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Integrated requirement of non-specific and sequence-specific DNA binding in MYC-driven transcription

ABSTRACT: Eukaryotic transcription factors recognize specific DNA sequence motifs, but are also endowed with generic, non-specific DNA-binding activity: how these binding modes are integrated to determine select transcriptional outputs remains unresolved. We addressed this question by site-directed mutagenesis of the Myc transcription factor. Impairment of non-specific DNA backbone contacts caused pervasive loss of genome interactions and gene regulation, associated with increased intra-nuclear mobility of the Myc protein in murine cells. In contrast, a mutant lacking base-specific contacts retained DNA binding and mobility profiles comparable to those of the wild-type protein, but failed to recognize its consensus binding motif (E-box) and could not activate Myc-target genes. Incidentally, this mutant gained weak affinity for an alternative motif, driving aberrant activation of different genes. Altogether, we conclude that non-specific DNA binding is required to engage onto genomic regulatory regions; sequence recognition in turn contributes to transcriptional activation, acting at distinct levels: stabilization and positioning of Myc onto DNA, and -unexpectedly- promotion of its transcriptional activity. Hence, pervasive genome interaction profiles actually encompass diverse DNA-binding modalities, driving defined, sequence-dependent transcriptional responses.

SUBMITTER: Dr. Paola Pellanda

PROVIDER: S-SCDT-EMBOJ-2020-105464 | biostudies-other |

REPOSITORIES: biostudies-other

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Integrated requirement of non-specific and sequence-specific DNA binding in Myc-driven transcription.

Project description:Eukaryotic transcription factors recognize specific DNA sequence motifs, but are also endowed with generic, non-specific DNA-binding activity. How these binding modes are integrated to determine select transcriptional outputs remains unresolved. We addressed this question by site-directed mutagenesis of the Myc transcription factor. Impairment of non-specific DNA backbone contacts caused pervasive loss of genome interactions and gene regulation, associated with increased intra-nuclear mobility of the Myc protein in murine cells. In contrast, a mutant lacking base-specific contacts retained DNA-binding and mobility profiles comparable to those of the wild-type protein, but failed to recognize its consensus binding motif (E-box) and could not activate Myc-target genes. Incidentally, this mutant gained weak affinity for an alternative motif, driving aberrant activation of different genes. Altogether, our data show that non-specific DNA binding is required to engage onto genomic regulatory regions; sequence recognition in turn contributes to transcriptional activation, acting at distinct levels: stabilization and positioning of Myc onto DNA, and-unexpectedly-promotion of its transcriptional activity. Hence, seemingly pervasive genome interaction profiles, as detected by ChIP-seq, actually encompass diverse DNA-binding modalities, driving defined, sequence-dependent transcriptional responses.

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Integrated requirement of non-specific and sequence-specific DNA binding for transcriptional regulation by Myc

Project description:This SuperSeries is composed of the SubSeries listed below.

2021-02-05 | GSE147639 | GEO

Integrated requirement of non-specific and sequence-specific DNA binding for transcriptional regulation by Myc (ChIP-seq)

Project description:Eukaryotic transcription factors recognize specific DNA sequence motifs, but are also endowed with generic, non-specific DNA-binding activity: how these binding modes are integrated to determine select transcriptional outputs remains unresolved. We designed mutants of the MYC transcription factor bearing substitutions in residues that contact either the DNA backbone or specific bases within the consensus motif (E-box), and profiled their DNA-binding and gene-regulatory properties in cultured cells. Our data reveal that non-specific DNA binding is required for MYC to target genomic regulatory regions, and a prerequisite for sequence recognition; beyond merely stabilizing MYC onto select target loci, sequence-specific binding contributes to its precise positioning and – most unexpectedly – to transcriptional activity per se. Hence, seemingly promiscuous genome interaction profiles encompass diverse binding modalities and drive defined, sequence-dependent transcriptional responses.

2021-02-05 | GSE147638 | GEO

Integrated requirement of non-specific and sequence-specific DNA binding for transcriptional regulation by Myc (RNA-seq)

2021-02-05 | GSE147637 | GEO

Integrated requirement of non-specific and sequence-specific DNA binding for transcriptional regulation by Myc

Project description:Integrated requirement of non-specific and sequence-specific DNA binding for transcriptional regulation by Myc

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Integrated requirement of non-specific and sequence-specific DNA binding for transcriptional regulation by Myc (ChIP-seq)

Project description:Integrated requirement of non-specific and sequence-specific DNA binding for transcriptional regulation by Myc (ChIP-seq)

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Integrated requirement of non-specific and sequence-specific DNA binding for transcriptional regulation by Myc (RNA-seq)

Project description:Integrated requirement of non-specific and sequence-specific DNA binding for transcriptional regulation by Myc (RNA-seq)

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Sequence-specific DNA binding by MYC/MAX to low-affinity non-E-box motifs.

Project description:The MYC oncoprotein regulates transcription of a large fraction of the genome as an obligatory heterodimer with the transcription factor MAX. The MYC:MAX heterodimer and MAX:MAX homodimer (hereafter MYC/MAX) bind Enhancer box (E-box) DNA elements (CANNTG) and have the greatest affinity for the canonical MYC E-box (CME) CACGTG. However, MYC:MAX also recognizes E-box variants and was reported to bind DNA in a "non-specific" fashion in vitro and in vivo. Here, in order to identify potential additional non-canonical binding sites for MYC/MAX, we employed high throughput in vitro protein-binding microarrays, along with electrophoretic mobility-shift assays and bioinformatic analyses of MYC-bound genomic loci in vivo. We identified all hexameric motifs preferentially bound by MYC/MAX in vitro, which include the low-affinity non-E-box sequence AACGTT, and found that the vast majority (87%) of MYC-bound genomic sites in a human B cell line contain at least one of the top 21 motifs bound by MYC:MAX in vitro. We further show that high MYC/MAX concentrations are needed for specific binding to the low-affinity sequence AACGTT in vitro and that elevated MYC levels in vivo more markedly increase the occupancy of AACGTT sites relative to CME sites, especially at distal intergenic and intragenic loci. Hence, MYC binds diverse DNA motifs with a broad range of affinities in a sequence-specific and dose-dependent manner, suggesting that MYC overexpression has more selective effects on the tumor transcriptome than previously thought.

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Potent stimulation of transcription-coupled DNA supercoiling by sequence-specific DNA-binding proteins.

Project description:Transcription by RNA polymerase can stimulate localized DNA supercoiling in Escherichia coli. In vivo, there is extensive experimental support for a "twin-domain" model in which positive DNA supercoils are generated ahead of a translocating RNA polymerase complex and negative supercoils are formed behind it. Negative supercoils accumulate in the template DNA because the positive supercoils are preferentially removed by cellular topoisomerase action. Yet, in vitro, clear and convincing support for the twin-domain mechanism has been lacking. In this article, we reconcile this inconsistency by showing that, in a defined in vitro system with plasmid DNA templates, a variety of sequence-specific DNA-binding proteins, such as the bacteriophage lambda O replication initiator or the E. coli lactose or galactose repressors, strikingly stimulate transcription-coupled DNA supercoiling. We demonstrate further that this stimulation requires the presence in the DNA template of a recognition sequence for the relevant DNA-binding protein and depends on the production of long RNA chains by an RNA polymerase. Our data are most consistent with a model in which specific DNA-binding proteins facilitate a twin-domain mechanism to enhance DNA supercoiling during transcription. More precisely, we suggest that some nucleoprotein complexes, perhaps those that contain sharply bent DNA, can form barriers that impede the diffusion and merger of independent chromosomal supercoil domains. Localization of DNA supercoils by nucleoprotein complexes may serve as a general mechanism for modulating DNA transactions that are sensitive to DNA superhelicity.

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