Authors: Cinghu S, Yang P, Kosak JP, Conway AE, Kumar D, Oldfield AJ, Adelman K, Jothi R
Journal: Mol Cell. 2017 Oct 5;68(1):104-117.e6. doi: 10.1016/j.molcel.2017.09.010
Eukaryotic gene transcription is regulated at many steps, including RNA polymerase II (Pol II) recruitment, transcription initiation, promoter-proximal Pol II pause release, and transcription termination; however, mechanisms regulating transcription during productive elongation remain poorly understood. Enhancers, which activate gene transcription, themselves undergo Pol II-mediated transcription, but our understanding of enhancer transcription and enhancer RNAs (eRNAs) remains incomplete. Here we show that transcription at intragenic enhancers interferes with and attenuates host gene transcription during productive elongation. While the extent of attenuation correlates positively with nascent eRNA expression, the act of intragenic enhancer transcription alone, but not eRNAs, explains the attenuation. Through CRISPR/Cas9-mediated deletions, we demonstrate a physiological role for intragenic enhancer-mediated transcription attenuation in cell fate determination. We propose that intragenic enhancers not only enhance transcription of one or more genes from a distance but also fine-tune transcription of their host gene through transcription interference, facilitating differential utilization of the same regulatory element for disparate functions.
Authors: Li P, Wang L, Bennett BD, Wang J, Li J, Qin Y, Takaku M, Wade PA, Wong J, Hu G
Journal: Nucleic Acids Res. 2017 Oct 11. doi: 10.1093/nar/gkx884. [Epub ahead of print]
Transposable elements, including endogenous retroviruses (ERVs), constitute a large fraction of the mammalian genome. They are transcriptionally silenced during early development to protect genome integrity and aberrant transcription. However, the mechanisms that control their repression are not fully understood. To systematically study ERV repression, we carried out an RNAi screen in mouse embryonic stem cells (ESCs) and identified a list of novel regulators. Among them, Rif1 displays the strongest effect. Rif1 depletion by RNAi or gene deletion led to increased transcription and increased chromatin accessibility at ERV regions and their neighboring genes. This transcriptional de-repression becomes more severe when DNA methylation is lost. On the mechanistic level, Rif1 directly occupies ERVs and is required for repressive histone mark H3K9me3 and H3K27me3 assembly and DNA methylation. It interacts with histone methyltransferases and facilitates their recruitment to ERV regions. Importantly, Rif1 represses ERVs in human ESCs as well, and the evolutionally-conserved HEAT-like domain is essential for its function. Finally, Rif1 acts as a barrier during somatic cell reprogramming, and its depletion significantly enhances reprogramming efficiency. Together, our study uncovered many previously uncharacterized repressors of ERVs, and defined an essential role of Rif1 in the epigenetic defense against ERV activation.
Authors: Venniro M, Caprioli D, Zhang M, Whitaker LR, Zhang S, Warren BL, Cifani C, Marchant NJ, Yizhar O, Bossert JM, Chiamulera C, Morales M, Shaham Y
Journal: Neuron. 2017 Oct 11;96(2):414-427.e8. doi: 10.1016/j.neuron.2017.09.024.
Despite decades of research on neurobiological mechanisms of psychostimulant addiction, the only effective treatment for many addicts is contingency management, a behavioral treatment that uses alternative non-drug reward to maintain abstinence. However, when contingency management is discontinued, most addicts relapse to drug use. The brain mechanisms underlying relapse after cessation of contingency management are largely unknown, and, until recently, an animal model of this human condition did not exist. Here we used a novel rat model, in which the availability of a mutually exclusive palatable food maintains prolonged voluntary abstinence from intravenous methamphetamine self-administration, to demonstrate that the activation of monosynaptic glutamatergic projections from anterior insular cortex to central amygdala is critical to relapse after the cessation of contingency management. We identified the anterior insular cortex-to-central amygdala projection as a new addiction- and motivation-related projection and a potential target for relapse prevention.
Authors: Ao M, Chavez MB, Chu EY, Hemstreet KC, Yin Y, Yadav MC, Millán JL, Fisher LW, Goldberg HA, Somerman MJ, Foster BL
Journal: Bone. 2017 Sep 1;105:134-147. doi: 10.1016/j.bone.2017.08.027. [Epub ahead of print]
Although acellular cementum is essential for tooth attachment, factors directing its development and regeneration remain poorly understood. Inorganic pyrophosphate (PPi), a mineralization inhibitor, is a key regulator of cementum formation: tissue-nonspecific alkaline phosphatase (Alpl/TNAP) null mice (increased PPi) feature deficient cementum, while progressive ankylosis protein (Ank/ANK) null mice (decreased PPi) feature increased cementum. Bone sialoprotein (Bsp/BSP) and osteopontin (Spp1/OPN) are multifunctional extracellular matrix components of cementum proposed to have direct and indirect effects on cell activities and mineralization. Studies on dentoalveolar development of Bsp knockout (Bsp-/-) mice revealed severely reduced acellular cementum, however underlying mechanisms remain unclear. The similarity in defective cementum phenotypes between Bsp-/- mice and Alpl-/- mice (the latter featuring elevated PPi and OPN), prompted us to examine whether BSP is operating by modulating PPi-associated genes. Genetic ablation of Bsp caused a 2-fold increase in circulating PPi, altered mRNA expression of Alpl, Spp1, and Ank, and increased OPN protein in the periodontia. Generation of a Bsp knock-out (KO) cementoblast cell line revealed significantly decreased mineralization capacity, 50% increased PPi in culture media, and increased Spp1 and Ank mRNA expression. While addition of 2μg/ml recombinant BSP altered Spp1, Ank, and Enpp1 expression in cementoblasts, changes resulting from this dose were not dependent on the integrin-binding RGD motif or MAPK/ERK signaling pathway. Decreasing PPi by genetic ablation of Ank on the Bsp-/- mouse background reestablished cementum formation, allowing >3-fold increased acellular cementum volume compared to wild-type (WT). However, deleting Ank did not fully compensate for the absence of BSP. Bsp-/-; Ank-/- double-deficient mice exhibited mean 20-27% reduced cementum thickness and volume compared to Ank-/- mice. From these data, we conclude that the perturbations in PPi metabolism are not solely driving the cementum pathology in Bsp-/- mice, and that PPi is more potent than BSP as a cementum regulator, as shown by the ability to override loss of BSP by lowering PPi. We propose that BSP and PPi work in concert to direct mineralization in cementum and likely other mineralized tissues.
Authors: McGowan CJ1, Kwok RK1, Engel LS1,2, Stenzel MR3, Stewart PA4, Sandler DP
Journal: Environ Health Perspect. 2017 Sep 15;125(9):097015. doi: 10.1289/EHP1677.
BACKGROUND: The large quantities of chemical oil dispersants used in the oil spill response and cleanup (OSRC) work following the Deepwater Horizon disaster provide an opportunity to study associations between dispersant exposure (Corexit™ EC9500A or EC9527A) and human health.
OBJECTIVES: Our objectives were to examine associations between potential exposure to the dispersants and adverse respiratory, dermal, and eye irritation symptoms.
METHODS: Using data from detailed Gulf Long-term Follow-up ( GuLF) Study enrollment interviews, we determined potential exposure to either dispersant from participant-reported tasks during the OSRC work. Between 27,659 and 29,468 participants provided information on respiratory, dermal, and eye irritation health. We estimated prevalence ratios (PRs) to measure associations with symptoms reported during the OSRC work and at study enrollment, adjusting for potential confounders including airborne total hydrocarbons exposure, use of cleaning chemicals, and participant demographics.
RESULTS: Potential exposure to either of the dispersants was significantly associated with all health outcomes at the time of the OSRC, with the strongest association for burning in the nose, throat, or lungs [adjusted PR (aPR)=1.61 (95% CI: 1.42, 1.82)], tightness in chest [aPR=1.58 (95% CI: 1.37, 1.81)], and burning eyes [aPR=1.48 (95% CI: 1.35, 1.64). Weaker, but still significant, associations were found between dispersant exposure and symptoms present at enrollment.
CONCLUSIONS: Potential exposure to Corexit™ EC9527A or EC9500A was associated with a range of health symptoms at the time of the OSRC, as well as at the time of study enrollment, 1-3 y after the spill. https://doi.org/10.1289/EHP1677.
Authors: Weng TY, Tsai SA, Su TP
Journal: J Biomed Sci. 2017 Sep 16;24(1):74. doi: 10.1186/s12929-017-0380-6.
The sigma-1 receptor (Sig-1R) is a chaperone that resides mainly at the mitochondrion-associated endoplasmic reticulum (ER) membrane (called the MAMs) and acts as a dynamic pluripotent modulator in living systems. At the MAM, the Sig-1R is known to play a role in regulating the Ca2+ signaling between ER and mitochondria and in maintaining the structural integrity of the MAM. The MAM serves as bridges between ER and mitochondria regulating multiple functions such as Ca2+ transfer, energy exchange, lipid synthesis and transports, and protein folding that are pivotal to cell survival and defense. Recently, emerging evidences indicate that the MAM is critical in maintaining neuronal homeostasis. Thus, given the specific localization of the Sig-1R at the MAM, we highlight and propose that the direct or indirect regulations of the Sig-1R on mitochondrial functions may relate to neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS). In addition, the promising use of Sig-1R ligands to rescue mitochondrial dysfunction-induced neurodegeneration is addressed.
Authors: Moon AF, Pryor JM, Ramsden DA, Kunkel TA, Bebenek K, Pedersen LC
Journal: Nucleic Acids Res. 2017 Sep 6;45(15):9138-9148. doi: 10.1093/nar/gkx527
While most DNA polymerases discriminate against ribonucleotide triphosphate (rNTP) incorporation very effectively, the Family X member DNA polymerase μ (Pol μ) incorporates rNTPs almost as efficiently as deoxyribonucleotides. To gain insight into how this occurs, here we have used X-ray crystallography to describe the structures of pre- and post-catalytic complexes of Pol μ with a ribonucleotide bound at the active site. These structures reveal that Pol μ binds and incorporates a rNTP with normal active site geometry and no distortion of the DNAsubstrate or nucleotide. Moreover, a comparison of rNTP incorporation kinetics by wildtype and mutant Pol μ indicates that rNTP accommodation involves synergistic interactions with multiple active site residues not found in polymerases with greater discrimination. Together, the results are consistent with the hypothesis that rNTP incorporation by Pol μ is advantageous in gap-filling synthesis during DNAdouble strand break repair by nonhomologous end joining, particularly in nonreplicating cells containing very low deoxyribonucleotide concentrations.
Authors: Zhang F, Zhu G, Jacobson O, Liu Y, Chen K, Yu G, Ni Q, Fan J, Yang Z, Xu F, Fu X, Wang Z, Ma Y, Niu G, Zhao X, Chen X
Journal: ACS Nano. 2017 Sep 26;11(9):8838-8848. doi: 10.1021/acsnano.7b03003. Epub 2017 Sep 6.
We report a camptothecin (CPT) prodrug that was well formulated in solution and rapidly transformed into long-circulating nanocomplexes in vivo for highly efficient drug delivery and effective cancer therapy. Specifically, using a redox-responsive disulfide linker, CPT was conjugated with an albumin-binding Evans blue (EB) derivative; the resulting amphiphilic CPT-ss-EB prodrug self-assembled into nanostructures in aqueous solution, thus conferring high solubility and stability. By binding CPT-ss-EB to endogenous albumin, the 80 nm CPT-ss-EB nanoparticles rapidly transformed into 7 nm albumin/prodrug nanocomplexes. CPT-ss-EB was efficient at intracellular delivery into cancer cells, released intact CPT in a redox-responsive manner, and exhibited cytotoxicity as potent as CPT. In mice, the albumin/CPT-ss-EB nanocomplex exhibited remarkably long blood circulation (130-fold greater than CPT) and efficient tumor accumulation (30-fold of CPT), which consequently contributed to excellent therapeutic efficacy. Overall, this strategy of transformative nanomedicine is promising for efficient drug delivery.
Authors: Schellenberg MJ, Lieberman JA, Herrero-Ruiz A, Butler LR, Williams JG, Muñoz-Cabello AM, Mueller GA, London RE, Cortés-Ledesma F, Williams RS
Journal: Science. 2017 Sep 29;357(6358):1412-1416. doi: 10.1126/science.aam6468. Epub 2017 Sep 14.
Topoisomerase 2 (TOP2) DNA transactions proceed via formation of the TOP2 cleavage complex (TOP2cc), a covalent enzyme-DNA reaction intermediate that is vulnerable to trapping by potent anticancer TOP2 drugs. How genotoxic TOP2 DNA-protein cross-links are resolved is unclear. We found that the SUMO (small ubiquitin-related modifier) ligase ZATT (ZNF451) is a multifunctional DNA repair factor that controls cellular responses to TOP2 damage. ZATT binding to TOP2cc facilitates a proteasome-independent tyrosyl-DNA phosphodiesterase 2 (TDP2) hydrolase activity on stalled TOP2cc. The ZATT SUMO ligase activity further promotes TDP2 interactions with SUMOylated TOP2, regulating efficient TDP2 recruitment through a "split-SIM" SUMO2 engagement platform. These findings uncover a ZATT-TDP2-catalyzed and SUMO2-modulated pathway for direct resolution of TOP2cc.
Authors: Chattaraj P, Munjal T, Honda K, Rendtorff ND, Ratay JS, Muskett JA, Risso DS, Roux I, Gertz EM, Schäffer AA, Friedman TB, Morell RJ, Tranebjærg L, Griffith AJ
Journal: J Med Genet. 2017 Aug 5. pii: jmedgenet-2017-104721. doi: 10.1136/jmedgenet-2017-104721.
BACKGROUND: Enlargement of the vestibular aqueduct (EVA) is the most common radiological abnormality in children with sensorineural hearing loss. Mutations in coding regions and splice sites of the SLC26A4 gene are often detected in Caucasians with EVA. Approximately one-fourth of patients with EVA have two mutant alleles (M2), one-fourth have one mutant allele (M1) and one-half have no mutant alleles (M0). The M2 genotype is correlated with a more severe phenotype.
METHODS: We performed genotype-haplotype analysis and massively parallel sequencing of the SLC26A4 region in patients with M1 EVA and their families.
RESULTS: We identified a shared novel haplotype, termed CEVA (Caucasian EVA), composed of 12 uncommon variants upstream of SLC26A4. The presence of the CEVA haplotype on seven of ten 'mutation-negative' chromosomes in a National Institutes of Health M1 EVA discovery cohort and six of six mutation-negative chromosomes in a Danish M1 EVA replication cohort is higher than the observed prevalence of 28 of 1006 Caucasian control chromosomes (p<0.0001 for each EVA cohort). The corresponding heterozygous carrier rate is 28/503 (5.6%). The prevalence of CEVA (11 of 126) is also increased among M0 EVA chromosomes (p=0.0042).
CONCLUSIONS: The CEVA haplotype causally contributes to most cases of Caucasian M1 EVA and, possibly, some cases of M0 EVA. The CEVA haplotype of SLC26A4 defines the most common allele associated with hereditary hearing loss in Caucasians. The diagnostic yield and prognostic utility of sequence analysis of SLC26A4 exons and splice sites will be markedly increased by addition of testing for the CEVA haplotype.