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Author(s): Jonathan J. Kotzin [1, 2]; Sean P. Spencer [1, 2]; Sam J. McCright [1, 2]; Dinesh B. Uthaya Kumar [3, 4]; Magalie A. Collet [3]; Walter K. Mowel [1, [...]

Author(s): Sydney M. Shaffer; Margaret C. Dunagin; Stefan R. Torborg; Eduardo A. Torre; Benjamin Emert; Clemens Krepler; Marilda Beqiri; Katrin Sproesser; Patricia A. Brafford; Min Xiao; Elliott Eggan; Ioannis N. [...]

Author(s): Sydney M. Shaffer [1, 2]; Margaret C. Dunagin [1]; Stefan R. Torborg [1, 3]; Eduardo A. Torre [1, 2]; Benjamin Emert [2, 4]; Clemens Krepler [5]; Marilda Beqiri [5]; [...]

Background: Long non-coding RNAs (lncRNAs) have been implicated in diverse biological processes. In contrast to extensive genomic annotation of lncRNA transcripts, far fewer have been characterized for subcellular localization and cell-to-cell variability. Addressing this requires systematic, direct visualization of lncRNAs in single cells at single-molecule resolution. Results: We use single-molecule RNA-FISH to systematically quantify and categorize the subcellular localization patterns of a representative set of 61 lncRNAs in three different cell types. Our survey yields high-resolution quantification and stringent validation of the number and spatial positions of these lncRNA, with an mRNA set for comparison. Using this highly quantitative image-based dataset, we observe a variety of subcellular localization patterns, ranging from bright sub-nuclear foci to almost exclusively cytoplasmic localization. We also find that the low abundance of lncRNAs observed from cell population measurements cannot be explained by high expression in a small subset of ‘jackpot’ cells. Additionally, nuclear lncRNA foci dissolve during mitosis and become widely dispersed, suggesting these lncRNAs are not mitotic bookmarking factors. Moreover, we see that divergently transcribed lncRNAs do not always correlate with their cognate mRNA, nor do they have a characteristic localization pattern. Conclusions: Our systematic, high-resolution survey of lncRNA localization reveals aspects of lncRNAs that are similar to mRNAs, such as cell-to-cell variability, but also several distinct properties. These characteristics may correspond to particular functional roles. Our study also provides a quantitative description of lncRNAs at the single-cell level and a universally applicable framework for future study and validation of lncRNAs. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0586-4) contains supplementary material, which is available to authorized users.

Cellular responses to environmental stimuli are typically thought to be governed by genetically encoded programs. We demonstrate that melanoma cells can form and maintain cellular memories during the acquisition of therapy resistance that exhibit characteristics of cellular learning and are dependent on the transcription factor AP-1. We show that cells exposed to a low dose of therapy adapt to become resistant to a high dose, demonstrating that resistance was not purely selective. The application of therapy itself results in the encoding of transient gene expression into cellular memory and that this encoding occurs for both transiently induced and probabilistically arising expression. Chromatin accessibility showed concomitant persistence. A two-color AP-1 reporter system showed that these memories are encoded in cis , constituting an example of activating cis epigenetics. Our findings establish the formation and maintenance of cellular memories as a critical aspect of gene regulation during the development of therapy resistance., Competing Interests: Declaration of interests A.R. receives royalties related to Stellaris RNA FISH probes. A.R. serves on the scientific advisory board of Spatial Genomics. All other authors declare no competing interests.

The ability of a virus to infect a cell type is at least in part determined by the presence of host factors required for the viral life cycle. However, even within cell types that express known factors needed for infection, not every cell is equally susceptible, suggesting that our knowledge of the full spectrum of factors that promote infection is incomplete. Profiling the most susceptible subsets of cells within a population may reveal additional factors that promote infection. However, because viral infection dramatically alters the state of the cell, new approaches are needed to reveal the state of these cells prior to infection with virus. Here, we used single-cell clone tracing to retrospectively identify and characterize lung epithelial cells that are highly susceptible to infection with SARS-CoV-2. The transcriptional state of these highly susceptible cells includes markers of retinoic acid signaling and epithelial differentiation. Loss of candidate factors identified by our approach revealed that many of these factors play roles in viral entry. Moreover, a subset of these factors exert control over the infectable cell state itself, regulating the expression of key factors associated with viral infection and entry. Analysis of patient samples revealed the heterogeneous expression of these factors across both cells and patients in vivo . Further, the expression of these factors is upregulated in particular inflammatory pathologies. Altogether, our results show that the variable expression of intrinsic cell states is a major determinant of whether a cell can be infected by SARS-CoV-2., Competing Interests: Competing Interests AR receives royalties related to Stellaris RNA FISH probes. All remaining authors declare no competing interests.

Pluripotency can be induced in somatic cells by the expression of the four "Yamanaka" factors OCT4, KLF4, SOX2, and MYC. However, even in homogeneous conditions, usually only a rare subset of cells admit reprogramming, and the molecular characteristics of this subset remain unknown. Here, we apply retrospective clone tracing to identify and characterize the individual human fibroblast cells that are primed for reprogramming. These fibroblasts showed markers of increased cell cycle speed and decreased fibroblast activation. Knockdown of a fibroblast activation factor identified by our analysis led to increased reprogramming efficiency, identifying it as a barrier to reprogramming. Changing the frequency of reprogramming by inhibiting the activity of LSD1 led to an enlarging of the pool of cells that were primed for reprogramming. Our results show that even homogeneous cell populations can exhibit heritable molecular variability that can dictate whether individual rare cells will reprogram or not., Competing Interests: Competing Interests AR receives royalties related to Stellaris RNA FISH probes. All remaining authors declare no competing interests.