How subtle changes in 3D structure can create large changes in transcription

Animal genomes are organized into topologically associated domains (TADs). TADs are thought to contribute to gene regulation by facilitating enhancer-promoter (E-P) contacts within a TAD preventing these contacts across TAD borders. However, the absolute difference in contact frequency across TAD boundaries is usually less than two-fold, even though disruptions of TAD borders can change gene expression by ten-fold. Existing models fail to explain this hypersensitive response. Here, we propose a futile cycle model of enhancer-mediated regulation that can exhibit hypersensitivity through bistability and hysteresis. Consistent with recent experiments, this regulation does not exhibit strong correlation between enhancer-promoter contact and promoter activity, even though regulation occurs through contact. Through mathematical analysis and stochastic simulation, we show that this system can create an illusion of enhancer-promoter biochemical specificity and explain the importance of weak TAD boundaries. It also offers a mechanism to reconcile apparently contradictory results from recent global TAD disruption with local TAD boundary deletion experiments. Together, these analyses advance our understanding of cis-regulatory contacts in controlling gene expression, and suggest new experimental directions.

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How subtle changes in 3D structure can create large changes in transcription

10.1101/2020.10.22.351395 ◽

2020 ◽

Author(s):

Jordan Xiao ◽

Antonina Hafner ◽

Alistair N. Boettiger

Keyword(s):

Gene Expression ◽

3D Structure ◽

Transcriptional Response ◽

Large Change ◽

Absolute Difference ◽

Cycle Model ◽

Futile Cycle ◽

Topologically Associated Domains ◽

Promoter Specificity ◽

Animal Genomes

AbstractAnimal genomes are organized into topologically associated domains (TADs), which exhibit more intra-domain than inter-domain contact. However, the absolute difference in contact is usually no more than twofold, even though disruptions to TAD boundaries can change gene expression by 8-10 fold. Existing models fail to explain this superlinear transcriptional response to changes in genomic contact. Here, we propose a futile cycle model where an enzyme stimulated by association with its products can exhibit bistability and hysteresis, allowing a small increase in enhancer-promoter contact to produce a large change in expression without obvious correlation between E-P contact and promoter activity. Through mathematical analysis and stochastic simulation, we show that this system can create an illusion of enhancer-promoter specificity and explain the importance of weak TAD boundaries. It also offers a mechanism to reconcile recent global cohesin loop disruption and TAD boundary deletion experiments. We discuss the model in the context of these recent controversial experiments. Together, these analyses advance our interpretation and understanding of cis-regulatory contacts in controlling gene expression, and suggest new experimental directions.

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Regulation of rpoS Gene Expression in Pseudomonas: Involvement of a TetR Family Regulator

Journal of Bacteriology ◽

10.1128/jb.183.12.3712-3720.2001 ◽

2001 ◽

Vol 183 (12) ◽

pp. 3712-3720 ◽

Cited By ~ 55

Author(s):

Milan Kojic ◽

Vittorio Venturi

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Quorum Sensing ◽

Stationary Phase ◽

Cell Density ◽

Promoter Activity ◽

Regulatory Protein ◽

Insertion Mutant ◽

Regulatory Cascade ◽

A Cell

ABSTRACT The rpoS gene encodes the sigma factor which was identified in several gram-negative bacteria as a central regulator during stationary phase. rpoS gene regulation is known to respond to cell density, showing higher expression in stationary phase. For Pseudomonas aeruginosa, it has been demonstrated that the cell-density-dependent regulation response known as quorum sensing interacts with this regulatory response. Using therpoS promoter of P. putida, we identified a genomic Tn5 insertion mutant of P. putida which showed a 90% decrease in rpoSpromoter activity, resulting in less RpoS being present in a cell at stationary phase. Molecular analysis revealed that this mutant carried a Tn5 insertion in a gene, designatedpsrA (Pseudomonas sigma regulator), which codes for a protein (PsrA) of 26.3 kDa. PsrA contains a helix-turn-helix motif typical of DNA binding proteins and belongs to the TetR family of bacterial regulators. The homolog of thepsrA gene was identified in P. aeruginosa; the protein showed 90% identity to PsrA ofP. putida. ApsrA::Tn5 insertion mutant ofP. aeruginosa was constructed. In bothPseudomonas species, psrA was genetically linked to the SOS lexA repressor gene. Similar to what was observed for P. putida, a psrA null mutant of P. aeruginosa also showed a 90% reduction inrpoS promoter activity; both mutants could be complemented for rpoS promoter activity when thepsrA gene was provided in trans.psrA mutants of both Pseudomonas species lost the ability to induce rpoS expression at stationary phase, but they retained the ability to produce quorum-sensing autoinducer molecules. PsrA was demonstrated to negatively regulatepsrA gene expression in Pseudomonas and in Escherichia coli as well as to be capable of activating the rpoS promoter in E. coli. Our data suggest that PsrA is an important regulatory protein ofPseudomonas spp. involved in the regulatory cascade controlling rpoS gene regulation in response to cell density.

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Characterization of Histone Deacetyalses Involved in γ-Globin Gene Regulation.

Blood ◽

10.1182/blood.v112.11.1869.1869 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1869-1869

Author(s):

Shalini A Muralidhar ◽

Sadeieh Nimer ◽

Betty Pace

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Promoter Activity ◽

Globin Gene ◽

Indirect Evidence ◽

K562 Cells ◽

Luciferase Reporter ◽

Globin Promoter ◽

Globin Gene Regulation ◽

Globin Gene Expression

Abstract Fetal hemoglobin (HbF; α2γ2) ameliorates vaso-occlusive symptoms in individuals with sickle cell disease (SCD) because of its ability to inhibit hemoglobin S polymerization. One mechanism for γ-globin reactivation likely involves chromatin modification and the release of repressor complexes in which histone deacetylases (HDACs) may be present. The objective of this study was to identify HDACs involved in γ-globin gene regulation. Experiments were performed in K562 cells to determine the ability of NaB (2mM), TSA (0.5μm) and the non-HDAC-inhibitor γ-globin activator hemin (50μM), to alter transcription levels of the HDAC genes during concomitant HbF induction. Gene expression was measured by reverse transcription (RT) of mRNA followed by quantitative PCR (qPCR) analysis using gene-specific primers. Treatment of K562 cells with TSA and NaB reduced transcription levels of both HDAC9 and HDRP (histone deacetylase related protein, a splice variant of HDAC9) from 20–80% as did the control agent hemin (p<0.05). By contrast, expression of HDAC7 and HDAC10 was enhanced in the presence of both HDAC inhibitors. The altered HDAC gene expression levels provided indirect evidence for a possible role in mechanisms of γ-globin response to drug inducers. Subsequent experiments were performed to delineate whether HDAC9 and HDRP are directly involved in γ-globin regulation. We performed siRNA knockdown of HDAC9 and HDRP in K562 cells to determine the effect on expression of endogenous γ-globin. siRNA oligonucleotides were transfected using Oligofectamine (Invitrogen) for 48 hrs and expression of targeted genes were quantified by RT-qPCR. siHDAC9 and siHDRP (Dharmacon) treatment resulted in dose-dependent γ-globin silencing and transactivation respectively at 80–320nM. Experiments were then performed with 160nM of siHDAC9 or siHDRP in the K562 cell lines which were stably transfected with a luciferase reporter (pGL4.17-Luc2-neo, Promega) under the control of Gγ-globin promoter (−1500 to +36) and the pGL4.17-Luc2-neo empty vector. We likewise observed a 30% decrease in luciferase activity with siHDAC9 and a 40% increase with siHDRP suggesting that HDAC9 and its variant are directly involved in regulation of γ-promoter activity. In summary, the effects on endogenous γ globin levels and Gγ globin promoter activity through HDAC9/HDRPspecific knockdown by siRNA experiments suggest that HDAC9 molecules play a role in regulating γ-globin gene expression. We conclude that HDAC9 and HDRP have opposite regulatory effect on γ-globin gene expression and may act by a feedback mechanism.

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Effects of the Rewiring of Promoter Activity States on Gene Expression

2018 37th Chinese Control Conference (CCC) ◽

10.23919/chicc.2018.8483048 ◽

2018 ◽

Author(s):

Heli Tan

Keyword(s):

Gene Expression ◽

Promoter Activity

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UBE2L6 is Involved in Cisplatin Resistance by Regulating the Transcription of ABCB6

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200424130934 ◽

2020 ◽

Vol 20 (12) ◽

pp. 1487-1496 ◽

Cited By ~ 1

Author(s):

Midori Murakami ◽

Hiroto Izumi ◽

Tomoko Kurita ◽

Chiho Koi ◽

Yasuo Morimoto ◽

...

Keyword(s):

Gene Expression ◽

Promoter Activity ◽

Anticancer Agent ◽

Cisplatin Resistance ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Molecular Target ◽

Transcriptional Level ◽

And Control ◽

Resistant Cells

Background: Cisplatin is an important anticancer agent in cancer chemotherapy, but when resistant cells appear, treatment becomes difficult, and the prognosis is poor. Objective: In this study, we investigated the gene expression profile in cisplatin sensitive and resistant cells, and identified the genes involved in cisplatin resistance. Methods: Comparison of gene expression profiles revealed that UBE2L6 mRNA is highly expressed in resistant cells. To elucidate whether UBE2L6 is involved in the acquisition of cisplatin resistance, UBE2L6- overexpressing cells established from cisplatin-sensitive cells and UBE2L6-silenced cells developed from cisplatin- resistant cells were generated, and the sensitivity of cisplatin was examined. Results: The sensitivity of the UBE2L6-overexpressing cells did not change compared with the control cells, but the UBE2L6-silenced cells were sensitized to cisplatin. To elucidate the mechanism of UBE2L6 in cisplatin resistance, we compared the gene expression profiles of UBE2L6-silenced cells and control cells and found that the level of ABCB6 mRNA involved in cisplatin resistance was decreased. Moreover, ABCB6 promoter activity was partially suppressed in UBE2L6-silenced cells. Conclusion: These results suggest that cisplatin-resistant cells have upregulated UBE2L6 expression and contribute to cisplatin resistance by regulating ABCB6 expression at the transcriptional level. UBE2L6 might be a molecular target that overcomes cisplatin resistance.

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Base-Pair Opening Dynamics Study of Fluoride Riboswitch in the Bacillus cereus CrcB Gene

International Journal of Molecular Sciences ◽

10.3390/ijms22063234 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3234

Author(s):

Juhyun Lee ◽

Si-Eun Sung ◽

Janghyun Lee ◽

Jin Young Kang ◽

Joon-Hwa Lee ◽

...

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Nmr Spectroscopy ◽

Bacillus Cereus ◽

Base Pair ◽

Noncoding Rna ◽

Hydrogen Exchange ◽

Magnesium Ion ◽

Fluoride Ion ◽

Base Pair Opening

Riboswitches are segments of noncoding RNA that bind with metabolites, resulting in a change in gene expression. To understand the molecular mechanism of gene regulation in a fluoride riboswitch, a base-pair opening dynamics study was performed with and without ligands using the Bacillus cereus fluoride riboswitch. We demonstrate that the structural stability of the fluoride riboswitch is caused by two steps depending on ligands. Upon binding of a magnesium ion, significant changes in a conformation of the riboswitch occur, resulting in the greatest increase in their stability and changes in dynamics by a fluoride ion. Examining hydrogen exchange dynamics through NMR spectroscopy, we reveal that the stabilization of the U45·A37 base-pair due to the binding of the fluoride ion, by changing the dynamics while maintaining the structure, results in transcription regulation. Our results demonstrate that the opening dynamics and stabilities of a fluoride riboswitch in different ion states are essential for the genetic switching mechanism.

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CFTR Cooperative Cis-Regulatory Elements in Intestinal Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22052599 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2599

Author(s):

Mégane Collobert ◽

Ozvan Bocher ◽

Anaïs Le Nabec ◽

Emmanuelle Génin ◽

Claude Férec ◽

...

Keyword(s):

Gene Expression ◽

Cystic Fibrosis ◽

Gene Regulation ◽

Genetic Diseases ◽

Regulatory Elements ◽

Cftr Gene ◽

Intestinal Cells ◽

Cooperative Effects ◽

Cis Acting ◽

Study Techniques

About 8% of the human genome is covered with candidate cis-regulatory elements (cCREs). Disruptions of CREs, described as “cis-ruptions” have been identified as being involved in various genetic diseases. Thanks to the development of chromatin conformation study techniques, several long-range cystic fibrosis transmembrane conductance regulator (CFTR) regulatory elements were identified, but the regulatory mechanisms of the CFTR gene have yet to be fully elucidated. The aim of this work is to improve our knowledge of the CFTR gene regulation, and to identity factors that could impact the CFTR gene expression, and potentially account for the variability of the clinical presentation of cystic fibrosis as well as CFTR-related disorders. Here, we apply the robust GWAS3D score to determine which of the CFTR introns could be involved in gene regulation. This approach highlights four particular CFTR introns of interest. Using reporter gene constructs in intestinal cells, we show that two new introns display strong cooperative effects in intestinal cells. Chromatin immunoprecipitation analyses further demonstrate fixation of transcription factors network. These results provide new insights into our understanding of the CFTR gene regulation and allow us to suggest a 3D CFTR locus structure in intestinal cells. A better understand of regulation mechanisms of the CFTR gene could elucidate cases of patients where the phenotype is not yet explained by the genotype. This would thus help in better diagnosis and therefore better management. These cis-acting regions may be a therapeutic challenge that could lead to the development of specific molecules capable of modulating gene expression in the future.

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Identification of the conserved long non-coding RNAs in myogenesis

BMC Genomics ◽

10.1186/s12864-021-07615-0 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Anupam Bhattacharya ◽

Simang Champramary ◽

Tanya Tripathi ◽

Debajit Thakur ◽

Ilya Ioshikhes ◽

...

Keyword(s):

Gene Regulation ◽

Muscle Development ◽

Three Dimensional ◽

C2c12 Cells ◽

Mouse Muscle ◽

Rna Molecules ◽

Expression Of Genes ◽

Novel Approach ◽

Non Coding Rna ◽

Topologically Associated Domains

Abstract Background Our understanding of genome regulation is ever-evolving with the continuous discovery of new modes of gene regulation, and transcriptomic studies of mammalian genomes have revealed the presence of a considerable population of non-coding RNA molecules among the transcripts expressed. One such non-coding RNA molecule is long non-coding RNA (lncRNA). However, the function of lncRNAs in gene regulation is not well understood; moreover, finding conserved lncRNA across species is a challenging task. Therefore, we propose a novel approach to identify conserved lncRNAs and functionally annotate these molecules. Results In this study, we exploited existing myogenic transcriptome data and identified conserved lncRNAs in mice and humans. We identified the lncRNAs expressing differentially between the early and later stages of muscle development. Differential expression of these lncRNAs was confirmed experimentally in cultured mouse muscle C2C12 cells. We utilized the three-dimensional architecture of the genome and identified topologically associated domains for these lncRNAs. Additionally, we correlated the expression of genes in domains for functional annotation of these trans-lncRNAs in myogenesis. Using this approach, we identified conserved lncRNAs in myogenesis and functionally annotated them. Conclusions With this novel approach, we identified the conserved lncRNAs in myogenesis in humans and mice and functionally annotated them. The method identified a large number of lncRNAs are involved in myogenesis. Further studies are required to investigate the reason for the conservation of the lncRNAs in human and mouse while their sequences are dissimilar. Our approach can be used to identify novel lncRNAs conserved in different species and functionally annotated them.

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