In vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation

ABSTRACTBetween birth and adulthood cardiomyocytes (CMs) undergo dramatic changes in size, ultrastructure, metabolism, and gene expression, in a process collectively referred to as CM maturation. The transcriptional network that coordinates CM maturation is poorly understood, creating a bottleneck for cardiac regenerative medicine. Forward genetic screens are a powerful, unbiased method to gain novel insights into transcriptional networks, yet this approach has rarely been used in vivo in mammals because of high resource demands. Here we utilized somatic mutagenesis to perform the first reported in vivo CRISPR genetic screen within a mammalian heart. We discovered and validated several novel transcriptional regulators of CM maturation. Among them were RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicated that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems.

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Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation

Nature Communications ◽

10.1038/s41467-021-24743-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Nathan J. VanDusen ◽

Julianna Y. Lee ◽

Weiliang Gu ◽

Catalina E. Butler ◽

Isha Sethi ◽

...

Keyword(s):

Gene Expression ◽

Genetic Screen ◽

Forward Genetics ◽

Epigenetic Mark ◽

Biological Processes ◽

Dynamic Changes ◽

Forward Genetic Screen ◽

High Resource ◽

Organ Systems

AbstractThe forward genetic screen is a powerful, unbiased method to gain insights into biological processes, yet this approach has infrequently been used in vivo in mammals because of high resource demands. Here, we use in vivo somatic Cas9 mutagenesis to perform an in vivo forward genetic screen in mice to identify regulators of cardiomyocyte (CM) maturation, the coordinated changes in phenotype and gene expression that occur in neonatal CMs. We discover and validate a number of transcriptional regulators of this process. Among these are RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicate that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems.

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An amiRNA screen uncovers redundant CBF & ERF34/35 transcription factors that differentially regulate arsenite and cadmium responses

10.1101/2020.12.30.424898 ◽

2021 ◽

Author(s):

Qingqing Xie ◽

Qi Yu ◽

Timothy O. Jobe ◽

Allis Pham ◽

Chennan Ge ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Gene Families ◽

Forward Genetics ◽

Homologous Gene ◽

Genetic Redundancy ◽

Cadmium Resistance ◽

Transcriptional Responses ◽

Forward Genetic Screens ◽

Erf Transcription Factors

AbstractArsenic stress causes rapid transcriptional responses in plants. However, transcriptional regulators of arsenic-induced gene expression in plants remain less well known. To date, forward genetic screens have proven limited for dissecting arsenic response mechanisms. We hypothesized that this may be due to the extensive genetic redundancy present in plant genomes. To overcome this limitation, we pursued a forward genetics screen for arsenite tolerance using a randomized library of plants expressing >2,000 artificial microRNAs (amiRNAs). This library was designed to knock-down diverse combinations of homologous gene family members within sub-clades of transcription factor and transporter gene families. We identified six transformant lines showing an altered response to arsenite in root growth assays. Further characterization of an amiRNA line targeting closely homologous CBF and ERF transcription factors show that the CBF1,2 and 3 transcription factors negatively regulate arsenite sensitivity. Furthermore, the ERF34 and ERF35 transcription factors are required for cadmium resistance. Generation of CRISPR lines, higher-order T-DNA mutants, and gene expression analyses, further support our findings. These ERF transcription factors differentially regulate arsenite sensitivity and cadmium tolerance.

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Gene expression profiles of RAW264.7 macrophages stimulated with preparations of LPS differing in isolation and purity

Innate Immunity ◽

10.1177/1753425910393540 ◽

2011 ◽

Vol 18 (1) ◽

pp. 80-88 ◽

Cited By ~ 14

Author(s):

Holly R Rutledge ◽

Weiwen Jiang ◽

Jun Yang ◽

Laura A Warg ◽

David A Schwartz ◽

...

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Differentially Expressed ◽

Transcriptional Networks ◽

Molecular Pathways ◽

Raw264.7 Macrophages ◽

Lps Treatment

Lipopolysaccharide is a major component of the cell wall of Gram-negative bacteria and a potent stimulator of innate immune response via TLR4. Studies on the LPS action both in vivo and in vitro have used different preparations of LPS, including ultra-pure LPS (LIST) and a less pure but less expensive form (Sigma) isolated from Escherichia coli serotype O111:B4. The difference between the effects of these compounds has not been well studied although this information is important in understanding TLR stimulation. In this study, we compared response of RAW264.7 macrophage cells treated LIST or Sigma LPS for 6 h and 24 h. Gene expression data were analyzed to identify specific genes and pathways that are in common and unique to the two LPS preparations. Seven hundred fifty-five genes were differentially expressed at 6 h in response to Sigma LPS and 973 were differentially expressed following LIST LPS treatment, with 503 in common. At 24 h, Sigma LPS induced or repressed 901 genes while 1646 genes were differentially regulated by LIST LPS treatment; 701 genes were shared by two forms of LPS. Although considerably more genes were differentially expressed in response to LIST LPS, similar molecular pathways and transcriptional networks were activated by the two LPS preparations. We also treated bone marrow-derived macrophages (BMMs) from three strains of mice with different concentrations of LIST and Sigma LPS and showed that BMMs produced more IL-6 and TNF-α in response to LIST LPS at low LPS concentrations but, at higher LPS concentrations, more cytokines were produced in response to stimulation by Sigma LPS. Together, these findings suggest that, despite activation of similar molecular pathways by LIST and Sigma LPS preparations, residual protein impurities in the Sigma LPS preparation may nevertheless influence the transcriptional profile attributed to TLR4 stimulation.

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Androgens regulate ovarian gene expression by balancing Ezh2-Jmjd3 mediated H3K27me3 dynamics

PLoS Genetics ◽

10.1371/journal.pgen.1009483 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1009483

Author(s):

Sambit Roy ◽

Binbin Huang ◽

Niharika Sinha ◽

Jianrong Wang ◽

Aritro Sen

Keyword(s):

Gene Expression ◽

Ovarian Function ◽

Critical Role ◽

Hypoxia Inducible Factor ◽

Female Fertility ◽

Epigenetic Mark ◽

Knockout Mouse Model ◽

Protein Levels ◽

Ovarian Granulosa Cell

Conventionally viewed as male hormone, androgens play a critical role in female fertility. Although androgen receptors (AR) are transcription factors, to date very few direct transcriptional targets of ARs have been identified in the ovary. Using mouse models, this study provides three critical insights about androgen-induced gene regulation in the ovary and its impact on female fertility. First, RNA-sequencing reveals a number of genes and biological processes that were previously not known to be directly regulated by androgens in the ovary. Second, androgens can also influence gene expression by decreasing the tri-methyl mark on lysine 27 of histone3 (H3K27me3), a gene silencing epigenetic mark. ChIP-seq analyses highlight that androgen-induced modulation of H3K27me3 mark within gene bodies, promoters or distal enhancers have a much broader impact on ovarian function than the direct genomic effects of androgens. Third, androgen-induced decrease of H3K27me3 is mediated through (a) inhibiting the expression and activity of Enhancer of Zeste Homologue 2 (EZH2), a histone methyltransferase that promotes tri-methylation of K27 and (b) by inducing the expression of a histone demethylase called Jumonji domain containing protein-3 (JMJD3/KDM6B), responsible for removing the H3K27me3 mark. Androgens through the PI3K/Akt pathway, in a transcription-independent fashion, increase hypoxia-inducible factor 1 alpha (HIF1α) protein levels, which in turn induce JMJD3 expression. Furthermore, proof of concept studies involving in vivo knockdown of Ar in the ovary and ovarian (granulosa) cell-specific Ar knockout mouse model show that ARs regulate the expression of key ovarian genes through modulation of H3K27me3.

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Comprehensive, high-resolution binding energy landscapes reveal context dependencies of transcription factor binding

10.1101/193904 ◽

2017 ◽

Cited By ~ 1

Author(s):

Daniel D. Le ◽

Tyler C. Shimko ◽

Arjun K. Aditham ◽

Allison M. Keys ◽

Yaron Orenstein ◽

...

Keyword(s):

Gene Expression ◽

Dna Sequences ◽

Large Fraction ◽

Binding Energies ◽

Transcriptional Networks ◽

Binding Behavior ◽

Biophysical Models ◽

Quantitative Measurements ◽

Biochemical Measurements

Transcription factors (TFs) are primary regulators of gene expression in cells, where they bind specific genomic target sites to control transcription. Quantitative measurements of TF-DNA binding energies can improve the accuracy of predictions of TF occupancy and downstream gene expression in vivo and further shed light on how transcriptional networks are rewired throughout evolution. Here, we present a novel sequencing-based TF binding assay and analysis pipeline capable of providing quantitative estimates of binding energies for more than one million DNA sequences in parallel at high energetic resolution. Using this platform, we measured the binding energies associated with all possible combinations of 10 nucleotides flanking the known consensus DNA target for two model yeast TFs, Pho4 and Cbf1. A large fraction of these flanking mutations change overall binding energies by an amount equal to or greater than consensus site mutations, suggesting that current definitions of TF binding sites may be too restrictive. By systematically comparing estimates of binding energies output by deep neural networks (NN) and biophysical models trained on these data, we establish that dinucleotide specificities are sufficient to explain essentially all variance in observed binding behavior, with Cbf1 binding exhibiting significantly more epistasis than Pho4. NN-derived binding energies agree with orthogonal biochemical measurements and reveal that dynamically occupied sites in vivo are both energetically and mutationally distant from the highest-affinity sites.

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A family of transcription factors that limit lifespan: ETS factors have conserved roles in longevity

10.1101/438879 ◽

2018 ◽

Author(s):

Adam J. Dobson ◽

Richard Boulton-McDonald ◽

Lara Houchou ◽

Ziyu Ren ◽

Mimoza Hoti ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Transcriptional Networks ◽

Regulation Of Transcription ◽

Detailed Knowledge ◽

Extended Lifespan ◽

Ets Family ◽

Ets Factors

ABSTRACTIncreasing average population age, and the accompanying burden of ill health, is one of the public health crises of our time. Understanding the basic biology of the ageing process may help ameliorate the pathologies that characterise old age. Ageing can be modulated, often through changes in gene expression where regulation of transcription plays a pivotal role. Activities of Forkhead transcription factors (TFs) are known to extend lifespan, but detailed knowledge of the broader transcriptional networks that promote longevity is lacking. This study focuses on the E twenty-six (ETS) family of TFs. This family of TFs is large, conserved across metazoa, and known to play roles in development and cancer, but the role of its members in ageing has not been studied extensively. InDrosophila, an ETS transcriptional repressor,Aop, and an ETS transcriptional activator,Pnt, are known to genetically interact withFoxoand activatingAopis sufficient to extend lifespan. Here, it is shown thatAopandFoxoeffect a related gene-expression programme. Additionally,Aopcan modulateFoxo’s transcriptional output to moderate or synergise withFoxoactivity depending on promoter context, bothin vitroandin vivo.In vivogenome-wide mRNA expression analysis in response toAop,PntorFoxoindicated, and further experiments confirmed, that combinatorial activities of the three TFs dictate metabolic status, and that direct reduction ofPntactivity is sufficient to promote longevity. The role of ETS factors in longevity was not limited toPntandAop. Knockdown ofEts21corEip74EFin distinct cell types also extended lifespan, revealing that lifespan is limited by transcription from the ETS binding site in multiple cellular contexts. Reducing the activity of theC. elegansETS TFLin-1also extended lifespan, a finding that corroborates established evidence of roles of this TF family in ageing. Altogether, these results reveal the ETS family of TFs as pervasive and evolutionarily conserved brokers of longevity.

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HIF1/2-exerted control over glycolytic gene expression is not functionally relevant for glycolysis in human leukemic stem/progenitor cells

Cancer & Metabolism ◽

10.1186/s40170-019-0206-y ◽

2019 ◽

Vol 7 (1) ◽

Cited By ~ 1

Author(s):

Albertus T. J. Wierenga ◽

Alan Cunningham ◽

Ayşegül Erdem ◽

Nuria Vilaplana Lopera ◽

Annet Z. Brouwers-Vos ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Lactate Production ◽

Glucose Consumption ◽

Transcriptional Networks ◽

Metabolic State ◽

Hematopoietic Stem ◽

Genome Wide ◽

Xenograft Mice

Abstract Background Hypoxia-inducible factors (HIF)1 and 2 are transcription factors that regulate the homeostatic response to low oxygen conditions. Since data related to the importance of HIF1 and 2 in hematopoietic stem and progenitors is conflicting, we investigated the chromatin binding profiles of HIF1 and HIF2 and linked that to transcriptional networks and the cellular metabolic state. Methods Genome-wide ChIPseq and ChIP-PCR experiments were performed to identify HIF1 and HIF2 binding sites in human acute myeloid leukemia (AML) cells and healthy CD34+ hematopoietic stem/progenitor cells. Transcriptome studies were performed to identify gene expression changes induced by hypoxia or by overexpression of oxygen-insensitive HIF1 and HIF2 mutants. Metabolism studies were performed by 1D-NMR, and glucose consumption and lactate production levels were determined by spectrophotometric enzyme assays. CRISPR-CAS9-mediated HIF1, HIF2, and ARNT−/− lines were generated to study the functional consequences upon loss of HIF signaling, in vitro and in vivo upon transplantation of knockout lines in xenograft mice. Results Genome-wide ChIP-seq and transcriptome studies revealed that overlapping HIF1- and HIF2-controlled loci were highly enriched for various processes including metabolism, particularly glucose metabolism, but also for chromatin organization, cellular response to stress and G protein-coupled receptor signaling. ChIP-qPCR validation studies confirmed that glycolysis-related genes but not genes related to the TCA cycle or glutaminolysis were controlled by both HIF1 and HIF2 in leukemic cell lines and primary AMLs, while in healthy human CD34+ cells these loci were predominantly controlled by HIF1 and not HIF2. However, and in contrast to our initial hypotheses, CRISPR/Cas9-mediated knockout of HIF signaling did not affect growth, internal metabolite concentrations, glucose consumption or lactate production under hypoxia, not even in vivo upon transplantation of knockout cells into xenograft mice. Conclusion These data indicate that, while HIFs exert control over glycolysis but not OxPHOS gene expression in human leukemic cells, this is not critically important for their metabolic state. In contrast, inhibition of BCR-ABL did impact on glucose consumption and lactate production regardless of the presence of HIFs. These data indicate that oncogene-mediated control over glycolysis can occur independently of hypoxic signaling modules.

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Single-cell analysis of human retina identifies evolutionarily conserved and species-specific mechanisms controlling development

10.1101/779694 ◽

2019 ◽

Cited By ~ 1

Author(s):

Yufeng Lu ◽

Fion Shiau ◽

Wenyang Yi ◽

Suying Lu ◽

Qian Wu ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cell Fate ◽

Single Cell Analysis ◽

Retinal Development ◽

Cell Types ◽

Retinal Cell ◽

Transcriptional Networks ◽

Species Specific

SummaryThe development of single-cell RNA-Sequencing (scRNA-Seq) has allowed high resolution analysis of cell type diversity and transcriptional networks controlling cell fate specification. To identify the transcriptional networks governing human retinal development, we performed scRNA-Seq over retinal organoid and in vivo retinal development, across 20 timepoints. Using both pseudotemporal and cross-species analyses, we examined the conservation of gene expression across retinal progenitor maturation and specification of all seven major retinal cell types. Furthermore, we examined gene expression differences between developing macula and periphery and between two distinct populations of horizontal cells. We also identify both shared and species-specific patterns of gene expression during human and mouse retinal development. Finally, we identify an unexpected role for ATOH7 expression in regulation of photoreceptor specification during late retinogenesis. These results provide a roadmap to future studies of human retinal development, and may help guide the design of cell-based therapies for treating retinal dystrophies.

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Comprehensive, high-resolution binding energy landscapes reveal context dependencies of transcription factor binding

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1715888115 ◽

2018 ◽

Vol 115 (16) ◽

pp. E3702-E3711 ◽

Cited By ~ 33

Author(s):

Daniel D. Le ◽

Tyler C. Shimko ◽

Arjun K. Aditham ◽

Allison M. Keys ◽

Scott A. Longwell ◽

...

Keyword(s):

Gene Expression ◽

Binding Energy ◽

Dna Sequences ◽

Large Fraction ◽

Binding Energies ◽

Transcriptional Networks ◽

Binding Behavior ◽

Biophysical Models ◽

Quantitative Measurements

Transcription factors (TFs) are primary regulators of gene expression in cells, where they bind specific genomic target sites to control transcription. Quantitative measurements of TF–DNA binding energies can improve the accuracy of predictions of TF occupancy and downstream gene expression in vivo and shed light on how transcriptional networks are rewired throughout evolution. Here, we present a sequencing-based TF binding assay and analysis pipeline (BET-seq, for Binding Energy Topography by sequencing) capable of providing quantitative estimates of binding energies for more than one million DNA sequences in parallel at high energetic resolution. Using this platform, we measured the binding energies associated with all possible combinations of 10 nucleotides flanking the known consensus DNA target interacting with two model yeast TFs, Pho4 and Cbf1. A large fraction of these flanking mutations change overall binding energies by an amount equal to or greater than consensus site mutations, suggesting that current definitions of TF binding sites may be too restrictive. By systematically comparing estimates of binding energies output by deep neural networks (NNs) and biophysical models trained on these data, we establish that dinucleotide (DN) specificities are sufficient to explain essentially all variance in observed binding behavior, with Cbf1 binding exhibiting significantly more nonadditivity than Pho4. NN-derived binding energies agree with orthogonal biochemical measurements and reveal that dynamically occupied sites in vivo are both energetically and mutationally distant from the highest affinity sites.

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