Unbiased Reconstruction of a Mammalian Transcriptional Network Mediating Pathogen Responses

Amit, Ido; Garber, Manuel; Chevrier, Nicolas; Leite, Ana Paula; Donner, Yoni; Eisenhaure, Thomas; Guttman, Mitchell; Grenier, Jennifer K.; Li, Weibo; Zuk, Or; Schubert, Lisa; Birditt, Brian; Shay, Tal; Goren, Alon; Zhang, Xiaolan; Smith, Zachary D.; Deering, Raquel P.; Mcdonald, R. C.; Cabili, Moran N.; Bernstein, B; Rinn, John L.; Meissner, Alex; Root, David E.; Hacohen, Nir; Regev, Aviv

doi:10.1126/science.1179050

Cited by 445 publications

(594 citation statements)

References 30 publications

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“…These data indicate that the α-and β-cell lines used are significantly different in baseline gene-expression patterns, despite being of closely related developmental origins and derived with an identical strategy of SV40 large T antigen overexpression (25,26). Compared with expression profiles of mouse cell lines derived from other tissues (27,28), α cells are approximately two-to threefold more similar to β cells than to bone marrow dendritic cells or embryonic stem cells (SI Appendix, Fig. S2B).…”

Section: Resultsmentioning

confidence: 90%

Chromatin-targeting small molecules cause class-specific transcriptional changes in pancreatic endocrine cells

Kubicek

Gilbert²,

Fomina-Yadlin³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Under the instruction of cell-fate-determining, DNA-binding transcription factors, chromatin-modifying enzymes mediate and maintain cell states throughout development in multicellular organisms. Currently, small molecules modulating the activity of several classes of chromatin-modifying enzymes are available, including clinically approved histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors. We describe the genome-wide expression changes induced by 29 compounds targeting HDACs, DNMTs, histone lysine methyltransferases (HKMTs), and protein arginine methyltransferases (PRMTs) in pancreatic α-and β-cell lines. HDAC inhibitors regulate several hundred transcripts irrespective of the cell type, with distinct clusters of dissimilar activity for hydroxamic acids and orthoamino anilides. In contrast, compounds targeting histone methyltransferases modulate the expression of restricted gene sets in distinct cell types. For example, we find that G9a/GLP methyltransferase inhibitors selectively up-regulate the cholesterol biosynthetic pathway in pancreatic but not liver cells. These data suggest that, despite their conservation across the entire genome and in different cell types, chromatin pathways can be targeted to modulate the expression of selected transcripts.histone modification | gene regulation | chemical epigenetics | beta cell biology | cholesterol pathway E pigenetic mechanisms mediated through chromatin control cell-state and cell-type decisions during development and in the adult (1, 2). Chromatin-modifying enzymes have been shown to function by interacting with master transcription factors, regulating the expression of key target genes and conferring epigenetic memory through the propagation of modifications to the chromatin template. These modifications include methylation of the DNA itself and a variety of posttranslational modifications to histones, the proteins most closely interacting with DNA. Depending on the type of modification, e.g., acetylation or methylation, and the exact position of the modified amino acid, these modifications can activate or repress transcription of the underlying DNA sequence (3).Small molecules targeting chromatin have mainly been developed for the treatment of cancer, justified by the identification of genetic aberrations leading to overexpression or activation of several chromatin-modifying enzymes (4-7). In contrast to clinically approved HDAC and DNMT inhibitors, fewer compounds targeting histone lysine methyltransferases (HKMTs) and protein arginine methyltransferases (PRMTs) are available. These small molecules are mainly used as chemical probe compounds in basic research, and toxicity is often limiting for testing in animal models. Interestingly, for most of the 50 HKMTs and 30 histone demethylases encoded in the human genome, no specific compounds are available. Even for HDACs, most compounds inhibit HDACs 1, 2, 3, and 6 (8), and HDAC8-specific compounds are only now emerging (9).Chromatin-modifying enzymes play important roles in normal development,...

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Section: Resultsmentioning

confidence: 90%

Chromatin-targeting small molecules cause class-specific transcriptional changes in pancreatic endocrine cells

Kubicek

Gilbert²,

Fomina-Yadlin³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Alternative promoter and polyadenylation usage are two key mechanisms by which expression can be controlled transcriptionally or post-transcriptionally (Elkon et al 2013;de Klerk and 't Hoen 2015). Although the response of mBMDCs to LPS has been extensively characterized (Amit et al 2009;Garber et al 2012;Rabani et al 2014;Jovanovic et al 2015), the exact contribution of these two mechanisms to the transcriptional response to LPS is largely unknown.…”

Section: A Methods Specifically Designed For End-sequence Quantificationmentioning

confidence: 99%

End Sequence Analysis Toolkit (ESAT) expands the extractable information from single-cell RNA-seq data

et al. 2016

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RNA-seq protocols that focus on transcript termini are well suited for applications in which template quantity is limiting. Here we show that, when applied to end-sequencing data, analytical methods designed for global RNA-seq produce computational artifacts. To remedy this, we created the End Sequence Analysis Toolkit (ESAT). As a test, we first compared endsequencing and bulk RNA-seq using RNA from dendritic cells stimulated with lipopolysaccharide (LPS). As predicted by the telescripting model for transcriptional bursts, ESAT detected an LPS-stimulated shift to shorter 3 ′ -isoforms that was not evident by conventional computational methods. Then, droplet-based microfluidics was used to generate 1000 cDNA libraries, each from an individual pancreatic islet cell. ESAT identified nine distinct cell types, three distinct β-cell types, and a complex interplay between hormone secretion and vascularization. ESAT, then, offers a much-needed and generally applicable computational pipeline for either bulk or single-cell RNA end-sequencing.

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“…This is especially important for TLR4, the cellular receptor for LPS, a molecular signature of the outer cell membrane of Gram-negative bacteria (6). Activation of TLR4 by LPS affects the regulation of more than 1000 genes (7). Additionally, TLR4 signaling has been implicated in sterile inflammation through activation of the receptor by endogenous ligands (8 -14).…”

mentioning

confidence: 99%

The Ectodomain of the Toll-like Receptor 4 Prevents Constitutive Receptor Activation

Panter

Jerala

2011

Journal of Biological Chemistry

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Toll-like receptor 4 (TLR4) is involved in activation of the innate immune response in a large number of different diseases.Despite numerous studies, the role of separate domains of TLR4 in the regulation of receptor activation is poorly understood. Replacement of the TLR4 ectodomain with LPS-binding proteins MD-2 or CD14 resulted in a robust ligand-independent constitutive activation comparable with the maximal stimulation of the receptor with LPS. The same effect was achieved by the replacement of the ectodomain with a monomeric fluorescent protein or a 24-kDa gyrase B fragment. This demonstrates an intrinsic dimerization propensity of the transmembrane and cytoplasmic domains of TLR4 and reveals a previously unknown function of the ectodomain in inhibiting spontaneous receptor dimerization. Constitutive activation was abolished by the replacement of the ectodomain by a bulkier protein ovalbumin. N-terminal deletion variants of TLR4 revealed that the smallest segment of the ectodomain that already prevents constitutive activity comprises only 90 residues (542 to 631) of the total 608 residues. We conclude that TLR4 represents a receptor with a low threshold of activation that can be rapidly activated by the release of inhibition exerted by its ectodomain. This is important for the sensitivity of TLR4 to activation by different agonists. The TLR4 ectodomain has multiple roles in enabling ligand regulated activation, providing proper localization while serving as an inhibitor to prevent spontaneous, ligand-independent dimerization.Pattern recognition receptors are proteins expressed by cells of the innate immune system and act as a first line of host defense against invading microorganisms, recognizing diverse but highly conserved structural pathogen-associated molecular patterns of bacteria, fungi, and viruses (1). Activation of pattern recognition receptors by their respective ligands triggers an immediate immune response, characterized by proinflammatory cytokine production (1, 2). Toll-like receptors (TLRs) 2 are a family of pattern recognition receptors central to the vertebrate innate immune response. Activation of TLRs links innate and adaptive immunity through proinflammatory signaling and induction of costimulatory molecules on immune cells (3, 4). However, activation of TLRs has to be a tightly regulated process. TLRs have to be able to mount an immediate immune response upon binding of the agonist, whereas in the absence of the ligand it is imperative that they remain in an inactive state to prevent unwanted activation that may lead to excessive inflammation and autoimmune disease (5). This is especially important for TLR4, the cellular receptor for LPS, a molecular signature of the outer cell membrane of Gram-negative bacteria (6). Activation of TLR4 by LPS affects the regulation of more than 1000 genes (7). Additionally, TLR4 signaling has been implicated in sterile inflammation through activation of the receptor by endogenous ligands (8 -14).TLR4 is a type I transmembrane glycoprotein. The ectodoma...

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Unbiased Reconstruction of a Mammalian Transcriptional Network Mediating Pathogen Responses

Cited by 445 publications

References 30 publications

Chromatin-targeting small molecules cause class-specific transcriptional changes in pancreatic endocrine cells

Chromatin-targeting small molecules cause class-specific transcriptional changes in pancreatic endocrine cells

End Sequence Analysis Toolkit (ESAT) expands the extractable information from single-cell RNA-seq data

The Ectodomain of the Toll-like Receptor 4 Prevents Constitutive Receptor Activation

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