7 research outputs found
Ste7 variants that promote pathway specificity in S. cerevisiae
No full textThe yeast S. cerevisiae must respond to a wide variety of external stimuli with the appropriate differential program to ensure survival. Yeast cells transmit signals from external environmental cues via signaling cascades to bring about proper transcriptional programs. One such signaling cascade, the Mitogen Activated Protein Kinase (MAPK) cascade, is conserved in all organisms. The MAPK cascade that contains the sequentially acting kinases Ste20, Ste11, Ste7 and Kss1 is used in both filamentous growth differentiation in response to limiting nutrients as well as differentiation in response to mating pheromone. Despite the shared components signaling fidelity remains. The central kinase in both the mating and filamentous growth differentiation pathways is Ste7. We used a constitutively active version of this kinase to examine if biological specificity is lost by removing the need for external stimulus. We found that the constitutively active Ste7 promoted filamentous growth while simultaneously failing to support mating differentiation. One version of constitutively active Ste7 that could not be feedback phosphorylated in a MAPK dependent manner was unable to bind to the MAPK Fus3 but retained the ability to bind to Ste5 and Kss1. Despite being able to bind to Ste5 this version of Ste7 was unable to activate Fus3 but retained the ability to activate Kss1. The activation of Kss1 by constitutively active Ste7 is independent of the scaffold protein Ste5. Unlike Kss1, Fus3 activation requires Ste5. We isolated a substitution mutation variant of Ste7 that was impaired for binding to Ste5 but not impaired for activation of the transcriptional reporter gene for filamentous growth. This variant of Ste7 neither activated Fus3 nor supported mating differentiation in response to pheromone. Yeast cells expressing this variant of Ste7 displayed higher levels of phosphorylated Mpk1, the MAPK that functions in response to plasma membrane distortions. Our findings uncovered a role for Ste5 in maintaining signal specificity by allowing for specific activation of Fus3 by Ste7 in response to mating pheromone. We also uncovered a novel role that Mpk1 may play in the transcriptional response during filamentous growth. These data suggest MAPK signaling occurs through a network rather than linear pathways
Native Elongating Transcript Sequencing Reveals Human Transcriptional Activity at Nucleotide Resolution
No full textSummaryMajor features of transcription by human RNA polymerase II (Pol II) remain poorly defined due to a lack of quantitative approaches for visualizing Pol II progress at nucleotide resolution. We developed a simple and powerful approach for performing native elongating transcript sequencing (NET-seq) in human cells that globally maps strand-specific Pol II density at nucleotide resolution. NET-seq exposes a mode of antisense transcription that originates downstream and converges on transcription from the canonical promoter. Convergent transcription is associated with a distinctive chromatin configuration and is characteristic of lower-expressed genes. Integration of NET-seq with genomic footprinting data reveals stereotypic Pol II pausing coincident with transcription factor occupancy. Finally, exons retained in mature transcripts display Pol II pausing signatures that differ markedly from skipped exons, indicating an intrinsic capacity for Pol II to recognize exons with different processing fates. Together, human NET-seq exposes the topography and regulatory complexity of human gene expression
Author Correction: Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade
No full textAbstract PR9: Loss of PD-1 promotes antitumor immunity by improving functions of both PD-1+ and PD-1- CD8+ T cells in the tumor microenvironment
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