1 inhibition in vivo, with treatment of a murine xenograft model showing rapid and complete tumor regression, without tumor regrowth upon treatment arrest. In conclusion, we identified RRM2 as a novel dependency gene in neuroblastoma and promising target for synergistic drug combinations with small compounds targeting DNA checkpoint regulators.

cancer biology↗

Recurrent chromosomal imbalances provide selective advantage to human embryonic stem cells under enhanced replicative stress conditions

Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress which is known to induce genomic instability causing chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea induced replicative stress versus normal growth conditions in an equally-mixed cell population of isogenic euploid and 17q+ hESCs. We could show that 17q+ hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.

cancer biology↗

Endogenous suppression of WNT signalling in human embryonic stem cells leads to low differentiation propensity towards definitive endoderm

SummaryLow differentiation propensity towards a targeted lineage can significantly hamper the utility of individual human pluripotent stem cell (hPSC) lines in biomedical applications. Here, we use monolayer and micropatterned cell cultures, as well as transcriptomic profiling, to investigate how variability in signalling pathway activity between human embryonic stem cell lines affects their differentiation efficiency towards definitive endoderm (DE). We show that endogenous suppression of WNT signalling in hPSCs at the onset of differentiation prevents the switch from self-renewal to DE specification. Gene expression profiling reveals that this inefficient switch is reflected in NANOG expression dynamics. Importantly, we demonstrate that higher WNT stimulation or inhibition of the PI3K/AKT signalling can overcome the DE commitment blockage. Our findings highlight that redirection of the activity of Activin/NODAL pathway by WNT signalling towards mediating DE fate specification is a vulnerable spot, as disruption of this process can result in poor hPSC specification towards DE.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

Ecological divergence of DNA methylation patterns at distinct spatial scales

Adaptive trait divergence between populations is regulated by genetic and non-genetic processes. Compared to genetic change, epigenetic change is unstable and short-lived, questioning its contribution to long-term adaptive potential. However, epigenetic change can accumulate over time, and may result in beneficial epigenetic memories where environments are heterogeneous. Diverging epigenetic memories have been observed across large spatial scales, and can persist through multiple generations even in the absence of the causative environmental stressor. It is unknown, however, how and to what extent epigenetic memories contribute to fine-scale population structure and evolution. Here, we performed whole genome bisulfite sequencing on 30 Fragaria vesca F1 plants originating from distinct ecological settings and grown in a controlled environment. Specifically, we compared methylation patterns between a steep, altitudinal gradient (<2 km) and a wide spatial gradient (>500 km). If epigenetic variation is random, arising from errors during replication and without evolutionary implications, one would expect similar amounts of epigenetic variation across populations and no spatial scale-effect. Here, we find that epigenetic memories arise even at fine spatial scale, and that both parallel and non-parallel biological processes underpin epigenetic divergence at distinct spatial scales. For example, demethylation of transposable elements consistently occurred at the large but not the small spatial scale, while methylation differentiation for most biological processes were shared between spatial scales. Acute drought stress did not result in significant epigenetic differentiation, indicating that repeated historical stress levels associated with heterogeneous environmental conditions are required for acquiring a stable epigenetic memory and for coping with future environmental change.

ecology↗

Analyzing data independent acquisition mass spectrometry with noiseless ion-networks

Data-independent acquisition (DIA) mass spectrometry (MS) has introduced deter-ministic, periodic and simultaneous acquisition of all fragment ions. Despite the chimeric side-effects associated with this unprecedented data integrity, DIA data analysis approaches still use conventional spectra and extracted ion chromatograms (XICs) that represent individual precursors and fragments. Here, we introduce ion-networks, an alternative data format wherein nodes correspond to reproducible fragment ions from multiple runs and edges correspond to consistent co-elution. Each ion-network represents a complete experiment and computationally eliminates chimericy based on reproducibility without sacrificing data integrity.

bioinformatics↗

Forensic STR profiling using Oxford Nanopore Technologies’ MinION sequencer

Forensic STR profiling using massively parallel sequencing (MPS) has gained much attention as an alternative for the traditional capillary electrophoresis (CE) methods. Oxford Nanopore Technologies recently developed the MinION, a pocket-sized nanopore sequencer with promising features that could be useful in the field of forensic genetics. We applied this technology for forensic sequencing in a pilot study. Using standard STR primers, originally designed for multiplex PCR and CE, we developed a library preparation method suited for nanopore sequencing. Several analysis approaches were evaluated to explore the usefulness of the generated data: we developed and applied both a sequence-based and an amplicon length-based analysis on data originating from a 14-loci multiplex PCR on a single contributor DNA sample. Despite the high sequencing error rate, the analyses yielded partial forensic profiles with some useful evidential value.

genomics↗

Various evolutionary trajectories lead to loss of the tobramycin-potentiating activity of the quorum sensing inhibitor baicalin hydrate in Burkholderia cenocepacia biofilms

Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant and -tolerant bacteria. As these potentiators typically do not interfere with essential processes of bacteria, it has been hypothesized that they are less likely to induce resistance than conventional antibiotics. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop resistance towards one such adjuvant, baicalin hydrate (BH), a quorum sensing inhibitor known to increase antibiotic-induced oxidative stress. Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After each cycle of treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to treatments with TOB and TOB+BH, compared to the start population, and the potentiating effect of BH towards TOB was lost. Whole genome sequencing was performed to probe which changes were involved in the reduced effect of BH and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter), as well as a partial deletion of two larger regions. No changes in the minimal inhibitory or minimal bactericidal concentration of TOB or changes in the number of persister cells were observed in the evolved populations. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that resistance towards antibiotic-potentiating activity can develop rapidly in B. cenocepacia J2315 biofilms and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as potential mechanisms.\n\nImportanceBacteria show a markedly reduced susceptibility to antibiotics when growing in a biofilm, which hampers effective treatment of biofilm-related infections. The use of potentiators that increase the activity of antibiotics against biofilms has been proposed as a solution to this problem, but it is unclear whether resistance to these potentiators could develop. Using an experimental evolution approach, we convincingly demonstrate that Burkholderia cenocepacia biofilms rapidly develop resistance towards the tobramycin-potentiating activity of baicalin hydrate. Whole genome sequencing revealed that there are different mechanisms that lead to this resistance, including mutations resulting in metabolic changes, changes in production of intracellular levels of reactive oxygen species, and differences in transporter-mediated tobramycin uptake. Our study suggests that this form of combination therapy is not evolution-proof and highlights the usefulness of experimental evolution to identify mechanisms of resistance and tolerance in biofilm-grown bacteria.

microbiology↗

Positive human health effects of sea spray aerosols: molecular evidence from exposed lung cell lines.

Sea spray aerosols (SSAs) have profound effects on climate and ecosystems. Furthermore, the presence of microbiota and biogenic molecules, produced by among others marine phytoplankton, in SSAs could lead to potential human health effects. Yet the exposure and effects of SSAs on human health remain poorly studied. Here, we exposed human epithelial lung cells to different concentrations of extracts of a natural sea spray aerosol (SSA), a laboratory-generated SSA, the marine algal toxin homoyessotoxin and a chemical mTOR inhibitor. The mTOR inhibitor was included as it has been hypothesized that natural SSAs may influence the mTOR cell signaling pathway. We observed significant effects on the mTOR pathway and PCSK9 in all exposures. Based on these expression patterns, a clear dose response relationship was observed. Our results indicate a potential for positive health effects when lung cells are exposed to environmentally relevant concentrations of natural SSAs, whereas potential negative effects were observed at high levels of the laboratory SSA and the marine algal toxin. Overall, these results provide a substantial molecular evidence base for potential positive health effects of SSAs at environmentally relevant concentrations through the mTOR pathway. The results provided here suggest that SSAs contain biomolecules with significant pharmaceutical potential in targeting PCSK9.

pharmacology and toxicology↗