Effective data sharing is key to accelerating research to improve diagnostic precision, treatment efficacy, and long-term survival in pediatric cancer and other childhood catastrophic diseases. We present St. Jude Cloud (https://www.stjude.cloud), a cloud-based data-sharing ecosystem for accessing, analyzing, and visualizing genomic data from >10,000 pediatric patients with cancer and long-term survivors, and >800 pediatric sickle cell patients. Harmonized genomic data totaling 1.25 petabytes are freely available, including 12,104 whole genomes, 7,697 whole exomes, and 2,202 transcriptomes. The resource is expanding rapidly, with regular data uploads from St. Jude's prospective clinical genomics programs. Three interconnected apps within the ecosystem—Genomics Platform, Pediatric Cancer Knowledgebase, and Visualization Community—enable simultaneously performing advanced data analysis in the cloud and enhancing the Pediatric Cancer knowledgebase. We demonstrate the value of the ecosystem through use cases that classify 135 pediatric cancer subtypes by gene expression profiling and map mutational signatures across 35 pediatric cancer subtypes. Significance: To advance research and treatment of pediatric cancer, we developed St. Jude Cloud, a data-sharing ecosystem for accessing >1.2 petabytes of raw genomic data from >10,000 pediatric patients and survivors, innovative analysis workflows, integrative multiomics visualizations, and a knowledgebase of published data contributed by the global pediatric cancer community. This article is highlighted in the In This Issue feature, p. 995

Approaches to vaccine-based immunotherapy of human cancer may ultimately require targets that are both tumour-specific and immunogenic. In order to generate specific antitumour immune responses to lung cancer, we have sought lung cancer-specific proteins that can be targeted for adjuvant vaccine therapy. By using a combination of cDNA subtraction and microarray analysis, we previously reported the identification of an RNA-binding protein within the KOC family, L523S, to be overexpressed in squamous cell cancers of the lung. We show here that L523S exhibits significant potential for vaccine immunotherapy of lung cancer. As an oncofetal protein, L523S is normally expressed in early embryonic tissues, yet it is re-expressed in a high percentage of nonsmall cell lung carcinoma. The specificity of L523S expression in lung cancer was demonstrated by both mRNA and protein measurements using real-time PCR, Western blot, and immunohistochemistry analyses. Furthermore, we show that immunological tolerance of L523S is naturally broken in lung cancer patients, as evidenced by detectable antibody responses to recombinant L523S protein in eight of 17 lung pleural effusions from lung cancer patients. Collectively, our studies suggest that L523S may be an important marker of malignant progression in human lung cancer, and further suggest that treatment approaches based on L523S as an immunogenic target are worthy of pursuit.

The general transcription factor IIB (TFIIB) is required for accurate and efficient transcription of protein-coding genes by RNA polymerase II (RNAPII). To define functional domains in the highly conserved Nterminal region of TFIIB, we have analyzed 14 site-directed substitution mutants of yeast TFIIB for their ability to support cell viability, transcription in vitro, accurate start site selection in vitro and in vivo, and to form stable complexes with purified RNAPII in vitro. Mutations impairing the formation of stable TFIIB⅐RNAPII complexes mapped to the zinc ribbon fold, whereas mutations conferring downstream shifts in transcription start site selection were identified at multiple positions within a highly conserved homology block adjacent and C-terminal to the zinc ribbon. These results demonstrate that the N-terminal region of yeast TFIIB contains two separable and adjacent functional domains involved in stable RNAPII binding and transcription start site selection, suggesting that downstream shifts in transcription start site selection do not result from impairment of stable TFIIB⅐RNAPII binding. We discuss models for yeast start site selection in which TFIIB may affect the ability of preinitiation complexes to interact with downstream DNA or to affect start site recognition by a scanning polymerase. Eukaryotic RNA polymerase II (RNAPII)1 requires the action of at least six accessory proteins to accurately initiate transcription. These accessory proteins, termed the general transcription factors (GTFs), have been the focus of much investigation and include TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. The GTFs and RNAPII assemble in an ordered stepwise fashion on a class II promoter in vitro to form a functional preinitiation complex (PIC) (reviewed in Ref. 1). Assembly is initiated by the binding of TFIID to the TATA element via the TATA-binding protein (TBP) subunit, in some cases assisted by TFIIA. This complex is recognized by TFIIB, which binds and recruits RNAPII and TFIIF. PIC formation is completed by the association of TFIIE and then TFIIH, and the resulting complex can hydrolyze ATP and initiate mRNA synthesis. In contrast to this ordered-assembly model for PIC formation, it has been proposed that a preassembled holoenzyme, consisting of RNAPII, most of the GTFs, and additional factors, is recruited in one step to promoter-bound TFIID in vivo (reviewed in Ref. 1).The general transcription factor TFIIB has an essential role in RNAPII transcription, and together with RNAPII and TBP, defines the minimal set of factors necessary for promoter-dependent transcription of a supercoiled DNA template in vitro (1). In both the ordered-assembly and holoenzyme-recruitment models of PIC formation, TFIIB recognizes promoter-bound TFIID and facilitates association of the remaining GTFs and RNAPII. Consistent with this role, TFIIB interacts with DNA adjacent to the TATA box (2) and binds to TBP (3-5), the TBP-associated factor TAF40 (6), RNAPII (4,7,8), and both subunits of TFIIF (4, 9). TFIIB may also play a r...

The general transcription factor IIB (TFIIB) plays an essential role in transcription of protein-coding genes by RNA polymerase II. We have used site-directed mutagenesis to assess the role of conserved amino acids in several important regions of yeast TFIIB. These include residues in the highly conserved amino-terminal region and basic residues in the D1 and E1 core domain ␣-helices. Acidic substitutions of residues K190 (D1) and K201 (E1) resulted in growth impairments in vivo, reduced basal transcriptional activity in vitro, and an inability to form stable TFIIB-TATA-binding protein-DNA (DB) complexes. Significantly, these mutants retained the ability to respond to acidic activators in vivo and to the Gal4-VP16 activator in vitro, supporting the view that these basic residues play a role in basal transcription. In addition, 14 single-amino-acid substitutions were introduced in the conserved amino-terminal region. Three of these mutants, the L50D, R64E, and R78L mutants, displayed altered growth properties in vivo and were compromised for supporting transcription in vitro. The L50D mutant was impaired for RNA polymerase II interaction, while the R64E mutant exhibited altered transcription start site selection both in vitro and in vivo and, surprisingly, was more active than the wild type in the formation of stable DB complexes. These results support the view that the amino-terminal domain is involved in the direct interaction between yeast TFIIB and RNA polymerase II and suggest that this domain may interact with DNA and/or modulate the formation of a DB complex.To specifically initiate transcription, eukaryotic RNA polymerase II (RNAPII) requires the assistance of at least six distinct auxiliary factors, commonly known as the general transcription factors (GTFs). The formation of an active preinitiation complex on the promoter of a protein-coding gene is thought to occur by the initial binding of transcription factor IID (TFIID) to the TATA element, followed either by an ordered assembly of the remaining GTFs and RNAPII or by the direct recruitment of a preassembled RNAPII holoenzyme (reviewed in reference 26). Some fundamental goals regarding the investigation of RNAPII transcription are to determine the structure and functions of the GTFs, elucidate the interactions between them and their mode of association, and determine the precise molecular mechanisms by which transcription complex assembly or recruitment and the ensuing processes of initiation and elongation are regulated.The general transcription factor TFIIB plays an essential role in RNAPII transcription (reviewed in reference 26). It has been proposed that the primary function of TFIIB is to act as a physical bridge between TFIID bound at the TATA element and the remainder of the preinitiation complex. Consistent with this view, biochemical studies have shown that TFIIB interacts with several components of the general transcription machinery, including TFIIF (13, 16), RNAPII (8, 16), TATAbinding protein (TBP) (6,16,24), and the TBP-associated facto...