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Research advances from the National Institutes of Health (NIH) Intramural Research Program (IRP) are often published in high-impact journals. Read some of our recent articles:

Authors: Tiano JP, Springer DA, Rane SG

Journal: J Biol Chem. 2015 Feb 3. pii: jbc.M114.617399. [Epub ahead of print]

Beige adipose cells are a distinct and inducible type of thermogenic fat cell that express the mitochondrial uncoupling protein-1 and thus represent a powerful target for treating obesity. Mice lacking the transforming growth factor-beta (TGF-β) effector protein SMAD3 are protected against diet-induced obesity due to browning of their white adipose tissue (WAT) leading to increased whole body energy expenditure. However, the role SMAD3 plays in WAT browning is not clearly understood. Irisin is an exercise-induced skeletal muscle hormone that induces WAT browning similar to that observed in SMAD3 deficient mice. Together these observations suggested that SMAD3 may negatively regulate irisin production and/or secretion from skeletal muscle. To address this question we used wild type and SMAD3 knockout (Smad3-/-) mice subjected to an exercise regime and C2C12 myotubes treated with TGF-β, a TGF-β receptor-1 pharmacological inhibitor, adenovirus expressing constitutively active SMAD3 or siRNA against SMAD3. We find that in Smad3-/- mice exercise increases serum irisin and skeletal muscle FNDC5 (irisin precursor) and its upstream activator peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) to a greater extent than in wild type mice. In C2C12 myotubes TGF-β suppresses FNDC5 and PGC-1α mRNA and protein levels via SMAD3 and promotes SMAD3 binding to the FNDC5 and PGC-1α promoters. These data establish that SMAD3 suppresses FNDC5 and PGC-1α in skeletal muscle cells. These findings shed light on the poorly understood regulation of irisin/FNDC5 by demonstrating a novel association between irisin and SMAD3 signaling in skeletal muscle.

Authors: Aroda VR, Christophi CA, Edelstein SL, Zhang P, Herman WH, Barrett-Connor E, Delahanty LM, Montez MG, Ackermann RT, Zhuo X, Knowler WC, Ratner RE; for the Diabetes Prevention Program Research Group

Journal: J Clin Endocrinol Metab. 2015 Feb 23:jc20143761. [Epub ahead of print]

Context: Gestational diabetes (GDM) confers a high risk of type 2 diabetes. In the Diabetes Prevention Program (DPP), intensive lifestyle (ILS) and metformin prevented or delayed diabetes in women with a history of GDM. Objective: The objective of the study was to evaluate the impact of ILS and metformin intervention over 10 years in women with and without a history of GDM in the DPP/Diabetes Prevention Program Outcomes Study. Design: This was a randomized controlled clinical trial with an observational follow-up. Setting: The study was conducted at 27 clinical centers. Participants: Three hundred fifty women with a history of GDM and 1416 women with previous live births but no history of GDM participated in the study. The participants had an elevated body mass index and fasting glucose and impaired glucose tolerance at study entry. Interventions: Interventions included placebo, ILS, or metformin. Outcomes Measure: Outcomes measure was diabetes mellitus. Results: Over 10 years, women with a history of GDM assigned to placebo had a 48% higher risk of developing diabetes compared with women without a history of GDM. In women with a history of GDM, ILS and metformin reduced progression to diabetes compared with placebo by 35% and 40%, respectively. Among women without a history of GDM, ILS reduced the progression to diabetes by 30%, and metformin did not reduce the progression to diabetes. Conclusions: Women with a history of GDM are at an increased risk of developing diabetes. In women with a history of GDM in the DPP/Diabetes Prevention Program Outcomes Study, both lifestyle and metformin were highly effective in reducing progression to diabetes during a 10-year follow-up period. Among women without a history of GDM, lifestyle but not metformin reduced progression to diabetes.

Authors: Naik S, Bouladoux N, Linehan JL, Han SJ, Harrison OJ, Wilhelm C, Conlan S, Himmelfarb S, Byrd AL, Deming C, Quinones M, Brenchley JM, Kong HH, Tussiwand R, Murphy KM, Merad M, Segre JA, Belkaid Y

Journal: Nature. 2015 Jan 5. doi: 10.1038/nature14052. [Epub ahead of print]

The skin represents the primary interface between the host and the environment. This organ is also home to trillions of microorganisms that play an important role in tissue homeostasis and local immunity. Skin microbial communities are highly diverse and can be remodelled over time or in response to environmental challenges. How, in the context of this complexity, individual commensal microorganisms may differentially modulate skin immunity and the consequences of these responses for tissue physiology remains unclear. Here we show that defined commensals dominantly affect skin immunity and identify the cellular mediators involved in this specification. In particular, colonization with Staphylococcus epidermidis induces IL-17A+ CD8+ T cells that home to the epidermis, enhance innate barrier immunity and limit pathogen invasion. Commensal-specific T-cell responses result from the coordinated action of skin-resident dendritic cell subsets and are not associated with inflammation, revealing that tissue-resident cells are poised to sense and respond to alterations in microbial communities. This interaction may represent an evolutionary means by which the skin immune system uses fluctuating commensal signals to calibrate barrier immunity and provide heterologous protection against invasive pathogens. These findings reveal that the skin immune landscape is a highly dynamic environment that can be rapidly and specifically remodelled by encounters with defined commensals, findings that have profound implications for our understanding of tissue-specific immunity and pathologies.

Authors: Piaggi P, Thearle MS, Bogardus C, Krakoff J

Journal: J Clin Endocrinol Metab. 2015 Jan 5:jc20143582. [Epub ahead of print]

Context: Increased adiposity and insulin resistance are associated with hyperglycemia and previous studies have reported that higher glucoses are associated with lower rates of weight gain. One possible mechanism is via increased energy expenditure (EE). Objective: To assess the relationships between changes in EE during spontaneous weight gain and concomitant changes in glucose levels. Design and Participants: Body composition, metabolic and glycemic data were available from non-diabetic Native Americans who underwent two measurements of 24-h EE during eucaloric feeding in a metabolic chamber (N=144; time between measurements: 5.0±3.3 years) or resting EE by ventilated hood system during the euglycemic-hyperinsulinemic clamp (N=261; 4.5±3.2 years). Long-term follow-up data (8.3±4.3 years) for weight and body composition were available in 131 and 122 subjects, respectively. Main Outcome Measures: 24-h EE and respiratory quotient (RQ), resting (RMR) and sleeping (SMR) metabolic rates, glucose and insulin levels, basal glucose output (BGO). Results: Weight gain-associated increase in fasting plasma glucose (FPG) levels was accompanied with decreased 24-h RQ (partial R=-0.24, P=0.002) and increased 24-h EE, RMR, SMR and fat oxidation after accounting for changes in body composition (partial R: 0.12 to 0.19, all P≤0.05). Upon weight gain, BGO tended to increase (P=0.07) while insulin infusion induced a decrease in EE (P=0.04). Higher baseline FPG predicted lower rates of future weight gain (partial R=-0.18, P=0.04). Conclusions: Higher FPG after weight gain was associated with greater-than-expected increase in EE. The rise in BGO and the insulin-induced EE suppression at follow-up indicate that increased hepatic gluconeogenesis may be an important mediator of EE changes associated with weight gain.

Authors: Andres SN, Appel CD, Westmoreland JW, Williams JS, Nguyen Y, Robertson PD, Resnick MA, Williams RS

Journal: Nat Struct Mol Biol. 2015 Jan 12. doi: 10.1038/nsmb.2945. [Epub ahead of print]

Ctp1 (also known as CtIP or Sae2) collaborates with Mre11-Rad50-Nbs1 to initiate repair of DNA double-strand breaks (DSBs), but its functions remain enigmatic. We report that tetrameric Schizosaccharomyces pombe Ctp1 contains multivalent DNA-binding and DNA-bridging activities. Through structural and biophysical analyses of the Ctp1 tetramer, we define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer-of-dimers (THDD) domain and a central intrinsically disordered region (IDR) linked to C-terminal 'RHR' DNA-interaction motifs. The THDD, IDR and RHR are required for Ctp1 DNA-bridging activity in vitro, and both the THDD and RHR are required for efficient DSB repair in S. pombe. Our results establish non-nucleolytic roles of Ctp1 in binding and coordination of DSB-repair intermediates and suggest that ablation of human CtIP DNA binding by truncating mutations underlie the CtIP-linked Seckel and Jawad syndromes.

Authors: Clausen AR, Lujan SA, Burkholder AB, Orebaugh CD, Williams JS, Clausen MF, Malc EP, Mieczkowski PA, Fargo DC, Smith DJ, Kunkel T

Journal: Nat Struct Mol Biol. 2015 Jan 26. doi: 10.1038/nsmb.2957. [Epub ahead of print]

Ribonucleotides are frequently incorporated into DNA during replication in eukaryotes. Here we map genome-wide distribution of these ribonucleotides as markers of replication enzymology in budding yeast, using a new 5' DNA end-mapping method, hydrolytic end sequencing (HydEn-seq). HydEn-seq of DNA from ribonucleotide excision repair-deficient strains reveals replicase- and strand-specific patterns of ribonucleotides in the nuclear genome. These patterns support the roles of DNA polymerases α and δ in lagging-strand replication and of DNA polymerase ɛ in leading-strand replication. They identify replication origins, termination zones and variations in ribonucleotide incorporation frequency across the genome that exceed three orders of magnitude. HydEn-seq also reveals strand-specific 5' DNA ends at mitochondrial replication origins, thus suggesting unidirectional replication of a circular genome. Given the conservation of enzymes that incorporate and process ribonucleotides in DNA, HydEn-seq can be used to track replication enzymology in other organisms.

Authors: Qiu C, McCann KL, Wine RN, Baserga SJ, Hall TM

Journal: Proc Natl Acad Sci U S A. 2014 Dec 15. pii: 201407634. [Epub ahead of print]

Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins bind sequence specifically to mRNA targets using a single-stranded RNA-binding domain comprising eight Pumilio (PUM) repeats. PUM repeats have now been identified in proteins that function in pre-rRNA processing, including human Puf-A and yeast Puf6. This is a role not previously ascribed to PUF proteins. Here we present crystal structures of human Puf-A that reveal a class of nucleic acid-binding proteins with 11 PUM repeats arranged in an "L"-like shape. In contrast to classical PUF proteins, Puf-A forms sequence-independent interactions with DNA or RNA, mediated by conserved basic residues. We demonstrate that equivalent basic residues in yeast Puf6 are important for RNA binding, pre-rRNA processing, and mRNA localization. Thus, PUM repeats can be assembled into alternative folds that bind to structured nucleic acids in addition to forming canonical eight-repeat crescent-shaped RNA-binding domains found in classical PUF proteins.

Authors: Nichols HB, DeRoo LA, Scharf DR, Sandler DP

Journal: J Natl Cancer Inst. 2014 Dec 3;107(1). pii: dju354. doi: 10.1093/jnci/dju354. Print 2015 Jan.

BACKGROUND:  Tamoxifen has been US Food and Drug Administration-approved for primary prevention of breast cancer since 1998 but has not been widely adopted, in part because of increased risk of serious side effects. Little is known about the risk-benefit profiles of women who use chemoprevention outside of a clinical trial. We examined characteristics associated with initiation and discontinuation of tamoxifen for primary prevention of breast cancer within a large cohort of women with a first-degree family history of breast cancer.
METHODS:  This research was conducted within The Sister Study, a cohort of 50884 US and Puerto Rican women age 35 to 74 years enrolled from 2003 to 2009. Eligible women were breast cancer-free at enrollment and had a sister who had been diagnosed with breast cancer. Participants reported tamoxifen use, ages started and stopped taking tamoxifen, and total duration of use at enrollment. We identified 788 tamoxifen users and 3131 nonusers matched on age and year of enrollment who had no history of contraindicating factors (stroke, transient ischemic attack, cataract, endometrial or uterine cancer). Characteristics associated with tamoxifen initiation were evaluated with multivariable conditional logistic regression. All statistical tests were two-sided.
RESULTS:  Based on published risk-benefit indices, 20% of women who used tamoxifen had insufficient evidence that the benefits of tamoxifen outweigh the risk of serious side effects. After 4.5 years, 46% of women had discontinued tamoxifen.
CONCLUSIONS:  While the majority of women who used tamoxifen for primary prevention of breast cancer were likely to benefit, substantial discontinuation of tamoxifen before five years and use by women at risk of serious side effects may attenuate benefits for breast cancer prevention.

Authors: Pratto F, Brick K, Khil P, Smagulova F, Petukhova GV, Camerini-Otero RD

Journal: Science. 2014 Nov 14;346(6211):1256442. doi: 10.1126/science.1256442

DNA double-strand breaks (DSBs) are introduced in meiosis to initiate recombination and generate crossovers, the reciprocal exchanges of genetic material between parental chromosomes. Here, we present high-resolution maps of meiotic DSBs in individual human genomes. Comparing DSB maps between individuals shows that along with DNA binding by PRDM9, additional factors may dictate the efficiency of DSB formation. We find evidence for both GC-biased gene conversion and mutagenesis around meiotic DSB hotspots, while frequent colocalization of DSB hotspots with chromosome rearrangement breakpoints implicates the aberrant repair of meiotic DSBs in genomic disorders. Furthermore, our data indicate that DSB frequency is a major determinant of crossover rate. These maps provide new insights into the regulation of meiotic recombination and the impact of meiotic recombination on genome function.

Authors: Freudenthal BD, Beard WA, Perera L, Shock DD, Kim T, Schlick T, and Wilson SH

Journal: Nature. 2014 Nov 17. doi: 10.1038/nature13886.

Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

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