Selective Activation of Alternative MYC Core Promoters by Wnt-Responsive Enhancers.

In Metazoans, transcription of most genes is driven by the use of multiple alternative promoters. Although the precise regulation of alternative promoters is important for proper gene expression, the mechanisms that mediates their differential utilization remains unclear. Here, we investigate how the two alternative promoters (P1, P2) that drive MYC expression are regulated. We find that P1 and P2 can be differentially regulated across cell-types and that their selective usage is largely mediated by distal regulatory sequences. Moreover, we show that in colon carcinoma cells, Wnt-responsive enhancers preferentially upregulate transcription from the P1 promoter using reporter assays and in the context of the endogenous Wnt induction. In addition, multiple enhancer deletions using CRISPR/Cas9 corroborate the regulatory specificity of P1. Finally, we show that preferential activation between Wnt-responsive enhancers and the P1 promoter is influenced by the distinct core promoter elements that are present in the MYC promoters. Taken together, our results provide new insight into how enhancers can specifically target alternative promoters and suggest that formation of these selective interactions could allow more precise combinatorial regulation of transcription initiation

Selective Activation of Alternative MYC Core Promoters by Wnt-Responsive Enhancers

In Metazoans, transcription of most genes is driven by the use of multiple alternative promoters. Although the precise regulation of alternative promoters is important for proper gene expression, the mechanisms that mediates their differential utilization remains unclear. Here, we investigate how the two alternative promoters (P1, P2) that drive MYC expression are regulated. We find that P1 and P2 can be differentially regulated across cell-types and that their selective usage is largely mediated by distal regulatory sequences. Moreover, we show that in colon carcinoma cells, Wnt-responsive enhancers preferentially upregulate transcription from the P1 promoter using reporter assays and in the context of the endogenous Wnt induction. In addition, multiple enhancer deletions using CRISPR/Cas9 corroborate the regulatory specificity of P1. Finally, we show that preferential activation between Wnt-responsive enhancers and the P1 promoter is influenced by the distinct core promoter elements that are present in the MYC promoters. Taken together, our results provide new insight into how enhancers can specifically target alternative promoters and suggest that formation of these selective interactions could allow more precise combinatorial regulation of transcription initiation

Metabolismo de hidrato de cloral en ratas con insuficiencia hepática inducida por insuficiencia hepática inducida por tetracloruro de carbono

Objetivo: Determinar el metabolismo del hidrato de cloral en ratas bajo condiciones normales y en ratas con insuficiencia hepática inducida con tetracloruro de carbono. Material y métodos: Se usaron ratas macho de la cepa Holtzman. El proceso de inducción de la insuficiencia hepática se realizó durante 3 días consecutivos; este efecto fue confirmado a través de cortes histológicos del hígado, administrándose luego hidrato de cloral por vía oral en ambos grupos. Posteriormente, se realizó el dosaje en plasma de tricloroetanol - metabolito activo del hidrato de cloral - por el método de cromatografía gaseosa con un detector de ionización de llama, realizándose la validación de dicho método. Resultados: Se encontró una diferencia significativa en la cantidad de tricloroetanol entre los grupos con y sin insuficiencia hepática. Se reporta los períodos de latencia, duración e intensidad del efecto del hidrato de cloral, en todos los grupos en estudio. Conclusiones: El metabolismo del hidrato de cloral se encuentra alterado en los grupos con insuficiencia hepática, el nivel de alteración del metabolismo puede ser cuantificado a través del dosaje de tricloroetanol, el cual fue realizado optimizando un método existente

New insights into the regulatory mechanisms of ppGpp and DksA on Escherichia coli RNA polymerase-promoter complex.

The stringent response modulators, guanosine tetraphosphate (ppGpp) and protein DksA, bind RNA polymerase (RNAP) and regulate gene expression to adapt bacteria to different environmental conditions. Here, we use Atomic Force Microscopy and in vitro transcription assays to study the effects of these modulators on the conformation and stability of the open promoter complex (RPo) formed at the rrnA P1, rrnB P1, its discriminator (dis) variant and λ pR promoters. In the absence of modulators, RPo formed at these promoters show different extents of DNA wrapping which correlate with the position of UP elements. Addition of the modulators affects both DNA wrapping and RPo stability in a promoter-dependent manner. Overall, the results obtained under different conditions of ppGpp, DksA and initiating nucleotides (iNTPs) indicate that ppGpp allosterically prevents the conformational changes associated with an extended DNA wrapping that leads to RPo stabilization, while DksA interferes directly with nucleotide positioning into the RNAP active site. At the iNTPs-sensitive rRNA promoters ppGpp and DksA display an independent inhibitory effect, while at the iNTPs-insensitive pR promoter DksA reduces the effect of ppGpp in accordance with their antagonistic role

New insights into the regulatory mechanisms of ppGpp and DksA on Escherichia coli RNA polymerase-promoter complex

The stringent response modulators, guanosine tetraphosphate (ppGpp) and protein DksA, bind RNA polymerase (RNAP) and regulate gene expression to adapt bacteria to different environmental conditions. Here, we use Atomic Force Microscopy and in vitro transcription assays to study the effects of these modulators on the conformation and stability of the open promoter complex (RPo) formed at the rrnA P1, rrnB P1, its discriminator (dis) variant and λ pR promoters. In the absence of modulators, RPo formed at these promoters show different extents of DNA wrapping which correlate with the position of UP elements. Addition of the modulators affects both DNA wrapping and RPo stability in a promoter-dependent manner. Overall, the results obtained under different conditions of ppGpp, DksA and initiating nucleotides (iNTPs) indicate that ppGpp allosterically prevents the conformational changes associated with an extended DNA wrapping that leads to RPo stabilization, while DksA interferes directly with nucleotide positioning into the RNAP active site. At the iNTPs-sensitive rRNA promoters ppGpp and DksA display an independent inhibitory effect, while at the iNTPs-insensitive pR promoter DksA reduces the effect of ppGpp in accordance with their antagonistic role

Rational design of a split-Cas9 enzyme complex.

Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications

Rational design of a split-Cas9 enzyme complex

Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications