Reactivation of telomerase reverse transcriptase (TERT) expression enables cells to overcome replicative senescence and escape apoptosis, fundamental steps in the initiation of human cancer. Multiple cancer types, including up to 83% of glioblastomas (GBM), harbor highly recurrent TERT promoter mutations of unknown function but specific to two nucleotide positions. We identify the functional consequence of these mutations in GBM to be recruitment of the multimeric GABP transcription factor specifically to the mutant promoter. Allelic recruitment of GABP is consistently observed across four cancer types, highlighting a shared mechanism underlying TERT reactivation. Tandem flanking native ETS motifs critically cooperate with these mutations to activate TERT, likely by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression in multiple cancers.

The G-rich single stranded DNA at the 3′ end of human telomeres can self-fold into G-quaduplex (GQ). However, telomere lengthening by telomerase or the recombination-based alternative lengthening of telomere (ALT) mechanism requires protein loading on the overhang. Using single molecule fluorescence spectroscopy we discovered that lengthening the telomeric overhang also increased the rate of dynamic exchanges between structural conformations. Overhangs with five to seven TTAGGG repeats, compared to four repeats, showed much greater dynamics and accessibility to telomerase binding and activity, and loading of the ALT-associated proteins RAD51, WRN and BLM. Although the eight repeats are highly dynamic, they can fold into two GQs, which limited protein accessibility. In contrast, the telomere-specific protein, POT1 is unique in that it binds independently of repeat number. Our results suggest that the telomeric overhang length and dynamics may contribute to the regulation of telomere extension via telomerase action and the ALT mechanism.

G-quadruplexes (GQs) are alternative DNA secondary structures that can form throughout the human genome and control the replication and transcription of important regulatory genes. Here, we established an ensemble fluorescence assay by employing two GQ-interacting compounds, N-methyl mesoporphyrin IX (NMM) and Crystal Violet (CV). This enables quantitative measurement of the GQ folding propensity and conformation specificity in both single strand (ss) and double strand (ds) DNA. Our GQ mapping indicates that the likelihood of GQ formation is substantially diminished in dsDNA, likely due to the competition from the Watson–Crick base pairing. Unlike GQ folding sequence in ssDNA which forms both parallel and antiparallel GQs, dsDNA displays only parallel folding. Additionally, we employed single molecule FRET to obtain a direct quantitation of stably formed-, weakly folded and unfolded GQ conformations. The findings of this study and the method developed here will enable identifying and classifying potential GQ-forming sequences in human genome.

G-quadruplex (GQ) is a four-stranded DNA structure that can be formed in guanine-rich sequences. GQ structures have been proposed to regulate diverse biological processes including transcription, replication, translation and telomere maintenance. Recent studies have demonstrated the existence of GQ DNA in live mammalian cells and a significant number of potential GQ forming sequences in the human genome. We present a systematic and quantitative analysis of GQ folding propensity on a large set of 438 GQ forming sequences in double-stranded DNA by integrating fluorescence measurement, single-molecule imaging and computational modeling. We find that short minimum loop length and the thymine base are two main factors that lead to high GQ folding propensity. Linear and Gaussian process regression models further validate that the GQ folding potential can be predicted with high accuracy based on the loop length distribution and the nucleotide content of the loop sequences. Our study provides important new parameters that can inform the evaluation and classification of putative GQ sequences in the human genome.

Reactivation of telomerase reverse transcriptase (TERT) expression enables cells to overcome replicative senescence and escape apoptosis, fundamental steps in the initiation of human cancer. Multiple cancer types, including up to 83% of glioblastomas (GBM), harbor highly recurrent mutations in the TERT promoter specific to two nucleotide positions. The common mutation sites, G228A and G250A, may create de-novo ETS family transcription factor binding sites, but the precise mechanism of how these mutations confer increased TERT expression has been elusive. Here, we demonstrate the de-novo ETS motif to be critical for mutant TERT activation by site directed mutagenesis. A focused siRNA screen of the many ETS transcription factors expressed in GBM identifies GABPA as the single ETS factor to selectively regulate the mutant but not the wild type TERT promoter. Single molecule binding assays and ChIP-qPCR analysis reveal that GABPA is exclusively recruited to the mutant allele in vitro and in vivo respectively. Furthermore, this allelic recruitment is consistent across four tested cancer types, highlighting a shared mechanism underlying mutant TERT promoter activation. Tandem flanking native ETS motifs critically cooperate with these mutations to activate TERT, likely by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression and may provide a novel therapeutic target for multiple cancers. Citation Format: Robert J.A Bell, H. Tomas Rube, Alex Kreig, Andrew Mancini, Shaun F. Fouse, Raman P. Nagarajan, Serah Choi, Chibo Hong, Daniel He, Melike Pekmezci, John K. Wiencke, Margaret R. Wrensch, Susan M. Chang, Kyle M. Walsh, Sua Myong, Jun S. Song, Joseph F. Costello. GABP selectively binds and activates the mutant TERT promoter across multiple cancer types. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B12.

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methylammonium, acetate and water [4]. With the removal of the previous ambiguity brought by using water as both the hydrogen-bond donor and acceptor fragment [2], improved screening results are seen using targets in the DUD database in the extended SILCS-Pharm method. Validations of the protocol using eight different protein targets show equal or better screening results when compared with results using common docking methods such as DOCK or Au-toDock, indicating the potential utility of the approach in rational drug design. Diverse classes of proteins such as molecular motors, enzymes, and active transmembrane transporters function through large-scale conformational changes. Computer simulations of these conformational transitions are challenging. A range of coarse-grained and biased simulation techniques have been used to generate individual transitions or ensembles of transition pathways but it has been difficult to compare pathways produced by different methods and so to assess their relative strengths. We introduce a comprehensive method (pathway similarity analysis, PSA) for quantitatively characterizing and comparing macromolecular pathways. The Hausdorff and Frechet metrics (known from computational geometry) are used to quantify the degree of similarity between piecewise-linear curves in configuration space. We tested PSA on a toy system to study the effect of temperature fluctuations (path roughness) and dimensionality. We compare a sample of publicly accessible transition pathway simulation servers and our own dynamic importance sampling (DIMS) MD method for the closed-to-open transitions of the apo enzyme adenylate kinase (AdK). PSA was applied to ensembles of hundreds of trajectories of the conformational transitions of the transporter Mhp1 and of AdK and diphtheria toxin, which were produced by DIMS MD and the Geometrical Pathways algorithm. Clustered PSA enabled the selection of a small subset of representative trajectories for further analysis. A strength of PSA is its use of the full information available from the 3N-dimensional configuration space trajectory, without requiring additional specific knowledge about the system. We show how trajectory analysis methods relying on pre-defined collective variables such as native contacts or geometric quantities can be used synergistically with PSA. We discuss the method's potential to enhance our understanding of transition path sampling methods, validate them, and ultimately help guide future research toward deeper physical insights into conformational transitions. 64-PlatComputational Design of Repeat-Proteins with a Predefined Geometry New protein design methods are needed to further improve the development of protein-binding scaffolds. Repeat proteins are linear tandem arrays of structurally similar building blocks, and they are established platforms for engineering proteins inhibitors and biosensors. However, current sequence-based engineering approaches lack the possibility of customizing the overall shape of a binder to its target molecule. Stru...

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