Responsiveness to perturbations is a hallmark of transcription factors that maintain cell identity in vitro

Mellis, Ian A.; Edelstein, Hailey I.; Truitt, Rachel; Goyal, Yogesh; Beck, Lauren E.; Symmons, Orsolya; Dunagin, Margaret C.; Linares-Saldana, Ricardo; Shah, Parisha P.; Pérez-Bermejo, Juan A.; Padmanabhan, Arun; Yang, Wenli; Jain, Rajan; Raj, Arjun

doi:10.1016/j.cels.2021.07.003

Cited by 17 publications

(23 citation statements)

References 79 publications

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“…While it is possible that this heterogeneity in repressive chromatin state has minimal functional consequence, cellular variation in chromatin state may also underlie future cell type-specific transcriptional responses. For example, Mellis et al recently found that cell type specificity was encoded not only in steady-state transcriptional output, but also in future responses to environmental perturbations 45 . Similarly, Shim et al identified that genes with heterogeneous H3K27me3 landscapes across distinct organ systems were strikingly enriched for key developmental, regulatory, and morphological functions 47 .…”

Section: Discussionmentioning

confidence: 99%

Characterizing cellular heterogeneity in chromatin state with scCUT&Tag-pro

Zhang

Srivastava

Mimitou

et al. 2021

Preprint

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New technologies that profile chromatin modifications at single-cell resolution offer enormous promise for functional genomic characterization. However, the sparsity of these measurements and the challenge of integrating multiple binding maps represent significant challenges. Here we introduce scCUT&Tag-pro, a multimodal assay for profiling protein-DNA interactions coupled with the abundance of surface proteins in single cells. In addition, we introduce scChromHMM, which integrates data from multiple experiments to infer and annotate chromatin states based on combinatorial histone modification patterns. We apply these tools to perform an integrated analysis across nine different molecular modalities in circulating human immune cells. We demonstrate how these two approaches can characterize dynamic changes in the function of individual genomic elements across both discrete cell states and continuous developmental trajectories, nominate associated motifs and regulators that establish chromatin states, and identify extensive and cell type-specific regulatory priming. Finally, we demonstrate how our integrated reference can serve as a scaffold to map and improve the interpretation of additional scCUT&Tag datasets.

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Section: Discussionmentioning

confidence: 99%

Characterizing cellular heterogeneity in chromatin state with scCUT&Tag-pro

Zhang

Srivastava

Mimitou

et al. 2021

Preprint

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“…As a metric of similarity, we chose to use the Jensen-Shannon distance, an entropy-based measurement that has been used previously to calculate cell type or tissue-specificity scores (Cabili et al 2011;Mellis et al 2021). The Jensen-Shannon distance metric (Fuglede and Topsoe 2004) quantifies the relative distance between 2 probability distributions (not assuming any ordinality to the individual bins), where a distance of 0 denotes 2 identical distributions and a distance of 1 denotes maximally different distributions.…”

Section: Related Hips Cells Sometimes Show Similar Expression States Upon Cardiac Directed Differentiationmentioning

confidence: 99%

“…To perform cardiac directed differentiation we adapted standard protocols as previously described (Mellis et al 2021;Shah et al 2021;Palpant et al 2017;Laflamme et al 2007). Briefly, we grew undifferentiated hiPS cells in feeder-free conditions for 4-5 days until they reached ~75% confluency.…”

Section: Cardiac Directed Differentiation Of Hips Cellsmentioning

confidence: 99%

“…Single-molecule RNA FISH TNNT2 and LUM probes were used as previously described (Mellis et al 2021;Padovan-Merhar et al 2015). For our other genes of interest, we designed complementary oligonucleotide probe sets using custom probe design software written in MATLAB as previously described (Rouhanifard et al 2018;Shaffer et al 2018;Emert et al 2021) and ordered oligonucleotides with a primary amine group on the 3' end from Biosearch Technologies (see Supplementary Table 2 for probe sequences).…”

Section: Computational Analyses Of Barcode Genomic Dna Sequencing Datamentioning

confidence: 99%

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Cell type determination for cardiac differentiation occurs soon after seeding of human induced pluripotent stem cells

Jiang

Goyal

Jain

et al. 2021

Preprint

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Cardiac directed differentiation of human induced pluripotent stem cells consistently produces a mixed population of cardiomyocytes and non-cardiac cell types even when using very well-characterized protocols. We wondered whether differentiated cell types might result from intrinsic differences in hiPS cells prior to the onset of differentiation. By associating individual differentiated cells that share a common hiPS cell precursor, we were able to test whether expression variability in differentiated cells was pre-determined from the hiPS cell state. Although within a single experiment, differentiated cells that share an hiPS cell progenitor were more transcriptionally similar to each other than to other cells in the differentiated population, when the same hiPS cells were differentiated in parallel, we did not observe high transcriptional similarity across differentiations. Additionally, we found that substantial cell death occurred during differentiation in a manner that suggested that all cells were equally likely to survive or die, suggesting that there was no intrinsic selection bias for cells descended from particular hiPS cell progenitors. These results led us to wonder about how cells grow out spatially during the directed differentiation process. Labeling cells by their expression of a few canonical cell type marker genes, we showed that cells expressing the same marker tended to occur in patches observable by visual inspection, suggesting that cell type determination across multiple cell types, once initiated, is maintained in a cell-autonomous manner for multiple divisions. Altogether, our results show that while there is substantial heterogeneity in the initial hiPS cell population, that heterogeneity is not responsible for heterogeneous outcomes, and that the window during which cell type specification occurs is likely to begin shortly after the seeding of hiPS cells for differentiation.

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“…Prior to extraction and library preparation, the samples were randomized to avoid any experimental and human biases. We aligned RNA-seq reads to the human genome (hg19) with STAR v2.5.2a and counted uniquely mapping reads with HTSeq v0.6.1 (Dobin et al 2013; Mellis et al 2021; Shaffer et al 2017) and outputs count matrix. The counts matrix was used to obtain tpm and other normalized values for each gene using scripts provided at: https://github.com/arjunrajlaboratory/RajLabSeqTools/tree/master/LocalComputerScripts…”

Section: Methodsmentioning

confidence: 99%

clampFISH 2.0 enables rapid, scalable amplified RNA detection in situ

Dardani

Emert

Goyal

et al. 2022

Preprint

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RNA labeling in situ has enormous potential to visualize transcripts and quantify their levels in single cells, but it remains challenging to produce high levels of signal while also enabling multiplexed detection of multiple RNA species simultaneously. Here, we describe clampFISH 2.0, a method that uses an inverted padlock design to efficiently detect and exponentially amplify signals from many RNA species at once, while also reducing time and cost compared to the prior clampFISH method. We leverage the increased throughput afforded by multiplexed signal amplification and sequential detection by demonstrating the ability to detect 10 different RNA species in over 1 million cells. We also show that clampFISH 2.0 works in tissue sections. We expect the advantages offered by clampFISH 2.0 will enable many applications in spatial transcriptomics.

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Responsiveness to perturbations is a hallmark of transcription factors that maintain cell identity in vitro

Cited by 17 publications

References 79 publications

Characterizing cellular heterogeneity in chromatin state with scCUT&Tag-pro

Characterizing cellular heterogeneity in chromatin state with scCUT&Tag-pro

Cell type determination for cardiac differentiation occurs soon after seeding of human induced pluripotent stem cells

clampFISH 2.0 enables rapid, scalable amplified RNA detection in situ

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