hnRNPA2 Mediated Acetylation Reduces Telomere Length in Response to Mitochondrial Dysfunction

Telomeres protect against chromosomal damage. Accelerated telomere loss has been associated with premature aging syndromes such as Werner’s syndrome and Dyskeratosis Congenita, while, progressive telomere loss activates a DNA damage response leading to chromosomal instability, typically observed in cancer cells and senescent cells. Therefore, identifying mechanisms of telomere length maintenance is critical for understanding human pathologies. In this paper we demonstrate that mitochondrial dysfunction plays a causal role in telomere shortening. Furthermore, hnRNPA2, a mitochondrial stress responsive lysine acetyltransferase (KAT) acetylates telomere histone H4at lysine 8 of (H4K8) and this acetylation is associated with telomere attrition. Cells containing dysfunctional mitochondria have higher telomere H4K8 acetylation and shorter telomeres independent of cell proliferation rates. Ectopic expression of KAT mutant hnRNPA2 rescued telomere length possibly due to impaired H4K8 acetylation coupled with inability to activate telomerase expression. The phenotypic outcome of telomere shortening in immortalized cells included chromosomal instability (end-fusions) and telomerase activation, typical of an oncogenic transformation; while in non-telomerase expressing fibroblasts, mitochondrial dysfunction induced-telomere attrition resulted in senescence. Our findings provide a mechanistic association between dysfunctional mitochondria and telomere loss and therefore describe a novel epigenetic signal for telomere length maintenance

HnRNPA2 is a Novel Histone Acetyltransferase That Mediates Mitochondrial Stress-Induced Nuclear Gene Expression

Reduced mitochondrial DNA copy number, mitochondrial DNA mutations or disruption of electron transfer chain complexes induce mitochondria-to-nucleus retrograde signaling, which induces global change in nuclear gene expression ultimately contributing to various human pathologies including cancer. Recent studies suggest that these mitochondrial changes cause transcriptional reprogramming of nuclear genes although the mechanism of this cross talk remains unclear. Here, we provide evidence that mitochondria-to-nucleus retrograde signaling regulates chromatin acetylation and alters nuclear gene expression through the heterogeneous ribonucleoprotein A2 (hnRNAP2). These processes are reversed when mitochondrial DNA content is restored to near normal cell levels. We show that the mitochondrial stress-induced transcription coactivator hnRNAP2 acetylates Lys 8 of H4 through an intrinsic histone lysine acetyltransferase (KAT) activity with Arg 48 and Arg 50 of hnRNAP2 being essential for acetyl-CoA binding and acetyltransferase activity. H4K8 acetylation at the mitochondrial stress-responsive promoters by hnRNAP2 is essential for transcriptional activation. We found that the previously described mitochondria-to-nucleus retrograde signaling-mediated transformation of C2C12 cells caused an increased expression of genes involved in various oncogenic processes, which is retarded in hnRNAP2 silenced or hnRNAP2 KAT mutant cells. Taken together, these data show that altered gene expression by mitochondria-to-nucleus retrograde signaling involves a novel hnRNAP2-dependent epigenetic mechanism that may have a role in cancer and other pathologies

A remote palm domain residue of RB69 DNA polymerase is critical for enzyme activity and influences the conformation of the active site

Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A), that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the PolD714A mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced kpol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction

A Distinct MaoC-like Enoyl-CoA Hydratase Architecture Mediates Cholesterol Catabolism in <i>Mycobacterium tuberculosis</i>

The <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) <i>igr</i> operon plays an essential role in <i>Mtb</i> cholesterol metabolism, which is critical for pathogenesis during the latent stage of <i>Mtb</i> infection. Here we report the first structure of a heterotetrameric MaoC-like enoyl-CoA hydratase, ChsH1-ChsH2, which is encoded by two adjacent genes from the <i>igr</i> operon. We demonstrate that ChsH1-ChsH2 catalyzes the hydration of a steroid enoyl-CoA, 3-oxo-4,17-pregnadiene-20-carboxyl-CoA, in the modified β-oxidation pathway for cholesterol side chain degradation. The ligand-bound and apoenzyme structures of ChsH1-ChsH2^N reveal an unusual, modified hot-dog fold with a severely truncated central α-helix that creates an expanded binding site to accommodate the bulkier steroid ring system. The structures show quaternary structure shifts that accommodate the four rings of the steroid substrate and offer an explanation for why the unusual heterotetrameric assembly is utilized for hydration of this steroid. The unique αβ heterodimer architecture utilized by ChsH1-ChsH2 to bind its distinctive substrate highlights an opportunity for the development of new antimycobacterial drugs that target a pathway specific to <i>Mtb</i>

Crystal structures of a catalytic complex of the Pol^Y567A/D714A mutant.

<p>(<b>A</b>) The active site of the mutant in ternary complex I. The end of the primer is shown with incoming dGpnpp (magenta), and bound metal ions: sodium (purple sphere), calcium (yellow sphere), and coordinating waters (red spheres). A simulated-annealing <i>F</i>_o-<i>F</i>_c omit map, contoured at 3σ, is shown in blue. (<b>B</b>) Overlay of ternary complex I (PDB ID code: 4I9Q) and the wild type (PDB ID code: 3NCI) polymerase active sites in their respective ternary complexes highlighting the different conformation of the phosphates of the incoming nucleotide, the metal ions and the flip of the 411 side chain. The D714 mutant structure is rendered with the same colors as in (<b>A</b>). The wild type structure is rendered in gray. The α-, β-, and γ-phosphates of the incoming nucleotide are marked with arrows. <b>C</b>) A network of interactions propagates the perturbation resulting from the absence of the 714 side chain into the active site, influencing the conformation of D411. The color scheme is as in (<b>B</b>). (<b>D</b>) The active site in ternary complex II. The DNA (yellow), incoming dGpnpp (cyan) and a calcium ion (yellow sphere) are shown. A simulated-annealing <i>F</i>_o-<i>F</i>_c omit map, contoured at 4σ, is shown in blue. (<b>E</b>) Overlay of ternary complex II (PDB ID code: 4KHN) and the wild type (PDB ID code: 3NCI) polymerase active sites, highlighting the large differences in the conformation of the β- and γ-phosphates of the incoming nucleotide. Like for ternary complex I, a flip in the D411 side chain can be observed relative to the wild-type structure. The α-, β-, and γ-phosphates of the incoming nucleotide are marked with arrows. (<b>F</b>) The absence of the 714 side chain in ternary complex II results in similar structural perturbations to those observed in ternary complex I (see C). Due to the lack of the electron density the side chain of E716 was not shown.</p

Pre-steady state kinetics of correct nucleotide incorporation by the Pol^D714AExo- mutant.

<p>Reactions to measure the incorporation of dATP opposite template T by RB69 DNA Pols were carried out at 10°C. A pre-incubated solution containing the enzyme (1 µM) and radiolabeled 13*/19-mer DNA substrate (100 nM) was mixed with 10mM MgSO₄ and dATP (0.001 mM – 1.5 mM). The reactions were quenched by addition of 0.5 M EDTA (pH 8.0) and analyzed on denaturing polyacrylamide gels. The data were fit to a single-exponential equation to obtain k_<i>obs</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076700#pone.0076700.s002" target="_blank">Figure S2</a>). These values were subsequently plotted as a function of dATP concentration for D714A and wild type RB69 DNA Pols and k_<i>pol</i> and K_{<i>d,dNTP</i>} were calculated as described in Materials and Methods. The standard deviations (SD) are shown as error bars (<b>A</b>) and ± values (<b>B</b>). A close-up of the mutant plot is depicted on the right. The catalytic efficiency of each polymerase was obtained by dividing its respective k_<i>pol</i> by K_{<i>d,dNTP</i>} (<b>B</b>).</p

DNA binding affinity and exonuclease activity of Pol^D714A on dsDNA.

<p>DNA binding affinities of the wild type RB69 and Pol^D714A polymerases (<b>A</b>), as well as their exonuclease deficient derivatives (<b>B</b>), were determined by DNA mobility-shift assays. A radiolabeled 20*/26-mer primer-template DNA substrate was incubated with increasing amounts of each polymerase, and the resulting DNA-protein complexes were analyzed on native 6% polyacrylamide gels (<b>A</b> and <b>B</b>). The amount of bound DNA substrate was quantified as an average from two independent experiments and plotted against protein concentration (<b>C</b> and <b>D</b>). (<b>E</b>) K_<i>Dapp</i> was calculated using Kaleidagraph (Synergy Software). The additional shifted DNA species visible for Exo⁺ variants at higher protein concentrations may correspond to more than one molecule of polymerase bound per oligonucleotide substrate. (<b>F</b>) A ³²P-labeled 20*/26-mer DNA substrate was incubated with the mutant or wild-type RB69 DNA Pol for 10, 20, 40, 80, 180 and 300 sec. at 37°C. Products of DNA degradation were analyzed on denaturing polyacrylamide gels and visualized on a phosphorimager. (<b>G</b>) The amount of undigested DNA substrate was calculated and plotted as a function of time. Data are averages from three independent experiments for each polymerase. Pol^D714AExo⁺ displays slightly elevated exonuclease activity, reflected in an only ~ 10% increase in DNA substrate consumption at the shortest incubation times. The observed difference between the exonucleolytic activities of the enzymes appears therefore to be negligible.</p