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Zinder, J. C.

Publications and source records attributed to Zinder, J. C..

3 recordsLinked to original sources

Allosteric interactions prime androgen receptor dimerization and activation

The androgen receptor (AR) is a steroid receptor and master transcription factor that governs gene expression programs required for luminal development of prostate epithelium, formation of muscle tissue and maintenance of the male phenotype. AR misregulation is a hallmark of multiple malignancies, including prostate cancer, where AR hyperactivation and expansion of its transcriptome occur in part through AR gene amplification and interaction with oncoprotein cofactors. Despite its biological importance, how ARs individual domains and its protein cofactors cooperate to bind DNA have remained elusive. Using a combination of reconstitution biochemistry and single particle cryo-electron microscopy (EM), we have isolated three conformational states of AR bound to DNA. We observe that AR forms a non-obligate dimer, with the buried dimer interface utilized by related ancestral nuclear receptors repurposed to facilitate cooperative DNA binding. We identify surfaces bridging ARs domains responsible for allosteric communication, that are compromised in partial androgen insensitivity syndrome (PAIS), and are reinforced by ARs oncoprotein cofactor, ERG, and DNA binding site motifs. Finally, we present evidence that this plastic dimer interface for transcriptional activation may have been adopted by AR at the expense of DNA binding. Our work highlights how fine-tuning of ARs cooperative interactions translate to consequences in development and disease.

biochemistry↗

Shelterin is a Dimeric Complex with Extensive Structural Heterogeneity

Human shelterin is a six-subunit complex - comprised of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1 - that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been generated of the individual structured domains within shelterin, the architecture and stoichiometry of the full complex are currently unknown. Here we report the purification of shelterin subcomplexes and reconstitution of the entire complex using full-length, recombinantly produced components. By combining negative-stain electron microscopy (EM), crosslinking mass spectrometry (XLMS), mass photometry, and native mass spectrometry (MS), we obtain stoichiometries as well as domain-scale architectures of shelterin subcomplexes and determine that they are extensively conformationally heterogenous. For POT1/TPP1 and POT1/TPP1/TIN2, we observe high variability in the positioning of the POT1 DNA-binding domain, the TPP1 OB fold, and the TIN2 TRFH domain with respect to the C-terminal domains of POT1. Truncation of unstructured linker regions in TIN2, TPP1, and POT1 did not reduce the conformational variability of the heterotrimer. Both shelterin and the TRF1/TIN2/TPP1/POT1 subcomplex primarily adopt fully dimeric complexes, even in the absence of DNA substrates. TRF1/TIN2/TPP1/POT1 and shelterin complex showed extensive conformational variability, regardless of the presence of DNA substrates. We conclude that shelterin adopts a multitude of conformations and argue that its unusual architectural variability is beneficial for its many functions at telomeres.

biochemistry↗

Cryo-EM structure of the human CST·Polα/Primase complex in a recruitment state

The CST*Pol/Primase complex is essential for telomere overhang maintenance and additionally functions to counteract resection at double-strand breaks. We report a 4.6-[A] resolution cryo-EM structure of CST*Pol/Primase, captured prior to catalysis in a recruitment state, which provides insights into the architecture and stoichiometry of the fill-in machinery. Our model informs on human disease mutations that cause Coats plus syndrome.

biochemistry↗