bims-ectoca 2021-08-15 papers

mBio. 2021 Aug 10. e0195921

Multifactorial Role of Mitochondria in Echinocandin Tolerance Revealed by Transcriptome Analysis of Drug-Tolerant Cells.

Rocio Garcia-Rubio, Cristina Jimenez-Ortigosa, Lucius DeGregorio, Christopher Quinteros, Erika Shor, David S Perlin.

Fungal infections cause significant mortality and morbidity worldwide, and the limited existing antifungal reservoir is further weakened by the emergence of strains resistant to echinocandins, a first line of antifungal therapy. Candida glabrata is an opportunistic fungal pathogen that rapidly develops mutations in the echinocandin drug target β-1,3-glucan synthase (GS), which are associated with drug resistance and clinical failure. Although echinocandins are considered fungicidal in Candida sp., a subset of C. glabrata cells survive echinocandin exposure, forming a drug-tolerant cell reservoir, from which resistant mutations are thought to emerge. Despite their importance, the physiology of rare drug-tolerant cells is poorly understood. We used fluorescence-activated cell sorting to enrich for echinocandin-tolerant cells, followed by modified single-cell RNA sequencing to examine their transcriptional landscape. This analysis identified a transcriptional signature distinct from the stereotypical yeast environmental stress response and characterized by upregulation of pathways involved in chromosome structure and DNA topology and downregulation of oxidative stress responses, of which the latter was observed despite increased levels of reactive oxygen species. Further analyses implicated mitochondria in echinocandin tolerance, wherein inhibitors of mitochondrial complexes I and IV reduced echinocandin-mediated cell killing, but mutants lacking various mitochondrial components all showed an echinocandin hypotolerant phenotype. Finally, GS enzyme complexes purified from mitochondrial mutants exhibited normal in vitro inhibition kinetics, indicating that mitochondrial defects influence cell survival downstream of the drug-target interaction. Together, these results provide new insights into the C. glabrata response to echinocandins and reveal a multifactorial role of mitochondria in echinocandin tolerance. IMPORTANCE Echinocandin drugs are a first-line therapy to treat invasive candidiasis, which is a major source of morbidity and mortality worldwide. The opportunistic fungal pathogen Candida glabrata is a prominent bloodstream fungal pathogen, and it is notable for rapidly developing echinocandin-resistant strains associated with clinical failure. Echinocandin resistance is thought to emerge within a small echinocandin-tolerant subset of C. glabrata cells that are not killed by drug exposure, but mechanisms underlying echinocandin tolerance are still unknown. Here, we describe the unique transcriptional signature of echinocandin-tolerant cells and the results of follow-up analyses, which reveal a multifactorial role of mitochondria in C. glabrata echinocandin tolerance. In particular, although chemical inhibition of respiratory chain enzymes increased echinocandin tolerance, deletion of multiple mitochondrial components made C. glabrata cells hypotolerant to echinocandins. Together, these results provide new insights into the C. glabrata response to echinocandins and reveal the involvement of mitochondria in echinocandin tolerance.

Keywords: Candida glabrata; antifungal drug tolerance; echinocandins; mitochondria; transcriptomics

DOI: https://doi.org/10.1128/mBio.01959-21

Nature. 2021 Aug 11.

Cycling cancer persister cells arise from lineages with distinct programs.

Non-genetic mechanisms have recently emerged as important drivers of cancer therapy failure1, where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment2. Although most cancer persisters remain arrested in the presence of the drug, a rare subset can re-enter the cell cycle under constitutive drug treatment. Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drugs. To study this rare, transiently resistant, proliferative persister population, we developed Watermelon, a high-complexity expressed barcode lentiviral library for simultaneous tracing of each cell's clonal origin and proliferative and transcriptional states. Here we show that cycling and non-cycling persisters arise from different cell lineages with distinct transcriptional and metabolic programs. Upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation are associated with persister proliferative capacity across multiple cancer types. Impeding oxidative stress or metabolic reprogramming alters the fraction of cycling persisters. In human tumours, programs associated with cycling persisters are induced in minimal residual disease in response to multiple targeted therapies. The Watermelon system enabled the identification of rare persister lineages that are preferentially poised to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence.

DOI: https://doi.org/10.1038/s41586-021-03796-6

J Med Chem. 2021 Aug 11.

Targeting Autophagy-Related Epigenetic Regulators for Cancer Drug Discovery.

Yang Li, Gaoxia Yang, Chengcan Yang, Pan Tang, Juncheng Chen, Jifa Zhang, Jie Liu, Liang Ouyang.

Existing evidence has demonstrated that epigenetic modifications (including DNA methylation, histone modifications, and microRNAs), which are associated with the occurrence and development of tumors, can directly or indirectly regulate autophagy. In particular, nuclear events induced by several epigenetic regulators can regulate the autophagic process and expression levels of tumor-associated genes, thereby promoting tumor progression. Tumor-associated microRNAs, including oncogenic and tumor-suppressive microRNAs, are of great significance to autophagy during tumor progression. Targeting autophagy with emerging epigenetic drugs is expected to be a promising therapeutic strategy for human tumors. From this perspective, we aim to summarize the role of epigenetic modification in the autophagic process and the underlying molecular mechanisms of tumorigenesis. Furthermore, the regulatory efficacy of epigenetic drugs on the autophagic process in tumors is also summarized. This perspective may provide a theoretical basis for the combined treatment of epigenetic drugs/autophagy mediators in tumors.

DOI: https://doi.org/10.1021/acs.jmedchem.1c00579

Oncogene. 2021 Aug 13.

Spontaneous and inherited TP53 genetic alterations.

Arnold J Levine.

The p53 protein is a transcription factor that prevents tumors from developing. In spontaneous and inherited cancers there are many different missense mutations in the DNA binding domain of the TP53 gene that contributes to tumor formation. These mutations produce a wide distribution in the transcriptional capabilities of the mutant p53 proteins with over four logs differences in the efficiencies of forming cancers in many diverse tissue types. These inherited and spontaneous TP53 mutations produce proteins that interact with both genetic and epigenetic cellular modifiers of p53 function and their inherited polymorphisms to produce a large number of diverse phenotypes in individual patients. This manuscript reviews these variables and discusses how the combinations of TP53 genetic alterations interact with genetic polymorphisms, epigenetic alterations, and environmental factors to begin predicting and modifying patient outcomes and provide a better understanding for new therapeutic opportunities.

DOI: https://doi.org/10.1038/s41388-021-01991-3

Methods Mol Biol. 2021 ;2351 307-320

Transcriptional Activation of Heterochromatin by Recruitment of dCas9 Activators.

Lukas Frank, Robin Weinmann, Fabian Erdel, Jorge Trojanowski, Karsten Rippe.

The transition from silenced heterochromatin to a biologically active state and vice versa is a fundamental part of the implementation of cell type-specific gene expression programs. To reveal structure-function relationships and dissect the underlying mechanisms, experiments that ectopically induce transcription are highly informative. In particular, the approach to perturb chromatin states by recruiting fusions of the catalytically inactive dCas9 protein in a sequence-specific manner to a locus of interest has been used in numerous applications. Here, we describe how this approach can be applied to activate pericentric heterochromatin (PCH) in mouse cells as a prototypic silenced state by providing protocols for the following workflow: (a) Recruitment of dCas9 fusion constructs with the strong transcriptional activator VPR to PCH. (b) Analysis of the resulting changes in chromatin compaction, epigenetic marks, and active transcription by fluorescence microscopy-based readouts. (c) Automated analysis of the resulting images with a set of scripts in the R programming language. Furthermore, we discuss how parameters for chromatin decondensation and active transcription are extracted from these experiments and can be combined with other readouts to gain insights into PCH activation.

Keywords: CRISPR; Chromocenter; Decondensation; Fluorescence microscopy; Image quantification; Mouse embryonic fibroblasts; Pericentric heterochromatin; dCas9

DOI: https://doi.org/10.1007/978-1-0716-1597-3_17

Methods Mol Biol. 2021 ;2351 181-199

Proximity Ligation-Assisted ChIP-Seq (PLAC-Seq).

Miao Yu, Ivan Juric, Armen Abnousi, Ming Hu, Bing Ren.

Proximity ligation-assisted ChIP-Seq (PLAC-Seq), also known as HiChIP, is a method to detect and quantify chromatin contacts anchored at genomic regions bound by specific proteins or histone modifications. By combining in situ Hi-C and chromatin immunoprecipitation (ChIP) using antibodies against transcription factors (TFs) or histone marks of interest, the method achieves targeted interrogation of chromatin organization at a subset of genomic regions. PLAC-Seq is able to identify long-range chromatin interactions at kilobase-scale resolution with significantly reduced sequencing cost.

Keywords: 3D genome; Chromatin immunoprecipitation; Hi-C; Long-range chromatin interaction; PLAC-Seq

DOI: https://doi.org/10.1007/978-1-0716-1597-3_10

Methods Mol Biol. 2021 ;2351 275-288

Using ChIP-SICAP to Identify Proteins That Co-localize in Chromatin.

Mahmoud-Reza Rafiee, Jeroen Krijgsveld.

Functionalization of the genome is carried out by proteins that bind to DNA to regulate gene expression. Since this process is highly dynamic, context-dependent, and rarely performed by single proteins alone, we here describe ChIP-SICAP to identify proteins that co-localize with a protein of interest on the genome. Benefiting from its nature as a dual purification approach via ChIP and DNA biotinylation, ChIP-SICAP distinguishes genuine chromatin-binders and is uniquely placed to identify novel players in genome regulation.

Keywords: Chromatin Immunoprecipitation (ChIP); Chromatin proteomics; SICAP

DOI: https://doi.org/10.1007/978-1-0716-1597-3_15

Mol Biol Rep. 2021 Aug 10.

Bidirectional promoters: an enigmatic genome architecture and their roles in cancers.

Sheikh Shafin Ahmad, Nure Sharaf Nower Samia, Auroni Semonti Khan, Rafeed Rahman Turjya, Md Abdullah-Al-Kamran Khan.

Bidirectional promoters are the transcription regulatory regions of genes positioned head-to-head on opposite strands. Specific sequence signals, chromatin modifications and three-dimensional structures of the transcription site facilitate the unconventional yet tightly regulated transcription proceeding in both directions from these promoters. Mutations or aberrant epigenetic changes can lead to abnormal enhanced or reduced expression from either of the bidirectionally transcribed genes resulting in tumorigenesis. Moreover, bidirectionally transcribed genes might also contribute towards the immune regulation in tumor microenvironment. In this review, we aimed to expound the characteristic features of bidirectional promoters alongside their transcriptional regulations, and ultimately, the association of these enigmatic genomic elements in different cancers.

Keywords: Bidirectional promoter; Bidirectional transcription; Cancers; Epigenetic regulation

DOI: https://doi.org/10.1007/s11033-021-06612-6

Stat Appl Genet Mol Biol. 2021 Aug 09.

Low variability in the underlying cellular landscape adversely affects the performance of interaction-based approaches for conducting cell-specific analyses of DNA methylation in bulk samples.

Richard Meier, Emily Nissen, Devin C Koestler.

Statistical methods that allow for cell type specific DNA methylation (DNAm) analyses based on bulk-tissue methylation data have great potential to improve our understanding of human disease and have created unprecedented opportunities for new insights using the wealth of publicly available bulk-tissue methylation data. These methodologies involve incorporating interaction terms formed between the phenotypes/exposures of interest and proportions of the cell types underlying the bulk-tissue sample used for DNAm profiling. Despite growing interest in such "interaction-based" methods, there has been no comprehensive assessment how variability in the cellular landscape across study samples affects their performance. To answer this question, we used numerous publicly available whole-blood DNAm data sets along with extensive simulation studies and evaluated the performance of interaction-based approaches in detecting cell-specific methylation effects. Our results show that low cell proportion variability results in large estimation error and low statistical power for detecting cell-specific effects of DNAm. Further, we identified that many studies targeting methylation profiling in whole-blood may be at risk to be underpowered due to low variability in the cellular landscape across study samples. Finally, we discuss guidelines for researchers seeking to conduct studies utilizing interaction-based approaches to help ensure that their studies are adequately powered.

Keywords: EWAS; cell fraction; cell proportion; interaction; methylation; variability

DOI: https://doi.org/10.1515/sagmb-2021-0004