Molecular mechanisms that distinguish TFIID housekeeping from regulatable SAGA promoters

An important distinction is frequently made between constitutively expressed housekeeping genes versus regulated genes. Although generally characterized by different DNA elements, chromatin architecture and cofactors, it is not known to what degree promoter classes strictly follow regulatability rules and which molecular mechanisms dictate such differences. We show that SAGA-dominated/wTATA-box promoters are more responsive to changes in the amount of activator, even compared to TFIID/TATA-like promoters that depend on the same activator Hsf1. Regulatability is therefore an inherent property of promoter class. Further analyses show that SAGA/TATA-box promoters are more dynamic because TATA-binding protein recruitment through SAGA is susceptible to removal by Mot1. In addition, the nucleosome configuration upon activator depletion shifts on SAGA/TATA-box promoters and seems less amenable to preinitiation complex formation. The results explain the fundamental difference between housekeeping and regulatable genes, revealing an additional facet of combinatorial control: an activator can elicit a different response dependent on core promoter class

Gcn4 misregulation reveals a direct role for the evolutionary conserved EKC/KEOPS in the t6A modification of tRNAs

The EKC/KEOPS complex is universally conserved in Archaea and Eukarya and has been implicated in several cellular processes, including transcription, telomere homeostasis and genomic instability. However, the molecular function of the complex has remained elusive so far. We analyzed the transcriptome of EKC/KEOPS mutants and observed a specific profile that is highly enriched in targets of the Gcn4p transcriptional activator. GCN4 expression was found to be activated at the translational level in mutants via the defective recognition of the inhibitory upstream ORFs (uORFs) present in its leader. We show that EKC/KEOPS mutants are defective for the N6-threonylcarbamoyl adenosine modification at position 37 (t6A37) of tRNAs decoding ANN codons, which affects initiation at the inhibitory uORFs and provokes Gcn4 de-repression. Structural modeling reveals similarities between Kae1 and bacterial enzymes involved in carbamoylation reactions analogous to t6A37 formation, supporting a direct role for the EKC in tRNA modification. These findings are further supported by strong genetic interactions of EKC mutants with a translation initiation factor and with threonine biosynthesis genes. Overall, our data provide a novel twist to understanding the primary function of the EKC/KEOPS and its impact on several essential cellular functions like transcription and telomere homeostasis

Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. As novel and improved immunotherapies may fill this need, we dissected the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 25 tumors (10 pre- and 15 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas were infiltrated by NK, T and B cells, and immunosuppressive myeloid populations. NK cells showed reduced cytotoxicity and T cells had a dysfunctional profile. Interaction analysis revealed a vast immunoregulatory network and identified NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduced neuroblastoma growth, with complete responses in vivo. Moreover, addition of TIGIT blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model significantly improved survival. Concluding, our integrative analysis of neuroblastoma’s vast immunoregulatory network provides novel targets and a rationale for immunotherapeutic combination strategies

Front of Stratenum building

<p>Front of Stratenum building</p