Publications by authors named "Benjamin R Houghtaling"

Determining mechanisms of drug action in human cells remains a major challenge. Here we describe an approach in which multiple-drug-resistant clones are isolated and transcriptome sequencing is used to find mutations in each clone. Further analysis of mutations common to more than one clone can identify a drug's physiological target and indirect resistance mechanisms, as indicated by our proof-of-concept studies of the cytotoxic anticancer drugs BI 2536 and bortezomib.

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Telomere length homeostasis is achieved by a balance of telomere shortening caused by DNA replication and nucleolytic attack and telomere lengthening by telomerase. The importance of telomere length maintenance to human health is best illustrated by dyskeratosis congenita (DC) a disease of telomere shortening caused by mutations in telomerase subunits. DC patients suffer stem cell depletion and die of bone marrow stem cell failure.

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Human telomere function is mediated by shelterin, a six-subunit complex that is required for telomere replication, protection, and cohesion. TIN2, the central component of shelterin, has binding sites to three subunits: TRF1, TRF2, and TPP1. Here we identify a fourth partner, heterochromatin protein 1γ (HP1γ), that binds to a conserved canonical HP1-binding motif, PXVXL, in the C-terminal domain of TIN2.

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Fluorescent speckle microscopy (FSM) is a live imaging and quantitative measurement technique used for analyzing motion and turnover of macromolecular assemblies in vivo and in vitro. It differs from related imaging techniques such as photobleaching and photoactivation in its use of substantially lower concentrations of fluorescently labeled assembly subunits. When small numbers of labeled subunits and large numbers of unlabeled subunits become randomly incorporated together into a macromolecular structure, the random distribution of fluorophores generates nonuniform fluorescence intensity patterns that appear as distinct puncta against low background fluorescence.

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Accuracy in chromosome segregation depends on the assembly of a bipolar spindle. Unlike mitotic spindles, which have roughly equal amounts of kinetochore microtubules (kMTs) and nonkinetochore microtubules (non-kMTs), vertebrate meiotic spindles are predominantly comprised of non-kMTs, a large subset of which forms an antiparallel "barrel" array at the spindle equator. Though kMTs are needed to drive chromosome segregation, the contributions of non-kMTs are more mysterious.

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Previous studies in human cells indicate that sister telomeres have distinct requirements for their separation at mitosis. In cells depleted for tankyrase 1, a telomeric poly(ADP-ribose) polymerase, sister chromatid arms and centromeres separate normally, but telomeres remain associated and cells arrest in mitosis. Here, we use biochemical and genetic approaches to identify proteins that might mediate the persistent association at sister telomeres.

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Bipolarity of the meiotic spindle, required for proper chromosome segregation, is maintained throughout cell division despite rapid microtubule turnover. How this is achieved has remained mysterious, as determining the organization of individual spindle microtubules has been difficult. Here, we develop single-fluorophore speckle imaging to examine microtubule organization in the vertebrate meiotic spindle.

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Background: Human telomeres are coated by the telomere repeat binding proteins TRF1 and TRF2, which are believed to function independently to regulate telomere length and protect chromosome ends, respectively.

Results: Here, we show that TRF1 and TRF2 are linked via TIN2, a previously identified TRF1-interacting protein, and its novel binding partner TINT1. TINT1 localized to telomeres via TIN2, where it functioned as a negative regulator of telomerase-mediated telomere elongation.

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