Kyle Eagen

All Publications

• Chromatin potentiates transcription. Proceedings of the National Academy of Sciences of the United States of America Nagai, S., Davis, R. E., Mattei, P. J., Eagen, K. P., Kornberg, R. D. 2017; 114 (7): 1536-1541

  Abstract

  Chromatin isolated from the chromosomal locus of the PHO5 gene of yeast in a transcriptionally repressed state was transcribed with 12 pure proteins (80 polypeptides): RNA polymerase II, six general transcription factors, TFIIS, the Pho4 gene activator protein, and the SAGA, SWI/SNF, and Mediator complexes. Contrary to expectation, a nucleosome occluding the TATA box and transcription start sites did not impede transcription but rather, enhanced it: the level of chromatin transcription was at least sevenfold greater than that of naked DNA, and chromatin gave patterns of transcription start sites closely similar to those occurring in vivo, whereas naked DNA gave many aberrant transcripts. Both histone acetylation and trimethylation of H3K4 (H3K4me3) were important for chromatin transcription. The nucleosome, long known to serve as a general gene repressor, thus also performs an important positive role in transcription.

  View details for DOI 10.1073/pnas.1620312114

  View details for PubMedID 28137832

  View details for PubMedCentralID PMC5320956

• Stable Chromosome Condensation Revealed by Chromosome Conformation Capture CELL Eagen, K. P., Hartl, T. A., Kornberg, R. D. 2015; 163 (4): 934-946

  Abstract

  Chemical cross-linking and DNA sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed with regions of little or no interaction, in interphase nuclei. We find that TADs and the regions between them correspond with the bands and interbands of polytene chromosomes of Drosophila. We further establish the conservation of TADs between polytene and diploid cells of Drosophila. From direct measurements on light micrographs of polytene chromosomes, we then deduce the states of chromatin folding in the diploid cell nucleus. Two states of folding, fully extended fibers containing regulatory regions and promoters, and fibers condensed up to 10-fold containing coding regions of active genes, constitute the euchromatin of the nuclear interior. Chromatin fibers condensed up to 30-fold, containing coding regions of inactive genes, represent the heterochromatin of the nuclear periphery. A convergence of molecular analysis with direct observation thus reveals the architecture of interphase chromosomes.

  View details for DOI 10.1016/j.cell.2015.10.026

  View details for Web of Science ID 000364829700018

  View details for PubMedID 26544940

  View details for PubMedCentralID PMC4639323

• Dihydropyrimidinone Positive Modulation of delta-Subunit-Containing gamma-Aminobutyric Acid Type A Receptors, Including an Epilepsy-Linked Mutant Variant BIOCHEMISTRY Lewis, R. W., Mabry, J., Polisar, J. G., Eagen, K. P., Ganem, B., Hess, G. P. 2010; 49 (23): 4841-4851

  Abstract

  Gamma-aminobutyric acid type A receptors (GABA(A) receptors) are ligand-gated chloride channels that play a central role in signal transmission within the mammalian central nervous system. Compounds that modulate specific GABA(A) receptor subtypes containing the delta-subunit are scarce but would be valuable research tools and starting points for potential therapeutic agents. Here we report a class of dihydropyrimidinone (DHPM) heterocycles that preferentially potentiate peak currents of recombinant GABA(A) receptor subtypes containing the delta-subunit expressed in HEK293T cells. Using the three-component Biginelli reaction, 13 DHPMs with structural features similar to those of the barbiturate phenobarbital were synthesized; one DHPM used (monastrol) is commercially available. An up to approximately 3-fold increase in the current from recombinant alpha1beta2delta receptors was observed with the DHPM compound JM-II-43A or monastrol when co-applied with saturating GABA concentrations, similar to the current potentiation observed with the nonselective potentiating compounds phenobarbital and tracazolate. No agonist activity was observed for the DHPMs at the concentrations tested. A kinetic model was used in conjunction with dose-dependent measurements to calculate apparent dissociation constant values for JM-II-43A (400 muM) and monastrol (200 microM) at saturating GABA concentrations. We examined recombinant receptors composed of combinations of subunits alpha1, alpha4, alpha5, alpha6, beta2, beta3, gamma2L, and delta with JM-II-43A to demonstrate the preference for potentiation of delta-subunit-containing receptors. Lastly, reduced currents from receptors containing the mutated delta(E177A) subunit, described by Dibbens et al. [(2004) Hum. Mol. Genet. 13, 1315-1319] as a heritable susceptibility allele for generalized epilepsy with febrile seizures plus, are also potentiated by these DHPMs.

  View details for DOI 10.1021/bi100119t

  View details for Web of Science ID 000278452300016

  View details for PubMedID 20450160

  View details for PubMedCentralID PMC5026497

• Structural and mechanistic exploration of acid resistance: Kinetic stability facilitates evolution of extremophilic behavior JOURNAL OF MOLECULAR BIOLOGY Kelch, B. A., Eagen, K. P., Erciyas, F. P., Humphris, E. L., Thomason, A. R., Mitsuiki, S., Agard, D. A. 2007; 368 (3): 870-883

  Abstract

  Kinetically stable proteins are unique in that their stability is determined solely by kinetic barriers rather than by thermodynamic equilibria. To better understand how kinetic stability promotes protein survival under extreme environmental conditions, we analyzed the unfolding behavior and determined the structure of Nocardiopsis alba Protease A (NAPase), an acid-resistant, kinetically stable protease, and compared these results with a neutrophilic homolog, alpha-lytic protease (alphaLP). Although NAPase and alphaLP have the same number of acid-titratable residues, kinetic studies revealed that the height of the unfolding free energy barrier for NAPase is less sensitive to acid than that of alphaLP, thereby accounting for NAPase's improved tolerance of low pH. A comparison of the alphaLP and NAPase structures identified multiple salt-bridges in the domain interface of alphaLP that were relocated to outer regions of NAPase, suggesting a novel mechanism of acid stability in which acid-sensitive electrostatic interactions are rearranged to similarly affect the energetics of both the native state and the unfolding transition state. An acid-stable variant of alphaLP in which a single interdomain salt-bridge is replaced with a corresponding intradomain NAPase salt-bridge shows a dramatic >15-fold increase in acid resistance, providing further evidence for this hypothesis. These observations also led to a general model of the unfolding transition state structure for alphaLP protease family members in which the two domains separate from each other while remaining relatively intact themselves. These results illustrate the remarkable utility of kinetic stability as an evolutionary tool for developing longevity over a broad range of harsh conditions.

  View details for DOI 10.1016/j.jmb.2007.02.032

  View details for Web of Science ID 000246060200022

  View details for PubMedID 17382344