scholarly journals The Firre locus produces a trans-acting RNA molecule that functions in hematopoiesis

2019 ◽  
Author(s):  
Jordan P. Lewandowski ◽  
James C. Lee ◽  
Taeyoung Hwang ◽  
Hongjae Sunwoo ◽  
Jill M. Goldstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
X Chromosome ◽  
Sex Chromosome ◽  
Noncoding Rnas ◽  
Regulatory Elements ◽  
Genetic Evidence ◽  
Loss Of Function ◽  
Lncrna Transcript ◽  
Dna And Rna

ABSTRACTRNA has been classically known to play central roles in biology, including maintaining telomeres1, protein synthesis2, and in sex chromosome compensation in certain species3,4. At the center of these important biological systems are noncoding RNAs. While thousands of long noncoding RNAs (lncRNAs) have been identified in mammalian genomes5–8, attributing RNA-based roles to lncRNA loci requires an assessment of whether the observed effect could be due to DNA regulatory elements, the act of transcription, or the lncRNA transcript. Here, we use the syntenically conserved lncRNA locus, Functional intergenic repeating RNA element (Firre), that is located on the X chromosome as a model to discriminate between DNA- and RNA-mediated effects in vivo. To this end, we generated genetically defined loss-of-function, gain-of-function, and rescue mouse models for Firre and provide genetic evidence that the Firre locus produces a trans-acting RNA. We report that: (i) Firre mutant mice have cell-specific defects during hematopoiesis and changes in gene expression that can be rescued by induction of Firre RNA from a transgene in the Firre knockout background, (ii) mice overexpressing Firre from a transgene exhibit increased levels of pro-inflammatory cytokines and impaired survival upon exposure to lipopolysaccharide, and (iii) deletion of the Firre locus did not result in changes in local gene expression on the X chromosome in 9 different biological contexts, suggesting that Firre does not function by cis-acting RNA or DNA elements. Together, our results provide genetic evidence that the Firre locus produces a trans-acting lncRNA that has physiological roles in hematopoiesis and immune function.

scholarly journals The Firre locus produces a trans-acting RNA molecule that functions in hematopoiesis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jordan P. Lewandowski ◽  
James C. Lee ◽  
Taeyoung Hwang ◽  
Hongjae Sunwoo ◽  
Jill M. Goldstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sex Chromosome ◽  
Noncoding Rnas ◽  
Regulatory Elements ◽  
Mediated Effects ◽  
Dna And Rna ◽  
Lncrna Locus ◽  
Dna Regulatory Elements ◽  
Pro Inflammatory Cytokines

Abstract RNA has been classically known to play central roles in biology, including maintaining telomeres, protein synthesis, and in sex chromosome compensation. While thousands of long noncoding RNAs (lncRNAs) have been identified, attributing RNA-based roles to lncRNA loci requires assessing whether phenotype(s) could be due to DNA regulatory elements, transcription, or the lncRNA. Here, we use the conserved X chromosome lncRNA locus Firre, as a model to discriminate between DNA- and RNA-mediated effects in vivo. We demonstrate that (i) Firre mutant mice have cell-specific hematopoietic phenotypes, and (ii) upon exposure to lipopolysaccharide, mice overexpressing Firre exhibit increased levels of pro-inflammatory cytokines and impaired survival. (iii) Deletion of Firre does not result in changes in local gene expression, but rather in changes on autosomes that can be rescued by expression of transgenic Firre RNA. Together, our results provide genetic evidence that the Firre locus produces a trans-acting lncRNA that has physiological roles in hematopoiesis.

scholarly journals Generation and validation of versatile inducible CRISPRi embryonic stem cell and mouse model

PLoS Biology ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. e3000749
Author(s):  
Rui Li ◽  
Xianyou Xia ◽  
Xing Wang ◽  
Xiaoyu Sun ◽  
Zhongye Dai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Model ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Loss Of Function ◽  
Transgenic Expression ◽  
Guide Rnas ◽  
High Throughput Screens

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) 9 has been widely used far beyond genome editing. Fusions of deactivated Cas9 (dCas9) to transcription effectors enable interrogation of the epigenome and controlling of gene expression. However, the large transgene size of dCas9-fusion hinders its applications especially in somatic tissues. Here, we develop a robust CRISPR interference (CRISPRi) system by transgenic expression of doxycycline (Dox) inducible dCas9-KRAB in mouse embryonic stem cells (iKRAB ESC). After introduction of specific single-guide RNAs (sgRNAs), the induced dCas9-KRAB efficiently maintains gene inactivation, although it modestly down-regulates the expression of active genes. The proper timing of Dox addition during cell differentiation or reprogramming allows us to study or screen spatiotemporally activated promoters or enhancers and thereby the gene functions. Furthermore, taking the ESC for blastocyst injection, we generate an iKRAB knock-in (KI) mouse model that enables the shutdown of gene expression and loss-of-function (LOF) studies ex vivo and in vivo by a simple transduction of gRNAs. Thus, our inducible CRISPRi ESC line and KI mouse provide versatile and convenient platforms for functional interrogation and high-throughput screens of specific genes and potential regulatory elements in the setting of development or diseases.

scholarly journals Generation and Validation of Versatile Inducible CRISPRi Embryonic Stem Cell and Mouse Models

2020 ◽  
Author(s):  
Rui Li ◽  
Xianyou Xia ◽  
Xing Wang ◽  
Xiaoyu Sun ◽  
Zhongye Dai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Loss Of Function ◽  
Transgenic Expression ◽  
Somatic Tissues ◽  
Gene Functions ◽  
High Throughput Screens

ABSTRACTCRISPR-Cas9 has been widely used far beyond genome editing. Fusions of deactivated Cas9 (dCas9) to transcription effectors enable interrogation of the epigenome and controlling of gene expression. However the large transgene size of dCas9-fusion hinders its applications especially in somatic tissues. Here, we develop a robust CRISPR interference (CRISPRi) system by transgenic expression of doxycycline (Dox) inducible dCas9-KRAB in mouse embryonic stem cells (iKRAB ESC). After introduction of specific gRNAs, the induced dCas9-KRAB efficiently maintains gene inactivation, though it exerts modest effects on active gene expression. Proper timing of Dox addition during cell differentiation or reprogramming allows us to study or screen spatiotemporally activated promoters or enhancers and thereby the gene functions. Furthermore, taking the ESC for blastocyst injection, we generate an iKRAB knockin (KI) mouse model that enables shut-down of gene expression and loss-of-function studies ex vivo and in vivo by a simple transduction of gRNAs. Thus, our inducible CRISPRi ESC line and KI mouse provide versatile and convenient platforms for functional interrogation and high-throughput screens of specific genes and potential regulatory elements in the setting of development or diseases.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

scholarly journals Synthetic gene regulatory networks in the opportunistic human pathogen Streptococcus pneumoniae

2019 ◽  
Author(s):  
Robin A. Sorg ◽  
Clement Gallay ◽  
Jan-Willem Veening
Keyword(s):  
Gene Expression ◽  
Streptococcus Pneumoniae ◽  
Regulatory Networks ◽  
Expression Patterns ◽  
Combinatorial Libraries ◽  
Regulatory Elements ◽  
Expression Control ◽  
Gene Expression Control

AbstractStreptococcus pneumoniae can cause disease in various human tissues and organs, including the ear, the brain, the blood and the lung, and thus in highly diverse and dynamic environments. It is challenging to study how pneumococci control virulence factor expression, because cues of natural environments and the presence of an immune system are difficult to simulate in vitro. Here, we apply synthetic biology methods to reverse-engineer gene expression control in S. pneumoniae. A selection platform is described that allows for straightforward identification of transcriptional regulatory elements out of combinatorial libraries. We present TetR- and LacI-regulated promoters that show expression ranges of four orders of magnitude. Based on these promoters, regulatory networks of higher complexity are assembled, such as logic AND and IMPLY gates. Finally, we demonstrate single-copy genome-integrated toggle switches that give rise to bimodal population distributions. The tools described here can be used to mimic complex expression patterns, such as the ones found for pneumococcal virulence factors, paving the way for in vivo investigations of the importance of gene expression control on the pathogenicity of S. pneumoniae.

Genetic evidence for the transcriptional-activating function of Homothorax during adult fly development

Development ◽  
2001 ◽  
Vol 128 (18) ◽  
pp. 3405-3413 ◽  
Author(s):  
Adi Inbal ◽  
Naomi Halachmi ◽  
Charna Dibner ◽  
Dale Frank ◽  
Adi Salzberg
Keyword(s):  
Transcriptional Activity ◽  
Target Genes ◽  
Genetic Evidence ◽  
In Vivo Studies ◽  
Loss Of Function ◽  
Native Form ◽  
Downstream Target ◽  
Activating Function

Homothorax (HTH) is a homeobox-containing protein, which plays multiple roles in the development of the embryo and the adult fly. HTH binds to the homeotic cofactor Extradenticle (EXD) and translocates it to the nucleus. Its function within the nucleus is less clear. It was shown, mainly by in vitro studies, that HTH can bind DNA as a part of ternary HTH/EXD/HOX complexes, but little is known about the transcription regulating function of HTH-containing complexes in the context of the developing fly. Here we present genetic evidence, from in vivo studies, for the transcriptional-activating function of HTH. The HTH protein was forced to act as a transcriptional repressor by fusing it to the Engrailed (EN) repression domain, or as a transcriptional activator, by fusing it to the VP16 activation domain, without perturbing its ability to translocate EXD to the nucleus. Expression of the repressing form of HTH in otherwise wild-type imaginal discs phenocopied hth loss of function. Thus, the repressing form was working as an antimorph, suggesting that normally HTH is required to activate the transcription of downstream target genes. This conclusion was further supported by the observation that the activating form of HTH caused typical hth gain-of-function phenotypes and could rescue hth loss-of-function phenotypes. Similar results were obtained with XMeis3, the Xenopus homologue of HTH, extending the known functional similarity between the two proteins. Competition experiments demonstrated that the repressing forms of HTH or XMeis3 worked as true antimorphs competing with the transcriptional activity of the native form of HTH. We also describe the phenotypic consequences of HTH antimorph activity in derivatives of the wing, labial and genital discs. Some of the described phenotypes, for example, a proboscis-to-leg transformation, were not previously associated with alterations in HTH activity. Observing the ability of HTH antimorphs to interfere with different developmental pathways may direct us to new targets of HTH. The HTH antimorph described in this work presents a new means by which the transcriptional activity of the endogenous HTH protein can be blocked in an inducible fashion in any desired cells or tissues without interfering with nuclear localization of EXD.

Abstract MP115: Transcriptional Patterning of the Ventricular Cardiac Conduction System

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Ozanna Burnicka-Turek ◽  
Michael Broman ◽  
Jeffrey D Steimle ◽  
Bastiaan Boukens ◽  
Nataliya B Petrenko ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ventricular Arrhythmias ◽  
Regulatory Elements ◽  
Conduction System ◽  
Mouse Genetics ◽  
Cardiac Conduction ◽  
Cardiac Conduction System ◽  
Gene Expression Network ◽  
T Box

The heart beats 2 billion times over the average human lifespan and is the manifestation of a pattern of cardiomyocyte depolarization organized by a specialized network of cardiomyocytes, the cardiac conduction system (CCS). Patterning of the CCS into atrial node versus ventricular conduction system (VCS) components with distinct physiology is essential for the normal heartbeat and has been recognized for more than a century. However molecular basis of this regional patterning is not well understood. Using mouse genetics, we found that the ratio between T-box transcriptional activator, Tbx5 , and T-box transcriptional repressor, Tbx3 , determines the molecular and functional output of VCS myocytes. Adult VCS-specific removal of Tbx5 or overexpression of Tbx3 re-patterned the fast VCS into slow, nodal-like cells based on molecular, cellular and functional criteria. Specifically, the ventricular fast conduction gene expression network was lost whereas the slow conduction nodal gene expression network was retained. Action potentials (APs) of Tbx5 -deficient VCS myocytes adopted nodal-specific characteristics, including increased AP duration and cellular automaticity. Removal of Tbx5 in-vivo precipitated inappropriate depolarizations in the His-bundle that initiated lethal ventricular arrhythmias. A T-box rheostat mechanism for CCS patterning was confirmed by Tbx5/Tbx3 genetic interaction studies. TBX5 bound and directly activated cis -regulatory elements at fast conduction loci genome-wide, defining the identity of the adult VCS. Furthermore, TBX5 bound and activated cis-regulatory elements at Tbx5 itself, describing a multi-tiered T-box-dependent fast conduction gene regulatory network (GRN). The hierarchical GRN established a bi-stable network in mathematical modeling, with only high or low fast conduction gene expression states, suggesting a genomic mechanism for the establishment of VCS versus nodal states in-vivo . Thus, the CCS is patterned entirely as a slow, nodal ground state, with a T-box dependent, physiologically dominant, fast conduction network driven specifically in the VCS. Disruption of the fast VCS GRN allowed nodal physiology to emerge, providing a molecular mechanism for some lethal ventricular arrhythmias.

scholarly journals The New Frontier of Functional Genomics: From Chromatin Architecture and Noncoding RNAs to Therapeutic Targets

2020 ◽  
Vol 25 (6) ◽  
pp. 568-580
Author(s):  
Natali Papanicolaou ◽  
Alessandro Bonetti
Keyword(s):  
Gene Expression ◽  
Association Studies ◽  
Noncoding Rnas ◽  
Regulatory Elements ◽  
Long Noncoding Rnas ◽  
Human Diseases ◽  
Genome Wide Association Studies ◽  
Complex Disorders ◽  
Chromatin Architecture ◽  
Common Diseases

Common diseases are complex, multifactorial disorders whose pathogenesis is influenced by the interplay of genetic predisposition and environmental factors. Genome-wide association studies have interrogated genetic polymorphisms across genomes of individuals to test associations between genotype and susceptibility to specific disorders, providing insights into the genetic architecture of several complex disorders. However, genetic variants associated with the susceptibility to common diseases are often located in noncoding regions of the genome, such as tissue-specific enhancers or long noncoding RNAs, suggesting that regulatory elements might play a relevant role in human diseases. Enhancers are cis-regulatory genomic sequences that act in concert with promoters to regulate gene expression in a precise spatiotemporal manner. They can be located at a considerable distance from their cognate target promoters, increasing the difficulty of their identification. Genomes are organized in domains of chromatin folding, namely topologically associating domains (TADs). Identification of enhancer–promoter interactions within TADs has revealed principles of cell-type specificity across several organisms and tissues. The vast majority of mammalian genomes are pervasively transcribed, accounting for a previously unappreciated complexity of the noncoding RNA fraction. Particularly, long noncoding RNAs have emerged as key players for the establishment of chromatin architecture and regulation of gene expression. In this perspective, we describe the new advances in the fields of transcriptomics and genome organization, focusing on the role of noncoding genomic variants in the predisposition of common diseases. Finally, we propose a new framework for the identification of the next generation of pharmacological targets for common human diseases.

scholarly journals Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression.

1996 ◽  
Vol 16 (7) ◽  
pp. 3245-3254 ◽  
Author(s):  
V Ngô ◽  
D Gourdji ◽  
J N Laverrière
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Cell Lines ◽  
Anterior Pituitary ◽  
Regulatory Elements ◽  
Specific Expression ◽  
Site Specific ◽  
Cpg Dinucleotides ◽  
Cpg Sites

The methylation patterns of the rat prolactin (rPRL) (positions -440 to -20) and growth hormone (rGH) (positions -360 to -110) promoters were analyzed by bisulfite genomic sequencing. Two normal tissues, the anterior pituitary and the liver, and three rat pituitary GH3 cell lines that differ considerably in their abilities to express both genes were tested. High levels of rPRL gene expression were correlated with hypomethylation of the CpG dinucleotides located at positions -277 and -97, near or within positive cis-acting regulatory elements. For the nine CpG sites analyzed in the rGH promoter, an overall hypomethylation-expression coupling was also observed for the anterior pituitary, the liver, and two of the cell lines. The effect of DNA methylation was tested by measuring the transient expression of the chloramphenicol acetyltransferase reporter gene driven by a regionally methylated rPRL promoter. CpG methylation resulted in a decrease in the activity of the rPRL promoter which was proportional to the number of modified CpG sites. The extent of the inhibition was also found to be dependent on the position of methylated sites. Taken together, these data suggest that site-specific methylation may modulate the action of transcription factors that dictate the tissue-specific expression of the rPRL and rGH genes in vivo.

scholarly journals Hepatic gene expression patterns in thyroid hormone-treated hypothyroid rats

2003 ◽  
Vol 31 (2) ◽  
pp. 291-303 ◽  
Author(s):  
JM Weitzel ◽  
S Hamann ◽  
M Jauk ◽  
M Lacey ◽  
A Filbry ◽  
...  
Keyword(s):  
Gene Expression ◽  
Thyroid Hormone ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Activation Mechanism ◽  
Gene Expression Patterns ◽  
Peroxisome Proliferator ◽  
Gene Promoters ◽  
Protein Levels

Thyroid hormone (T3) is essential for normal development, differentiation and metabolic balance. We have performed DNA microarray experiments using hepatic RNA from hypothyroid and T3-treated hypothyroid rats in order to characterize T3-induced gene expression patterns after various time points (6, 24 and 48 h after the administration of the hormone). Sixty-two of 4608 different genes displayed a reproducible T3-response, and cluster analysis divided these differentially regulated genes into six expression patterns. Thirty-six genes were not significantly regulated within the first 24 h. Transient transfection experiments of eight late-induced gene promoters failed to detect a thyroid hormone response element within their regulatory elements, suggesting an indirect activation mechanism(s). In search for an intermediate factor of T3 action, we examined whether various rather ubiquitous transcription factors, peroxisome proliferator-activated receptors (PPARs) and coactivators of the PPARgamma coactivator 1 family (PGC-1) are regulated by T3. Only PPARgamma and PERC/PGC-1beta exhibit a significant T3-response within the first 6 h after treatment, identifying these factors as candidate components for mediating the late-induced expression pattern. Regulation of early-induced genes within the first 6 h after administration of T3 on transcript levels correlates with altered protein levels after 24 and 48 h in vivo.

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