scholarly journals Enhancer RNAs predict enhancer–gene regulatory links and are critical for enhancer function in neuronal systems

2020 ◽  
Vol 48 (17) ◽  
pp. 9550-9570 ◽  
Author(s):  
Nancy V N Carullo ◽  
Robert A Phillips III ◽  
Rhiana C Simon ◽  
Salomon A Roman Soto ◽  
Jenna E Hinds ◽  
...  
Keyword(s):  
Target Genes ◽  
Creb Binding Protein ◽  
Disease States ◽  
Mrna Induction ◽  
Genome Wide ◽  
Neuronal Gene ◽  
Neuronal Systems ◽  
And Function ◽  
Acetyltransferase Domain ◽  
Transcriptional Output

Abstract Genomic enhancer elements regulate gene expression programs important for neuronal fate and function and are implicated in brain disease states. Enhancers undergo bidirectional transcription to generate non-coding enhancer RNAs (eRNAs). However, eRNA function remains controversial. Here, we combined Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) and RNA-Seq datasets from three distinct neuronal culture systems in two activity states, enabling genome-wide enhancer identification and prediction of putative enhancer–gene pairs based on correlation of transcriptional output. Notably, stimulus-dependent enhancer transcription preceded mRNA induction, and CRISPR-based activation of eRNA synthesis increased mRNA at paired genes, functionally validating enhancer–gene predictions. Focusing on enhancers surrounding the Fos gene, we report that targeted eRNA manipulation bidirectionally modulates Fos mRNA, and that Fos eRNAs directly interact with the histone acetyltransferase domain of the enhancer-linked transcriptional co-activator CREB-binding protein (CBP). Together, these results highlight the unique role of eRNAs in neuronal gene regulation and demonstrate that eRNAs can be used to identify putative target genes.

scholarly journals Enhancer RNAs predict enhancer-gene regulatory links and are critical for enhancer function in neuronal systems

2018 ◽  
Author(s):  
Nancy V. N. Carullo ◽  
Robert A. Phillips ◽  
Rhiana C. Simon ◽  
Salomon A. Roman Soto ◽  
Jenna E. Hinds ◽  
...  
Keyword(s):  
Target Genes ◽  
Disease States ◽  
Mrna Induction ◽  
Neuronal Fate ◽  
Genome Wide ◽  
Neuronal Gene ◽  
Neuronal Systems ◽  
And Function ◽  
Acetyltransferase Domain ◽  
Transcriptional Output

Genomic enhancer elements regulate gene expression programs important for neuronal fate and function and are implicated in brain disease states. Enhancers undergo bidirectional transcription to generate non-coding enhancer RNAs (eRNAs). However, eRNA function remains controversial. Here, we combined ATAC-Seq and RNA-Seq datasets from three distinct neuronal culture systems in two activity states, enabling genome-wide enhancer identification and prediction of putative enhancer-gene pairs based on correlation of transcriptional output. Notably, stimulus-dependent enhancer transcription preceded mRNA induction, and CRISPR- based activation of eRNA synthesis increased mRNA at paired genes, functionally validating enhancer-gene predictions. Focusing on enhancers surrounding the Fos gene, we report that targeted eRNA manipulation bidirectionally modulates Fos mRNA, and that Fos eRNAs directly interact with the histone acetyltransferase domain of the enhancer-linked transcriptional co-activator CBP. Together, these results highlight the unique role of eRNAs in neuronal gene regulation and demonstrate that eRNAs can be used to identify putative target genes.

Analysis of MicroRNA Transcriptomes Insights into the miR-148a-3p Inducer of Rumen Development in Goats

2020 ◽  
Author(s):  
Tao Zhong ◽  
Cheng Wang ◽  
Jiangtao Hu ◽  
Xiaoyong Chen ◽  
Lili Niu ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Target Genes ◽  
Genetic Regulation ◽  
Mapk Signaling ◽  
Regulation Mechanism ◽  
Ras Signaling ◽  
Genome Wide ◽  
A Genome ◽  
Differentially Expressed Mirnas ◽  
And Function

Abstract Background: Rumen is an important digestive organ of ruminant. From fetal to adult stage, the morphology, structure and function of rumen have changed significantly. But the intrinsic genetic regulation is still limited. We previously reported a genome-wide expression profile of miRNAs in prenatal goat rumens. In the present study, we rejoined analyzed the transcriptomes of rumen miRNAs during prenatal (E60 and E135) and postnatal (D30 and D150) stages.Results: A total of 66 differentially expressed miRNAs (DEMs) were identified in the rumen tissues from D30 and D150 goats. Of these, 17 DEMs were consistently highly expressed in the rumens at the preweaning stages (E60, E135 and D30), while down-regulated at D150. Noteworthy, annotation analysis revealed that the target genes regulated by the DEMs were mainly enriched in MAPK signaling pathway, Jak-STAT signaling pathway and Ras signaling pathway. Interestingly, the expression of miR-148a-3p was significantly high in the embryonic stage and down-regulated at D150. The potential binding sites between miR-148a-3p and QKI were predicted by the TargetScan and verified by the dual luciferase report assay. The co-localization of miR-148a-3p and QKI was observed not in intestinal tracts but in rumen tissues by in situ hybridization. Moreover, the expression of miR-148a-3p in the epithelium was significantly higher than that in the other layers, suggesting that miR-148a-3p involve in the development of rumen epithelial cells by targeting QKI. Subsequently, miR-148a-3p inhibitor was found to induce the proliferation of GES-1 cells.Conclusions: Taken together, these results identified the DEMs involved in the development of rumen and provided an insight into the regulation mechanism of goat rumens during development.

scholarly journals Role of co-regulators in metabolic and transcriptional actions of thyroid hormone

2016 ◽  
Vol 56 (3) ◽  
pp. R73-R97 ◽  
Author(s):  
Inna Astapova
Keyword(s):  
Thyroid Hormone ◽  
Transcriptional Repression ◽  
Target Genes ◽  
Thyroid Hormone Receptor ◽  
Retinoic Acid Receptor ◽  
Specific Response ◽  
Physiological Processes ◽  
Genome Wide ◽  
Wide Range ◽  
And Function

Thyroid hormone (TH) controls a wide range of physiological processes through TH receptor (TR) isoforms. Classically, TRs are proposed to function as tri-iodothyronine (T3)-dependent transcription factors: on positively regulated target genes, unliganded TRs mediate transcriptional repression through recruitment of co-repressor complexes, while T3binding leads to dismissal of co-repressors and recruitment of co-activators to activate transcription. Co-repressors and co-activators were proposed to play opposite roles in the regulation of negative T3target genes and hypothalamic–pituitary–thyroid axis, but exact mechanisms of the negative regulation by TH have remained elusive. Important insights into the roles of co-repressors and co-activators in different physiological processes have been obtained using animal models with disrupted co-regulator function. At the same time, recent studies interrogating genome-wide TR binding have generated compelling new data regarding effects of T3, local chromatin structure, and specific response element configuration on TR recruitment and function leading to the proposal of new models of transcriptional regulation by TRs. This review discusses data obtained in various mouse models with manipulated function of nuclear receptor co-repressor (NCoR or NCOR1) and silencing mediator of retinoic acid receptor and thyroid hormone receptor (SMRT or NCOR2), and family of steroid receptor co-activators (SRCs also known as NCOAs) in the context of TH action, as well as insights into the function of co-regulators that may emerge from the genome-wide TR recruitment analysis.

scholarly journals An improved CRISPR/dCas9 interference tool for neuronal gene suppression

2020 ◽  
Author(s):  
Corey G. Duke ◽  
Svitlana V. Bach ◽  
Jasmin S. Revanna ◽  
Faraz A. Sultan ◽  
Nicholas T. Southern ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Repression ◽  
Expression System ◽  
Genetic Material ◽  
Gene Promoters ◽  
Disease States ◽  
Transcriptional Inhibition ◽  
Health And Disease ◽  
Neuronal Systems ◽  
And Function

The expression of genetic material governs brain development, differentiation, and function, and targeted manipulation of gene expression is required to understand contributions of gene function to health and disease states. Although recent improvements in CRISPR/dCas9 interference (CRISPRi) technology have enabled targeted transcriptional repression at selected genomic sites, integrating these techniques for use in non-dividing neuronal systems remains challenging. Previously, we optimized a dual lentivirus expression system to express CRISPR-based activation machinery in post-mitotic neurons. Here we used a similar strategy to adapt an improved dCas9-KRAB-MeCP2 repression system for robust transcriptional inhibition in neurons. We find that lentiviral delivery of a dCas9-KRAB-MeCP2 construct driven by the neuron-selective promoter human synapsin 1 enabled transgene expression in primary rat neurons. Next, we demonstrate transcriptional repression using CRISPR sgRNAs targeting diverse gene promoters, and show superiority of this system in neurons compared to existing RNA interference methods for robust transcript specific manipulation at the complex Brain-derived neurotrophic factor (Bdnf) gene. Our findings advance this improved CRISPRi technology for use in neuronal systems for the first time, potentially enabling improved ability to manipulate gene expression states in the nervous system.

scholarly journals Comparison of MicroRNA Transcriptomes Reveals the Association between MiR-148a-3p Expression and Rumen Development in Goats

Animals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1951
Author(s):  
Tao Zhong ◽  
Cheng Wang ◽  
Jiangtao Hu ◽  
Xiaoyong Chen ◽  
Lili Niu ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Target Genes ◽  
Genetic Regulation ◽  
Mapk Signaling ◽  
Regulation Mechanism ◽  
Ras Signaling ◽  
Genome Wide ◽  
A Genome ◽  
Rumen Development ◽  
And Function

The rumen is an important digestive organ of ruminants. From the fetal to adult stage, the morphology, structure and function of the rumen change significantly. However, the knowledge of the intrinsic genetic regulation of these changes is still limited. We previously reported a genome-wide expression profile of miRNAs in pre-natal goat rumens. In this study, we combined and analyzed the transcriptomes of rumen miRNAs during pre-natal (E60 and E135) and post-natal (D30 and D150) stages. A total of 66 differentially expressed miRNAs (DEMs) were identified in the rumen tissues from D30 and D150 goats. Of these, 17 DEMs were consistently highly expressed in the rumens at the pre-weaning stages (E60, E135 and D30), while down-regulated at D150. Noteworthy, annotation analysis revealed that the target genes regulated by the DEMs were mainly enriched in MAPK signaling pathway, Jak-STAT signaling pathway and Ras signaling pathway. Interestingly, the expression of miR-148a-3p was significantly high in the embryonic stage and down-regulated at D150. The potential binding sites of miR-148a-3p in the 3′-UTR of QKI were predicted by the TargetScan and verified by the dual luciferase report assay. The co-localization of miR-148a-3p and QKI through in situ hybridization was observed in the rumen tissues but not in the intestinal tracts. Moreover, the expression of miR-148a-3p in the epithelium was significantly higher than that in the other layers of the rumen, suggesting that miR-148a-3p is involved in the development of the rumen epithelial cells by targeting QKI. Subsequently, miR-148a-3p inhibitor was found to induce the proliferation of GES-1 cells. Taken together, our study identified DEMs involved in the development of the rumen and provides insights into the regulation mechanism of rumen development in goats.

scholarly journals Genome-Wide Screening Reveals CSDE1 as a Novel Regulator of the LDL Receptor

2020 ◽  
Author(s):  
Geoffrey A. Smith ◽  
Akhil Pampana ◽  
Pradeep Natarajan ◽  
Kevan M. Shokat ◽  
John S. Chorba
Keyword(s):  
Translational Control ◽  
Rna Binding ◽  
Human Genetics ◽  
Ldl Receptor ◽  
Experimental System ◽  
Disease States ◽  
Genome Wide ◽  
Bona Fide ◽  
And Function

AbstractIn humans, clearance of LDL cholesterol, which causes atherosclerotic heart disease, is mediated by the hepatic LDL receptor (LDLR)1. As a result, therapies that upregulate the LDLR are highly effective treatments for atherosclerosis2. Since cardiovascular disease remains the leading cause of death in Western countries3, we sought to identify regulators of the LDLR beyond the known genetic causes of familial hypercholesterolemia. Here we show that CSDE1, an RNA-binding protein involved in mRNA stability4, enhances LDLR mRNA degradation to modulate LDLR expression and function. Using parallel phenotypic genome-wide screens, based on the CRISPR interference platform5, we identified over 100 specific regulators of surface LDLR expression in HepG2 cells, characterized their effects on LDLR function, and leveraged pharmacologic strategies to probe their mechanistic pathways. Among our hits, we found that CSDE1 participates in post-translational control of the LDLR independent from well-established, and clinically exploited, transcriptional and lysosomal regulatory mechanisms. Overall, our results reveal a network of novel LDLR modulators left undiscovered by human genetics, many of which have phenotypic strengths similar to bona fide targets in the clinic, offering hope for new therapeutic strategies against atherosclerosis. We anticipate that our approach of modelling a clinically relevant phenotype in an in vitro experimental system amenable to a forward genetic screen, followed by high throughput validation and mechanistic pharmacologic dissection, will serve as a template for the identification of novel therapeutic targets for other disease states.Graphical Abstract

Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3164-3173 ◽  
Author(s):  
Masao Mizuki ◽  
Joachim Schwäble ◽  
Claudia Steur ◽  
Chunaram Choudhary ◽  
Shuchi Agrawal ◽  
...  
Keyword(s):  
Target Genes ◽  
Wild Type ◽  
Transcriptional Program ◽  
Serine Threonine Kinase ◽  
Myeloid Progenitor ◽  
Genome Wide ◽  
Flt3 Mutations ◽  
Myeloid Progenitor Cells ◽  
And Function ◽  
Microarray Expression

Abstract The receptor tyrosine kinase Flt3 is expressed and functionally important in early myeloid progenitor cells and in the majority of acute myeloid leukemia (AML) blasts. Internal tandem duplications (ITDs) in the juxtamembrane domain of the receptor occur in 25% of AML cases. Previously, we have shown that these mutations activate the receptor and induce leukemic transformation. In this study, we performed genome-wide parallel expression analyses of 32Dcl3 cells stably transfected with either wild-type or 3 different ITD isoforms of Flt3. Comparison of microarray expression analyses revealed that 767 of 6586 genes differed in expression between FLT3-WT– and FLT3-ITD–expressing cell lines. The target genes of mutationally activated Flt3 resembled more closely those of the interleukin 3 (IL-3) receptor than those of ligand-activated Flt3. The serine-threonine kinase Pim-2 was up-regulated on the mRNA and the protein level in Flt3-ITD–expressing cells. Further experiments indicated that Pim-2 function was important for clonal growth of 32D cells. Several genes repressed by the mutations were found to be involved in myeloid gene regulation. Pu.1 and C/EBPα, both induced by ligand-activation of wild-type Flt3, were suppressed in their expression and function by the Flt3 mutations. In conclusion, internal tandem duplication mutations of Flt3 activate transcriptional programs that partially mimic IL-3 activity. Interestingly, other parts of the transcriptional program involve novel, IL-3–independent pathways that antagonize differentiation-inducing effects of wild-type Flt3. The identification of the transcriptional program induced by ITD mutations should ease the development of specific therapies.

scholarly journals Co-regulation of transcription by BRG1 and BRM, two mutually exclusive SWI/SNF ATPase subunits

2017 ◽  
Author(s):  
Jesse R. Raab ◽  
John S. Runge ◽  
Camarie C. Spear ◽  
Terry Magnuson
Keyword(s):  
Gene Expression ◽  
Tumor Formation ◽  
Regulation Of Transcription ◽  
Functional Relationships ◽  
Genome Wide ◽  
Atpase Subunits ◽  
Series Of Experiments ◽  
And Function ◽  
Transcriptional Output

AbstractBackgroundSWI/SNF is a large heterogenous multi-subunit chromatin remodeling complex. It consists of multiple sets of mutually exclusive components. Understanding how loss of one sibling of a mutually exclusive pair affects the occupancy and function of the remaining complex is needed to understand how mutations in a particular subunit might affect tumor formation. Recently, we showed that the members of the ARID family of SWI/SNF subunits (ARID1A, ARID1B, and ARID2) had complex transcriptional relationships including both antagonism and cooperativity. However, it remains unknown how loss of the catalytic subunit(s) affects the binding and genome-wide occupancy of the remainder complex and how changes in occupancy affect transcriptional output.ResultsWe addressed this gap by depleting BRG1 and BRM, the two ATPase subunits in SWI/SNF, and characterizing the changes to chromatin occupancy of the remaining subunit and related this to transcription changes induced by loss of the ATPase subunits. We show that depletion of one subunit frequently leads to loss of the remaining subunit. This could cause either positive or negative changes in gene expression. At a subset of sites the sibling subunit is either retained or gained. Additionally, we show genome-wide that BRG1 and BRM have both cooperative and antagonistic interactions with respect to transcription. Importantly, at genes where BRG1 and BRM antagonise one another we observe a nearly complete rescue of gene expression changes in the combined BRG/BRM double knockdown.ConclusionThis series of experiments demonstrate that mutually exclusive SWI/SNF complexes have heterogenous functional relationships and highlights the importance of considering the role of the remaining SWI/SNF complexes following loss or depletion of a single subunit.

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