Biology subjects

Kaczorowski, D.

Publications and source records attributed to Kaczorowski, D..

9 recordsLinked to original sources

bioRxiv2024

Protein Translation Dysregulation and Immune Cell Evasion Define Metastatic Clones in HPV-related Cancer of the Oropharynx

Head and neck cancers, representing the seventh most common malignancy globally, have seen a shift in causative factors from traditional smoking and alcohol use to human papillomavirus (HPV) infection, now accounting for up to 80% of oropharyngeal cancers. We identify the cellular and clonal mechanisms underlying immune avoidance and metastasis by analysing single-cell and spatial genomic data from primary and metastatic cancers. We first map the clonal evolution of malignant cells based on the accumulation of mutations. We identify metastasising clones based on mutational similarity scores between cells in the primary and lymph node metastasis. Genomic analysis of metastasising and non-metastasising clones identified virally mediated protein translation relief (P=4.24x10-24) pathway underlying metastatic expansion. We show that in metastatic clones, this process is driven through upregulation of transition-initiating factors, EIF4E (P=1.5x10-13) and EIFG1 (P<2.22x10-16), and suppression of regulatory kinases EIF4EBP1 (P=2.1x10), EIF2AK2 (P<2.22x10-16), and EIF2S1 (P<2.22x10-16). We subsequently identify that metastatic clones have a corresponding downregulation of the JAK/STAT pathway and immunoproteasome genes PSMB8 (P<2.22x10- 16) and PSMB9 (P<2.22x10-16), suggesting these clones escape immune surveillance through decreased INF inflammatory response and antigen presentation. We validate these results using spatial RNA-seq data, where metastatic cancer clones show decreased cell-to-cell interactions with CD4 T-effector memory cells (CD4TEM) (P=0.0077), CD8 T-exhausted cells (CD8Ex) (P=0.0191), and innate lymphoid cells (ILC) (P=0.04). Finally, we demonstrate that the upregulation of cap-independent translational drives cell proliferation in metastatic clones through the expression of translation initiation factors (EIF4G1: P<2.22x10-16). Our results provide evidence of the mechanisms by which virally induced cancer clones lead to advanced disease and poor prognosis in patients.

cancer biology↗

bioRxiv2023

snPATHO-seq: unlocking the pathology archives

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low DNA and RNA quality. In this study, leveraging recent single-cell genomic technology advances, we introduce a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples.

molecular biology↗

bioRxiv2023

An experimental comparison of the Digital Spatial Profiling and Visium spatial transcriptomics technologies for cancer research

BackgroundSpatial transcriptomic technologies are powerful tools for resolving the spatial heterogeneity of gene expression in tissue samples. However, little evidence exists on relative strengths and weaknesses of the various available technologies for profiling human tumour tissue. In this study, we aimed to provide an objective assessment of two common spatial transcriptomics platforms, 10X Genomics Visium and Nanostrings GeoMx DSP. MethodThe abilities of the DSP and Visium platforms to profile transcriptomic features were compared using matching cell line and primary breast cancer tissue samples. A head-to-head comparison was conducted using data generated from matching samples and synthetic tissue references. Platform specific features were also assessed according to manufacturers recommendations to evaluate the optimal usage of the two technologies. ResultsWe identified substantial variations in assay design between the DSP and Visium assays such as transcriptomic coverage and composition of the transcripts detected. When the data was standardised according to manufacturers recommendations, the DSP platform was more sensitive in gene expression detection. However, its specificity was diminished by the presence of non-specific detection. Our results also confirmed the strength and weakness of each platform in characterising spatial transcriptomic features of tissue samples, in particular their application to hypothesis generation versus hypothesis testing. ConclusionIn this study, we share our experience on both DSP and Visium technologies as end users. We hope this can guide future users to choose the most suitable platform for their research. In addition, this dataset can be used as an important resource for the development of new analysis tools.

cancer biology↗

bioRxiv2022

Multimodal single cell analysis of the paediatric lower airway reveals novel immune cell phenotypes in early life health and disease

Inflammation is a key driver of cystic fibrosis (CF) lung disease, not addressed by current standard care. Improved understanding of the mechanisms leading to aberrant inflammation may assist the development of effective anti-inflammatory therapy. Single-cell RNA sequencing (scRNA-seq) allows profiling of cell composition and function at previously unprecedented resolution. Herein, we seek to use multimodal single-cell analysis to comprehensively define immune cell phenotypes, proportions and functional characteristics in preschool children with CF. We analyzed 42,658 cells from bronchoalveolar lavage of 11 preschool children with CF and a healthy control using scRNA-seq and parallel assessment of 154 cell surface proteins. Validation of cell types identified by scRNA-seq was achieved by assessment of samples by spectral flow cytometry. Analysis of transcriptome expression and cell surface protein expression, combined with functional pathway analysis, revealed 41 immune and epithelial cell populations in BAL. Spectral flow cytometry analysis of over 256,000 cells from a subset of the same patients revealed high correlation in major cell type proportions across the two technologies. Macrophages consisted of 13 functionally distinct sub populations, including previously undescribed populations enriched for markers of vesicle production and regulatory/repair functions. Other novel cell populations included CD4 T cells expressing inflammatory IFN/{beta} and NF{kappa}B signalling genes. Our work provides a comprehensive cellular analysis of the pediatric lower airway in preschool children with CF, reveals novel cell types and provides a reference for investigation of inflammation in early life CF.

systems biology↗

bioRxiv2021

High temporal resolution RNA-seq time course data reveals mammalian lncRNA activationmirrors neighbouring protein-coding genes

BackgroundThe advent of next-generation sequencing revealed extensive transcription beyond protein-coding genes, identifying tens of thousands of long non-coding RNAs (lncRNAs). Selected functional examples raised the possibility that lncRNAs, as a class, may maintain broad regulatory roles. Compellingly, lncRNA expression is strongly linked with adjacent protein-coding gene expression, suggesting a potential cis-regulatory function. Evidence for these regulatory roles may be obtained through careful examination of the precise timing of lncRNA expression relative to adjacent protein-coding genes. ResultsWhere causal cis-regulatory relationships exist, lncRNA activation is expected to precede changes in adjacent target gene expression. Using an RNA-seq time course of uniquely high temporal resolution, we profiled the expression dynamics of several thousand lncRNAs and protein-coding genes in synchronized, transitioning human cells. Our findings reveal lncRNAs are expressed synchronously with adjacent protein-coding genes. Analysis of lipopolysaccharide-activated mouse dendritic cells revealed the same temporal relationship observed in transitioning human cells. ConclusionOur findings suggest broad-scale cis-regulatory roles for lncRNAs are not common. The strong association between lncRNAs and adjacent genes may instead indicate an origin as transcriptional by-products from active protein-coding gene promoters and enhancers.

bioinformatics↗

bioRxiv2020

Subcellular relocalization and nuclear redistribution of the RNA methyltransferases TRMT1 and TRMT1L upon neuronal activation

RNA modifications are dynamic chemical entities that regulate RNA fate, and an avenue for environmental response in neuronal function. However, which RNA modifications may be playing a role in neuronal plasticity and environmental responses is largely unknown. Here we characterize the biochemical function and cellular dynamics of two human RNA methyltransferases previously associated with neurological dysfunction, TRMT1 and its homolog, TRMT1-like (TRMT1L). Using a combination of next-generation sequencing, LC-MS/MS, patient-derived cell lines and knockout mouse models, we confirm the previously reported dimethylguanosine (m 2,2 G) activity of TRMT1 in tRNAs, as well as reveal that TRMT1L, whose activity was unknown, is responsible for methylating a subset of cytosolic tRNA Ala (AGC) isoacceptors at position 26. Using a cellular in vitro model that mimics neuronal activation and long term potentiation, we find that both TRMT1 and TRMT1L change their subcellular localization upon neuronal activation. Specifically, we observe a major subcellular relocalization from mitochondria and other cytoplasmic domains (TRMT1) and nucleoli (TRMT1L) to different small punctate compartments in the nucleus, which are as yet uncharacterized. This phenomenon does not occur upon heat shock, suggesting that the relocalization of TRMT1 and TRMT1L is not a general reaction to stress, but rather a specific response to neuronal activation. Our results suggest that subcellular relocalization of RNA modification enzymes play a role in neuronal plasticity and transmission of information, presumably by addressing new targets.

molecular biology↗

bioRxiv2020

DNA methylation is required to maintain DNA replication timing precision and 3D genome integrity

DNA replication timing and three-dimensional (3D) genome organisation occur across large domains associated with distinct epigenome patterns to functionally compartmentalise genome regulation. However, it is still unclear if alternations in the epigenome, in particular cancer-related DNA hypomethylation, can directly result in alterations to cancer higher order genome architecture. Here, we use Hi-C and single cell Repli-Seq, in the colorectal cancer DNMT1 and DNMT3B DNA methyltransferases double knockout model, to determine the impact of DNA hypomethylation on replication timing and 3D genome organisation. First, we find that the hypomethylated cells show a striking loss of replication timing precision with gain of cell-to-cell replication timing heterogeneity and loss of 3D genome compartmentalisation. Second, hypomethylated regions that undergo a large change in replication timing also show loss of allelic replication timing, including at cancer-related genes. Finally, we observe the formation of broad ectopic H3K4me3-H3K9me3 domains across hypomethylated regions where late replication is maintained, that potentially prevent aberrant transcription and loss of genome organisation after DNA demethylation. Together, our results highlight a previously underappreciated role for DNA methylation in maintenance of 3D genome architecture.

molecular biology↗

bioRxiv2019

Targeted, High-Resolution RNA Sequencing Of Non-Coding Genomic Regions Associated With Neuropsychiatric Functions

The human brain is one of the last frontiers of biomedical research. Genome-wide association studies (GWAS) have succeeded in identifying thousands of haplotype blocks associated with a range of neuropsychiatric traits, including disorders such as schizophrenia, Alzheimers and Parkinsons disease. However, the majority of single nucleotide polymorphisms (SNPs) that mark these haplotype blocks fall within non-coding regions of the genome, hindering their functional validation. While some of these GWAS loci may contain cis-acting regulatory DNA elements such as enhancers, we hypothesized that many are also transcribed into non-coding RNAs that are missing from publicly available transcriptome annotations. Here, we use targeted RNA capture ( RNA CaptureSeq) in combination with nanopore long-read cDNA sequencing to transcriptionally profile 1,023 haplotype blocks across the genome containing non-coding GWAS SNPs associated with neuropsychiatric traits, using post-mortem human brain tissue from three neurologically healthy donors. We find that the majority (62%) of targeted haplotype blocks, including 13% of intergenic blocks, are transcribed into novel, multi-exonic RNAs, most of which are not yet recorded in GENCODE annotations. We validated our findings with short-read RNA-seq, providing orthogonal confirmation of novel splice junctions and enabling a quantitative assessment of the long-read assemblies. Many novel transcripts are supported by independent evidence of transcription including cap analysis of gene expression (CAGE) data and epigenetic marks, and some show signs of potential functional roles. We present these transcriptomes as a preliminary atlas of non-coding transcription in human brain that can be used to connect neurological phenotypes with gene expression.

genomics↗

bioRxiv2016

High temporal resolution of gene expression dynamics in developing mouse embryonic stem cells

Investigations of transcriptional responses during developmental transitions typically use time courses with intervals that are not commensurate with the timescales of known biological processes. Moreover, such experiments typically focus on protein-coding transcripts, ignoring the important impact of long noncoding RNAs. We evaluated coding and noncoding expression dynamics at high temporal resolution (6-hourly) in differentiating mouse embryonic stem cells and report the effects of increased temporal resolution on the characterization of the underlying molecular processes. We present a refined resolution of global transcriptional alterations, including regulatory network interactions, coding and noncoding gene expression changes as well as alternative splicing events, many of which cannot be resolved by existing coarse developmental time-{-}-courses. We describe novel short lived and cycling patterns of gene expression and temporally dissect ordered gene expression at bidirectional promoters and responses to transcription factors. These findings demonstrate the importance of temporal resolution for understanding gene interactions in mammalian systems.\n\nLinks to dataData has been deposited into GEO: The Reviewer access link is: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=cnglummejbkltyj@acc=GSE75028

systems biology↗