The recent boom in microfluidics and combinatorial indexing strategies, combined with low sequencing costs, has empowered single-cell sequencing technology. Thousands-or even millions-of cells ...analyzed in a single experiment amount to a data revolution in single-cell biology and pose unique data science problems. Here, we outline eleven challenges that will be central to bringing this emerging field of single-cell data science forward. For each challenge, we highlight motivating research questions, review prior work, and formulate open problems. This compendium is for established researchers, newcomers, and students alike, highlighting interesting and rewarding problems for the coming years.
Resistant tumours are thought to arise from the action of Darwinian selection on genetically heterogenous cancer cell populations. However, simple clonal selection is inadequate to describe the late ...relapses often characterising luminal breast cancers treated with endocrine therapy (ET), suggesting a more complex interplay between genetic and non-genetic factors. Here, we dissect the contributions of clonal genetic diversity and transcriptional plasticity during the early and late phases of ET at single-cell resolution. Using single-cell RNA-sequencing and imaging we disentangle the transcriptional variability of plastic cells and define a rare subpopulation of pre-adapted (PA) cells which undergoes further transcriptomic reprogramming and copy number changes to acquire full resistance. We find evidence for sub-clonal expression of a PA signature in primary tumours and for dominant expression in clustered circulating tumour cells. We propose a multi-step model for ET resistance development and advocate the use of stage-specific biomarkers.
The transcription factor ΔNp63 regulates epithelial stem cell function and maintains the integrity of stratified epithelial tissues by acting as transcriptional repressor or activator towards a ...distinct subset of protein-coding genes and microRNAs. However, our knowledge of the functional link between ∆Np63 transcriptional activity and long non-coding RNAs (lncRNAs) expression is quite limited. Here, we show that in proliferating human keratinocytes ∆Np63 represses the expression of the lncRNA NEAT1 by recruiting the histone deacetylase HDAC1 to the proximal promoter of NEAT1 genomic locus. Upon induction of differentiation, ∆Np63 down-regulation is associated by a marked increase of NEAT1 RNA levels, resulting in an increased assembly of paraspeckles foci both in vitro and in human skin tissues. RNA-seq analysis associated with global DNA binding profile (ChIRP-seq) revealed that NEAT1 associates with the promoter of key epithelial transcription factors sustaining their expression during epidermal differentiation. These molecular events might explain the inability of NEAT1-depleted keratinocytes to undergo the proper formation of epidermal layers. Collectively, these data uncover the lncRNA NEAT1 as an additional player of the intricate network orchestrating epidermal morphogenesis.
Background Enhancer reprogramming plays a significant role in the heterogeneity of cancer. However, we have limited knowledge about the impact of chromatin remodeling in B-Cell Precursor Acute ...Lymphoblastic Leukemia (BCP-ALL) patients, and how it affects tumorigenesis and drug response. Our research focuses on investigating the role of enhancers in sustaining oncogenic transformation in children with BCP-ALL. Methods We used ATAC-seq to study the accessibility of chromatin in pediatric BCP-ALL at three different stages--onset, remission, and relapse. Using a combination of computational and experimental methods, we were able to analyze the accessibility landscape and focus on the most significant cis-regulatory sites. These sites were then functionally validated through the use of Promoter capture Hi-C in a primary cell line model called LAL-B, followed by RNA-seq and genomic deletion of target sites using CRISPR-Cas9 editing. Results We found that enhancer activity changes during cancer progression and is mediated by the production of enhancer RNAs (eRNAs). CRISPR-Cas9-mediated validation of previously unknown eRNA productive enhancers demonstrated their capability to control the oncogenic activities of the MYB and DCTD genes. Conclusions Our findings directly support the notion that productive enhancer engagement is a crucial determinant of the BCP-ALL and highlight the potential of enhancers as therapeutic targets in pediatric BCP-ALL. Keywords: Cis-regulatory elements, BCP-ALL, CRISPR/Cas-9, MYB, DCTD
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. However, little is known of the relative contribution to pro-senescence signal propagation provided ...by microRNAs shuttled by extracellular vesicles (EVs) released from senescent cells. Analysis of microRNA and DNA methylation profiles in non-senescent (control) and senescent (SEN) human umbilical vein endothelial cells (HUVECs), and microRNA profiling of their cognate small EVs (sEVs) and large EVs demonstrated that SEN cells released a significantly greater sEV number than control cells. sEVs were enriched in miR-21-5p and miR-217, which target DNMT1 and SIRT1. Treatment of control cells with SEN sEVs induced a miR-21/miR-217-related impairment of DNMT1-SIRT1 expression, the reduction of proliferation markers, the acquisition of a senescent phenotype and a partial demethylation of the locus encoding for miR-21. MicroRNA profiling of sEVs from plasma of healthy subjects aged 40-100 years showed an inverse U-shaped age-related trend for miR-21-5p, consistent with senescence-associated biomarker profiles. Our findings suggest that miR-21-5p/miR-217 carried by SEN sEVs spread pro-senescence signals, affecting DNA methylation and cell replication.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Current popular variant calling pipelines rely on the mapping coordinates of each input read to a reference genome in order to detect variants. Since reads deriving from variant loci that diverge in ...sequence substantially from the reference are often assigned incorrect mapping coordinates, variant calling pipelines that rely on mapping coordinates can exhibit reduced sensitivity.
In this work we present GeDi, a suffix array-based somatic single nucleotide variant (SNV) calling algorithm that does not rely on read mapping coordinates to detect SNVs and is therefore capable of reference-free and mapping-free SNV detection. GeDi executes with practical runtime and memory resource requirements, is capable of SNV detection at very low allele frequency (<1%), and detects SNVs with high sensitivity at complex variant loci, dramatically outperforming MuTect, a well-established pipeline.
By designing novel suffix-array based SNV calling methods, we have developed a practical SNV calling software, GeDi, that can characterise SNVs at complex variant loci and at low allele frequency thus increasing the repertoire of detectable SNVs in tumour genomes. We expect GeDi to find use cases in targeted-deep sequencing analysis, and to serve as a replacement and improvement over previous suffix-array based SNV calling methods.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background Understanding how cancer signaling pathways promote an immunosuppressive program which sustains acquired or primary resistance to immune checkpoint blockade (ICB) is a crucial step in ...improving immunotherapy efficacy. Among the pathways that can affect ICB response is the interferon (IFN) pathway that may be both detrimental and beneficial. The immune sensor retinoic acid-inducible gene I (RIG-I) induces IFN activation and secretion and is activated by actin cytoskeleton disturbance. The actin cytoskeleton regulatory protein hMENA, along with its isoforms, is a key signaling hub in different solid tumors, and recently its role as a regulator of transcription of genes encoding immunomodulatory secretory proteins has been proposed. When hMENA is expressed in tumor cells with low levels of the epithelial specific hMENA 11a isoform, identifies non-small cell lung cancer (NSCLC) patients with poor prognosis. Aim was to identify cancer intrinsic and extrinsic pathways regulated by hMENA 11a downregulation as determinants of ICB response in NSCLC. Here, we present a potential novel mechanism of ICB resistance driven by hMENA 11a downregulation. Methods Effects of hMENA 11a downregulation were tested by RNA-Seq, ATAC-Seq, flow cytometry and biochemical assays. ICB-treated patient tumor tissues were profiled by Nanostring IO 360 Panel enriched with hMENA custom probes. OAK and POPLAR datasets were used to validate our discovery cohort. Results Transcriptomic and biochemical analyses demonstrated that the depletion of hMENA 11a induces IFN pathway activation, the production of different inflammatory mediators including IFNβ via RIG-I, sustains the increase of tumor PD-L1 levels and activates a paracrine loop between tumor cells and a unique macrophage subset favoring an epithelial-mesenchymal transition (EMT). Notably, when we translated our results in a clinical setting of NSCLC ICB-treated patients, transcriptomic analysis revealed that low expression of hMENA 11a , high expression of IFN target genes and high macrophage score identify patients resistant to ICB therapy. Conclusions Collectively, these data establish a new function for the actin cytoskeleton regulator hMENA 11a in modulating cancer cell intrinsic type I IFN signaling and extrinsic mechanisms that promote protumoral macrophages and favor EMT. These data highlight the role of actin cytoskeleton disturbance in activating immune suppressive pathways that may be involved in resistance to ICB in NSCLC.
According to the cancer stem cells (CSCs) hypothesis, a population of cancer cells with stem cell properties is responsible for tumor propagation, drug resistance, and disease recurrence. Study of ...the mechanisms responsible for lung CSCs propagation is expected to provide better understanding of cancer biology and new opportunities for therapy.
The Lung Adenocarcinoma (LUAD) NCI-H460 cell line was grown either as 2D or as 3D cultures. Transcriptomic and genome-wide chromatin accessibility studies of 2D vs. 3D cultures were carried out using RNA-sequencing and Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq), respectively. Reverse transcription polymerase chain reaction (RT-PCR) was also carried out on RNA extracted from primary cultures derived from malignant pleural effusions to validate RNA-seq results.
RNA-seq and ATAC-seq data disentangled transcriptional and genome accessibility variability of 3D vs. 2D cultures in NCI-H460 cells. The examination of genomic landscape of genes upregulated in 3D vs. 2D cultures led to the identification of 2D cultures led to the identification of Beta-1,4-galactosyltranferase 1 (B4GALT1) as the top candidate. B4GALT1 as the top candidate. B4GALT1 was validated as a stemness factor, since its silencing caused strong inhibition of 3D spheroid formation.
Combined transcriptomic and chromatin accessibility study of 3D vs. 2D LUAD cultures led to the identification of B4GALT1 as a new factor involved in the propagation and maintenance of LUAD CSCs.
Several kinds of stress promote the formation of three‐stranded RNA:DNA hybrids called R‐loops. Insufficient clearance of these structures promotes genomic instability and DNA damage, which ...ultimately contribute to the establishment of cancer phenotypes. Paraspeckle assemblies participate in R‐loop resolution and preserve genome stability, however, the main determinants of this mechanism are still unknown. This study finds that in Multiple Myeloma (MM), AATF/Che‐1 (Che‐1), an RNA‐binding protein fundamental to transcription regulation, interacts with paraspeckles via the lncRNA NEAT1_2 (NEAT1) and directly localizes on R‐loops. We systematically show that depletion of Che‐1 produces a marked accumulation of RNA:DNA hybrids. We provide evidence that such failure to resolve R‐loops causes sustained activation of a systemic inflammatory response characterized by an interferon (IFN) gene expression signature. Furthermore, elevated levels of R‐loops and of mRNA for paraspeckle genes in patient cells are linearly correlated with Multiple Myeloma progression. Moreover, increased interferon gene expression signature in patients is associated with markedly poor prognosis. Taken together, our study indicates that Che‐1/NEAT1 cooperation prevents excessive inflammatory signaling in Multiple Myeloma by facilitating the clearance of R‐loops. Further studies on different cancer types are needed to test if this mechanism is ubiquitously conserved and fundamental for cell homeostasis.
Synopsis
Che‐1/AATF (Che‐1) is a nuclear protein involved in cellular stress response and neoplastic transformation in Multiple Myeloma. Here, Che‐1 is identified as a component of paraspeckles and a key factor in cell stress resolution in Multiple Myeloma.
Che‐1 interacts with NEAT1 lncRNA and localizes to nuclear paraspeckles.
Che‐1 and NEAT1 prevent ER‐stress‐dependent R‐loop accumulation in MM cells.
Che‐1‐mediated R‐loop suppression prevents interferon (IFN) response activation.
Elevated R‐loops levels and expression of paraspeckle genes are associated with MM progression in patient tissues.
Nucleolar protein Che‐1 interacts with NEAT1 lncRNA to minimize RNA:DNA hybrid formation and cGAS‐dependent interferon responses caused by the unfolded protein response in Multiple Myeloma cells.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The degree of intrinsic and interpatient phenotypic heterogeneity and its role in tumor evolution is poorly understood. Phenotypic drifts can be transmitted via inheritable transcriptional programs. ...Cell-type specific transcription is maintained through the activation of epigenetically defined regulatory regions including promoters and enhancers. Here we have annotated the epigenome of 47 primary and metastatic estrogen-receptor (ERα)-positive breast cancer clinical specimens and inferred phenotypic heterogeneity from the regulatory landscape, identifying key regulatory elements commonly shared across patients. Shared regions contain a unique set of regulatory information including the motif for transcription factor YY1. We identify YY1 as a critical determinant of ERα transcriptional activity promoting tumor growth in most luminal patients. YY1 also contributes to the expression of genes mediating resistance to endocrine treatment. Finally, we used H3K27ac levels at active enhancer elements as a surrogate of intra-tumor phenotypic heterogeneity to track the expansion and contraction of phenotypic subpopulations throughout breast cancer progression. By tracking the clonality of SLC9A3R1-positive cells, a bona fide YY1-ERα-regulated gene, we show that endocrine therapies select for phenotypic clones under-represented at diagnosis. Collectively, our data show that epigenetic mechanisms significantly contribute to phenotypic heterogeneity and evolution in systemically treated breast cancer patients.
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