A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

Epigenetic mechanisms are essential to establish and safeguard cellular identities in mammals. They dynamically regulate the expression of genes, transposable elements, and higher-order chromatin structures. Expectedly, these chromatin marks are indispensable for mammalian development and alterations often lead to diseases such as cancer. Molecularly, epigenetic mechanisms rely on factors to establish patterns, interpret them into a transcriptional output, and maintain them across cell divisions. A global picture of these phenomena has started to emerge over the years, yet many of the molecular actors remain to be discovered. In this context, we have developed a reporter system sensitive to epigenetic perturbations to report on repressive pathways based on Dazl, which is normally repressed in mouse ES cells. We used this system for a genome-wide CRISPR knock-out screen, which yielded expected hits (DNMT1, UHRF1, MGA), as well as novel candidates. We prioritized the candidates by secondary screens, and led further experiments on 6 of them: ZBTB14, KDM5C, SPOP, MCM3AP, BEND3, and KMT2D. Our results show that all 6 candidates regulate the expression of germline genes. In addition, we find that removal of ZBTB14, KDM5C, SPOP and MCM3AP led to similar transcriptional responses, including a reactivation of the 2-cell like cell (2CLC) signature. Therefore, our genetic screen has identified new regulators of key cellular states.

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Hoxa1 and TALE proteins display cross-regulatory interactions and form a combinatorial binding code on Hoxa1 targets

10.1101/092296 ◽

2016 ◽

Author(s):

Bony De Kumar ◽

Hugo J. Parker ◽

Ariel Paulson ◽

Mark E. Parrish ◽

Irina Pushel ◽

...

Keyword(s):

Es Cells ◽

Hox Proteins ◽

Binding Motifs ◽

Functional Roles ◽

Regulatory Interactions ◽

Mouse Es Cells ◽

Genome Wide ◽

A Genome ◽

Combinatorial Interactions ◽

Insight Into

AbstractHoxa1 has diverse functional roles in differentiation and development. We have identified and characterized properties of regions bound by Hoxa1 on a genome-wide basis in differentiating mouse ES cells. Hoxa1 bound regions are enriched for clusters of consensus binding motifs for Hox, Pbx and Meis and many display co-occupancy of Pbx and Meis. Pbx and Meis are members of the TALE family and genome-wide analysis of multiple TALE members (Pbx, Meis, TGIF, Prep1 and Prep2) show that nearly all Hoxa1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins defines distinct classes of Hoxa1 targets and indicates a role as cofactors in modulating the specificity of Hox proteins. We also discovered extensive auto- and cross-regulatory interactions among the Hoxa1 and TALE genes. This study provides new insight into a regulatory network involving combinatorial interactions between Hoxa1 and TALE proteins.

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A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

10.1242/prelights.29562 ◽

2021 ◽

Author(s):

María Mariner-Faulí

Keyword(s):

Epigenetic Silencing ◽

Knock Out ◽

Cell State ◽

Genome Wide ◽

A Genome ◽

Germline Genes

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BIOM-32. ENDOPLASMIC RETICULUM PROTEIN SSR3 DETERMINES AND PREDICTS RESPONSE TO PACLITAXEL IN BREAST CANCER AND GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noab196.063 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi17-vi18

Author(s):

Crismita Dmello ◽

Aarón Sonabend ◽

Víctor Arrieta ◽

Daniel Zhang ◽

Deepak Kanojia ◽

...

Keyword(s):

Breast Cancer ◽

Endoplasmic Reticulum ◽

Glioma Cells ◽

Predictive Biomarkers ◽

Predictive Biomarker ◽

Precision Oncology ◽

Patient Response ◽

Knock Out ◽

Genome Wide ◽

A Genome

Abstract Paclitaxel (PTX) is one the most potent and commonly used chemotherapies for breast and pancreatic cancer. Given the potency of this drug for glioblastomas (GBM) several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood-brain barrier for this disease. In spite of the efficacy of PTX, individual tumors exhibit variable susceptibility to this drug, with response rate in the range of 30%-60%. To identify predictive biomarkers for response to PTX, we performed a genome-wide CRISPR knock-out screen using human glioma cells. The most enriched genes in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort. This led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with response to PTX in breast cancer cells, glioma cells, in multiple intracranial glioma xenografts and in GBM patient derived explant cultures. Knockout of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. In gliomas, SSR3-mediated susceptibility to PTX relates to modulation of phosphorylation of ER stress sensor IRE1α. Thus, by using genome-wide screen combined with patient response data, we discovered a biomarker that demonstrates causal and correlative relationship with response to PTX in breast cancer and GBM. Prospective validation of this biomarker is warranted for its broad implementation for precision oncology.

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Crosstalk in oxygen homeostasis networks: SKN-1/NRF inhibits the HIF-1 hypoxia-inducible factor in Caenorhabditis elegans

10.1101/2021.03.12.435071 ◽

2021 ◽

Author(s):

Dingxia Feng ◽

Zhiwei Zhai ◽

Zhiyong Shao ◽

Yi Zhang ◽

Jo Anne Powell-Coffman

Keyword(s):

Oxidative Stress ◽

Hypoxia Inducible Factor ◽

Regulatory Sequences ◽

Low Oxygen ◽

Transcriptional Responses ◽

C Elegans ◽

Protein Levels ◽

Oxygen Homeostasis ◽

Genome Wide ◽

A Genome

AbstractDuring development, homeostasis, and disease, organisms must balance responses that allow adaptation to low oxygen (hypoxia) with those that protect cells from oxidative stress. The evolutionarily conserved hypoxia-inducible factors are central to these processes, as they orchestrate transcriptional responses to oxygen deprivation. Here, we employ genetic strategies in C. elegans to identify stress-responsive genes and pathways that modulate the HIF-1 hypoxia-inducible factor and facilitate oxygen homeostasis. Through a genome-wide RNAi screen, we show that RNAi-mediated mitochondrial or proteasomal dysfunction increases the expression of hypoxia-responsive reporter Pnhr-57:GFP in C. elegans. Interestingly, only a subset of these effects requires hif-1. Of particular importance, we found that skn-1 RNAi increases the expression of hypoxia-responsive reporter Pnhr-57:GFP and elevates HIF-1 protein levels. The SKN-1/NRF transcription factor has been shown to promote oxidative stress resistance. We present evidence that the crosstalk between HIF-1 and SKN-1 is mediated by EGL-9, the prolyl hydroxylase that targets HIF-1 for oxygen-dependent degradation. Treatment that induces SKN-1, such as heat, increases expression of a Pegl-9:GFP reporter, and this effect requires skn-1 function and a putative SKN-1 binding site in egl-9 regulatory sequences. Collectively, these data support a model in which SKN-1 promotes egl-9 transcription, thereby inhibiting HIF-1. We propose that this interaction enables animals to adapt quickly to changes in cellular oxygenation and to better survive accompanying oxidative stress.

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Genome-Wide Effects of Selenium and Translational Uncoupling on Transcription in the Termite Gut Symbiont Treponema primitia

mBio ◽

10.1128/mbio.00869-13 ◽

2013 ◽

Vol 4 (6) ◽

Cited By ~ 1

Author(s):

Eric G. Matson ◽

Adam Z. Rosenthal ◽

Xinning Zhang ◽

Jared R. Leadbetter

Keyword(s):

Protein Synthesis ◽

Large Body ◽

Transcriptional Responses ◽

Translational Coupling ◽

Genome Wide ◽

A Genome ◽

Protein Encoding ◽

Termite Gut ◽

Inhibiting Protein ◽

Encoding Genes

ABSTRACTWhen prokaryotic cells acquire mutations, encounter translation-inhibiting substances, or experience adverse environmental conditions that limit their ability to synthesize proteins, transcription can become uncoupled from translation. Such uncoupling is known to suppress transcription of protein-encoding genes in bacteria. Here we show that the trace element selenium controls transcription of the gene for the selenocysteine-utilizing enzyme formate dehydrogenase (fdhFSec) through a translation-coupled mechanism in the termite gut symbiontTreponema primitia, a member of the bacterial phylumSpirochaetes. We also evaluated changes in genome-wide transcriptional patterns caused by selenium limitation and by generally uncoupling translation from transcription via antibiotic-mediated inhibition of protein synthesis. We observed that inhibiting protein synthesis inT. primitiainfluences transcriptional patterns in unexpected ways. In addition to suppressing transcription of certain genes, the expected consequence of inhibiting protein synthesis, we found numerous examples in which transcription of genes and operons is truncated far downstream from putative promoters, is unchanged, or is even stimulated overall. These results indicate that gene regulation in bacteria allows for specific post-initiation transcriptional responses during periods of limited protein synthesis, which may depend both on translational coupling and on unclassified intrinsic elements of protein-encoding genes.IMPORTANCEA large body of literature demonstrates that the coupling of transcription and translation is a general and essential method by which bacteria regulate gene expression levels. However, the potential role of noncanonical amino acids in regulating transcriptional output via translational control remains, for the most part, undefined. Furthermore, the genome-wide transcriptional state in response to translational decoupling is not well quantified. The results presented here suggest that the noncanonical amino acid selenocysteine is able to tune transcription of an important metabolic gene via translational coupling. Furthermore, a genome-wide analysis reveals that transcriptional decoupling produces a wide-ranging effect and that this effect is not uniform. These results exemplify how growth conditions that impact translational processivity can rapidly feed back on transcriptional productivity of prespecified groups of genes, providing bacteria with an efficient response to environmental changes.

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TERRA regulate the transcriptional landscape of pluripotent cells through TRF1-dependent recruitment of PRC2

eLife ◽

10.7554/elife.44656 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 12

Author(s):

Rosa María Marión ◽

Juan J Montero ◽

Isabel López de Silanes ◽

Osvaldo Graña-Castro ◽

Paula Martínez ◽

...

Keyword(s):

Cell Fate ◽

Es Cells ◽

Epigenetic Changes ◽

Pluripotent Cells ◽

Telomere Repeat ◽

Mouse Es Cells ◽

Genome Wide ◽

Binding Factor ◽

Transcriptional Landscape ◽

Pluripotency Genes

The mechanisms that regulate pluripotency are still largely unknown. Here, we show that Telomere Repeat Binding Factor 1 (TRF1), a component of the shelterin complex, regulates the genome-wide binding of polycomb and polycomb H3K27me3 repressive marks to pluripotency genes, thereby exerting vast epigenetic changes that contribute to the maintenance of mouse ES cells in a naïve state. We further show that TRF1 mediates these effects by regulating TERRA, the lncRNAs transcribed from telomeres. We find that TERRAs are enriched at polycomb and stem cell genes in pluripotent cells and that TRF1 abrogation results in increased TERRA levels and in higher TERRA binding to those genes, coincidental with the induction of cell-fate programs and the loss of the naïve state. These results are consistent with a model in which TRF1-dependent changes in TERRA levels modulate polycomb recruitment to pluripotency and differentiation genes. These unprecedented findings explain why TRF1 is essential for the induction and maintenance of pluripotency.

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Genome-wide decoupling of H2Aub and H3K27me3 in early mouse development

10.1101/2021.03.23.436550 ◽

2021 ◽

Author(s):

Yezhang Zhu ◽

Jiali Yu ◽

Yan Rong ◽

Yun-Wen Wu ◽

Heng-Yu Fan ◽

...

Keyword(s):

Polycomb Group ◽

Mammalian Development ◽

Mouse Development ◽

Chromatin Regulators ◽

Genome Wide ◽

A Genome ◽

Independent Functions ◽

Preimplantation Mouse ◽

Early Mouse ◽

Early Mammalian Development

Polycomb group (PcG) proteins are crucial chromatin regulators during development. H2Aub and H3K27me3 are catalyzed by Polycomb-repressive Complex 1 and 2 (PRC1/2) respectively, and largely overlap in the genome due to mutual recruitment of the two complexes. However, whether PRC1/H2Aub and PRC2/H3K27me3 can function independently remains obscure. Here we uncovered a genome-wide decoupling of H2Aub and H3K27me3 in preimplantation mouse embryos, at both canonical PcG targets and broad distal domains. H2Aub represses future bivalent genes without H3K27me3 but does not contribute to maintenance of H3K27me3-dependent non-canonical imprinting. Our study thus revealed their distinct and independent functions in early mammalian development.

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Tracking the embryonic stem cell transition from ground state pluripotency

10.1101/092510 ◽

2016 ◽

Cited By ~ 4

Author(s):

Tüzer Kalkan ◽

Nelly Olova ◽

Mila Roode ◽

Carla Mulas ◽

Heather J. Lee ◽

...

Keyword(s):

Ground State ◽

Es Cells ◽

Single Cells ◽

Embryonic Stem ◽

List Type ◽

Es Cell ◽

Genome Wide ◽

Developmental Progression ◽

A Genome ◽

Lineage Priming

SummaryMouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition of ES cells. The population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naive status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state exhibit global transcriptome features consistent with features of early post-implantation epiblast and distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naïve cells transition to a discrete formative phase of pluripotency preparatory to lineage priming.HighlightsThe Rex1 destabilized GFP reporter demarcates naive pluripotency.Exit from the naive state is asynchronous in the population.Transition is relatively acute in individual cells and precedes lineage priming.Transcriptome and DNA methylome reflect events in the pre-gastrulation embryo.

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A Genome-Wide View of Transcriptional Responses during Aphis glycines Infestation in Soybean

International Journal of Molecular Sciences ◽

10.3390/ijms21155191 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5191

Author(s):

Luming Yao ◽

Biyun Yang ◽

Xiaohong Ma ◽

Shuangshuang Wang ◽

Zhe Guan ◽

...

Keyword(s):

Transcription Factor ◽

Expression Patterns ◽

Aphis Glycines ◽

Limiting Factors ◽

Small Subset ◽

Transcriptional Responses ◽

Callose Deposition ◽

Aphid Infestation ◽

Genome Wide ◽

A Genome

Soybean aphid (Aphis glycines Matsumura) is one of the major limiting factors in soybean production. The mechanism of aphid resistance in soybean remains enigmatic as little information is available about the different mechanisms of antibiosis and antixenosis. Here, we used genome-wide gene expression profiling of aphid susceptible, antibiotic, and antixenotic genotypes to investigate the underlying aphid–plant interaction mechanisms. The high expression correlation between infested and non-infested genotypes indicated that the response to aphid was controlled by a small subset of genes. Plant response to aphid infestation was faster in antibiotic genotype and the interaction in antixenotic genotype was moderation. The expression patterns of transcription factor genes in susceptible and antixenotic genotypes clustered together and were distant from those of antibiotic genotypes. Among them APETALA 2/ethylene response factors (AP2/ERF), v-myb avian myeloblastosis viral oncogene homolog (MYB), and the transcription factor contained conserved WRKYGQK domain (WRKY) were proposed to play dominant roles. The jasmonic acid-responsive pathway was dominant in aphid–soybean interaction, and salicylic acid pathway played an important role in antibiotic genotype. Callose deposition was more rapid and efficient in antibiotic genotype, while reactive oxygen species were not involved in the response to aphid attack in resistant genotypes. Our study helps to uncover important genes associated with aphid-attack response in soybean genotypes expressing antibiosis and antixenosis.

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QSER1 protects DNA methylation valleys from de novo methylation

Science ◽

10.1126/science.abd0875 ◽

2021 ◽

Vol 372 (6538) ◽

pp. eabd0875 ◽

Cited By ~ 1

Author(s):

Gary Dixon ◽

Heng Pan ◽

Dapeng Yang ◽

Bess P. Rosen ◽

Therande Jashari ◽

...

Keyword(s):

Dna Methylation ◽

De Novo ◽

Embryonic Stem ◽

Central Question ◽

Mammalian Development ◽

Uncharacterized Gene ◽

Pathological Conditions ◽

Genome Wide ◽

A Genome ◽

De Novo Methylation

DNA methylation is essential to mammalian development, and dysregulation can cause serious pathological conditions. Key enzymes responsible for deposition and removal of DNA methylation are known, but how they cooperate to regulate the methylation landscape remains a central question. Using a knockin DNA methylation reporter, we performed a genome-wide CRISPR-Cas9 screen in human embryonic stem cells to discover DNA methylation regulators. The top screen hit was an uncharacterized gene, QSER1, which proved to be a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys (or canyons). We further demonstrate genetic and biochemical interactions of QSER1 and TET1, supporting their cooperation to safeguard transcriptional and developmental programs from DNMT3-mediated de novo methylation.

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