scholarly journals Homologous BHLH transcription factors induce distinct deformations of torsionally-stressed DNA: a potential transcription regulation mechanism.

2022 ◽  
Author(s):  
Johanna Hörberg ◽  
Kevin Moreau ◽  
Anna Reymer

Changing torsional restraints on DNA is essential for the regulation of transcription. Torsional stress, introduced by RNA polymerase, can propagate along chromatin facilitating topological transitions and modulating the specific binding of transcription factors (TFs) to DNA. Despite the importance, the mechanistic details on how torsional stress impacts the TFs-DNA complexation remain scarce. Herein we address the impact of torsional stress on DNA complexation with homologous human basic-helix-loop-helix (BHLH) hetero- and homodimers: MycMax, MadMax, and MaxMax. The three TF dimers exhibit specificity towards the same DNA consensus sequences, the E-box response element, while regulating different transcriptional pathways. Using microseconds-long atomistic molecular dynamics simulations together with the torsional restraint that controls DNA total helical twist, we gradually over- and underwind naked and complexed DNA to a maximum of ±5°/b.p. step. We observe that the binding of the BHLH dimers results in a similar increase in DNA torsional rigidity. However, under torsional stress the BHLH dimers induce distinct DNA deformations, characterised by changes in DNA grooves geometry and a significant asymmetric DNA bending. Supported by bioinformatics analyses, our data suggest that torsional stress may contribute to the execution of differential transcriptional programs of the homologous TFs by modulating their collaborative interactions.

Author(s):  
Johanna Hörberg ◽  
Anna Reymer

ABSTRACTTorsional stress on DNA, introduced by molecular motors, constitutes an important regulatory mechanism of transcriptional control. Torsional stress can modulate specific binding of transcription factors to DNA and introduce local conformational changes that facilitate the opening of promoters and nucleosome remodeling. Using all-atom microsecond scale molecular dynamics simulations together with a torsional restraint that controls the total helical twist of a DNA fragment, we addressed the impact of torsional stress on DNA complexation with a human BZIP transcription factor, MafB. We gradually over- and underwind DNA alone and in complex with MafB by 5° per dinucleotide step, monitoring the evolution of the protein-DNA contacts at different degrees of torsional strain. Our computations show that MafB changes the DNA sequence-specific response to torsional stress. The dinucleotide steps that are susceptible to absorb most of the torsional stress become more torsionally rigid, as they are involved in the protein-DNA contacts. Also, the protein undergoes substantial conformational changes to follow the stress-induced DNA deformation, but mostly maintains the specific contacts with DNA. This results in a significant asymmetric increase of free energy of DNA twisting transitions, relative to free DNA, where overtwisting is more energetically unfavorable. Our data suggest that MafB could act as a torsional stress insulator, modulating the propagation of torsional stress along the chromatin fiber, which might promote cooperative binding of other transcription factors.


2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Johanna Hörberg ◽  
Anna Reymer

Abstract Torsional stress on DNA, introduced by molecular motors, constitutes an important regulatory mechanism of transcriptional control. Torsional stress can modulate specific binding of transcription factors to DNA and introduce local conformational changes that facilitate the opening of promoters and nucleosome remodelling. Using all-atom microsecond scale molecular dynamics simulations together with a torsional restraint that controls the total twist of a DNA fragment, we address the impact of torsional stress on DNA complexation with a human BZIP transcription factor, MafB. We gradually over- and underwind DNA alone and in complex with MafB by 0.5° per dinucleotide step, starting from the relaxed state to a maximum of 5° per dinucleotide step, monitoring the evolution of the protein-DNA contacts at different degrees of torsional strain. Our computations show that MafB changes the DNA sequence-specific response to torsional stress. The dinucleotide steps that are susceptible to absorbing most of the torsional stress become more torsionally rigid, as they are involved in protein-DNA contacts. Also, the protein undergoes substantial conformational changes to follow the stress-induced DNA deformation, but mostly maintains the specific contacts with DNA. This results in a significant asymmetric increase of free energy of DNA twisting transitions, relative to free DNA, where overtwisting is more energetically unfavourable. Our data suggest that specifically bound BZIP factors could act as torsional stress insulators, modulating the propagation of torsional stress along the chromatin fibre, which might promote cooperative binding of collaborative DNA-binding factors.


2020 ◽  
Vol 21 (4) ◽  
pp. 1337 ◽  
Author(s):  
Weida Lin ◽  
Yueling Li ◽  
Qiuwei Lu ◽  
Hongfei Lu ◽  
Junmin Li

To assess changes of metabolite content and regulation mechanism of the phenolic acid biosynthesis pathway at different developmental stages of leaves, this study performed a combined metabolome and transcriptome analysis of Cyclocarya paliurus leaves at different developmental stages. Metabolite and transcript profiling were conducted by ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometer and high-throughput RNA sequencing, respectively. Transcriptome identification showed that 58 genes were involved in the biosynthesis of phenolic acid. Among them, 10 differentially expressed genes were detected between every two developmental stages. Identification and quantification of metabolites indicated that 14 metabolites were located in the phenolic acid biosynthetic pathway. Among them, eight differentially accumulated metabolites were detected between every two developmental stages. Association analysis between metabolome and transcriptome showed that six differentially expressed structural genes were significantly positively correlated with metabolite accumulation and showed similar expression trends. A total of 128 transcription factors were identified that may be involved in the regulation of phenolic acid biosynthesis; these include 12 MYBs and 10 basic helix–loop–helix (bHLH) transcription factors. A regulatory network of the phenolic acid biosynthesis was established to visualize differentially expressed candidate genes that are involved in the accumulation of metabolites with significant differences. The results of this study contribute to the further understanding of phenolic acid biosynthesis during the development of leaves of C. paliurus.


2020 ◽  
Vol 61 (6) ◽  
pp. 1041-1053 ◽  
Author(s):  
Shunya Hayashi ◽  
Mutsumi Watanabe ◽  
Makoto Kobayashi ◽  
Takayuki Tohge ◽  
Takashi Hashimoto ◽  
...  

Abstract The toxic alkaloid nicotine is produced in the roots of Nicotiana species and primarily accumulates in leaves as a specialized metabolite. A series of metabolic and transport genes involved in the nicotine pathway are coordinately upregulated by a pair of jasmonate-responsive AP2/ERF-family transcription factors, NtERF189 and NtERF199, in the roots of Nicotiana tabacum (tobacco). In this study, we explored the potential of manipulating the expression of these transcriptional regulators to alter nicotine biosynthesis in tobacco. The transient overexpression of NtERF189 led to alkaloid production in the leaves of Nicotiana benthamiana and Nicotiana alata. This ectopic production was further enhanced by co-overexpressing a gene encoding a basic helix-loop-helix-family MYC2 transcription factor. Constitutive and leaf-specific overexpression of NtERF189 increased the accumulation of foliar alkaloids in transgenic tobacco plants but negatively affected plant growth. By contrast, in a knockout mutant of NtERF189 and NtERF199 obtained through CRISPR/Cas9-based genome editing, alkaloid levels were drastically reduced without causing major growth defects. Metabolite profiling revealed the impact of manipulating the nicotine pathway on a wide range of nitrogen- and carbon-containing metabolites. Our findings provide insights into the biotechnological applications of engineering metabolic pathways by targeting transcription factors.


2019 ◽  
Author(s):  
Manuela Malsy ◽  
Bernhard Graf ◽  
Anika Bundscherer

Abstract Background Pancreatic adenocarcinoma is one of the most lethal cancers worldwide with very poor long-term survival. The treatment of choice next to chemotherapy or radiation treatment is surgical removal of the tumor. However, medication, surgery, and perioperative immunosuppression induce the constitutive activation of important signaling pathways and change the regulation of transcription factors, which may facilitate tumor progression and metastasis. Recent research has identified the transcription factors NFATc2 and Sp1 as key regulators in the carcinogenesis of pancreatic carcinoma. It is still unclear to what extent the transcription factors NFATc2 and Sp1 are influenced by analgesics given via peridural anesthetics or lidocaine infusions administered in perioperative settings or as postoperative pain therapy.Aims To conduct an in vitro analysis of the impact of clinically achievable dosages of ketamine, s-ketamine, metamizole, and paracetamol as well as of sufentanil, ropicavaine, and lidocaine on pancreatic carcinoma cells in dependency of NFATc2 and Sp1.Methods Analgesic stimulation and its effects on the expression of NFATc2 and Sp1 were investigated with immunoblot. Cell proliferation was measured with the ELISA BrdU assay.Results In PaTu8988t pancreatic carcinoma cells, 48h stimulation with ketamine and s-ketamine significantly inhibited proliferation and contemporaneously decreased endogen expression of NFATc2 in the nucleus. The addition of metamizole and lidocaine to PaTu8988t cells reduced proliferation after 48h.Conclusions New treatment concepts target the efficient modulation of specific signaling and transcription pathways. The extent to which drugs influence these mechanisms in vulnerable phases of pancreatic carcinoma cells needs to be investigated in future studies. The basis of novel therapeutic approaches to any disease is detailed knowledge of the carcinogenesis and profound molecular and biological understanding of the mechanisms.


2019 ◽  
Author(s):  
Manuela Malsy ◽  
Bernhard Graf ◽  
Anika Bundscherer

Abstract Background Pancreatic adenocarcinoma is one of the most lethal cancers worldwide with very poor long-term survival. The treatment of choice next to chemotherapy or radiation treatment is surgical removal of the tumor. However, medication, surgery, and perioperative immunosuppression induce the constitutive activation of important signaling pathways and change the regulation of transcription factors, which may facilitate tumor progression and metastasis. Recent research has identified the transcription factors NFATc2 and Sp1 as key regulators in the carcinogenesis of pancreatic carcinoma. It is still unclear to what extent the transcription factors NFATc2 and Sp1 are influenced by analgesics given via peridural anesthetics or lidocaine infusions administered in perioperative settings or as postoperative pain therapy.Aims To conduct an in vitro analysis of the impact of clinically achievable dosages of ketamine, s-ketamine, metamizole, and paracetamol as well as of sufentanil, ropicavaine, and lidocaine on pancreatic carcinoma cells in dependency of NFATc2 and Sp1.Methods Analgesic stimulation and its effects on the expression of NFATc2 and Sp1 were investigated with immunoblot. Cell proliferation was measured with the ELISA BrdU assay.Results In PaTu8988t pancreatic carcinoma cells, 48h stimulation with ketamine and s-ketamine significantly inhibited proliferation and contemporaneously decreased endogen expression of NFATc2 in the nucleus. The addition of metamizole and lidocaine to PaTu8988t cells reduced proliferation after 48h.Conclusions New treatment concepts target the efficient modulation of specific signaling and transcription pathways. The extent to which drugs influence these mechanisms in vulnerable phases of pancreatic carcinoma cells needs to be investigated in future studies. The basis of novel therapeutic approaches to any disease is detailed knowledge of the carcinogenesis and profound molecular and biological understanding of the mechanisms.


2020 ◽  
Vol 117 (9) ◽  
pp. 4885-4893 ◽  
Author(s):  
David A. Anderson ◽  
Theresa L. Murphy ◽  
Robert N. Eisenman ◽  
Kenneth M. Murphy

We previously found that MYCL is required by aBatf3-dependent classical dendritic cell subset (cDC1) for optimal CD8 T cell priming, but the underlying mechanism has remained unclear. The MAX-binding proteins encompass a family of transcription factors with overlapping DNA-binding specificities, conferred by a C-terminal basic helix-loop-helix domain, which mediates heterodimerization. Thus, regulation of transcription by these factors is dependent on divergent N-terminal domains. The MYC family, including MYCL, has actions that are reciprocal to the MXD family, which is mediated through the recruitment of higher-order activator and repressor complexes, respectively. As potent proto-oncogenes, models of MYC family function have been largely derived from their activity at supraphysiological levels in tumor cell lines. MYC and MYCN have been studied extensively, but empirical analysis of MYCL function had been limited due to highly restricted, lineage-specific expression in vivo. Here we observed thatMyclis expressed in immature cDC1s but repressed on maturation, concomitant withMxd1induction in mature cDC1s. We hypothesized that MYCL and MXD1 regulate a shared, but reciprocal, transcriptional program during cDC1 maturation. In agreement, immature cDC1s inMycl−/−-deficient mice exhibited reduced expression of genes that regulate core biosynthetic processes. Mature cDC1s fromMxd1−/−mice exhibited impaired ability to inhibit the transcriptional signature otherwise supported by MYCL. The present study reveals LMYC and MXD1 as regulators of a transcriptional program that is modulated during the maturation ofBatf3-dependent cDC1s.


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