Hierarchical TAF1-dependent co-translational assembly of the basal transcription factor TFIID.

Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunit basal transcription factor TFIID nucleates the RNA polymerase II preinitiation complex at gene promoters. Here, by combining systematic RNA-immunoprecipitation (RIP) experiments, single-molecule imaging, proteomics and structure-function analyses, we show that human TFIID biogenesis occurs co-translationally. We discovered that all protein heterodimerization steps happen during protein synthesis. We identify TAF1-the largest protein in the complex-as a critical factor for TFIID assembly. TAF1 acts as a flexible scaffold that drives the co-translational recruitment of TFIID submodules preassembled in the cytoplasm. Altogether, our data suggest a multistep hierarchical model for TFIID biogenesis that culminates with the co-translational assembly of the complex onto the nascent TAF1 polypeptide. We envision that this assembly strategy could be shared with other large heteromeric protein complexes.

Hierarchical TAF1-dependent co-translational assembly of the basal transcription factor TFIID.

Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunit basal transcription factor TFIID nucleates RNA polymerase II preinitiation complex at gene promoters. Here, by combining systematic RNA-immunoprecipitation (RIP) experiments, single-molecule imaging, proteomics and structure-function analyses, we show that TFIID biogenesis occurs co-translationally. We discovered that all protein heterodimerization steps happen during protein synthesis. We identify TAF1 - the largest protein in the complex - as a critical factor for TFIID assembly. TAF1 acts as a flexible scaffold that drives the co-translational recruitment of TFIID submodules preassembled in the cytoplasm. Altogether, our data suggest a multistep hierarchical model for TFIID biogenesis that culminates with the co-translational assembly of the complex onto the nascent TAF1 polypeptide. We envision that this assembly strategy could be shared with other large heteromeric protein complexes.

A new method of cryopreserving colorectal carcinoma cells for patient derived xenograft model generation.

Patient derived xenograft (PDX) models provide an efficient way to study anti-tumor drug efficacy. In this respect, it is essential to study the optimal method needed to cryopreserve the starting cells obtained from tumor samples for PDX model generation. Cryopreservation of cells prior to xenografting is necessary for cross-verification of results obtained by xenografting and also for practical planning of experiments. In the present work, we studied the cryopreservation of colorectal carcinoma (CRC) cells isolated from patient tumor samples for generating their patient derived xenograft models. CRC therapeutics study is essential for early stage intervention and treatment of the disease. CRC cell lines do not ideally depict the molecular characteristics of patient CRC tumor samples. This necessitates the generation of CRC PDX models for drug discovery. We show that CRC cells isolated from patient tumor samples have comparable recovery, viability and growth with both conventional cryopreservation methods as well as Fibulas BioFlash Drive™. However, xenograft tumor formation was much more effective with Fibulas BioFlash Drive™ cryopreserved cells than with cells cryopreserved with conventional methods. Therefore, we put forward an effective way to cryopreserve primary cells obtained from patient tumor samples for PDX model generation in this study.

Engagement of Components of DNA-Break Repair Complex and NFκB in Hsp70A1A Transcription Upregulation by Heat Shock.

An involvement of components of DNA-break repair (DBR) complex including DNA-dependent protein kinase (DNA-PK) and poly-ADP-ribose polymerase 1 (PARP-1) in transcription regulation in response to distinct cellular signalling has been revealed by different laboratories. Here, we explored the involvement of DNA-PK and PARP-1 in the heat shock induced transcription of Hsp70A1A. We find that inhibition of both the catalytic subunit of DNA-PK (DNA-PKc), and Ku70, a regulatory subunit of DNA-PK holo-enzyme compromises transcription of Hsp70A1A under heat shock treatment. In immunoprecipitation based experiments we find that Ku70 or DNA-PK holoenzyme associates with NFκB. This NFκB associated complex also carries PARP-1. Downregulation of both NFκB and PARP-1 compromises Hsp70A1A transcription induced by heat shock treatment. Alteration of three bases by site directed mutagenesis within the consensus κB sequence motif identified on the promoter affected inducibility of Hsp70A1A transcription by heat shock treatment. These results suggest that NFκB engaged with the κB motif on the promoter cooperates in Hsp70A1A activation under heat shock in human cells as part of a DBR complex including DNA-PK and PARP-1.