Profiling of Pluripotency Factors in Single Cells and Early Embryos

Hainer, Sarah J.; Bošković, Ana; McCannell, Kurtis N.; Rando, Oliver J.; Fazzio, Thomas G.

doi:10.1016/j.cell.2019.03.014

Cited by 166 publications

(129 citation statements)

References 58 publications

(73 reference statements)

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“…In the future, scRNA-seq analyses reported in this study, together with single-cell epigenetic approaches (Buenrostro et al, 2015;Hainer et al, 2019;Huan et al, 2018;Lai et al, 2018) and methods with spatial information using in situ reverse transcription, can help characterize the spatiotemporal transcriptome atlas of plants. Such studies will not only promote the understanding of plant cellular and developmental biology at single-cell resolution but also help breed better crops against environmental stresses (Rhee et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Single-cell transcriptome analyses recapitulate the cellular and developmental responses to abiotic stresses in rice

Wang

Huan

Chu

et al. 2020

Preprint

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The metabolism and reproduction of plants depend on the division of labors among cells. However, cells with various functions are often studied as a bulk where their specificities were diluted. Here, we apply single-cell RNA sequencing to the aerial part of rice seedlings growing in various environments. We capture the transcriptomes of thousands of cells, identify all major cell types, and reconstruct their developmental trajectories. We find that abiotic stresses not only affect gene expression in a cell-type-specific manner but also have impacts on the physical size of cells and the composition of cell populations. We validate some of these conclusions with microscopy and provide developmental mechanisms with computational analyses. Collectively, our study represents a benchmark-setting data resource of single-cell transcriptome atlas in rice and an illustration of exploiting such resource to drive discoveries in plant biology.

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Section: Discussionmentioning

confidence: 99%

Single-cell transcriptome analyses recapitulate the cellular and developmental responses to abiotic stresses in rice

Wang

Huan

Chu

et al. 2020

Preprint

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“…CUT&RUN uses a protein A-micrococcal nuclease fusion protein to cleave nuclear DNA in the vicinity of antibody-TF complexes, eliminating the need for crosslinking or chromatin shearing. This approach has been used to determine TF occupancy in individual mouse embryonic stem cells (Hainer et al, 2019) and could potentially identify binding sites for TFs in discrete brain regions and cell types.…”

Section: Gonadal Steroid Hormones Direct Sexual Differentiation Of mentioning

confidence: 99%

“…Previous attempts at studying these mechanisms by ChIP-seq have been stymied by the high number of cells required for the method, which prohibits analysis of specific brain areas. Newer approaches such as CUT&RUN (Box 1) use low cell numbers, even single cells, to identify genomic sites of histone modifications or TF binding (Hainer, Boskovic, Rando, & Fazzio, 2019;Skene, Henikoff, & Henikoff, 2018) and could potentially revolutionize the study of gene regulation in the brain.…”

Section: Sex Differences Revealed Through Single-cell Rna-seqmentioning

confidence: 99%

Signatures of sex: Sex differences in gene expression in the vertebrate brain

Gegenhuber

Tollkühn

2019

WIREs Developmental Biology

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Women and men differ in disease prevalence, symptoms, and progression rates for many psychiatric and neurological disorders. As more preclinical studies include both sexes in experimental design, an increasing number of sex differences in physiology and behavior have been reported. In the brain, sex‐typical behaviors are thought to result from sex‐specific patterns of neural activity in response to the same sensory stimulus or context. These differential firing patterns likely arise as a consequence of underlying anatomic or molecular sex differences. Accordingly, gene expression in the brains of females and males has been extensively investigated, with the goal of identifying biological pathways that specify or modulate sex differences in brain function. However, there is surprisingly little consensus on sex‐biased genes across studies and only a handful of robust candidates have been pursued in the follow‐up experiments. Furthermore, it is not known how or when sex‐biased gene expression originates, as few studies have been performed in the developing brain. Here we integrate molecular genetic and neural circuit perspectives to provide a conceptual framework of how sex differences in gene expression can arise in the brain. We detail mechanisms of gene regulation by steroid hormones, highlight landmark studies in rodents and humans, identify emerging themes, and offer recommendations for future research. This article is categorized under: Nervous System Development > Vertebrates: General Principles Gene Expression and Transcriptional Hierarchies > Regulatory Mechanisms Gene Expression and Transcriptional Hierarchies > Sex Determination

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“…To identify the specific genomic regions affected by the disrupted heterochromatin structure, using CUT&RUN (Hainer et al, 2019;Skene and Henikoff, 2017), we profiled native (no crosslinking) chromatin structure of intestinal epithelial cells brushed from normal mouse intestine of wild-type and age-matched Apc Min/+ mice at 6 weeks, which is in early carcinogenesis when tissue still appears histologically normal. CUT&RUN is a newly developed genome-scale protein localization technique that serves as an alternative to chromatin immunoprecipitation (ChIP) but provides lower background signal.…”

Section: Disrupted Heterochromatin Coincides With Reduced Occupancy Omentioning

confidence: 99%

“…After scraping villi cells from mouse intestine, cells were pelleted, washed with cold PBS, and nuclei were extracted in a hypotonic buffer (NE buffer; 20mM HEPES-KOH pH 7.9, 10mM KCl, 0.5mM Spermidine, 0.1% TritonX-100, 20% glycerol, and freshly added protease inhibitors). CUT&RUN was performed as previously described (Hainer et al 2019). Briefly, lectin-coated Concanavalin A beads (Polysciences) were prepared using binding buffer (20mM HEPES-KOH pH 7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2), and nuclei were bound to beads.…”

Section: Cutandrunmentioning

confidence: 99%

Super-resolution imaging reveals the evolution of higher-order chromatin folding in early carcinogenesis

et al. 2019

Preprint

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SUMMARYAberrant chromatin structure is a hallmark in cancer cells and has long been used for clinical diagnosis of cancer. However, underlying higher-order chromatin folding during malignant transformation remains elusive, due to the lack of molecular scale resolution. Using optimized stochastic optical reconstruction microscopy (STORM) for pathological tissue (PathSTORM), we uncovered a gradual decompaction and fragmented higher-order chromatin folding throughout all stages of carcinogenesis in multiple tumor types, even prior to the tumor formation. Our integrated imaging, genomic, and transcriptomic analyses reveal the functional consequences in enhanced formation of transcription factories, spatial juxtaposition with relaxed nanosized chromatin domains and impaired genomic stability. We also demonstrate the potential of imaging higher-order chromatin decompaction to detect high-risk precursors that cannot be distinguished by conventional pathology. Taken together, our findings reveal the gradual decompaction and fragmentation of higher-order chromatin structure as an enabling characteristic in early carcinogenesis to facilitate malignant transformation, which may improve cancer diagnosis, risk stratification, and prevention.SIGNIFICANCEGenomic DNA is folded into a higher-order structure that regulates transcription and maintains genomic stability. Although much progress has been made on understanding biochemical characteristics of epigenetic modifications in cancer, the higher-order folding of chromatin structure remains largely unknown. Using optimized super-resolution microscopy, we uncover de-compacted and fragmented chromatin folding in tumor initiation and stepwise progression in multiple tumor types, even prior to the presence of tumor cells. This study underlines the significance of unfolding higher-order chromatin structure as an enabling characteristic to promote tumorigenesis, which may facilitate the development and evaluation of new preventive strategies. The potential of imaging higher-order chromatin folding to improve cancer detection and risk stratification is demonstrated by detecting high-risk precursors that cannot be distinguished by conventional pathology.

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Profiling of Pluripotency Factors in Single Cells and Early Embryos

Abstract: Highlights d A modified CUT&RUN method allows chromatin profiling from small numbers of cells d Transcription factors are profiled from single cells and blastocyst-stage embryos

Cited by 166 publications

References 58 publications

Single-cell transcriptome analyses recapitulate the cellular and developmental responses to abiotic stresses in rice

Single-cell transcriptome analyses recapitulate the cellular and developmental responses to abiotic stresses in rice

Signatures of sex: Sex differences in gene expression in the vertebrate brain

Super-resolution imaging reveals the evolution of higher-order chromatin folding in early carcinogenesis

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