Long noncoding RNAs transcribed downstream of the human β-globin locus regulate β-globin gene expression

2021 ◽  
Author(s):  
Miki Higashi ◽  
Tsuyoshi Ikehara ◽  
Takeya Nakagawa ◽  
Mitsuhiro Yoneda ◽  
Naoko Hattori ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Locus ◽  
Globin Gene ◽  
Noncoding Rnas ◽  
K562 Cells ◽  
In Vitro Transcription ◽  
Long Noncoding Rnas ◽  
Transcription Assay ◽  
Nuclear Rna

Abstract The five β-like globin genes (ε, Gγ, Aγ, δ, and β) at the human β-globin gene locus are known to be expressed at specific developmental stages, although details of the underlying mechanism remain to be uncovered. Here we used an in vitro transcription assay to clarify the mechanisms that control this gene expression. We first tested nuclear RNA from HeLa cells using RT-qPCR and discovered a long noncoding RNAs (lncRNAs) within a 5.2-kb region beginning 4.4 kb downstream of the β-globin gene coding region. We investigated nuclear RNA from K562 cells using a primer-extension assay and determined the transcription start sites (TSSs) of these lncRNAs. To clarify their functional role, we performed knockdown (KD) of these lncRNAs in K562 cells. Hydroxyurea, which induces differentiation of K562 cells, increased hemoglobin peptide production, and the effect was enhanced by KD of these lncRNAs, which also enhanced upregulation of the γ-globin expression induced by hydroxyurea. To confirm these results, we performed an in vitro transcription assay. Noncoding single-stranded RNAs inhibited β-globin expression, which was upregulated by GATA1. Furthermore, lncRNAs interacted with GATA1 without sequence specificity and inhibited its binding to its target DNA response element in vitro. Our results suggest that lncRNAs downstream of the β-globin gene locus are key factors regulating globin gene ex pression.

scholarly journals In vitro transcription from the human A gamma-globin gene promoter

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1344-1350 ◽  
Author(s):  
M Castle ◽  
D O'Neill ◽  
A Bank
Keyword(s):  
Gene Transcription ◽  
Globin Gene ◽  
Gene Promoter ◽  
K562 Cells ◽  
In Vitro Transcription ◽  
Systematic Analysis ◽  
Gamma Globin ◽  
Octamer Motif

Abstract We report enhanced transcription from the human A gamma-globin gene promoter in nuclear extracts (NE) of erythroleukemia (K562) cells compared with that in HeLa NE. We do not observe differences in transcription levels in the two extracts with nonglobin promoter templates. Our findings, indicating preferential recognition of the globin gene promoter by nuclear factors in K562 cells, are consistent with results of studies previously reported by ourselves and others. A novel finding described here is that the addition of a double-stranded octamer motif oligonucleotide to K562 NE increases the level of transcription from the A gamma-globin gene promoter, suggesting a potential role for an octamer motif-binding factor in the repression of A gamma-globin gene transcription. A cosmid construct containing extensive human gamma- and beta-globin gene promoter and structural sequences as well as upstream control sequences also exhibits higher levels of globin gene transcription in K562 NE than in HeLa NE. Our demonstration of the feasibility of efficient, globin promoter-specific in vitro transcription of this complex template offers a novel approach for the systematic analysis of the effects of putative regulatory factors on globin gene expression in vitro in the context of a genetic environment approximating that found in vivo.

scholarly journals In vitro transcription from the human A gamma-globin gene promoter

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1344-1350 ◽  
Author(s):  
M Castle ◽  
D O'Neill ◽  
A Bank
Keyword(s):  
Gene Transcription ◽  
Globin Gene ◽  
Gene Promoter ◽  
K562 Cells ◽  
In Vitro Transcription ◽  
Systematic Analysis ◽  
Gamma Globin ◽  
Octamer Motif

We report enhanced transcription from the human A gamma-globin gene promoter in nuclear extracts (NE) of erythroleukemia (K562) cells compared with that in HeLa NE. We do not observe differences in transcription levels in the two extracts with nonglobin promoter templates. Our findings, indicating preferential recognition of the globin gene promoter by nuclear factors in K562 cells, are consistent with results of studies previously reported by ourselves and others. A novel finding described here is that the addition of a double-stranded octamer motif oligonucleotide to K562 NE increases the level of transcription from the A gamma-globin gene promoter, suggesting a potential role for an octamer motif-binding factor in the repression of A gamma-globin gene transcription. A cosmid construct containing extensive human gamma- and beta-globin gene promoter and structural sequences as well as upstream control sequences also exhibits higher levels of globin gene transcription in K562 NE than in HeLa NE. Our demonstration of the feasibility of efficient, globin promoter-specific in vitro transcription of this complex template offers a novel approach for the systematic analysis of the effects of putative regulatory factors on globin gene expression in vitro in the context of a genetic environment approximating that found in vivo.

scholarly journals Publisher Correction: Exogenously overexpressed intronic long noncoding RNAs activate host gene expression by affecting histone modification in Arabidopsis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Zhang-Wei Liu ◽  
Nan Zhao ◽  
Yin-Na Su ◽  
Shan-Shan Chen ◽  
Xin-Jian He
Keyword(s):  
Gene Expression ◽  
Histone Modification ◽  
Noncoding Rnas ◽  
Long Noncoding Rnas ◽  
Host Gene ◽  
Host Gene Expression

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Long Noncoding RNAs in Plants

2021 ◽  
Vol 72 (1) ◽  
Author(s):  
Andrzej T. Wierzbicki ◽  
Todd Blevins ◽  
Szymon Swiezewski
Keyword(s):  
Gene Expression ◽  
Nuclear Dna ◽  
Noncoding Rnas ◽  
Chromatin Accessibility ◽  
Long Noncoding Rnas ◽  
Annual Review ◽  
Publication Date ◽  
Protein Coding ◽  
Ribonucleic Acids ◽  
Coding Potential

Plants have an extraordinary diversity of transcription machineries, including five nuclear DNA-dependent RNA polymerases. Four of these enzymes are dedicated to the production of long noncoding RNAs (lncRNAs), which are ribonucleic acids with functions independent of their protein-coding potential. lncRNAs display a broad range of lengths and structures, but they are distinct from the small RNA guides of RNA interference (RNAi) pathways. lncRNAs frequently serve as structural, catalytic, or regulatory molecules for gene expression. They can affect all elements of genes, including promoters, untranslated regions, exons, introns, and terminators, controlling gene expression at various levels, including modifying chromatin accessibility, transcription, splicing, and translation. Certain lncRNAs protect genome integrity, while others respond to environmental cues like temperature, drought, nutrients, and pathogens. In this review, we explain the challenge of defining lncRNAs, introduce the machineries responsible for their production, and organize this knowledge by viewing the functions of lncRNAs throughout the structure of a typical plant gene. Expected final online publication date for the Annual Review of Plant Biology, Volume 72 is May 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Ligand-dependent, Pit-1/growth hormone factor-1 (GHF-1)-independent transcriptional stimulation of rat growth hormone gene expression by thyroid hormone receptors in vitro

1993 ◽  
Vol 13 (3) ◽  
pp. 1719-1727
Author(s):  
C S Suen ◽  
W W Chin
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Thyroid Hormone ◽  
Promoter Activity ◽  
Hormone Receptors ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Stimulation Of ◽  
Transcriptional Stimulation

The expression of the rat growth hormone (rGH) gene in the anterior pituitary gland is modulated by Pit-1/GHF-1, a pituitary-specific transcription factor, and by other more widely distributed factors, such as the thyroid hormone receptors (TRs), Sp1, and the glucocorticoid receptor. Thyroid hormone (T3)-mediated transcriptional stimulation of rGH gene expression has been extensively studied in vivo and in vitro including the measurements of (i) rGH mRNA by blot hybridization, (ii) transcriptional rate of rGH gene by nuclear run-on, and (iii) reporter gene expression in which a chimeric plasmid containing 5'-flanking sequences of the rGH gene linked to a reporter gene has been transfected either stably or transiently into pituitary and/or nonpituitary cells. From these studies, it has been suggested that the Pit-1/GHF-1 binding site is necessary for full T3 action. We developed a cell-free in vitro transcription system to examine further the roles of the TRs and Pit-1/GHF-1 in rGH gene activation. Using GH3 nuclear extract as a source of TRs and Pit-1/GHF-1, this in vitro transcription assay showed that T3 stimulation of rGH promoter activity is dependent on the addition of T3 to the GH3 nuclear extract. This transcriptional stimulation was augmented with increasing concentrations of ligand and was T3, but not T4 or reverse T3, specific. T3-mediated stimulation of rGH promoter activity was completely abolished by preincubation of the nuclear extract with rGH-thyroid hormone response element (-200 to -160) but not with Pit-1/GHF-1 (-137 to -65) oligonucleotides. Further, neither deletion of both Pit-1/GHF-1 binding sites nor mutation of the proximal Pit-1/GHF-1 binding site from the rGH promoter abrogated the T3 effect. These results provide evidence that T3-stimulated rGH promoter activity is independent of Pit-1/GHF-1 and raise the possibility that the stimulation of rGH gene expression by T3 might involve direct interaction of TRs with the general transcriptional apparatus.

scholarly journals Regulation of protamine gene expression in an in vitro homologous system.

1996 ◽  
Vol 43 (2) ◽  
pp. 369-377 ◽  
Author(s):  
J M Jankowski ◽  
P D Cannon ◽  
F Van der Hoorn ◽  
L D Wasilewska ◽  
N C Wong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulatory Elements ◽  
In Vitro Transcription ◽  
Tata Box ◽  
Transcription System ◽  
Proposed Model ◽  
Binding Factor ◽  
Gc Box ◽  
Responsive Element

An in vitro transcription system from the trout testis nuclei was developed to study trout protamine gene expression. The protamine promoter contains, among others, two regulatory elements: 1) a cAMP-responsive element or CRE element (TGACGTCA) which is present in position 5' to TATA box, and 2) GC box (CCGCCC) which is present in position 3' to TATA box. The removal of the CRE-binding protein by titration (by the addition of appropriate oligonucleotides to the incubation mixture) resulted in a decrease in transcription of the protamine gene. These results were confirmed by experiments in which the pure CRE-binding factor (TPBP1) was used, as well as by those where a stimulatory effect of cAMP on protamine promoter transcription was observed. On the other hand, addition of oligonucleotides containing the GC-box sequence enhanced the protamine gene transcription indicating that the protein (Sp1 like) which binds to this sequence acts as a repressor of protamine gene expression. These results confirm the previously proposed model which suggested that the GC box played a role in negative regulation of the protamine gene expression. Involvement of some other factors in this process was also discussed.

scholarly journals The GAGA factor is required in the early Drosophila embryo not only for transcriptional regulation but also for nuclear division

Development ◽  
1996 ◽  
Vol 122 (4) ◽  
pp. 1113-1124 ◽  
Author(s):  
K.M. Bhat ◽  
G. Farkas ◽  
F. Karch ◽  
H. Gyurkovics ◽  
J. Gausz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chromatin Remodeling ◽  
In Vitro Transcription ◽  
Drosophila Embryo ◽  
Gaga Factor ◽  
Early Drosophila Embryo ◽  
Hypersensitive Sites ◽  
And Function ◽  
Stimulatory Factor

The GAGA protein of Drosophila was first identified as a stimulatory factor in in vitro transcription assays using the engrailed and Ultrabithorax promoters. Subsequent studies have suggested that the GAGA factor promotes transcription by blocking the repressive effects of histones; moreover, it has been shown to function in chromatin remodeling, acting together with other factors in the formation of nuclease hypersensitive sites in vitro. The GAGA factor is encoded by the Trithorax-like locus and in the studies reported here we have used the maternal effect allele Trl13C to examine the functions of the protein during embryogenesis. We find that GAGA is required for the proper expression of a variety of developmental loci that contain GAGA binding sites in their upstream regulatory regions. The observed disruptions in gene expression are consistent with those expected for a factor involved in chromatin remodeling. In addition to facilitating gene expression, the GAGA factor appears to have a more global role in chromosome structure and function. This is suggested by the spectrum of nuclear cleavage cycle defects observed in Trl13C embryos. These defects include asynchrony in the cleavage cycles, failure in chromosome condensation, abnormal chromosome segregation and chromosome fragmentation. These defects are likely to be related to the association of the GAGA protein with heterochromatic satellite sequences which is observed throughout the cell cycle.

scholarly journals Identification of Novel Long Noncoding RNAs and Their Role in Abdominal Aortic Aneurysm

2020 ◽  
Vol 2020 ◽  
pp. 1-22
Author(s):  
Abulaihaiti Maitiseyiti ◽  
Hongbo Ci ◽  
Qingbo Fang ◽  
Sheng Guan ◽  
Alimujiang Shawuti ◽  
...  
Keyword(s):  
Abdominal Aortic Aneurysm ◽  
Aortic Aneurysm ◽  
Expression Profiles ◽  
Noncoding Rnas ◽  
Long Noncoding Rnas ◽  
Functional Enrichment ◽  
Pcr Analysis ◽  
Control Group ◽  
Abdominal Aortic

Objective. Long noncoding RNAs (lncRNAs) have emerged as critical molecular regulators in various diseases. However, the potential regulatory role of lncRNAs in the pathogenesis of abdominal aortic aneurysm (AAA) remains elusive. The aim of this study was to identify crucial lncRNAs associated with human AAA by comparing the lncRNA and mRNA expression profiles of patients with AAA with those of control individuals. Materials and Methods. The expression profiles of lncRNAs and mRNAs were analyzed in five dilated aortic samples from AAA patients and three normal aortic samples from control individuals using microarray technology. Functional annotation of the screened lncRNAs based on the differentially expressed genes was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Results. Microarray results revealed 2046 lncRNAs and 1363 mRNAs. Functional enrichment analysis showed that the mRNAs significantly associated with AAA were enriched in the NOD-like receptor (NLR) and nuclear factor kappa-B (NF-κB) signaling pathways and in cell adhesion molecules (CAMs), which are closely associated with pathophysiological changes in AAA. The lncRNAs identified using microarray analysis were further validated using quantitative real-time polymerase chain reaction (qRT-PCR) analysis with 12 versus 11 aortic samples. Finally, three key lncRNAs (ENST00000566954, ENST00000580897, and T181556) were confirmed using strict validation. A coding-noncoding coexpression (CNC) network and a competing endogenous RNA (ceRNA) network were constructed to determine the interaction among the lncRNAs, microRNAs, and mRNAs based on the confirmed lncRNAs. Conclusions. Our microarray profiling analysis and validation of significantly expressed lncRNAs between patients with AAA and control group individuals may provide new diagnostic biomarkers for AAA. The underlying regulatory mechanisms of the confirmed lncRNAs in AAA pathogenesis need to be determined using in vitro and in vivo experiments.

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