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Kub5-Hera, the human Rtt103 homolog, playsdual functional roles in transcription terminationand DNA repairJulio C. Morales1, Patricia Richard2, Amy Rommel3, Farjana J. Fattah1,Edward A. Motea1, Praveen L. Patidar1, Ling Xiao1, Konstantin Leskov4,Shwu-Yuan Wu1, Walter N. Hittelman5, Cheng-Ming Chiang1, James L. Manley2andDavid A. Boothman1,*1Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75390-8807, USA,2Department of Biological Sciences, Columbia University, New York, NY 10027, USA,3Laboratory of Genetics,Salk Institute of Biological Studies, La Jolla, CA 92037, USA,4Department of Radiation Oncology, CaseWestern Reserve University, Cleveland, OH 44106, USA and5Department of Experimental Therapeutics, M.D.Anderson Cancer Center, Houston, TX 77030, USAReceived November 20, 2013; Revised February 3, 2014; Accepted February 5, 2014ABSTRACTFunctions of Kub5-Hera (In Greek Mythology Heracontrolled Artemis) (K-H), the human homolog of theyeast transcription termination factor Rtt103, remainundefined. Here, we show that K-H has functions inboth transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct proteincomplexes with factors that repair DSBs (e.g. Ku70,Ku86, Artemis) and terminate transcription (e.g. RNApolymerase II). K-H loss resulted in increased basalR-looplevels,DSBs,responses andenhancedSignificantly lowered Artemis protein levels weredetected in K-H knockdown cells, which wererestored with specific K-H cDNA re-expression. K-Hdeficient cells were hypersensitive to cytotoxicagents that induce DSBs, unable to reseal complexDSB ends, and showed significantly delayed c-H2AXand 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repairfunction and resistance to DSB-inducing agents.However,R loops persistedconsistent withdual rolesofK-HintranscriptionterminationandDSBrepair.activatedgenomicDNA-damageinstability.INTRODUCTIONMaintaining genomic stability through progressive cell-cycle divisions is essential for survival. Genomic instabilitycan arise by several different mechanisms (1), ultimatelyleading to mutations and/or chromosomal rearrangementsthat contribute to disease states, such as cancer (2). Oneunrepaired DNA double-strand break (DSB) can causelethality (3). Mis-repaired DSBs are also a prominentsource of chromosomal rearrangements, resulting in trans-locations within the genome (4). Thus, DSBs are aconstant threat to genomic stability and can arise natur-ally during normal metabolic, replication and/or develop-mental processes (5). Another prominent, yet at this pointunderstudied, mechanism for genomic instability isthrough the formation of persistent RNA:DNA hybrids,known as R loops (6). R loops are an evolutionarilyconserved consequence of transcription that form undera variety of conditions, and if not properly resolved leadto DSBs and genetic instability (6,7). In transient forms,R-loop formation is an essential process in numerousnormal cellular processes, such as class switch recombin-ation (8), and may also contributes to normal transcrip-tion termination by RNA Polymerase II (RNAPII) (9–11).Transcription termination by RNAPII is a complexprocessrequiringmultipleInterestingly, termination factors are linked to several dif-ferent disease states. Senataxin, a putative DNA:RNAhelicase,ishighlyassociatedpathologies, such as amyotrophic lateral sclerosis 4 andataxia with oculomotor apraxia 2 (13,14). Polymorphismsin Xrn2, a 50–30exoribonuclease, are associated with casesof spontaneous lung cancer in non-smokers (15). PSF,together with p54(nrb), functions in the recruitment ofXrn2 (16) and is critical for cellular survival in colonand prostate cancers (17,18). p54(nrb) is highly expressedand required for development and progression of malig-nant melanoma (19).proteinfactors(12).withneurological*To whom correspondence should be addressed. Tel: +1 214 645 6371; Fax: +1 214 645 6347; Email: email@example.com Acids Research, 2014, 1–11doi:10.1093/nar/gku160? The Author(s) 2014. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), whichpermits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research Advance Access published March 3, 2014 at UT Southwestern on March 4, 2014http://nar.oxfordjournals.org/Downloaded from
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Along with roles in transcription termination, PSF,p54(nrb) and Senataxin are also implicated in the DNA-damage response (DDR), particularly in response toDSBs. PSF and p54(nrb) have direct functional roles inboth the non-homologous end-joining (NHEJ) and hom-ologous recombination (HR) pathways of DSB repair(20–22). Loss of either PSF or p54(nrb) abrogates DNA-repair kinetics and leads to increased chromosomal aber-rations (21,23). Senataxin is also implicated in the DDR,and in particular, in response to DSBs created by R loopsformed during various stages of transcriptional pausing(24–26).In light of the complex nature of the RNAPII termin-ation process, it is likely that factors in addition to thosementioned above are involved. In yeast cells, an additionalfactor, Rtt103 contributes to transcription termination(27). Rtt103 interacts with both RNAPII, via theC-terminal domain (CTD) of its largest subunit, andRat1, the yeast homolog of Xrn2. Rtt103 facilitates theexonuclease activity of Rat1 in termination (27). Thehuman homolog of Rtt103 is Ku70 binding protein #5-Hera (K-H), also known as RPRD1B or CREPT (28,29).Similar to Rtt103, K-H interacts with RNAPII (29). K-Halso mediates expression of Cyclin D1 and RNAPII occu-pancy at the 30-end of genes (28). Rtt103 has also beenimplicated in the DDR, yet its functions and involvementin specific DNA-repair pathways remain unknown (30).While K-H overexpression may also promote cell prolif-eration and tumorigenesis (28), these functions within thecell also remain undefined.Here, we employ genetic and biochemical techniques touncover novel functions of K-H. We report that K-Hfunctions in RNAPII regulation, and aids in stabilizinginteractions between transcription termination factors,localizing Xrn2 to the 30-end of genes and ultimately sup-pressing R-loop formation. Importantly, we report thatK-H forms additional complexes and has additional andseparate functions in DSB repair through stabilization ofthe DNA-repair factor, Artemis.MATERIALS AND METHODSCell cultureshScr, shk-h and all variations of cells were grown inDMEM with15% FBS,hygromycin and 1mg/ml puromycin in a 10% CO2–90%O2air humidified atmosphere at 37?C. shScr and shk-hknockdown cells were grown as described above, exceptunder selection with 1mg/ml puromycin. shScr and shk-h-MDA231 and all variations of cells were grown in DMEMwith 5% FBS and L-glutamine in a 10% CO2–90% O2airhumidified atmosphere at 37?C.L-glutamine, 100mg/mlMouse embryonic fibroblast productionK-H heterozygote males and females were used for timedmating. At 12.5p.c., a female mouse was sacrificed andembryos removed. Individual embryos were then mincedand treated with trypsin at 37?C for 1h to separate cells.The cell slurry was then plated into a T75 tissue cultureflask and allowed to attach and grow. These cells weremaintained in DMEM with 5% FBS and at 10% CO2.Antibodies usedAn antibody recognizing H2AX (a-total H2AX, BL179)and 53BP1 (A300-272A) was purchased from BethylLaboratories (Montgomery, TX). g-H2AX phospho-specific antibody (JBW301) was obtained from Millipore(Billerica, MA). a-Artemis antibodies (E-18, K-14), andactin (C-11) were obtained from Santa Cruz Biotech(Santa Cruz, CA). a-Ku70 and a-Ku70/80 heterodimerspecific antibodies (N3H10 and 162, respectively) werepurchased from Genetex (Irvine, CA). K-H monoclonalantibody was producedatUniversity. K-H polyclonal antibody (SAB1102247) waspurchased from Sigma. S9.6, an antibody specific forR loops (RNA:DNA hybrids) was provided by DrStephen H. Leppla (NIH, Bethesda, MD). PSF (A301-320A)andXrn2 (A301-103A)Laboratories (Montgomery, TX). RNAPII antibodies(sc-56767) is from Santa Cruz Biotech (Santa Cruz, CA).CaseWesternReserveare fromBethylColony-forming assaysshScr, shScr-MDA231, shk-h and shk-h-MDA231 and allvariations of these cells were plated onto 60-mm tissue-culture plates and allowed to grow for 2 days. Cells werethen exposed to IR (at various doses as indicated), allowedto grow for 7 days, washed with PBS and stained withcrystal violet solution. Colonies with >50 normal appear-ing cells were counted and percent survival calculated andgraphed with dose.Neutral comet assayAssays were performed as per manufacturer's instructions(Trevigen) (Catalog# 4250-050-K). Briefly, cells weremock or IR treated and allowed to recover for indicatedtimes. After harvesting, cells were mixed with low meltingagarose. Cells were then exposed to electrophoresis andstained with SYBR?-green to detect released DNA.Cells were then imaged and comet tail lengths measuredby NIH Image J.ImmunofluorescenceIn order to visualize 53BP1, g-H2AX and Artemis, cellswere plated and grown to 70% confluence on glass cover-slips and either mock- or IR-treated. Cells were thenwashed once with PBS, permeabilized and fixed inmethanol/acetone (70/30, v/v). Cells were then blockedin PBS containing 5% FBS for 30min at room tempera-ture. Cells were then washed three times with PBS andexposed to primary antibody for 1h at room temperatureas indicated. Cells were washed three times with PBS,exposed to secondary antibody for 30min at room tem-perature, washed three times with PBS and mounted ontoglass slides. Detection of R loops using the S9.6 antibodywas performed as previously described (31). Visualizationwas performed using a 100X oil objective with fluores-cence on a Nikon microscope.2 Nucleic AcidsResearch, 2014 at UT Southwestern on March 4, 2014http://nar.oxfordjournals.org/Downloaded from
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Plasmid re-ligation assaysThe pYes2 plasmid DNA from Invitrogen was digestedwith the following restriction enzymes: BamHI, SacI,PvuII and BamHI+SacI at 37?C for 45min. Cut anduncut DNAs were purified from Agarose gels usingQiagen Extraction kits. Linearized and control circularplasmid DNAs were then used in PEG/LiAc-based yeasttransformationsfromClontechTransformation System 2). Overnight YPDA cultures(25ml) were grown to an OD600of 0.25, harvested andre-suspended in 50ml YPDA and incubated for 4h at30?C until OD600 of 0.4–0.5 were reached. Competentyeast cells were generated as described (Clontech) andtransformed with equivalent amounts of uncut or restric-tion enzyme-digested pYes2 plasmid DNA as indicated.Transformation reactions were performed using plasmidDNA concentrations of 10mg in 50ml competent rtt103D,hdf1D, wild-type (WT) and rtt103D yeast stably correctedwith yeast RTT103 and human K-H cDNA (hK-H).Briefly, after 42?C heat shock to promote DNA trans-formation, yeast were harvested and resuspended in0.5ml 0.9% NaCl and plated onto SD–URA selectionplates. Colonies were scored 3 days later and valuesgraphed as means±standard errors for three separatetransformation experiments.(YeastmakerYeastMammalian plasmid re-ligation assaysThe pEGFP-Pem1 plasmid was digested with HindIII orI-SceI for 8–12h to generate free DNA ends. pCherryplasmid was co-transfected with linearized DNA tocontrol for transfection efficiency. shScr and shk-h cellswere transfected at roughly 20–25% confluency andallowed to grow for 3 days. Transfections were performedusing Lipofectamine-2000 using manufacturer's instruc-tions. Flow cytometry was performed using a Beckman–Coulter Cytomic FC 500.Metaphase spreads and chromosome aberration analysesExponentially growing cells were mock- or IR-treated.Cells were returned to 37?C for 30min to allow formitotic exit (particularly IR-exposed cells) and thenincubated with colcemid (1mg/ml) for 2h for mitotic cellselection before harvest. Harvested cells were fixed inhypotonic solution containing 75mM KCl and fixed inmethanol:acetic acid (1:1 v/v). Metaphase spreads wereprepared, stained with Giemsa and examined by light mi-croscopy. Metaphase spreads (>50) were then scored forchromosome breaks, gaps and aberrations.HeLa whole-cell extract preparation and gel-filtrationchromatographyHeLa cells were cultured in two 150mm2dishes (up to?80% confluency) in DMEM supplemented with 5%FBS and 1mM L-glutamine in a 5% CO2humidified at-mosphere at 37?C. Cells were trypsinized, harvested bycentrifugation and washed with cold 1X PBS. Cells wereresuspended in 1ml extraction buffer [25mM Tris–HCl(pH7.7), 2mMMgCl2,b-glycerophosphate, 5mM NaF, 0.5mM Na3VO4, 10%100mM NaCl, 10mMglycerol, 0.1% NP-40, 1X protease inhibitor cocktail(Sigma), 100 units of turbonuclease (Fisher) and 1mMDTT]. The cell suspension was incubated on ice for5min and passed through 1-ml syringe with 27G needleuntil homogeneous suspensionsuspension was incubated on ice for 30min followed by10min at 37?C. The cell lysate was centrifuged at14000rpm for 30min at 4?C using a microfuge. Thesupernatant was carefully collected as whole cell lysate andused for gel-filtration chromatography. Chromatographysteps were carried out using AKTA Purifier 10 (GEHealthcare). For the fractionation of whole cell lysate,?3.0mg of protein was loaded onto a 24-ml Superose 6HR 10/30 column (GE Healthcare) pre-equilibrated withchromatography buffer [25mM Tris–HCl (pH 7.7),100mM NaCl, 5% glycerol and 1mM DTT] and run inthe same buffer at a flow rate of 0.5ml/min. Molecularweight standards (Pharmacia Biotech) were used to cali-brate the column (as indicated in Figure 1A).was obtained.TheNuclear extract preparationCell pellets were re-suspended in Buffer A [10mM Hepes(pH 7.9), 10mM KCl, 0.1mM EDTA (pH 8.0), 0.1mMEGTA, 1.0mM DTT, 0.5mM PMSF] and allowed toswell for 10min, 4?C. NP-40 was then added to cell solu-tions to a final concentration of 0.5% and vortexed at lowintensity for 30sec. Isolated nuclei were then harvested bycentrifugation (2000? g), the nuclear pellets were resus-pended in Buffer C [20mM Hepes (pH 7.9), 0.4M NaCl,1.0mM EDTA, 1.0mM EGTA, 1.0mM DTT, 0.5mMPMSF] for 15min at 4?C. Nuclear extracts were thenCWB: K-HWB: Ku70EtBrIP: K-H-IgG+ 5%InputWB: ArtWB: K-H M.W. (kDa) Fraction # WB: Ku70WB: Ku86 WB: RNA Pol II 669 158 43 WB: p15RS 2000 WB: Art (High exposure) #n o i t carFECW16272829222120191872712625247332 3630 2928 353433 3231BAWB: K-H Figure 1. K-H forms a complex with NHEJ factors. (A and B) Tointerrogate potential in vivo K-H interacting partners exponentiallygrowing HeLa cells were collected and lysed for FPLC. Individual frac-tions were separated by SDS-PAGE and western blot techniques wereused to probe for indicated proteins. (C) To validate K-H and NHEJcomplex member interactions nuclear extracts from shScr cells wereused to perform co-immunoprecipitation (co-IPs) experiments withand without EtBr (5 ?M), to check for the contribution of DNA tothese interactions. Co-IPs were separated by SDS-PAGE followed bywestern blot analysis for indicated NHEJ factor.Nucleic Acids Research, 20143 at UT Southwestern on March 4, 2014http://nar.oxfordjournals.org/Downloaded from
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isolated by centrifugation (25000? g) for 15min, andassessed for protein concentrations by Bradford assays.Immunoprecipitation and Chromatin ImmunoprecipitationOf nuclear extract, 0.5 to 1mg was incubated with 5mgof specified primary antibody conjugated to Protein A/Gbeads.Eachexperimentwith NETN solution [20mM Tris–HCL (pH 8.0), 0.1MNaCl, 1mMEDTA, 0.05%each sample was separated on 8% SDS-polyacrylamide gel.Immunoprecipitation and Chromatin Immunoprecipitation(ChIP) experiments were performed using previouslydescribed methods (16).was washedthree timesNP-40]. After washesStatisticsAll experiments were performed three or more times intriplicate.Westernand immunofluorescenceshown, are representative of these findings. Means andstandard errors were calculated and differences betweentreatments were determined by confidence limit calcula-tions using student's t-tests. P-values (0.01 and 0.05) for99%and 95%confidencesignificant.imageslimitswere consideredRESULTSK-H biochemically interacts with NHEJ factorsKu70 binding protein #5-Hera (K-H) was isolated in ascreenintended to identifyinvolved in NHEJ using a similar yeast two-hybridstrategy as described (32). The association of K-H withKu70 in these assays was relatively strong, comparable tothe association of p53 with SV40 large T as measured bygrowth on selection media and beta-galactosidase activity(Supplementary Table S1). Deletion analyses indicatedthat a coiled-coil domain in K-H interacted with a com-parable coiled-coil domain within Ku70, also known toassociate with nuclear clusterin (33). K-H has two highlyconserved functional domains: an amino-terminal CTD-interacting domain (CID), that mediates interactions withthe CTD of RNAPII (27,29) and a carboxy-terminalcoiled-coil domain (Supplementary Figure S1A), whichis required for Ku70 interaction. A specific pointmutation, L276A, within the coiled-coil domain of K-Habolished its binding to Ku70 (Supplementary Table S1).Human (hK-H) and mouse (mK-H) K-H share significantregions of homology with yeast Rtt103 as noted by ClustalOmega analyses (Supplementary Figure 1A).Due to its association with Ku70, we used a plasmid-based DSB-repair assaydeficiencies inRtt103Dyeast.growth on Uracil-deficient medium (UraA cDNAs) weredigested with BamHI (50-overhangs), SacI (30-overhangs),a combination of BamHI and SacI, (BamHI/SacI, yieldingincompatible 50- and 30-end overhangs) or PvuII (bluntends) (Supplementary Figure S1B). Unlike yeast deficientin Hdf1 (yKu70, the canonical yeast NHEJ protein), thatwere unable to repair any type of introduced DSB,Rtt103D yeast lacked the specific ability to repair DSBsKu70-bindingpartnerstodefinePlasmidspossiblerepairconferringwith incompatible ends (i.e., complex or blunt DNA ends),which require additional DNA end-processing (Supple-mentary Figure S1B). Unlike prior reports stating thatRtt103 is not required for the end-joining process (30),our data strongly suggest that Rtt103 deficient cellsexhibit a specific defect in the repair of incompatible orblunt DSB ends. Importantly, re-expression of either yeastRtt103 or human K-H (hK-H) cDNAs restored the DNArepair capabilities of Rtt103D yeast (SupplementaryFigure S1B). Interestingly, the DNA-repair defect notedin Rtt103D yeast closely resembled the DNA-repair defi-ciency of yeast lacking Exo1, a 50–30exonuclease that isrequired for DNA end-processing during NHEJ (34), asimilar but non-overlapping function performed by thehuman Artemis protein (35).To confirmand explorepartners, we performed gel filtration on HeLa whole cellextracts. Interestingly, two separate complexes containingK-H were readily detectable, suggesting relatively separateassociations with RNAPII (fractions 16–22) (Figure 1A)and another complex closely associated with p15RS (frac-tions 27–35) (Figure 1A); an association between K-H andp15RS was previously reported (29). Additionally, K-Hco-eluted with several known NHEJ factors, includingKu70, Ku86 and Artemis (fractions 27–29) (Figure 1Aand B), consistent with yeast two-hybrid data suggestingan association of K-H with Ku70 (SupplementaryTable S1). To visualize Artemis, fractions 27–29 wereconcentrated and probed for levels of K-H and Artemisby western blot analyses (Figure 1B). Importantly,co-immunoprecipitation (co-IP) analyses using a K-H-specific antibody generated in our laboratory, which rec-ognizes K-H but, not the closely related p15RS protein(SupplementaryFigureS2A),associated with Ku70 and Artemis. Furthermore, thisassociation was not due to interactions with DNA, sinceco-IPs were noted in the presence or absence of ethidiumbromide (5 ?M) (Figure 1C). DNase treatments also didnot affect the co-IPs (not shown). These data confirm theinitial yeast two-hybrid observation that K-H interactedwith Ku70, and show that Ku86 and Artemis are presentin higher molecular weight protein complexes with K-H,separate from RNAPII.additionalK-H-bindingrevealedthatK-HIncreased genomic instability and basal DSB formationdue to K-H lossTo examine possible functions of K-H in DNA repair, wegenerated stable shRNA-K-H knockdown cell lines usinghuman foreskin fibroblast (shk-h) and triple negative,metastaticMDA-MB-231MDA231) cells. Non-targeted shRNA-Scr fibroblast(shScr) and MDA-MB-231 (shScr-MDA231) controlcells were generated at the same time. K-H-targetedshRNA knockdown was directed to a 50-untranslatedregion (UTR) sequence to facilitate reconstitution ofcells with human K-H cDNA. We also generated mouseembryonic fibroblasts (MEFs) from K-H wild type (mk-h+/+) or heterozygote (het) mice (mk-h+/?). We confirmedstable loss of K-H in knockdown or mk-h+/?het cells, andshowed that levels of Ku70 remained unchanged (Figurebreastcancer(shk-h-4Nucleic AcidsResearch, 2014 at UT Southwestern on March 4, 2014http://nar.oxfordjournals.org/Downloaded from
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2A). We used mk-h+/?MEFs in our studies since completeloss of K-H led to early embryonic lethality (data notshown) and mk-h+/?cells are haplo-insufficient for pheno-types examined below. In all three cell systems, loss of K-H led to increased basal levels of g-H2AX and 53BP1 fociformed without genomic insult (Figure 2B and C andSupplementary Figure S2B). Furthermore, K-H deficientcells showed delayed disappearance of DDR biomarkersfollowing exposure to IR (Figure 2D and E andSupplementary Figure 2C). The delay in DDR foci regres-sion was confirmed by a delay in neutral comet tail regres-sion after IR treatment in shk-h-MDA231 cells comparedto shScr-MDA231 or K-H reconstituted shk-h-MDA231cells (Supplementary Figure 2D). As neutral cometanalysis examines DSBs specifically, we concluded thatloss of K-H impaired the ability of affected cells torepair DSB lesions. We also observed significant increasesin chromosomal aberrations, by metaphase spreads,before and after exposure to IR (Figure 2F andSupplementary Figure 3A) in shk-h knockdown fibro-blasts compared to shScr cells. Elevated levels of chroma-tid aberrations were also noted in irradiated shk-hfibroblasts compared to shScr control cells (Figure 2F).In contrast, levels of other genomic aberrations, such asdi-centric, tri- and tetra-radial chromosomes, were notstatistically different between shScr and shk-h fibroblastcells (Supplementary Table S2). Collectively, these datasuggested that K-H plays a role in mediating specificDSB-repair processes. However, direct or indirect role(s)for K-H in HR repair of DSBs in mammalian cells couldnot be ruled out, since increases in chromatid and chromo-some breaks after IR were observed with loss of either HRor NHEJ DSB-repair pathways (36). Yet, the lack ofradial chromosomes, commonly seen in cells that havelost the ability to perform HR; thus reverting to NHEJfor DSB repair (36,37), in shk-h cells suggest that theprimary DSB repair defect in K-H-lacking cells is in theNHEJ DSB-repair pathway, and not HR.K-H loss sensitizes cells to chemotherapeutics thatinduce DSBsCells deficient in specific DSB-repair pathways typicallyshow unique hypersensitivitiesagents in long-term colony forming (survival) assays(38,39); for example, NHEJ-deficient Ku70 knockoutcells are hypersensitive to agents that specifically createDSBs (40). In general, all cells deficient in K-H(Figure2A) werehypersensitivecompared to mammalian Scr or wild-type (mk-h+/+)MEF cells (Figure 3A and Supplementary Figure 4Aand B). Furthermore, shk-h-MDA231 cells were alsohypersensitiveto allDSB-inducingincluding cisplatin, H2O2, Etoposide, Doxorubicin andTopotecan compared to genetically matched shScr-MDA231 cells (Figure 3B and C and SupplementaryFigure 4C–E, respectively). In contrast, shk-h-MDA231cellswere notsensitive(Figure 3D). To control for shRNA off-target effects, wereconstituted shk-h-MDA-231 cells with human K-HcDNA and observed that such cells were restored for re-sistance to IR, cisplatin and H2O2treatments to levelscomparable to shScr-MDA231 cells (Figure 3A–D).These data demonstrate that K-H is intimately involvedin mediating the repair of DSBs and not UV-inducedDNA lesions, such as UV-induced thymine dimers and6–4 photoproducts (41).to specificcytotoxictoIR treatmentsagents tested,to ultraviolet(UV) lightThe CID domain of K-H is required for Artemis proteinstabilizationAs shown above, K-H displays a biochemical associationwith NHEJ factors, Ku70, Ku86 and Artemis (Figure 1).To determine whether loss of K-H had any effect on theexpression levels of NHEJ proteins, we examined theirsteady state levels in stable shScr compared to shk-hknockdown fibroblasts. Significantly, loss of K-H led todecreased levels of Artemis, but not Ku70 or Ku86.Importantly, Artemis protein levels were restored uponre-expression of human K-H cDNA, monitored by bothwestern blotting and immunofluorescence (IF) (Figure 4Aand B). Since Artemis mRNA levels remained unchangedafter K-H loss (Supplementary Figure S5A) or K-H res-toration, we suspected that loss of Artemis was due toprotein instability, and that the K-H protein stabilizesCBAFEDWB: K-HWB: Ku70231WB: mK-HWB: Ku70shk-h+_mk-h +/+ +/-WB: K-HWB: Ku70fibro**********************Figure 2. Loss of K-H leads to increased DSBs and genomic instabil-ity. (A) shScr, shk-h, shScr-MDA231, shk-h-MDA231, mk-h+/+andmk-h+/–cells were used to interrogate the degree of K-H loss eachcell system. (B and C) Basal levels of the DNA damage indicatorg-H2AX were measured in shScr-MDA231, shk-h-MDA231, mk-h+/+and mk-h+/–cells by immunofluorescence (IF). (D and E) Rates ofg-H2AX disappearance were measured in shScr-MDA231, shk-h-MDA231,shk-h-MDA231+K-H,indicated times after ionizing radiation (IR) exposure by IF, the firsttime point is 0.5h after IR exposure. (F) Amounts of genomic aberra-tions were quantitated in shScr and shk-h cells with and withoutexposure to IR (2Gy). (**P<0.01).mk-h+/+andmk-h+/–cellsatNucleic Acids Research, 2014 5 at UT Southwestern on March 4, 2014http://nar.oxfordjournals.org/Downloaded from
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Artemis by protein–protein interaction. To determineregions of K-H required for Artemis stabilization, wegenerated various deletion mutants of K-H spanningspecific domains within the protein (Figure 4C) andoverexpressed each of them in 293T cells deficient in en-dogenous K-H expression by prior exposure to siRNAspecific for the 30-UTR region of K-H mRNA. K-H andArtemis mRNA and steady state protein levels were con-firmed by qRT-PCR (data not shown) and westernblotting, respectively. Lamin B protein levels served asloading controls (Figure 4D). Along with full-length K-H, fragments F1, F3 and F4 of K-H caused stabilizationof Artemis steady state protein levels (compare lanes 1, 2,4 and 5 to siRNAk-h+empty vector, lane 6, Figure 4D).In contrast, overexpression of K-H fragment F2 was notable to stabilize steady state levels of Artemis (lane 3)(Figure 4D). These data strongly suggested that the N-terminal CID domain of K-H (minimally represented bythe region indicated by K-H fragment F3) was requiredfor Artemis stabilization (Figure 4D).Artemis overexpression rescues DNA repair defects inK-H deficient cellsLoss of Artemis impairs DNA repair (particularly in thevery late phase of DSB repair noted by foci regression),resulting in genomic instability and sensitivity to IR (42).Because K-H loss led to a concomitant loss of Artemisprotein expression (Figures 4D and 5A), we examinedthe effects of Artemis re-expression on the DNA-repaircapacity of K-H deficient fibroblasts. We transfectedshk-h knockdown fibroblasts with Artemis cDNA (shk-h+Art), which restored Artemis protein expression tolevels found in shScr fibroblast cells transfected with theempty vector (Figure 5A and Supplementary Figure 5B).Strikingly, Artemisre-expression in shk-hcellsdramaticallycompared to non-transfected or vector-alone transfectedshk-h cells (Figure 5A). Artemis re-expression in shk-hcells also restored resistance to IR and rates of 53BP1foci regression similar to shScr cells (Figure 5B and C).We then examined whether loss of K-H expression alsoaffected NHEJ capacity in shScr, shk-h and shk-h+Artfibroblasts using a plasmid-based NHEJ assay developedas described (43). These assays employ an mCherryexpression reporter plasmid linearized by restrictiondigestion with HindIII (compatible DNA ends) or I-SceI(incompatible DNA ends) digestion (43). While cellsknocked down for K-H expression showed only a slightdecrease in the ability to repair compatible ends, therewas a more pronounced deficiency in the ability of thesecells to repair incompatible DNA ends, similar to resultsfound in Rtt103D yeast (Figure 5D and SupplementaryFigure 1B).Intriguingly, we also noted that the DNA-repairkinetics, monitored by 53BP1 and g-H2AX-foci regres-sion, in shk-h knockdown cells were significantly and fun-damentally different from the DNA-repair kinetic-defectsreported in Artemis-deficient cells (42). To explore themechanistic basis for this difference, we compared theDNA-repair kinetics of wild-type mouse (mk-h+/+), het-erozygote mouse K-H (mk-h+/?) and mouse Artemis?/?knockout (mart?/?) MEFs. mk-h+/?cells demonstrated alarge increase in basal DSB levels compared to mart?/?cells (Figure 5E). In contrast, mart?/?cells exhibited basalDSB levels (averaging 2–4 foci/nucleus) similar to mk-h+/+lowered basalDSB levelsdetectedA+---K-H cDNA:+shk-h:+ Art K-H Ku70 Ku86FLF1F2F3F4BD0755025100Relative Art levels (% T/C) WB: Art WB: Myc WB: Lamin Bsik-huntreatedFLF1F2F3F4Mockempty vector+ + + + + + --++-+--+---+----+-----+------+-----------------------------------C**3261631CID**154327 86Figure 4. K-H CID domain is essential for stabilization of Artemis.(A) shScr, shk-h and shk-h+K-H were monitored for changes inKu70, Ku86, Artemis, and K-H expression by western blot. (B)shScr, shk-h and shk-h+K-H cells were assessed for changes inArtemis nuclear location monitored by immunofluorescence. (C)Schematic of K-H deletion mutants tested. (D) K-H domainsrequired for Artemis expression were monitored in 293T cells trans-fected with a K-H specific siRNA designed to the 30-UTR, that werethen transfected with wt or mutant K-H plasmid constructs. Cells wereharvested and separated by SDS-PAGE and monitored for Artemis,c-Myc and Lamin B by western blot. (*P<0.05).Figure 3. Loss of K-H sensitizes MDA-MB-231 cells to variouschemotherapeutic agents. (A–D) shScr-MDA231, shk-h-MDA231 andshk-h-MDA231+K-H mock untreated or (A) IR, (B) cisplatin, (C)H2O2, (D) ultraviolent light (UV) exposed cells were monitored forsurvival by colony formation assay. Colonies were determined as accu-mulations of at least 50 cells. (**P<0.01).6Nucleic AcidsResearch, 2014 at UT Southwestern on March 4, 2014http://nar.oxfordjournals.org/Downloaded from
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cells. After IR treatment (1Gy was an equitoxic dose formart?/?and mk-h+/?cells), mk-h+/?cells showed adramatic impediment in regression of 53BP1 foci/nucleusover time compared to mart?/?or wild-type mk-h+/+cells,which are wild-type for Artemis expression (Figure 5F).As previously reported, mart?/?cells showed similar53BP1 foci/nucleus regression as wild-type mk-h+/+cells,except that mart?/?MEFs exhibited a defect in the slowphase of DSB repair, noted at >5h post-irradiation(Figure 5F). This defect is similar to previously publishedresults using Artemis-deficient human cells (42,44), andstrongly suggested that Artemis loss alone was not suffi-cient to explain the defective DNA-repair phenotype(monitored as foci regression) (Figure 5C and F)observed in mk-h+/?cells (Figure 5F).Loss of K-H leads to R-loop formationGiven previous observations linking K-H to RNAPIItranscriptional regulation (28,29), we then examinedwhether by-products of mis-regulated transcription, incombination with loss of Artemis expression, mayexplainthe qualitativeandbetween the DNA-repair capacities of K-H-deficient andArtemis knockout cells. One such transcriptional by-product is an R loop. Since prolonged R-loop formationleads to increased basal DSBs (45), we monitored R-loopfoci/nuclei formation in shk-h and shScr cells by immuno-fluorescence (IF) using the S9.6 antibody that specificallyrecognizes RNA:DNA hybrids (31). A considerableincrease (?4-fold) in the number of basal R-loop foci/nucleus was detected in shk-h compared to shScr cells(Figure 6A and Supplementary Figure S6). We alsofound that Artemis reconstitution had no effect on thelevel of R loops formed with K-H loss, while R loopswere lostwhen GFP-RNase(Figure 6A). Importantly, overexpression of GFP-RNaseH, which alleviated R-loop formation similar to priorreports (46,47), significantly decreased basal 53BP1 foci/nuclei in shk-h knockdown fibroblasts to levels detected inshScr cells, with or without RNase H overexpression(Figure 6B): similar effects of RNase H overexpressionon R-loop formation and foci representing DSBs werepreviously reported (46,47). In contrast, transfection ofshScr and shk-h cells with a GFP-control plasmid didnot alter the increased basal 53BP1 foci/nuclei levels(Figure 6B) noted in shk-h knockdown compared toshScr cells.To uncouple the contributions of R-loop formationversus Artemis loss and its accompanying specific defectin delayed DSB repair in K-H-deficient cells, weoverexpressed GFP or GFP-RNase H in shScr and shk-h cells and exposed these cells to IR (1Gy). We then moni-toredg-H2AX foci-regressionknockdown cells transfected with GFP alone had similardefective DNA-repair kinetics associated with K-H loss,cells transfected with GFP-RNase H had a distinctly dif-ferent DNA-repair-kinetic profile (Figure 6F), similar tocells defective in Artemis expression alone (compare repairkinetics in Figure 6F with Artemis-deficient cells inFigure 5F). The DNA-repair kinetics in GFP-RNase Htransfected shk-h knockdown cells were similar to GFP-RNase H shScr cells 3h after IR treatment, but at 6h asmall portion of residual DSBs remained in GFP-RNaseH transfected shk-h cells. This response was very similarto that observed with Artemis loss in human cells (42,44).Collectively, our data strongly suggest that R loopsdirectly affect the rate of 53BP1 and g-H2AX foci regres-sion in early phases of DNA DSB repair in K-H-deficientcells, providing an explanation as to why there is a differ-ence in DNA-repair kinetics in cells with loss of K-Hcompared to cells deficient in Artemis expression andfunction.Yeast Rtt103 interacts with Rat1, the yeast homolog ofXrn2 (27). Rat1 and Xrn2 are 50–30RNA exonucleasesrequired for efficient RNA-transcription termination(48). Accordingly, we noted that the Xrn2–RNAPII–PSF complex observed in shScr fibroblasts was dramatic-ally disrupted in shk-h knockdown cells as monitored byco-immunoprecipitationanalysesdirected toward RNAPII or PSF, a known modulator ofquantitativedifferencesH was expressedrates. Whileshk-husingantibodiesFigure 5. Artemis is required for the DNA-repair capability in K-H defi-cient cells. (A) Basal levels of the DNA-damage repair biomarker 53BP1were measured in shScr, shk-h and shk-h cells stably over-expressingArtemis (Art), (shk-h+Art), by immunofluorescence (IF). (B) Sensitivityto IR in shScr, shk-h and shk-h+Art, mock untreated or IR exposed cellswas monitored by colony forming assay. (C) Disappearance of 53BP1 fociwas measured in shScr, shk-h and shk-h+Art at indicated times after IR,first time point measured is 0.5h after IR exposure. (D) The ability toperform NHEJ in shScr, shk-h and shk-h+Art was monitored by aplasmid-based NHEJ assay with plasmids digested with either HindIII tostudy compatible end re-ligation, or I-SceI to study incompatible end re-ligation.(E)Basallevelsof53BP1fociweremeasuredinwildtype(mk-h+/+),K-H heterozygote (mk-h+/–) and Artemis deficient (mart–/–) MEFs by IF.(F) Rate of 53BP1 foci disappearance after IR at times indicated wasmeasured in mk-h+/+, mk-h+/–and mart–/–MEFs by IF, first time point is0.5h after IR. Cells (300) were visualized for 53BP1 foci. Colonies weredetermined as ?50 normal-appearing cells in a 7-day period. Events(10000) were counted by flow cytometry. (**P<0.01, *P<0.05).Nucleic Acids Research, 20147 at UT Southwestern on March 4, 2014http://nar.oxfordjournals.org/Downloaded from
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Xrn2 (16) (Figure 6C). Kaneko et al. (2007) found thatXrn2 localization to the 30-end of genes is facilitated byPSF (16). In yeast, Rtt103 and Rat1 interact andco-localize to the 30-end of genes, mediating transcriptiontermination (27). K-H localizes to the 30-end of the b-actingene, and loss of K-H leads to increased RNAPII occu-pancy downstream of the poly A cleavage site of theb-actin gene, suggesting a possible termination defect(28). Loss of Xrn2 also leads to higher RNAPII occu-pancy at the 30-end of the b-actin gene (16). We, therefore,assessed whether loss of K-H affected Xrn2 localizationalong the b-actin gene, contributing to this terminationdefect. By Chromatin-immunoprecipitation (ChIP) experi-ments along the b-actin gene we noted that loss of K-Hlowered the level of Xrn2 localizing to the 30-end of thegene (Figure 6D). Therefore, appropriate K-H levels areessential for localizing Xrn2 to transcription-terminationregions of DNA. To determine whether loss of othertranscription termination factors led to an accumulationof basal DSB formation in our cell model system, wetransfected shScr cells with siRNA-targeting p54(nrb),which functions with PSF in termination (16). Indeed,loss of p54(nrb) led to increased basal DSB levels(Figure 6E), similar to previously published results (21).DISCUSSIONConsistent with previously published data (30), we dem-onstrate that Rtt103D yeast cells can not repair incompat-ible DSBs. In contrast, these cells were competent atrepairing DNA lesions with compatible ends. In humancells, we demonstrate that K-H-deficient cells are also in-capable of repairing incompatible DNA ends (Figure 5).Furthermore, we demonstrate that the repair defect in K-H-deficient cells is restored by Artemis re-expression. Thissuggests that, similar to K-H, the Rtt103 protein mightinteract with a corresponding yeast protein importantfor incompatible DSB end processing. While we have nodirect evidence for an interaction between these twoproteins, yeast defective in Exo1 display a similar DNA-repair defect (34) as noted in Rtt103D yeast. That is,Rtt103D and Exo1D yeast can fully repair plasmid DNAwith compatible DNA ends, but are incapable of repairingDSBs with incompatible ends (supplementary Figure 1)(34). Our laboratory is currently investigating whetherRtt103 and Exo1 interact in a manner similar to the asso-ciation of Artemis with K-H.K-H-deficient cells demonstrated increased sensitivityto genotoxic stresses that specifically induce DSBs,whereas no hypersensitivity to UV irradiation was noted(Figure 3). Loss of K-H expression greatly suppressedDNA-repair foci regression after a genomic insult withIR, increased genomic instability (Figure 2), and causeda dramatic decrease in the cell's ability to specificallyperform NHEJ (Figure 5). Loss of K-H expression wasalso accompanied by increased basal DSBs (Figure 2).RNase H overexpression in shk-h cells suppressed basalDSBs (Figure 6), indicating a role for RNA metabolism inthe genomic instability observed in these cells.K-H is known to interact with the CTD of RNAPII(29), and loss of K-H leads to higher RNAPII occupancyat the 30-end of genes (28). We showed that K-H mediatesPSF-Xrn2-RNAPII interactions (Figure 6) and that lossof K-H disrupts these leading to a decrease in Xrn2recruitment to the 30-end of genes (Figure 6). These dataprovide evidence that the RNA PolII termination defectnoted in K-H-deficient cells is most likely due to loss ofXrn2 localization.Importantly, the function of Xrn2 is not limited to the30-end of genes. Xrn2 also functions in the premature ter-mination of elongating RNAPII transcription (49,50).Thus, it is not known if the accumulation of persistentand stable R loops is at sites of transcription terminationper se or at mis-regulated premature termination events.Another interesting possibility is that R loops observed inK-H-deficient cells may be accumulating in the nucleolus.Two interesting studies recently demonstrated that loss oftranscriptional regulation of ribosomal DNA leads to R-****BA**************RNAPIIPSFIP: RNAPIIK-HCXrn2IgG + -IP: PSFK-HIgG PSF Xrn2 RNAPIID+ -WB:WB:FE****Figure 6. Loss of K-H disrupts transcription termination complexesand leads to R-loop formation. (A) Level of RNA:DNA hybrids(R loops) in shScr and shk-h cells was measured by immunofluores-cence (IF). Basal levels of 53BP1 foci in shScr and shk-h cells trans-fected with either a GFP-expressing plasmid or a GFP-RNaseH-expressing plasmid were measured by IF. (C) Interactions betweentranscription termination factors PSF and Xrn2 and RNAPII weremonitored in shScr and shk-h by co-immunoprecipitation (co-IP) ex-periments. Co-IPs were performed with RNAPII and PSF specificantibodies and separated by SDS-PAGE. Western blots were then per-formed with indicated antibodies. (D) ChIP were performed usingXrn2-specific antibody in HeLa cells treated with K-H-specific siRNAor control siRNA at the promoter (prom) and designated pointsbeyond the polyadenylation site. (E) Basal levels of 53BP1-foci forma-tion were measured in shScr, shk-h and shScr cells transfected withp54nrb specific siRNA by IF. (F) Rates of 53BP1 foci disappearanceafter IR exposure at times indicated were monitored in shScr and shk-hcell transfected with a GFP or and GFP-RNase H expressing plasmidby IF, first time point measure is 0.5h after IR exposure. (**P<0.05).8Nucleic AcidsResearch, 2014 at UT Southwestern on March 4, 2014http://nar.oxfordjournals.org/Downloaded from
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loop formation and subsequent DSB formation (10,24).Given that Xrn2 plays an integral role in ribosomalRNA trimming (51), it is highly conceivable that the Rloops we observe in K-H-deficient cells are accumulatingin the nucleolus.Our data demonstrate that expression of the NHEJprotein, Artemis, was reduced after K-H depletion bytransient or stable siRNA knockdown, or throughsomatic haplo-insufficiency. Since Artemis mRNA levelswere not altered in these cells and re-expression of K-HcDNAs containing specific wild-type CIDs restorednormal Artemis level, we concluded that K-H is necessaryto stabilize Artemis. Interestingly, overexpression ofArtemisto over-ride normalcaused by K-H loss, restored the DNA-repair capabilitiesand sensitivities to DNA-damaging agents in K-H-defi-cient cells to levels comparable to wild-type cells. Thus,Artemis expression can repair DSBs created from persist-ent R-loop formation in K-H deficient cells. This providesinsight into the nature of DSB ends created due toR loops,sinceArtemissmall subset of DSBs with known substrate specificity,including stem and heterologous loops, flap and gappedsubstrates (24).Artemis overexpressionDSBs, but did not affect persistent R loops (Figure 6) inK-H-deficient cells. We conclude that Artemis acts down-stream of RNA:DNA hybrid formation, on DSBs pre-sumably created by DNA or RNA polymerase collisionswith R-loop roadblocks. Interestingly, persistent R-loopformation correlates with Histone H3 S10 phosphoryl-ation and chromatin condensation (52), suggesting thatthe localized chromatin compartment surrounding theR loop is more heterochromatic than euchromatic.Given that Artemis is required for the repair of DSBs inheterochromatic regions of DNA (44), these findingssupport our data that DSBs created in response to persist-ent R-loop formation would require Artemis for repair.Furthermore, RNase H overexpression restored DNA-repair kinetics in shk-h knockdown cells to relativelynormal rates similar to those found in Artemis-deficientcells after IR. These data suggest that removal of R loopsresults only in a defect in the slow phase of DSB repair,similar to that of Artemis-deficient cells. It is known thatDSBs in heterochromatic regions of DNA are repaired ata slower rate than DSBs in euchromatin after IR (53). K-H-deficient cells also lose Artemis expression, which isrequired for efficient slow phase repair of DSBs in hetero-chromatin (44). R-loop-mediated chromatin condensationwould also increase in K-H-deficient cells (52). Couple thiswith the fact that IR does not adversely affect global tran-scription rates (54), one would anticipate that cells defi-cient in K-H expression will have a higher proportion ofDSBs with slower DNA-repair kinetics.Our data provide evidence that K-H exists in multipleproteincomplexesand withproperties. K-H associates with several different DNA-repair proteins, such as Ku70, Ku86 and Artemis. Inseparate complexes, K-H associates with RNAPII andfactors that function in transcription termination. Whilethe amino acid sequence of K-H does not appear todegradation processesprimarilyfunctionsonaresolvedR-loop-mediatedtwo clearfunctionalcontain any obvious functional enzymatic domains, itdoescontain importantdomains, suggesting that it may function as a molecularmatchmaker, or a protein important for functional scaf-folding. The coiled-coil region of K-H appears to be dis-pensable for Artemis stabilization and mediates aninteraction with Ku70. Importantly, the CID region ofK-H was necessary and sufficient for stabilization of theArtemis protein. CID-domain-containing proteins primar-ily interact with the CTD of RNAPII (55). Indeed, the CIDdomain of K-H can associate with the CTD of RNAPII(29). Yet, our data suggest that CID domains may mediateprotein–protein interactions independent of RNAPII. Thenature of how the CID domain of K-H stabilizes Artemisthrough protein–protein interactions is unknown and iscurrently under investigation in our laboratory.Taken together, our data strongly suggest that loss ofK-H copy number and corresponding steady state proteinlevel leads to simultaneous R-loop-derived DSBs and lossof DNA-repair capacity of specific DSBs with complexends. We theorize that this combination confers a chromo-somal 'mutator' phenotype on cells and likely contributesto carcinogenesis, since K-H knockdown led to chromatinaberrations. Our data demonstrate that K-H is importantin mediating NHEJ, through stabilization of Artemis. Inmouse models, Artemis loss was linked to the formation ofseveral different tumor types ranging from pro-B celllymphomas to sarcomas and malignant gliomas (56,57).Consistent with these mouse data, loss of NHEJ capabilityandchromosomaltranslocationsassociated with the formation of many different tumortypes in humans (58). Interestingly, overexpression ofK-H has also been observed in multiple tumor types,such as liver, lung, prostate and colon (28), althoughcurrent data on K-H deficiency in cancers are extremelylimited. In tumors with overexpressed K-H, then, onemight expect heightened resistance to genotoxic agentsand possibly enhanced cancer progression, particularly ifcomplexes needed in RNA transcription or DNA repair(shown in Figure 1) are functionally altered. Data pre-sented in this article suggest that K-H expression mustbe critically controlled in the cell for balanced andnormal cell growth. Altering its expression may alter itsroles as a potential scaffolding protein mediating abalance between transcription termination, genetic in-stability and Artemis-dependent DSB repair. Loss orgain of expression of K-H may then lead to a chromo-somal-level 'mutator' phenotype.protein–protein interactionare commonlySUPPLEMENTARY DATASupplementary Data are available at NAR Online.ACKNOWLEDGEMENTSWe thank Dr Randy J. Legerski and Dr Robert J. Crouchforproviding uswithoverexpression plasmids respectively. We also thank DrStephen H. Leppla for providing us with antibodiesdirected against RNA:DNA hybrids (R loops) (S9.6).ArtemisandRNaseHNucleic Acids Research, 20149 at UT Southwestern on March 4, 2014http://nar.oxfordjournals.org/Downloaded from
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We also thank Dr. Hongtao Yu and his lab members forhelp with optimizing gel filtration chromatography. Wealso thank the members of the Boothman lab and DrsDavid Guis, Jessica Tyler and Shigeki Miyamoto forcritical reading of the article.FUNDINGNational Institutes of Health [grants CA139217 to D.A.B,GM28983 to J.L.M. and CA103867 to C.-M.C.]; CPRIT[grant RP110471 to C,-M.C.]; Welch Foundation [grant I-1805 to C.-M.C.]; Cancer Biology T32 training grantT32CA124334-06to E.A.M.,Funding for open access charges: NIH [CA139217 toD.A.B.].PI: Dr.JerryShay.Conflict of interest statement. None declared.REFERENCES1. Ciccia,A. and Elledge,S.J. (2010) The DNA damage response:making it safe to play with knives. Mol. Cell, 40, 179–204.2. Zhang,Y., Gostissa,M., Hildebrand,D.G., Becker,M.S.,Boboila,C., Chiarle,R., Lewis,S. and Alt,F.W. (2010) Chapter 4 -The Role of Mechanistic Factors in Promoting ChromosomalTranslocations Found in Lymphoid and Other Cancers.In: Frederick,W.A. 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(2013) DNAdouble-strand break repair as determinant of cellularradiosensitivity to killing and target in radiation therapy. Front.Oncol., 3, 113.39. Chalasani,P. and Livingston,R. (2013) Differentialchemotherapeutic sensitivity for breast tumors with ''BRCAness'':a review. The Oncologist, 18, 909–916.40. Mahaney,B.L., Meek,K. and Lees-miller,S.P. (2009) Repair ofionizing radiation-induced DNA double-strand breaks by non-homologous end-joining. Biochem. J., 417, 639–650.41. Douki,T. (2013) The variety of UV-induced pyrimidine dimericphotoproducts in DNA as shown by chromatographicquantification methods. Photochem. Photobiol. Sci., 12,1286–1302.42. Evans,P.M., Woodbine,L., Riballo,E., Gennery,A.R., Hubank,M.and Jeggo,P.A. (2006) Radiation-induced delayed cell death in ahypomorphic Artemis cell line. Hum. Mol. Genet., 15, 1303–1311.43. Fattah,F., Lee,E.H., Weisensel,N., Wang,Y., Lichter,N. andHendrickson,E.A. (2010) Ku regulates the non-homologous endjoining pathway choice of DNA double-strand break repair inhuman somatic cells. PLoS Genet, 6, e1000855.44. Woodbine,L., Brunton,H., Goodarzi,A.A., Shibata,A. andJeggo,P.A. (2011) Endogenously induced DNA double strandbreaks arise in heterochromatic DNA regions and require ataxiatelangiectasia mutated and Artemis for their repair. Nucleic AcidsRes., 39, 6986–6997.45. Kim,N. and Jinks-Robertson,S. (2012) Transcription as a sourceof genome instability. Nat. Rev. Genet., 13, 204–214.46. Sordet,O., Redon,C.E., Gulroullh-Barbat,J., Smith,S., Soller,S.,Douarre,C., Conti,C., Nakamura,A.J., Das,B.B., Nicolas,E. et al.(2009) Ataxia telangiectasia mutated activation by transcription-and topoisomerase I-induced DNA double-strand breaks. EMBO,10, 887–893.47. Yu ¨ ce,O¨. and West,S.C. (2013) Senataxin, defective in theneurodegenerative disorder Ataxia with Oculomotor Apraxia 2,lies at the interface of transcription and the DNA damageresponse. Mol. Cell. Biol., 33, 406–417.48. West,S., Gromak,N. and Proudfoot,N.J. (2004) Human 50–30exonuclease Xrn2 promotes transcription termination at co-transcriptional cleavage sites. Nature, 432, 522–525.49. Brannan,K., Kim,H., Erickson,B., Glover-Cutter,K., Kim,S.,Fong,N., Kiemele,L., Hansen,K., Davis,R., Lykke-Andersen,J.et al. (2012) mRNA decapping factors and the exonuclease Xrn2function in widespread premature termination of RNApolymerase II transcription. Mol. Cell, 46, 311–324.50. Wagschal,A., Rousset,E., Basavarajaiah,P., Contreras,X.,Harwig,A., Laurent-Chabalier,S., Nakamura,M., Chen,X.,Zhang,K., Meziane,O. et al. (2012) Microprocessor, Setx, Xrn2,and Rrp6 co-operate to induce premature termination oftranscription by RNAPII. Cell, 150, 1147–1157.51. Preti,M., O'Donohue,M.-F., Montel-Lehry,N., Bortolin-Cavaille ´ ,M.-L., Choesmel,V. and Gleizes,P.-E. (2013) Gradualprocessing of the ITS1 from the nucleolus to the cytoplasmduring synthesis of the human 18S rRNA. 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text:
DEPARTMENT OF HEALTH AND HUMAN SERVICES ADMINISTRATION FOR CHILDREN AND FAMILIES, SEATTLE, WASHINGTON and NATIONAL TREASURY EMPLOYEES UNION
|DEPARTMENT OF HEALTH AND HUMAN SERVICES ADMINISTRATION FOR CHILDREN AND FAMILIES, SEATTLE, WASHINGTON Respondent and NATIONAL TREASURY EMPLOYEES UNION Charging Party
Case No. SF-CA-01-0307
Yolanda Shepherd Eckford, Esquire For the General Counsel
Before: WILLIAM B. DEVANEY Administrative Law Judge
The Complaint and Notice of Hearing in this case issued on July 31, 2001, and set the hearing for October 26, 2001. The Answer was due to be received, if filed in person, or postmarked, no later than August 27, 2001. Respondent did not file an Answer by August 27, 2001, and the General Counsel on October 5, 2001, filed a Motion For Summary Judgement which was served by certified mail on Respondent on October 5, 2001. Respondent has filed no response to the motion for summary judgement. Accordingly, the General Counsel's motion for summary judgement is Granted.
I. Standard For Granting Summary Judgement
Section 2423.27 of the Authority's Rules and Regulations
codifies the summary judgement procedures adopted by the Authority
in earlier cases, in which the Authority adopted the requirements
of Rule 56 of the Federal Rules of Civil Procedure. E.g.
Department of the Navy, U.S. Naval Ordinance Station, Lousiville,
Kentucky, 33 FLRA 3, 4-6 (1988). Thus, under §2423.27(a),
moving party's motion "shall be supported by documents, affidavits,
applicable precedent, or other appropriate materials" and,
consistent with Rule 56(c), the motion is to be granted if the
"pleadings, depositions, answers to interrogatories, and admissions
on file, together with the affidavits, if any, show that there is
no genuine issue as to any material fact and that the moving party
is entitled to a judgement as a matter of law." Rule 56(c).
Section 2423.20(b) of the Authority's Rules and Regulations provides in part that, "Absent a showing of good cause to the contrary, failure to file an answer or respond to any allegation shall constitute an admission." Respondent has failed to answer the allegations in the Complaint or respond to the allegations of the Complaint. Accordingly, the factual allegations in this Complaint must be deemed to be admitted in their entirety. The only issue for determination, therefore, is the legal issue as to whether Respondent's implementation of Team Administrator positions and refusal to bargain regarding the matter violated section 7116(a)(1) and (5) of the Statute.
II. Findings
Based on the Complaint, testimony provided by affiants and Respondent's failure to answer the July 31, 2001 Complaint, the following material of facts are not in dispute:
1. The Department of Health and Human Services, Administration for Children and Families, Seattle Washington, is an agency under 5 U.S.C. § 7103(a)(3). (Complaint ¶ 2).
2. The National Treasury Employees Union (Union) is a labor
organization under 5 U.S.C. §7103(a)(4) and is the certified
exclusive representative of an appropriate nationwide unit of
employees of the Administration for Children and Families,
including employees at Respondent. (Complaint ¶ 3).
3. The charge was filed by the Charging Party with the San Francisco Regional Director on February 12, 2001 and a copy of the charge was served on the Respondent. (Complaint ¶ 4, 5).
4. During the time period covered by this complaint, the persons listed below occupied the positions opposite their names (Complaint ¶ 7, 8):
Steve Henigson Regional Administrator
Nancy Hutchinson Assistant Regional Administrator
Vince Herberholt Assistant Regional Administrator
Diane Brockington Labor Relations Specialist
5. During the time period covered by this complaint, Steve Henigson, Nancy Hutchinson, Vince Herberholt and Diane Brockington were supervisors and/or management officials under the Statute, and were acting on behalf of Respondent. (Complaint ¶ 6, 7, 8, 9).
6. Respondent is organized into five teams, each responsible for different programs administered by the Respondent: Head Start Childcare; Child Welfare; TANF, which deals with welfare to work issues; Tribal; and Child Support. Each team has a designated team leader who serves as a technical expert, providing program information and resources to members of the team. The team leaders, like employees on the team, are classified at the GS-13 level. (Affidavit of Ann Snead, Ex 4, p. 2).
7. In or about November 2000, Respondent established a new GS-14 Team Administrator position. At the time, Respondent selected three employees to serve as Team Administrators with responsibility for the five program teams and for the Administrative Support Team. (Complaint ¶ 10; Affidavit of Ann Snead, Ex 4, p. 3). The Team Administrators have authority to approve bargaining unit employees' leave requests, make recommendations for employee awards and for changes in work processes. Team Administrators also will have input into bargaining unit employees' performance elements, performance plans and performance appraisals. (Affidavit of Ann Snead, Ex 4, p. 4(1); Ex 5(a), p. 2, numbers 1 and 6). Respondent established these positions without affording the Charging Party prior notice or the opportunity to bargain regarding the matter. (Complaint ¶ 10, 13, Affidavit of Ann Snead, Ex 4).
8. On December 8, 2000, the Charging Party requested that Respondent negotiate over the impact and implementation of the establishment of Team Administrator positions. (Complaint ¶ 11; Affidavit of Carol Perkins, Ex 3).
9. On February 12, 2001, Respondent, by Brockington, responded to the Charging Party's request, refusing to bargain concerning the impact and implementation of its decision to establish Team Administrator positions. (Complaint ¶ 12; Affidavit of Carol Perkins, Ex 3).
The right of Respondent to establish the Team Administrator
positions is not at issue in this case; nor is the inclusion, or
exclusion, of the Team Administrator position in a bargaining unit
under the Statute at issue in this case. I fully agree with the
General Counsel that the duty to bargain arises because of the
impact of this new position on bargaining unit employees who are on
teams under the Team Administrator's responsibility.
An agency has the duty to give a union prior notice and the opportunity to bargain when it makes a change in working conditions which has a reasonably foreseeable impact which is more than de minimis. Department of Health and Human Services, Social Security Administration, 24 FLRA 403 (1986) (SSA); Social Security Administration, Gilroy Branch Office, Gilroy, California, 53 FLRA 1358 (1998). This obligation to engage in impact and implementation bargaining with the exclusive representative extends to organizational changes that have a reasonably foreseeable impact which is more than de minimis. See U.S. Government Printing Office, 13 FLRA 203 (1983)(where the Authority upheld the ALJ's determination that an organizational change that affected sick leave procedures had more than de minimis impact on bargaining unit employees and brought about a bargaining obligation).
Applying the standard established by the Authority in SSA to the present case, I conclude that the establishment of Team Administrator positions at Respondent had a reasonably foreseeable impact on bargaining unit employees which was more than de minimis. Respondent has determined that Team Administrators will have input into employees' performance plans and performance appraisals, impacting on employees' work requirements and performance expectations, as well as adding another level of review of bargaining unit employees work. The importance of the appraisal process to an employee's conditions of employment may not be questioned. An employee's performance appraisal impacts awards and promotional opportunities, and forms the basis for continued employment. In light of the significance of the appraisal process to employees' conditions of employment, Respondent's determination that Team Administrators would have input into employees' work plans and performance appraisals, had more than de minimis impact on conditions of employment of the affected bargaining unit employees. Clearly, when an agency decides to make a change which affects such a basic aspect of employment, the exclusive representative of employees should have the opportunity to address the change, including such issues as notice to employees of the change and how the change will be implemented, procedural matters which are negotiable under the Statute. Patent Office Professional Association and U.S. Department of Commerce, Patent and Trademark Office, Washington, D.C., 48 FLRA 129, 142-43 (1993)(procedural matters regarding performance appraisal systems which do not interfere with the right to assign work are within the duty to bargain).
Further, the establishment of Team Administrator positions affected the procedure for requesting leave at Respondent by adding an official with leave approval authority to the leave approval process. It is not unreasonable to assume that whether an employee is granted leave depends upon an employee following the correct procedure in requesting leave and thus, the addition of Team Administrators to the process, without clarification of their authority or the procedure to be followed, has impact on the bargaining unit employees. Cf. Defense Logistics Agency, Defense Depot Tracy, Tracy, California, 14 FLRA 475 (1984)(unilateral change in sick leave call in procedure was ULP).
In light of the above, I find that establishment of the Team Administrator positions constituted a change which reasonably would have more than de minimis impact on bargaining unit employees. Accordingly, Respondent's unilateral implementation of Team Administrator positions and refusal to bargain concerning the matter violated section 7116(a)(1) and (5) of the Statute. Department of Health and Human Services, Social Security Administration, 24 FLRA 403 (1986). Moreover, Respondent's unilateral implementation of the change in conditions of employment and Respondent's refusal to negotiate on the impact and implementation of the change warrants a status quo ante remedy. Federal Correctional Institution, 8 FLRA 604, 606 (1982); U.S. Department of Justice, Immigration and Naturalization Service, 55 FLRA 892, 902-03 (1999).
Accordingly, the General Counsel is entitled to Summary Judgement. U.S. Department of the Air Force, Lowry Air Force Base, Denver, Colorado, 36 FLRA 183 (1990); Department of Veterans Affairs, Veterans Affairs Medical Center,
Nashville, Tennessee, 50 FLRA 220 (1995).
Having found that Respondent violated §§16(a)(5) and (1) of the
Statute, it is recommended that the Authority adopt the following
Pursuant to section 2423.41(c) of the Authority's Rules and Regulations, 5 C.F.R. § 2423.41(c), and section 7118 of the Federal Service Labor-Management Relations Statute, 5 U.S.C. § 7118, it is hereby ordered that the Department of Health and Human Services, Administration for Children and Families, Seattle, Washington, shall:
1. Cease and desist from:
(a) Unilaterally implementing changes in working conditions of unit employees by establishing Team Administrator's positions without first notifying the National Treasury Employees Union (NTEU), of the change and providing NTEU an opportunity to bargain over procedures for implementing the change and/or appropriate arrangements for employees adversely affected by the change.
(b) Refusing to bargain with NTEU over procedures for implementing the decision to establish Team Administrator's and/or appropriate arrangements for employees adversely affected by the decision to establish Team Administrators.
(c) In any like, or related, manner, interfering with, restraining or coercing its employees in the exercise of their rights assured by the Federal Service Labor-Management Relations Statute (Statute).
2. Take the following affirmative action in order to effectuate the purposes and policies of the Statute:
(a) Withdraw and rescind the implementation of the Team Administrator positions which it unlawfully implemented without notice to NTEU and without bargaining over the impact and implementation of the change.
(b) Give NTEU notice of any future intention to implement Team Administrator positions and, upon request, bargain in good faith with NTEU concerning procedures for implementation and/or appropriate arrangements for employees adversely affected before implementing any decision to establish Team Administrators.
(c) Post at its Seattle, Washington facilities, copies of the attached Notice on forms to be furnished by the Federal Labor Relations Authority. Upon receipt of such forms, they shall be signed by the Regional Administrator, and shall be posted and maintained for 60 consecutive days thereafter, in conspicuous places, including all bulletin boards and other places where notices to employees are customarily posted. Reasonable steps shall be taken to ensure that such Notices are not altered, defaced, or covered by any other material.
(d) Pursuant to section 2423.41(e) of the Authority's Rules and
Regulations, 5 C.F.R. § 2423.41(e), notify the Regional Director,
San Francisco Region, Federal Labor Relations Authority, in
writing, within 30 days from the date of this Order, as to what
steps have been taken to comply with this Order.
Administrative Law Judge
Dated: October 29, 2001
Washington, D.C.

subset: commoncrawl


text:
Health Care Technology
Company Overview of Cambio Healthcare Systems AB
BOARD MEMBERS AFFILIATED WITH Eva Lindqvist M.Sc. (Eng.), MBA*
Business Partner
MOST CONNECTED PEOPLE TO Eva Lindqvist M.Sc. (Eng.), MBA
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To contact Cambio Healthcare Systems AB, please visit . Company data is provided by Capital IQ. Please use this form to report any data issues.
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text:
Werewolf attack by ShockboltWatch
©2009-2016 Shockbolt
Illustration painted for a game called MageBattle , created & published by 4GlobalPlayers.
Painted in Photoshop CS 2 using a Wacom Intuos 3 tablet.
Do not use for any reason!
Painted in Photoshop CS 2 using a Wacom Intuos 3 tablet.
Do not use for any reason!
great work
Oh wow, awesome work. I really like the perspective. What period is the armour from?
Oh, I didn't paint the armor with a specific country or time period in mind as it's a fantasy image
The soldierd don't have a chance...
I love the werewolf there, very dynamic looking.
i bet that's a viedo game.
What power what amazing power! This just amazing!!! My gosh this crazy good I love this!!!
Whoa, pretty awesome picture! I love that face of the werewolf and the way how the moon shows behind it's back. ~
This is bloody awesome, but that werewolf had better kick that guy's ARSE!
"Now 'Aaahh'... wider... wider. Good. I see you've been flossing, we're just going to scrape off a little plaque and give you a nice grin."
I like tension in the werewolfs face. Kinda gets me stressed out
Amazing work with the fur and lighting!
Damn, I love that crazed, but super awesome face. I find it interesting how, although he's very terrifying, the werewolf still manages to pull off the shining-in-the-moonlight-look
Fantastic, I always had a thing for weres...
Thanks! Even for wererats?
that is so amazing!
Thanks yet again!
You're making me blush here with all those nice
comments about my images
You're making me blush here with all those nice
comments about my images
Wow! That's an amazing fuzzy fur!


text:
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Rutgers University (Biological Science)
My name is Alec. I just graduated Rutgers New Brunswick with a Biological Sciences degree and a 3.61 GPA. Currently, I am applying to medical school and want to use my time in helping students succeed in the sciences. I tutored ORGANIC CHEMISTRY, PHYSICS, SYSTEMS PHYSIOLOGY, and GENERAL CHEMISTRY for ODASIS at Rutgers and was apart of ODASIS myself. I scored both A's in General Chemistry and Organic Chemistry I and II. I scored a 100 on my first exam and all above 90s on the other exams. I know the concept and material very well especially the way the professor is going to ask the questions because I am familiar in teaching it to other students.
I am an experienced tutor with many hours of tutoring. I have tutored AP CHEMISTRY and took the exam myself. I have experience in SAT MATH, GENETICS, MICROBIOLOGY, MCAT BIOLOGICAL SCIENCES, GENERAL CHEMISTRY I AND II, ORGANIC CHEMISTRY I AND II, and PHYSICS I AND II scoring all academic classes in college of B+ or higher. In addition, I also tutor the Biological Sciences portion of the MCAT scoring a 13 out of 15 in that part.
My method of teaching is to teach the student the skills and concepts in understanding basic steps to conquering difficult problems. In order for a student to achieve full success, I believe that they should know the basics vividly. This helps then attack harder problems by simply seeing that in fact it has a pattern. I use a combination of methods in preparing a student to see that each problem is not as difficult as it look as long as they completely understand the concept. Teaching them the concept will also lead to future success so the skill is used for the future. I have been taught this way before from tutors and I hope I can expand my skills to other students so they can achieve the same success I did. I hope that future students will be able to attack problems feeling confident not nervous or scared. Having that strength and confidence will give the student the power to succeed.
Let me know if you further want to discuss anything and email me back. Thanks! My name is Alec. I just graduated Rutgers New Brunswick with a Biological Sciences degree and a 3.61 GPA. Currently, I am applying to medical school and want to use my time in helping students succeed in the sciences. I tutored ORGANIC CHEMISTRY, … Read more
He is an awesome tutor! He knows all the material and doesn't even have to reference the book! I would recommend him for anyone struggling with organic chemistry!
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I have previously taught and tutored college level Chemistry for the ODASIS program at Rutgers. This was a bi-weekly sessions in which I taught all aspects of Chemistry that students where required to know. In addition, sessions included mock exams every other week.
I took a general Genetics course at Rutgers University and scored a B+ I am very familiar with the basic multiple choice questions and the open ended questions. Topics I am familiar with include Introduction to Molecular Genetics, DNA Structure and Manipulation, Mendelian Genetics, Sex-chromosomes and Sex-linkage, Genetic, Linkage and Chromosome Mapping, DNA Replication and Recombination, Molecular Organization of Chromosomes, Human Karyotypes and Chromosome Behavior, Microbial Genetics, Gene Expression, Regulation of Gene Expression, Genomics, Proteomics and Transgenics, Genetic Control of Development, Mutations and DNA Repair, Genetics of Cell Cycle and Cancer, Mitochondrial DNA and Extranuclear Inheritance, Population Genetics, and lastly Quantitative Genetics. I know the concepts in the multiple choice questions and how to conquer the open ended questions. Learning the concept and understanding the basics is the first thing to know in attacking these exam questions. Once you have successfully conquered those basic questions, the more difficult questions will be easy to attack. Understanding the key concept is how I was able to achieve success and earn a B+ in my overall grade in Genetics.
I took the MCATs and received a 31 on the exam. I scored 13 in biological sciences, 9 verbal reasoning, 9 writing, and 9 in physical sciences. I believe the best way to prepare for the MCAT is learning the skills in test taking and practice. The best way I teach people to prepare for the MCAT is to start learning the material from the first day by understanding the exam itself, how it works and by learning the skills to multiple choice. The best way to learn is to review the materials periodically. I learned best this way by constantly preparing and practicing with also learning the techniques to conquering the multiple choice questions. I scored a 31 on my first time and being above average I did not retake it.
I took Microbiology at Rutgers University and received a B+ in the class. Topics that I am familiar with and know the concepts very well include the history of microbiology, eukaryotes, archaea and bacteria, structure and function of membranes, transport cell walls, motility, organelles, endospores, microbial nutrition, metabolism, energy generation, electron transport, proton motive force, citric acid cycle, alternative modes of catabolism, biosynthesis, regulation of enzyme activity, feedback inhibition, and microbial growth. In addition, I am also familiar with DNA structure and synthesis, transcription, protein synthesis, gene regulation, viruses, bacteriophages, animal viruses viroids, prions, mutation, recombination, transformation, transduction, plasmids, conjugation, transposons, genetics engineering, microbial genomics, microbial evolution and systematics, survery of bacteria, photoautotrophy, chemolithotrophy, nitrogen fixation, anaerobic respiration, microbial ecosystems, and biogeochemical cycles. Furthermore, I understand the concepts of symbioses, industrial microbiology, water water purification, food microbiology, control of microorganisms, antibiotics, human-microbial interactions, normal flora, virulence, toxins, food poisoning, host defenses, essentials of immunology, molecular immunology, immunity and prevention of infectious diseases, immunodiagnostics, immunoassays, epidemiology, and microbial diseases. I know the material very well and know the skills into successfully attempting the exam questions. As long as the basics are taught the harder questions will be easily manageable. The material is very straight through, but the key is understanding the concept to each subject. Overall, this is how I was able to succeed and get a B+ in the subject.
I tutored Organic Chemistry for Rutgers students, and I myself scored A's in both Organic Chemistry I and Organic Chemistry II. I know the material and concepts very well, and understand the subject very thoroughly.
I have taught and tutored various classes under the physical sciences, such as chemistry and physics, at the college level. I taught weekly sessions that consisted of a small group of students. In addition, I also conducted mock exams for these subjects.
I have previously taught and tutored college-level Physiology for the ODASIS program at Rutgers. This was a weekly session in which I taught all aspects of the subject that students where required to know. In addition, sessions included mock exams every other week.
I have teaching experience in math stemming from volunteer experience. I am part of a high school program that assists under-represented and under-privileged high school students in various subjects. I taught and assisted students with mathematics, which ranged from algebra to trigonometry, in addition to SAT mathematics.


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Doggies Blog: Recent Posts
West Virginia: Shetland Sheepdog Breeders
Below is a sample search of our Shetland Sheepdog breeders with puppies for sale. The search tool above returns a list of breeders located listings located nearest to the zip or postal code you enter. From there you can see photos of their pups, find out detailed information on their web page and contact them by email, phonoe or even get driving directions.
(Detailed contact information has been omitted Use Enter your zip or postal code above to access our up to date directory).
This is a FREE search tool! Use the form at the top to get complete listings for breeders in your area!
Casia Shelties
Additional Information: We are a small kennel breeding for good temperament and sound bodies. All puppies are sold on spay/neuter contracts to approved homes. Contact us for information on upcoming litters.
Valley View Shelties
Additional Information: 2 litters possible in 3/16.PLEASE CALL 540-489-4809. Raising AKC Shelties since 2007. Puppies are raised with children & are loved & spoiled from birth! 1-4 litters for 2016. We strive for quality!
NO EMAIL!!! PLEASE CALL 540-420-8305 or 540-489-4809. We do keep a waiting list, so contact us with the specifics of the puppy you are looking... ...
Eiryk Loch-Mere Shelties
Additional Information: We breed for intelligence, conformation, agility and obedience. Our AKC shelties are born and raised inside with children and cats. We do all applicable health testing. Text or call 540-243-0470
We do have a Tri male puppy available. $1200 Domino is a gorgeous Tri boy with an impressive pedigree. He is available to a companion or... ...
Five Blessings Farm
Additional Information: We are a small, hobby breeder located in Central Virginia. Our puppies are raised in our home with our five children, and get lots of attention and TLC. We breed for pets.
We believe that everyone should have the opportunity to own a well bred puppy. We abide by the AKC rules and standards, and register with... ...
Oak Glen
Additional Information: Near Lynchburg, VA and 1 hr from NC border off US-29 434-546-3059. We are a retired USMC family & shelties are a big part of us.
If you have more than a couple of questions please call. I would rather talk to you. We breed strictly AKC shelties. We are a retired USMC family... ...
Whiteleaf Shelties
Additional Information: Whiteleaf Shelties have been breeding since the year 2000. All my shelties are akc regestered, I have all colors, and have a few litters a year. Visit my site for current puppy pictures.
... ...
Wind Dancer Shelties
Additional Information: Located in NE Ohio on six acres of land, fenced in for the shelties' pleasure is Wind Dancer Shelties. Wind Dancer is the home to the beautiful and intelligent Shetland sheepdog breed. We take pride in our dogs, show them in UKC and AKC venues and are proud of our breeding standards. Our dogs are shown in confirmation and bred for exceptional quality and temperament. Breedings are well planned out and are done with the sole purpose of improving the breed as a whole. We have health testing done to ensure that our puppies are free from hereditary defects and any incapacitating faluts. We follow the Shetland sheepdog breed standard and do not breed animals that are oversized or undersized, nor do we breed animals with any...
Wind Dancer Shelties, located in NE Ohio, strives to produce quality shetland sheepdogs for both personal pets and show/breeding.... ...
Twice Upon A Puppy
Additional Information: Raising AKC and CKC Sheltie puppies since 2007. We usually have 2-3 litters per year, all colors. Microchipped, one year health guarantee, pre-spoiled. Shipping/delivery available.
These puppies belong to my friend Tyffani Knott, and they are located in Martinsville, Virginia. Puppies were born 12-16-15 to Bella... ...
Additional Information: We are a small showing and breeding hobby kennel. I am a member of the American Shetland Sheepdog Association. All puppies are from champion lines, well socialized and vet checked.
Glen-Lakin Shelties
Additional Information: Welcome to Glen-Lakin Shelties - a small hobby kennel in Central Maryland. Our Shelties are sable and white from championship bloodlines suitable for conformation, agility and companions.
Grassy Creek Shelties
Additional Information: all puppies have been sold
Additional Information: Please visit my website, puppies are AKC reg. Check out my facebook page to see video of pups. www.bestpups.org or call me at 804-226-8963 hor 804-305-2106 cell
I am expecting puppies in February 2016 out of Precious & Checkers. Shelties are wonderful dogs for all ages. I have been breeding for 26... ...
Emperor Shelties
Additional Information: Emperor Shelties breed InternationalAKC Champions Conformation,Performance,Therapy&Loving Pets.Home raised, 35yrs an ASSA member. Puppies now Please 'Like' Emperor Shelties Littlestown on Facebook
Emperor Shelties has been breeding and showing Shetland Sheepdogs over 30 yrs and is a member of the American Shetland Sheepdog... ...
Additional Information: Pashwood Shelties has produced multiple AKC champions and Grand Champions. We have a sable and white male "pet" puppy available and are expecting an AOAC litter December 2014!
Wild Hair Acres
Additional Information: Welcome to Wild Hair Acres Shelties (Shetland Sheepdogs). In addition to AKC papers our puppies come with complete health records and puppy packs. We have over 20 years of experience raising & showing
Welcome to Wild Hair Acre Shelties (Shetland Sheepdogs). All of our puppies & dogs are up to date and... ...
Additional Information: Member of American Shetland Sheepdog Association. Info and pictures of some of our Shelties on www.lynroseshelties.com .
Candescent Shetland Sheepdogs
Additional Information: We are a hobby/show breeder active in conformation and performance. Genetic testing for hips, eyes, thyroid, and vWD is completed on all breeding animals. Visit our website!
We are dedicated to providing each one of our Shelties with the best care possible. We participate in Conformation and Agility, because we... ...
Wistwin Shelties
Additional Information: We are a show kennel, however we usually have pet puppies (or sometimes adults)available, (neutering required.) Member, American Shetland Sheepdog Assn.; Triangle SSC. AKC liclensed judge.
Additional Information: A small hobby breeder located in central PA. AKC Champion lines.AKC Breeder of MERITEXPECTING SABLE PUPPIES DEC 2015
CASMOR is a small hobby breeder located between Hershey and Harrisburg, PA. We are members of Harrisburg Shetland Sheepdog Club, Greater... ...
Smiley Acres Shelties
Additional Information: Raising since 1972, mostly for disposition&soundness.Bloodlines include Banchory,Cerarhope&Jade Mist. Colors of Tri,Bi Black,Sable&Blues.Showing in Obed.,Herding&Agility.My puppies will make you Smile
A litter of Sables was born on June 10, 2015. (All SOLD) Thanks, Patty ...
Additional Information: AKC registered shetland sheepdogs since 1989. litter born 1/7/16 to chassis by chumlee. 6 pups...1 female, 5 males. $500
litter born 1/7/16 to chassis by chumlee... 6 pups, 5 males, 1 female..will be ready to go first week of march. $500. i DO NOT ship puppies. ...
Fishers Sheltiepoos
Additional Information: WE HAVE PUPPIES AVAILABLE NOW! they are vet checked , Up to Date on their Vaccines, and Dewormed. They are very well socialized and love children!
Call us today to schedule a time to come meet your new family member 717-529-4066 ...
Additional Information: At Carlyle only healthy dogs with outgoing temperaments are used for breeding and competing. Referral service available. We currently do not have any puppies, but welcomeinquires
. Since 1989 Shelties have captured our hearts at Carlyle. We are breeders of AKC Champions and... ...
Additional Information: ABC kennel is a hobby kennel of 3 families that have over 100 years of combined experience showing, performance and breeding shelties and collies, including 2 veterinarians and a professional groomer.
We currently do not have any puppies available. ...
Fox Point Farm
Additional Information: We are a small private breeder of the finest small, mini and toy Shelties and Poshies for sale. All puppies are raised in our home with loving care. Our business is horses, our passion is shelties.
Additional Information: Welcome to Grand Jarvis Shelties, located in Greenville, NC. All of our dogs are AKC registered Shetland Sheepdogs.
Our puppies are well socialized and have lots of champions in their pedigrees.Puppies are Vet checked with their dew claws removed shortly after... ...
Country Shenanigans
Additional Information: We are a small breeder focusing on temperment and breed standard. Our puppies are born and raised in our home with plenty of love and socialization.
Updated 11/9/15 We have puppies!! Misty and Ramsey's litter were born on 10/30/15. I bi-blue male, 1 bi-black female, 1 blue... ...
Swansen Shelties
Additional Information: Sable Sheltie pups with great personalities! We are expecting a litter, hopefully, this spring.
~~~~~ Gorgeous Sheltie Pups! ~~~~~~ We are expecting a litter Christmas 2014. (Updated November 6, 2013) ...
Skye Shelties
Additional Information: Available pups as of September 2015: 1 blue merle female pup, 2 sable female and 1 male sable pup. They must be spayed/neutered, sold on AKC limited contract. $800-$1000.
Both the AOAC litter and the Sable litter were born on the same day: June 11, 2015 so the pups are 13 weeks old. They've had 2 vaccines, been... ...
Additional Information: 2 AKC Reg female pups will be ready 7 FEB (just in time for Valentines Da)y. Raised inside smoke free home. Vet shots, checked and dewclawed. Pets for forever homes only. See Macshelties
***EXTRA CUTE AKC Registered Shetland Sheepdog puppies born to Roxy and Prince Nathan our family pets on 15 December 2015.... ...
Important: breeders.NET has not screened the listed breeders and accepts no responsibility for their reputation or quality. It is your responsibility to investigate. See How to Find a Dog.
New Articles
NUTRITIONWith the wide variety of food at Thanksgiving dinner, chances are you'll want to give your dog something special, too. If you're contemplating what to feed your dog for the holiday, here is a guide to a great Canine Thanksgiving Feast.
DOG HEALTH So you have your new puppy picked out. There are quite a few shots, treatments and examinations that will keep the newest member of your family healthy.
DOG FUN Walking your dog is not only crucial to keeping him healthy and happy, it strengthens the bond between your canine friend and his caregiver. There are a lot of obstacles out there. Don't forget these simple tips to keep your walk fun and safe in the outside world.
HEALTH The same techniques that physiotherapists use to treat a variety of injuries and conditions in humans have been adapted to suit animals with great success. Family pets, show dogs, and working dogs can all benefit greatly from physiotherapy. Dogs whose activities involve a lot of agility are especially susceptible to the types of problems that physiotherapy can address.
FIRST TIME OWNERSBringing a dog into your family is a decision where many people don't realize it's magnitude until after they have the dog. There are a number of things that you need to research before you decide to purchase a dog, and it starts right in your own home.
HEALTH Many believe that a dog and a new baby cannot happily coexist, so therefore the dog has to go. This is not necessarily the case. A new baby does not mean you have to abandon your dog.
DOG TRAINING It's not enough to simply walk the dog through the front door and hope he will acclimate to his new surroundings; you have to teach him how to interact with the new people and items in his environment.
PET-NEWSWORTHY If you're considering adding a pet to your home in 2015, you're in very good company. As many as 71.4 million homes are expected to include a pet in 2015. According to the American Pet Product Association, pet families have increased from 56% of households in 1988 to 62% this year.


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Large and Small BGE, and a baby black Kub.
And all the toys to make me look like a Gizmo Chef.
Chattanooga, TN.
"You are who you are when nobody is looking"
Alcohol does not leave any taste on the food.No way would I use any type of liquid fire starter, leaves too much off taste to food.
"Entrepreneurs are simply those who understand that there is little difference between obstacle and opportunity and are able to turn both to their advantage."
I think your onto something about keeping it drier, thanks.My wife saves paper towels that she has used for bacon to soak up grease. She puts them in a ziplock in the fridge and keeps me supplied. I take a bacon grease soaked paper towel and tear it into three strips. I roll these strips up into a "candle" - and poke them down into my lump in three places. I use a butane grill lighter to ignite them. They really do burn a long time with the grease in them - just like candles. As they burn down, they ignite the lump down into the pile, closer to the air source. I have found this method to work extremely well - and it is cheap!As for taking longer to get your lump lit... I'm betting it is from moisture collecting inside your Egg between cooks. After your fires are snuffed out and the temp drops to zero, open the bottom vent all the way, but leave the screen in place to keep bugs out. This will help air circulate in the Egg between cooks. I have started doing this since about May and have found the interior has stayed much drier, the lump lights and temps rise much quicker without the excess moisture in it.