Diverse control of metabolism and other cellular processes in Streptomyces coelicolor by the PhoP transcription factor: genome-wide identification of in vivo targets

DNA-protein interactions (DPIs) are central to such fundamental cellular processes as transcription and chromosome maintenance and organization. The spatiotemporal dynamics of these interactions dictate their functional consequences; therefore, there is great interest in facile methods for defining the sites of DPI within cells. Here, we present a general method for mapping DPI sites in vivo using the double stranded DNA-specific cytosine deaminase toxin DddA. Our approach, which we term DddA-sequencing (3D-seq), entails generating a translational fusion of DddA to a DNA binding protein of interest, inactivating uracil DNA glycosylase, modulating DddA activity via its natural inhibitor protein, and DNA sequencing for genome-wide DPI detection. We successfully applied this method to three Pseudomonas aeruginosa transcription factors that represent divergent protein families and bind variable numbers of chromosomal locations. 3D-seq offers several advantages over existing technologies including ease of implementation and the possibility to measure DPIs at single-cell resolution.

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Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID

eLife ◽

10.7554/elife.47864 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 22

Author(s):

Andrea Mair ◽

Shou-Ling Xu ◽

Tess C Branon ◽

Alice Y Ting ◽

Dominique C Bergmann

Keyword(s):

Transcription Factor ◽

Protein Interactions ◽

Protein Complexes ◽

Cell Types ◽

Specific Protein ◽

Cell Type ◽

Subcellular Compartment ◽

Cellular Processes ◽

Highly Active

Defining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurbo), allowed us to address two challenging questions in plants: (1) what are in vivo partners of a low abundant key developmental transcription factor and (2) what is the nuclear proteome of a rare cell type? Proteins identified with FAMA-TurboID include known interactors of this stomatal transcription factor and novel proteins that could facilitate its activator and repressor functions. Directing TurboID to stomatal nuclei enabled purification of cell type- and subcellular compartment-specific proteins. Broad tests of TurboID and miniTurbo in Arabidopsis and Nicotiana benthamiana and versatile vectors enable customization by plant researchers.

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The Flo8 Transcription Factor Is Essential for Hyphal Development and Virulence inCandida albicans

Molecular Biology of the Cell ◽

10.1091/mbc.e05-06-0502 ◽

2006 ◽

Vol 17 (1) ◽

pp. 295-307 ◽

Cited By ~ 146

Author(s):

Fang Cao ◽

Shelley Lane ◽

Prashna Pala Raniga ◽

Yang Lu ◽

Zhou Zhou ◽

...

Keyword(s):

Transcription Factor ◽

Systemic Infection ◽

Hyphal Growth ◽

Specific Gene ◽

Transcription Profiling ◽

Hyphal Development ◽

Genome Wide ◽

Pathway Regulation ◽

Pka Pathway

The transcription factor Flo8 is essential for filamentous growth in Saccharomyces cerevisiae and is regulated under the cAMP/protein kinase A (PKA) pathway. To determine whether a similar pathway/regulation exists in Candida albicans, we have cloned C. albicans FLO8 by its ability to complement S. cerevisiae flo8. Deleting FLO8 in C. albicans blocked hyphal development and hypha-specific gene expression. The flo8/flo8 mutant is avirulent in a mouse model of systemic infection. Genome-wide transcription profiling of efg1/efg1 and flo8/flo8 using a C. albicans DNA microarray suggests that Flo8 controls subsets of Efg1-regulated genes. Most of these genes are hypha specific, including HGC1 and IHD1. We also show that Flo8 interacts with Efg1 in yeast and hyphal cells by in vivo immunoprecipitation. Similar to efg1/efg1, flo8/flo8 and cdc35/cdc35 show enhanced hyphal growth under an embedded growth condition. Our results suggest that Flo8 may function downstream of the cAMP/PKA pathway, and together with Efg1, regulates the expression of hypha-specific genes and genes that are important for the virulence of C. albicans.

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ScbR- and ScbR2-mediated signal transduction networks coordinate complex physiological responses in Streptomyces coelicolor

Scientific Reports ◽

10.1038/srep14831 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 23

Author(s):

Xiao Li ◽

Juan Wang ◽

Shanshan Li ◽

Junjie Ji ◽

Weishan Wang ◽

...

Keyword(s):

Streptomyces Coelicolor ◽

Model Organism ◽

Morphological Differentiation ◽

Signalling Molecules ◽

Cellular Processes ◽

Signal Transduction Networks ◽

Primary And Secondary Metabolism ◽

A Genome ◽

Regulatory Cascades

Abstract In model organism Streptomyces coelicolor, γ-butyrolactones (GBLs) and antibiotics were recognized as signalling molecules playing fundamental roles in intra- and interspecies communications. To dissect the GBL and antibiotic signalling networks systematically, the in vivo targets of their respective receptors ScbR and ScbR2 were identified on a genome scale by ChIP-seq. These identified targets encompass many that are known to play important roles in diverse cellular processes (e.g. gap1, pyk2, afsK, nagE2, cdaR, cprA, cprB, absA1, actII-orf4, redZ, atrA, rpsL and sigR) and they formed regulatory cascades, sub-networks and feedforward loops to elaborately control key metabolite processes, including primary and secondary metabolism, morphological differentiation and stress response. Moreover, interplay among ScbR, ScbR2 and other regulators revealed intricate cross talks between signalling pathways triggered by GBLs, antibiotics, nutrient availability and stress. Our work provides a global view on the specific responses that could be triggered by GBL and antibiotic signals in S. coelicolor, among which the main echo was the change of production profile of endogenous antibiotics and antibiotic signals manifested a role to enhance bacterial stress tolerance as well, shedding new light on GBL and antibiotic signalling networks widespread among streptomycetes.

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A systematic genome-wide account of binding sites for the model transcription factor Gcn4

Genome Research ◽

10.1101/gr.276080.121 ◽

2021 ◽

pp. gr.276080.121

Author(s):

Christopher T Coey ◽

David J. Clark

Keyword(s):

Gene Regulation ◽

Transcription Factor ◽

Binding Sites ◽

Yeast Genome ◽

High Affinity ◽

First Approximation ◽

Genome Wide ◽

Insight Into

Sequence-specific DNA-binding transcription factors are central to gene regulation. They are often associated with consensus binding sites that predict far more genomic sites than are bound in vivo. One explanation is that most sites are blocked by nucleosomes, such that only sites in nucleosome-depleted regulatory regions are bound. We compared the binding of the yeast transcription factor Gcn4 in vivo using published ChIP-seq data (546 sites) and in vitro, using a modified SELEX method ("G-SELEX"), which utilizes short genomic DNA fragments to quantify binding at all sites. We confirm that Gcn4 binds strongly to an AP-1-like sequence (TGACTCA) and weakly to half-sites. However, Gcn4 binds only some of the 1078 exact matches to this sequence, even in vitro. We show that there are only 166 copies of the high-affinity RTGACTCAY site (exact match) in the yeast genome, all occupied in vivo, largely independently of whether they are located in nucleosome-depleted or nucleosomal regions. Generally, RTGACTCAR/YTGACTCAY sites are bound much more weakly and YTGACTCAR sites are unbound, with biological implications for determining induction levels. We conclude that, to a first approximation, Gcn4 binding can be predicted using the high-affinity site, without reference to chromatin structure. We propose that transcription factor binding sites should be defined more precisely using quantitative data, allowing more accurate genome-wide prediction of binding sites and greater insight into gene regulation.

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Proximity labeling of protein complexes and cell type-specific organellar proteomes in Arabidopsis enabled by TurboID

10.1101/629675 ◽

2019 ◽

Cited By ~ 3

Author(s):

Andrea Mair ◽

Shou-Ling Xu ◽

Tess C. Branon ◽

Alice Y. Ting ◽

Dominique C. Bergmann

Keyword(s):

Transcription Factor ◽

Protein Interactions ◽

Protein Complexes ◽

Cell Types ◽

Specific Protein ◽

Cell Type ◽

Subcellular Compartment ◽

Cellular Processes ◽

Highly Active

AbstractDefining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurboID), allowed us to address two challenging questions in plants: (1) what are in vivo partners of a low abundant key developmental transcription factor and (2) what is the nuclear proteome of a rare cell type? Proteins identified with FAMA-TurboID include known interactors of this stomatal transcription factor and novel proteins that could facilitate its activator and repressor functions. Directing TurboID to stomatal nuclei enabled purification of cell type- and subcellular compartment-specific proteins. Broad tests of TurboID and miniTurboID in Arabidopsis and N. benthamiana and versatile vectors enable customization by plant researchers.

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Genome-Wide Mutagenesis Links Multiple Metabolic Pathways with Actinorhodin Production inStreptomyces coelicolor

Applied and Environmental Microbiology ◽

10.1128/aem.03005-18 ◽

2019 ◽

Vol 85 (7) ◽

Cited By ~ 6

Author(s):

Zhong Xu ◽

Yuanyuan Li ◽

Yemin Wang ◽

Zixin Deng ◽

Meifeng Tao

Keyword(s):

Metabolic Pathways ◽

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Acid Biosynthesis ◽

Content Type ◽

Cellular Processes ◽

Genome Wide ◽

A Genome ◽

Wide Perspective ◽

Actinorhodin Production

ABSTRACTStreptomycesspecies are important antibiotic-producing organisms that tightly regulate their antibiotic production. Actinorhodin is a typical antibiotic produced by the model actinomyceteStreptomyces coelicolor. To discover the regulators of actinorhodin production, we constructed a library of 50,000 independent mutants with hyperactive Tn5transposase-based transposition systems. Five hundred fifty-one genes were found to influence actinorhodin production in 988 individual mutants. Genetic complementation suggested that most of the insertions (76%) were responsible for the changes in antibiotic production. Genes involved in diverse cellular processes such as amino acid biosynthesis, carbohydrate metabolism, cell wall homeostasis, and DNA metabolism affected actinorhodin production. Genome-wide mutagenesis can identify novel genes and pathways that impact antibiotic levels, potentially aiding in engineering strains to optimize the production of antibiotics inStreptomyces.IMPORTANCEPrevious studies have shown that various genes can influence antibiotic production inStreptomycesand that intercommunication between regulators can complicate antibiotic production. Therefore, to gain a better understanding of antibiotic regulation, a genome-wide perspective on genes that influence antibiotic production was needed. We searched for genes that affected production of the antibiotic actinorhodin using a genome-wide gene disruption system. We identified 551 genes that altered actinorhodin levels, and more than half of these genes were newly identified effectors. Some of these genes may be candidates for engineeringStreptomycesstrains to improve antibiotic production levels.

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Functions and properties of nuclear lncRNAs—from systematically mapping the interactomes of lncRNAs

Journal of Biomedical Science ◽

10.1186/s12929-020-00640-3 ◽

2020 ◽

Vol 27 (1) ◽

Cited By ~ 5

Author(s):

Chia-Yu Guh ◽

Yu-Hung Hsieh ◽

Hsueh-Ping Chu

Keyword(s):

Protein Complexes ◽

Nuclear Organization ◽

Regulation Of Gene Expression ◽

Nuclear Architecture ◽

Living Cells ◽

Cellular Processes ◽

Double Stranded Dna ◽

Genome Wide ◽

Non Coding Rnas

AbstractProtein and DNA have been considered as the major components of chromatin. But beyond that, an increasing number of studies show that RNA occupies a large amount of chromatin and acts as a regulator of nuclear architecture. A significant fraction of long non-coding RNAs (lncRNAs) prefers to stay in the nucleus and cooperate with protein complexes to modulate epigenetic regulation, phase separation, compartment formation, and nuclear organization. An RNA strand also can invade into double-stranded DNA to form RNA:DNA hybrids (R-loops) in living cells, contributing to the regulation of gene expression and genomic instability. In this review, we discuss how nuclear lncRNAs orchestrate cellular processes through their interactions with proteins and DNA and summarize the recent genome-wide techniques to study the functions of lncRNAs by revealing their interactomes in vivo.

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Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis Shows that WhiB Is a Transcription Factor That Cocontrols Its Regulon with WhiA To Initiate Developmental Cell Division inStreptomyces

mBio ◽

10.1128/mbio.00523-16 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 43

Author(s):

Matthew J. Bush ◽

Govind Chandra ◽

Maureen J. Bibb ◽

Kim C. Findlay ◽

Mark J. Buttner

Keyword(s):

Transcription Factor ◽

Cell Division ◽

Dna Binding ◽

Chromatin Immunoprecipitation ◽

Sequencing Analysis ◽

Founding Member ◽

Genome Wide ◽

Biochemical Function ◽

Chromatin Immunoprecipitation Sequencing

ABSTRACTWhiB is the founding member of a family of proteins (theWhiB-like [Wbl] family) that carry a [4Fe-4S] iron-sulfur cluster and play key roles in diverse aspects of the biology of actinomycetes, including pathogenesis, antibiotic resistance, and the control of development. InStreptomyces, WhiB is essential for the process of developmentally controlled cell division that leads to sporulation. The biochemical function of Wbl proteins has been controversial; here, we set out to determine unambiguously if WhiB functions as a transcription factor using chromatin immunoprecipitation sequencing (ChIP-seq) inStreptomyces venezuelae. In the first demonstration ofin vivogenome-wide Wbl binding, we showed that WhiB regulates the expression of key genes required for sporulation by binding upstream of ~240 transcription units. Strikingly, the WhiB regulon is identical to the previously characterized WhiA regulon, providing an explanation for the identical phenotypes ofwhiAandwhiBmutants. Using ChIP-seq, we demonstrated thatin vivoDNA binding by WhiA depends on WhiB and vice versa, showing that WhiA and WhiB function cooperatively to control expression of a common set of WhiAB target genes. Finally, we show that mutation of the cysteine residues that coordinate the [4Fe-4S] cluster in WhiB prevents DNA binding by both WhiB and WhiAin vivo.IMPORTANCEDespite the central importance ofWhiB-like (Wbl) proteins in actinomycete biology, a conclusive demonstration of their biochemical function has been elusive, and they have been difficult to study, particularlyin vitro, largely because they carry an oxygen-sensitive [4Fe-4S] cluster. Here we used genome-wide ChIP-seq to investigate the function ofStreptomycesWhiB, the founding member of the Wbl family. The advantage of this approach is that the oxygen sensitivity of the [4Fe-4S] cluster becomes irrelevant once the protein has been cross-linked to DNAin vivo. Our data provide the most compellingin vivoevidence to date that WhiB, and, by extension, probably all Wbl proteins, function as transcription factors. Further, we show that WhiB does not act independently but rather coregulates its regulon of sporulation genes with a partner transcription factor, WhiA.

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Homotypic cooperativity and collective binding are determinants of bHLH specificity and function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1818015116 ◽

2019 ◽

Vol 116 (32) ◽

pp. 16143-16152 ◽

Cited By ~ 7

Author(s):

Christian A. Shively ◽

Jiayue Liu ◽

Xuhua Chen ◽

Kaiser Loell ◽

Robi D. Mitra

Keyword(s):

Transcription Factor ◽

Dna Sequences ◽

Dna Binding Proteins ◽

Eukaryotic Cells ◽

Sequence Information ◽

Genome Wide ◽

Important Open Problem ◽

And Function

Eukaryotic cells express transcription factor (TF) paralogues that bind to nearly identical DNA sequences in vitro but bind at different genomic loci and perform different functions in vivo. Predicting how 2 paralogous TFs bind in vivo using DNA sequence alone is an important open problem. Here, we analyzed 2 yeast bHLH TFs, Cbf1p and Tye7p, which have highly similar binding preferences in vitro, yet bind at almost completely nonoverlapping target loci in vivo. We dissected the determinants of specificity for these 2 proteins by making a number of chimeric TFs in which we swapped different domains of Cbf1p and Tye7p and determined the effects on in vivo binding and cellular function. From these experiments, we learned that the Cbf1p dimer achieves its specificity by binding cooperatively with other Cbf1p dimers bound nearby. In contrast, we found that Tye7p achieves its specificity by binding cooperatively with 3 other DNA-binding proteins, Gcr1p, Gcr2p, and Rap1p. Remarkably, most promoters (63%) that are bound by Tye7p do not contain a consensus Tye7p binding site. Using this information, we were able to build simple models to accurately discriminate bound and unbound genomic loci for both Cbf1p and Tye7p. We then successfully reprogrammed the human bHLH NPAS2 to bind Cbf1p in vivo targets and a Tye7p target intergenic region to be bound by Cbf1p. These results demonstrate that the genome-wide binding targets of paralogous TFs can be discriminated using sequence information, and provide lessons about TF specificity that can be applied across the phylogenetic tree.

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