The poly (ADP-ribose) polymerase (PARP) inhibitor olaparib is FDA approved for the treatment of BRCA-mutated breast, ovarian and pancreatic cancers. Olaparib inhibits PARP1/2 enzymatic activity and ...traps PARP1 on DNA at single-strand breaks, leading to replication-induced DNA damage that requires BRCA1/2-dependent homologous recombination repair. Moreover, DNA damage response pathways mediated by the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia mutated and Rad3-related (ATR) kinases are hypothesised to be important survival pathways in response to PARP-inhibitor treatment. Here, we show that olaparib combines synergistically with the ATR-inhibitor AZD6738 (ceralasertib), in vitro, leading to selective cell death in ATM-deficient cells. We observe that 24 h olaparib treatment causes cells to accumulate in G2-M of the cell cycle, however, co-administration with AZD6738 releases the olaparib-treated cells from G2 arrest. Selectively in ATM-knockout cells, we show that combined olaparib/AZD6738 treatment induces more chromosomal aberrations and achieves this at lower concentrations and earlier treatment time-points than either monotherapy. Furthermore, single-agent olaparib efficacy in vitro requires PARP inhibition throughout multiple rounds of replication. Here, we demonstrate in several ATM-deficient cell lines that the olaparib and AZD6738 combination induces cell death within 1-2 cell divisions, suggesting that combined treatment could circumvent the need for prolonged drug exposure. Finally, we demonstrate in vivo combination activity of olaparib and AZD6738 in xenograft and PDX mouse models with complete ATM loss. Collectively, these data provide a mechanistic understanding of combined PARP and ATR inhibition in ATM-deficient models, and support the clinical development of AZD6738 in combination with olaparib.
A comprehensive map of transcription start sites (TSSs) across the highly AT-rich genome of P. falciparum would aid progress toward deciphering the molecular mechanisms that underlie the timely ...regulation of gene expression in this malaria parasite. Using high-throughput sequencing technologies, we generated a comprehensive atlas of transcription initiation events at single-nucleotide resolution during the parasite intra-erythrocytic developmental cycle. This detailed analysis of TSS usage enabled us to define architectural features of plasmodial promoters. We demonstrate that TSS selection and strength are constrained by local nucleotide composition. Furthermore, we provide evidence for coordinate and stage-specific TSS usage from distinct sites within the same transcription unit, thereby producing transcript isoforms, a subset of which are developmentally regulated. This work offers a framework for further investigations into the interactions between genomic sequences and regulatory factors governing the complex transcriptional program of this major human pathogen.
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•A genome-wide map of transcription start sites (TSSs) for P. falciparum•Sequence and chromatin features for core promoters of malaria parasites are defined•Dynamic study of TSS usage identifies developmentally regulated transcript isoforms
The genome of P. falciparum is characterized by an extremely AT-biased nucleotide composition. Adjalley et al. conducted a survey of transcription initiation events throughout the malaria parasite’s blood cycle. They detect coordinate and stage-specific transcription initiation and extract sequence and chromatin signatures from promoter regions.
In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes ...involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome maintenance.
Intra-tumor hypoxia is a common feature in many solid cancers. Although transcriptional targets of hypoxia-inducible factors (HIFs) have been well characterized, alternative splicing or processing of ...pre-mRNA transcripts which occurs during hypoxia and subsequent HIF stabilization is much less understood. Here, we identify many HIF-dependent alternative splicing events after whole transcriptome sequencing in pancreatic cancer cells exposed to hypoxia with and without downregulation of the aryl hydrocarbon receptor nuclear translocator (ARNT), a protein required for HIFs to form a transcriptionally active dimer. We correlate the discovered hypoxia-driven events with available sequencing data from pan-cancer TCGA patient cohorts to select a narrow set of putative biologically relevant splice events for experimental validation. We validate a small set of candidate HIF-dependent alternative splicing events in multiple human gastrointestinal cancer cell lines as well as patient-derived human pancreatic cancer organoids. Lastly, we report the discovery of a HIF-dependent mechanism to produce a hypoxia-dependent, long and coding isoform of the UDP-N-acetylglucosamine transporter SLC35A3.
•Identification of alternative RNA isoforms of unknown function specifically produced during cellular hypoxia.•Computational validation of the presence of isoforms in TCGA, with survival and hypoxic status associations.•Experimental validation of promising isoforms in multiple in vitro systems.•Characterization of a hypoxia-dependent long isoform of the glucosamine transporter SLC35A3
Cryptic transcription is widespread and generates a heterogeneous group of RNA molecules of unknown function. To improve our understanding of cryptic transcription, we investigated their ...transcription start site (TSS) usage, chromatin organization, and posttranscriptional consequences in
We show that TSSs of chromatin-sensitive internal cryptic transcripts retain comparable features of canonical TSSs in terms of DNA sequence, directionality, and chromatin accessibility. We define the 5' and 3' boundaries of cryptic transcripts and show that, contrary to RNA degradation-sensitive ones, they often overlap with the end of the gene, thereby using the canonical polyadenylation site, and associate to polyribosomes. We show that chromatin-sensitive cryptic transcripts can be recognized by ribosomes and may produce truncated polypeptides from downstream, in-frame start codons. Finally, we confirm the presence of the predicted polypeptides by reanalyzing N-terminal proteomic data sets. Our work suggests that a fraction of chromatin-sensitive internal cryptic promoters initiates the transcription of alternative truncated mRNA isoforms. The expression of these chromatin-sensitive isoforms is conserved from yeast to human, expanding the functional consequences of cryptic transcription and proteome complexity.
Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) or microarray hybridization (ChIP-on-chip) are standard methods for the study of transcription factor binding sites and histone ...chemical modifications. However, these approaches only allow profiling of a single factor or protein modification at a time.In this chapter, we present Bar-ChIP, a higher throughput version of ChIP-Seq that relies on the direct ligation of molecular barcodes to chromatin fragments. Bar-ChIP enables the concurrent profiling of multiple DNA-protein interactions and is therefore amenable to experimental scale-up, without the need for any robotic instrumentation.
Next generation sequencing protocols such as RNA-seq have made the genome-wide characterization of the transcriptome a crucial part of many research projects in biology. Analyses of the resulting ...data provide key information on gene expression and in certain cases on exon or isoform usage. The emergence of transcript quantification software such as Salmon has enabled researchers to efficiently estimate isoform and gene expressions across the genome while tremendously reducing the necessary computational power. Although overall gene expression estimations were shown to be accurate, isoform expression quantifications appear to be a more challenging task. Low expression levels and uneven or insufficient coverage were reported as potential explanations for inconsistent estimates. Here, through the example of the ketohexokinase (
Khk) gene in mouse, we demonstrate that the use of an incorrect gene annotation can also result in erroneous isoform quantification results. Manual correction of the input
Khk gene model provided a much more accurate estimation of relative
Khk isoform expression when compared to quantitative PCR (qPCR measurements). In particular, removal of an unexpressed retained intron and a proper adjustment of the 5' and 3' untranslated regions both had a strong impact on the correction of erroneous estimates. Finally, we observed a better concordance in isoform quantification between datasets and sequencing strategies when relying on the newly generated
Khk annotations. These results highlight the importance of accurate gene models and annotations for correct isoform quantification and reassert the need for orthogonal methods of estimation of isoform expression to confirm important findings.
We present a modified approach of chromatin immuno‐precipitation followed by sequencing (ChIP‐Seq), which relies on the direct ligation of molecular barcodes to chromatin fragments, thereby ...permitting experimental scale‐up. With Bar‐ChIP now enabling the concurrent profiling of multiple DNA–protein interactions, we report the simultaneous generation of 90 ChIP‐Seq datasets without any robotic instrumentation. We demonstrate that application of Bar‐ChIP to a panel of Saccharomyces cerevisiae chromatin‐associated mutants provides a rapid and accurate genome‐wide overview of their chromatin status. Additionally, we validate the utility of this technology to derive novel biological insights by identifying a role for the Rpd3S complex in maintaining H3K14 hypo‐acetylation in gene bodies. We also report an association between the presence of intragenic H3K4 tri‐methylation and the emergence of cryptic transcription in a Set2 mutant. Finally, we uncover a crosstalk between H3K14 acetylation and H3K4 methylation in this mutant. These results show that Bar‐ChIP enables biological discovery through rapid chromatin profiling at single‐nucleosome resolution for various conditions and protein modifications at once.
Synopsis
A new approach provides a rapid and accurate genome‐wide overview of the chromatin status of multiple yeast chromatin‐associated mutants at once. The simultaneous profiling of epigenetic marks in the mutants is achieved by multiplex immuno‐precipitation of barcoded chromatin samples.
Bar‐ChIP is based on the immuno‐precipitation of barcoded chromatin and permits sample multiplexing, thereby increasing the throughput of ChIP‐Seq experiments.
Application of the method to yeast chromatin‐associated mutants enabled the concurrent generation of 90 ChIP‐Seq datasets without the need for robotic instrumentation.
The rapid chromatin profiling of the mutants at single‐nucleosome resolution uncovered an association between intragenic H3K4 tri‐methylation and cryptic transcription in set2∆.
A new approach provides a rapid and accurate genome‐wide overview of the chromatin status of multiple yeast chromatin‐associated mutants at once. The simultaneous profiling of epigenetic marks in the mutants is achieved by multiplex immuno‐precipitation of barcoded chromatin samples.
The genome-wide study of epigenetic states requires the integrative analysis of histone modification ChIP-seq data. Here, we introduce an easy-to-use analytic framework to compare profiles of ...enrichment in histone modifications around classes of genomic elements, e.g. transcription start sites (TSS). Our framework is available via the user-friendly R/Bioconductor package DChIPRep. DChIPRep uses biological replicate information as well as chromatin Input data to allow for a rigorous assessment of differential enrichment. DChIPRep is available for download through the Bioconductor project at http://bioconductor.org/packages/DChIPRep. Contact. DChIPRep@gmail.com.
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