Enhancer Sharing Promotes Neighborhoods of Transcriptional Regulation Across Eukaryotes

Enhancers physically interact with transcriptional promoters, looping over distances that can span multiple regulatory elements. Given that enhancer–promoter (EP) interactions generally occur via common protein complexes, it is unclear whether EP pairing is predominantly deterministic or proximity guided. Here, we present cross-organismic evidence suggesting that most EP pairs are compatible, largely determined by physical proximity rather than specific interactions. By reanalyzing transcriptome datasets, we find that the transcription of gene neighbors is correlated over distances that scale with genome size. We experimentally show that nonspecific EP interactions can explain such correlation, and that EP distance acts as a scaling factor for the transcriptional influence of an enhancer. We propose that enhancer sharing is commonplace among eukaryotes, and that EP distance is an important layer of information in gene regulation.

Cell-free translation is more variable than transcription

ABSTRACTAlthough RNA synthesis can be reliably controlled with different T7 transcriptional promoters during cell-free gene expression with the PURE system, protein synthesis remains largely unaffected. To better control protein levels, a series of ribosome binding sites (RBS) was investigated. While RBS strength did strongly affect protein synthesis, the RBS sequence could explain less than half of the variability of the data. Protein expression was found to depend on other factors besides the strength of the RBS, including GC content. The complexity of protein synthesis in comparison to RNA synthesis was observed by the higher degree of variability associated with protein expression. This variability was also observed in an E. coli cell extract-based system. However, the coefficient of variation was larger with E. coli RNA polymerase than with T7 RNA polymerase, consistent with the increased complexity of E. coli RNA polymerase.

Enhancer sharing promotes neighborhoods of transcriptional regulation across eukaryotes

Enhancers physically interact with transcriptional promoters, looping over distances that can span multiple regulatory elements. Given that enhancer-promoter (EP) interactions generally occur via common protein complexes, it is unclear whether EP pairing is predominantly deterministic or proximity guided. Here we present cross-organismic evidence suggesting that most EP pairs are compatible, largely determined by physical proximity rather than specific interactions. By re-analyzing transcriptome datasets, we find that the transcription of gene neighbors is correlated over distances that scale with genome size. We experimentally show that non-specific EP interactions can explain such correlation, and that EP distance acts as a scaling factor for the transcriptional influence of an enhancer. We propose that enhancer sharing is commonplace among eukaryotes, and that EP distance is an important layer of information in gene regulation.

Downregulation of vaccinia virus intermediate and late promoters by host transcription factor YY1

Approximately half of the intermediate and late gene transcriptional promoters of vaccinia virus have a binding site for the cellular transcription factor YY1 that overlaps the initiator elements. Depletion of YY1 using RNA interference enhanced the activity of these promoters, while overexpression of YY1 repressed their activity. Viral promoter nucleotide replacements that specifically impair the binding of YY1 mostly alleviated the transcriptional repression and correlated with the ability of YY1 to stably interact with the initiator DNAs in vitro. The transcriptional repression activity was localized to the C-terminal DNA-binding domain of the protein. These results indicate that YY1 functions to negatively regulate these vaccinia virus promoters by binding to their initiator elements.

Redundancy of Enzymes for Formaldehyde Detoxification in Pseudomonas putida

ABSTRACT Pseudomonas putida KT2440 exhibits redundant formaldehyde dehydrogenases and formate dehydrogenases that contribute to the detoxification of formaldehyde, a highly toxic compound. Physical and transcriptional analyses showed that the open reading frame (ORF) PP0328, encoding one of the formaldehyde dehydrogenases, is self-sufficient, whereas the other functional formaldehyde dehydrogenase gene (ORF PP3970) forms an operon with another gene of unknown function. Two formate dehydrogenase gene clusters (PP0489 to PP0492 and PP2183 to PP2186) were identified, and genes in these clusters were found to form operons. All four transcriptional promoters were mapped by primer extension and revealed the presence of noncanonical promoters expressed at basal level in the exponential growth phase and at a higher level in the stationary phase regardless of the presence of extracellular formaldehyde or formate. These promoters were characterized by a 5′-AG-CCA-C/A-CT-3′ conserved region between −7 and −16. To determine the contribution of the different gene products to formaldehyde and formate mineralization, mutants with single and double mutations of formaldehyde dehydrogenases were generated, and the effect of the mutations on formaldehyde catabolism was tested by measuring 14CO2 evolution from 14C-labeled formaldehyde. The results showed that both enzymes contributed to formaldehyde catabolism. A double mutant lacking these two enzymes still evolved CO2 from formaldehyde, suggesting the presence of one or more still-unidentified formaldehyde dehydrogenases. Mutants with single and double mutations in the clusters for formate dehydrogenases were also generated, and all of them were able to metabolize [14C]formate to 14CO2, suggesting a redundancy of functions that was not limited to only the annotated genes. Single and double mutants deficient in formaldehyde dehydrogenases and formate dehydrogenases exhibited longer lag phases than did the parental strain when confronted with concentrations of formaldehyde close to the MICs. This suggests a role for the detoxification system in tolerance to sublethal concentrations of formaldehyde.