scholarly journals LmbU directly regulates SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780, which are located outside the lmb cluster and inhibit lincomycin biosynthesis in Streptomyces lincolnensis

2021 ◽  
Author(s):  
Bingbing Hou ◽  
Xianyan Zhang ◽  
Yue Mao ◽  
Ruida Wang ◽  
Jiang Ye ◽  
...  
Keyword(s):  
Binding Sites ◽  
Biosynthetic Gene Cluster ◽  
Regulatory Mechanisms ◽  
Whole Genome ◽  
Biosynthetic Gene ◽  
Qrt Pcr ◽  
Dna Binding Sites ◽  
Reporter Assays ◽  
Streptomyces Lincolnensis

The productions of antibiotics are usually regulated by cluster-situated regulators (CSRs), which can directly regulate the genes within the corresponding biosynthetic gene cluster (BGC). However, few studies have looked into the regulation of CSRs on the targets outside the BGC. Here, we screened the targets of LmbU in the whole genome of S. lincolnensis, and found 14 candidate targets, among of which, 8 targets can bind to LmbU by EMSAs. Reporter assays in vivo revealed that LmbU repressed transcription of SLINC_RS02575 and SLINC_RS05540, while activated transcription of SLINC_RS42780. In addition, disruptions of SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 promoted the production of lincomycin, and qRT-PCR showed that SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 inhibited transcription of the lmb genes, indicating that all the three regulators can negatively regulate lincomycin biosynthesis. What's more, the homologues of LmbU and its targets SLINC_RS02575, SLINC_RS05540 and SLINC_RS42780 are widely found in actinomycetes, while the distributions of DNA-binding sites (DBS) of LmbU are diverse, indicating the regulatory mechanisms of LmbU homologues in various strains are different and complicated.

scholarly journals Refactoring the formicamycin biosynthetic gene cluster to make high-level producing strains and new molecules

2020 ◽  
Author(s):  
Rebecca Devine ◽  
Hannah McDonald ◽  
Zhiwei Qin ◽  
Corinne Arnold ◽  
Katie Noble ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Large Scale ◽  
Liquid Culture ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Resistant Bacteria ◽  
Drug Resistant ◽  
Biosynthetic Gene ◽  
Biosynthetic Genes

AbstractThe formicamycins are promising antibiotics with potent activity against Gram-positive pathogens including VRE and MRSA and display a high barrier to selection of resistant isolates. They were first identified in Streptomyces formicae KY5, which produces the formicamycins at low levels on solid agar but not in liquid culture, thus hindering further investigation of these promising antibacterial compounds. We hypothesised that by understanding the organisation and regulation of the for biosynthetic gene cluster, we could rationally refactor the cluster to increase production levels. Here we report that the for biosynthetic gene cluster consists of 24 genes expressed on nine transcripts. Seven of these transcripts, including those containing all the major biosynthetic genes, are repressed by the MarR-regulator ForJ which also controls the expression of the ForGF two-component system that initiates biosynthesis. A third cluster-situated regulator, ForZ, autoregulates and controls production of the putative MFS transporter ForAA. Consistent with these findings, deletion of forJ increased formicamycin biosynthesis 5-fold, while over-expression of forGF in the ΔforJ background increased production 10-fold compared to the wild-type. De-repression by deleting forJ also switched on biosynthesis in liquid-culture and induced the production of two novel formicamycin congeners. By combining mutations in regulatory and biosynthetic genes, six new biosynthetic precursors with antibacterial activity were also isolated. This work demonstrates the power of synthetic biology for the rational redesign of antibiotic biosynthetic gene clusters both to engineer strains suitable for fermentation in large scale bioreactors and to generate new molecules.ImportanceAntimicrobial resistance is a growing threat as existing antibiotics become increasingly ineffective against drug resistant pathogens. Here we determine the transcriptional organisation and regulation of the gene cluster encoding biosynthesis of the formicamycins, promising new antibiotics with activity against drug resistant bacteria. By exploiting this knowledge, we construct stable mutant strains which over-produce these molecules in both liquid and solid culture whilst also making some new compound variants. This will facilitate large scale purification of these molecules for further study including in vivo experiments and the elucidation of their mechanism of action. Our work demonstrates that understanding the regulation of natural product biosynthetic pathways can enable rational improvement of the producing strains.

Genetic localization and in vivo characterization of a Monascus azaphilone pigment biosynthetic gene cluster

2013 ◽  
Vol 97 (14) ◽  
pp. 6337-6345 ◽  
Author(s):  
Bijinu Balakrishnan ◽  
Suman Karki ◽  
Shih-Hau Chiu ◽  
Hyun-Ju Kim ◽  
Jae-Won Suh ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Genetic Localization ◽  
In Vivo Characterization ◽  
Azaphilone Pigment

scholarly journals An effective approach for identification of in vivo protein-DNA binding sites from paired-end ChIP-Seq data

2010 ◽  
Vol 11 (1) ◽  
pp. 81 ◽  
Author(s):  
Congmao Wang ◽  
Jie Xu ◽  
Dasheng Zhang ◽  
Zoe A Wilson ◽  
Dabing Zhang
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Dna Binding Sites

scholarly journals Functional Interactions between NURF and Ctcf Regulate Gene Expression

2014 ◽  
Vol 35 (1) ◽  
pp. 224-237 ◽  
Author(s):  
Zhijun Qiu ◽  
Carolyn Song ◽  
Navid Malakouti ◽  
Daniel Murray ◽  
Aymen Hariz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Nucleosome Occupancy ◽  
Cell Types ◽  
Ctcf Binding ◽  
Nucleosome Remodeling ◽  
Functional Interactions ◽  
Chromatin Remodeling Complexes ◽  
Reporter Assays

Gene expression frequently requires chromatin-remodeling complexes, and it is assumed that these complexes have common gene targets across cell types. Contrary to this belief, we show by genome-wide expression profiling that Bptf, an essential and unique subunit of the nucleosome-remodeling factor (NURF), predominantly regulates the expression of a unique set of genes between diverse cell types. Coincident with its functions in gene expression, we observed that Bptf is also important for regulating nucleosome occupancy at nucleosome-free regions (NFRs), many of which are located at sites occupied by the multivalent factors Ctcf and cohesin. NURF function at Ctcf binding sites could be direct, because Bptf occupies Ctcf binding sitesin vivoand has physical interactions with CTCF and the cohesin subunit SA2. Assays of several Ctcf binding sites using reporter assays showed that their regulatory activity requires Bptf in two different cell types. Focused studies atH2-K1showed that Bptf regulates the ability of Klf4 to bind near an upstream Ctcf site, possibly influencing gene expression. In combination, these studies demonstrate that gene expression as regulated by NURF occurs partly through physical and functional interactions with the ubiquitous and multivalent factors Ctcf and cohesin.

scholarly journals Genome-wide analysis of the role of the antibiotic biosynthesis regulator AbsA2 inStreptomyces coelicolorA3(2)

2018 ◽  
Author(s):  
Richard A. Lewis ◽  
Abdul Wahab ◽  
Giselda Bucca ◽  
Emma E. Laing ◽  
Carla Möller-Levet ◽  
...  
Keyword(s):  
Large Scale ◽  
Time Course ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Negative Influence ◽  
Antibiotic Biosynthesis ◽  
Biosynthetic Gene

AbstractThe AbsA1-AbsA2 two component signalling system ofStreptomyces coelicolorhas long been known to exert a powerful negative influence on the production of the antibiotics actinorhodin, undecylprodiginine and the Calcium-Dependent Antibiotic (CDA). Here we report the analysis of aΔabsA2deletion strain, which exhibits the classic precocious antibiotic hyper-production phenotype, and its complementation by an N-terminal triple-FLAG-tagged version of AbsA2. The complemented and non-complementedΔabsA2mutant strains were used in large-scale microarray-based time-course experiments to investigate the effect of deletingabsA2on gene expression and to identify thein vivoAbsA2 DNA-binding target sites using ChIP-on chip. We show that in addition to binding to the promoter regions ofredZandactII-orfIVAbsA2 binds to several previously unidentified sites within thecdabiosynthetic gene cluster within and/or upstream ofSCO3215-SCO3216,SCO3217,SCO3229-SCO3230, andSCO3226, and we relate the pattern of AbsA2 binding to the results of the transcriptomic study and antibiotic phenotypic assays. Interestingly, dual ‘biphasic’ ChIP peaks were observed with AbsA2 binding across the regulatory genesactII-orfIVandredZand theabsA2gene itself, while more conventional single promoter-proximal peaks were seen at the CDA biosynthetic genes suggesting a different mechanism of regulation of the former loci. Taken together the results shed light on the complex mechanism of regulation of antibiotic biosynthesis inStreptomyces coelicolorand the important role of AbsA2 in controlling the expression of three antibiotic biosynthetic gene clusters.

scholarly journals Accurate and sensitive quantification of protein-DNA binding affinity

2018 ◽  
Vol 115 (16) ◽  
pp. E3692-E3701 ◽  
Author(s):  
Chaitanya Rastogi ◽  
H. Tomas Rube ◽  
Judith F. Kribelbauer ◽  
Justin Crocker ◽  
Ryan E. Loker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Sites ◽  
Biophysical Model ◽  
Regulatory Sequences ◽  
Binding Modes ◽  
Perfect Agreement ◽  
Dna Binding Sites

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

scholarly journals LncRNA SNHG6 promotes chemoresistance through ULK1-induced autophagy by sponging miR-26a-5p in colorectal cancer cells

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xinke Wang ◽  
Zhixian Lan ◽  
Juan He ◽  
Qiuhua Lai ◽  
Xiang Yao ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Western Blotting ◽  
Target Genes ◽  
Bioinformatics Analysis ◽  
Chemotherapy Resistance ◽  
Micro Rna ◽  
Luciferase Reporter ◽  
Qrt Pcr ◽  
Reporter Assays

Abstract Background Chemotherapy resistance is one of the main causes of recurrence in colorectal cancer (CRC) patients and leads to poor prognosis. Long noncoding RNAs (lncRNAs) have been reported to regulate chemoresistance. We aimed to determine the role of the lncRNA small nucleolar RNA host gene 6 (SNHG6) in CRC cell chemoresistance. Methods Cell drug sensitivity tests and flow cytometry were performed to analyze CRC cell chemoresistance. Animal models were used to determine chemoresistance in vivo, and micro RNA (miRNA) binding sites were detected by dual-luciferase reporter assays. Bioinformatics analysis was performed to predict miRNAs binding to SNHG6 and target genes of miR-26a-5p. SNHG6/miR-26a-5p/ULK1 axis and autophagy-related proteins were detected by qRT-PCR and western blotting. Furthermore, immunofluorescence was employed to confirm the presence of autophagosomes. Results SNHG6 enhanced CRC cell resistance to 5-fluorouracil (5-FU), promoted autophagy, inhibited 5-FU-induced apoptosis, and increased 5-FU resistance in vivo. Bioinformatics analysis showed that miR-26a-5p might bind to SNHG6 and target ULK1, and dual-luciferase reporter assays confirmed this activity. qRT-PCR and western blotting showed that SNHG6 was able to negatively regulate miR-26a-5p but correlated positively with ULK1. Conclusion SNHG6 may promote chemoresistance through ULK1-induced autophagy by sponging miR-26a-5p in CRC cells.

scholarly journals Properties of initiator-associated transcription mediated by GAL4-VP16.

1993 ◽  
Vol 13 (12) ◽  
pp. 7469-7475 ◽  
Author(s):  
C Chang ◽  
J D Gralla
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Dna Binding Sites ◽  
Terminal Deoxynucleotide Transferase ◽  
Initiator Element ◽  
High Level ◽  
Primary Activation ◽  
Initial Assembly

Transcription associated with a terminal deoxynucleotide transferase gene initiator element is shown to respond to the transcription factor GAL4-VP16 both in vivo and in vitro. High-level transcription requires both an intact initiator element and bound activator. Transcription from this initiator-directed promoter is synergistic in vivo in that five GAL4 DNA binding sites yield 36 times the expression of a single site. Promoters dominated by initiator and TATA elements respond similarly to several GAL4-based activators, including GAL4-Sp1, GAL4-CTF, GAL4(1-147), GAL4-p53, GAL4-C/EBP, and GAL4-ER(EF), as well as GAL4-VP16 and Sp1. These and other similarities suggest that primary activation of TATA- and initiator-dominated promoters occurs at common steps. Since the initial assembly steps do not appear to be common for the two promoter types, the results place interesting constraints on models for how activation occurs.

scholarly journals Reprogramming of DNA methylation at NEUROD2-bound sequences during cortical neuron differentiation

2019 ◽  
Vol 5 (10) ◽  
pp. eaax0080 ◽  
Author(s):  
Maria A. Hahn ◽  
Seung-Gi Jin ◽  
Arthur X. Li ◽  
Jiancheng Liu ◽  
Zhijun Huang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Binding Sites ◽  
Bisulfite Sequencing ◽  
Dna Demethylation ◽  
Whole Genome ◽  
Dna Hypomethylation ◽  
Embryonic Neurogenesis ◽  
Genome Bisulfite Sequencing ◽  
Developing Neurons

The characteristics of DNA methylation changes that occur during neurogenesis in vivo remain unknown. We used whole-genome bisulfite sequencing to quantitate DNA cytosine modifications in differentiating neurons and their progenitors isolated from mouse brain at the peak of embryonic neurogenesis. Localized DNA hypomethylation was much more common than hypermethylation and often occurred at putative enhancers within genes that were upregulated in neurons and encoded proteins crucial for neuronal differentiation. The hypomethylated regions strongly overlapped with mapped binding sites of the key neuronal transcription factor NEUROD2. The 5-methylcytosine oxidase ten-eleven translocation 2 (TET2) interacted with NEUROD2, and its reaction product 5-hydroxymethylcytosine accumulated at the demethylated regions. NEUROD2-targeted differentially methylated regions retained higher methylation levels in Neurod2 knockout mice, and inducible expression of NEUROD2 caused TET2-associated demethylation at its in vivo binding sites. The data suggest that the reorganization of DNA methylation in developing neurons involves NEUROD2 and TET2-mediated DNA demethylation.

scholarly journals In Vivo Analysis of the Regulatory Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455 Reveals Their Differential Control Over Antibiotic Biosynthesis

2004 ◽  
Vol 186 (5) ◽  
pp. 1345-1354 ◽  
Author(s):  
Olga N. Sekurova ◽  
Trygve Brautaset ◽  
Håvard Sletta ◽  
Sven E. F. Borgos ◽  
Øyvind M. Jakobsen ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Regulatory Genes ◽  
Antibiotic Biosynthesis ◽  
Biosynthetic Gene ◽  
Promoter Regions ◽  
Streptomyces Noursei ◽  
In Vivo Analysis ◽  
Differential Control

ABSTRACT Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of nystatin biosynthesis.

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