cv and references maynard dec30 2015
TRANSCRIPT
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Curriculum Vitae
Scott Maynard, Ph.D. Contact information: Danish Cancer Society Research Center, Strandboulevarden 49, Copenhagen, Denmark 2100 Phone: 011- 45-50146031 Email: [email protected] or [email protected] Education: Ph.D. York University, Toronto, Canada. PhD granted Oct 2004. Dissertation: The role of C/EBP family members in v-src transformation
and stress response in embryo fibroblasts; regulation of GABARAP during autophagy and apoptosis.
M.Sc. University of Saskatchewan, SK. Canada Thesis: Detection and quantitation of S. nodorum blotch on wheat stubble by PCR
B.Sc. University of Guelph, Guelph, Canada. Major in Biochemistry, Major in Molecular Biology, Minor in Microbiology Professional experience: Postdoctoral Fellow Nov. 2014-present Danish Cancer Society Research Center, Denmark (Jiri Bartek group) Summary: DNA repair and mitochondrial defects in premature aging diseases Research Assistant Professor (non-tenured) Jan. 2010-Oct 2014 Center for Healthy Aging, University of Copenhagen, Denmark (Vilhelm Bohr group) Summary: Investigation of DNA repair and mitochondrial dysfunction in blood cells of human patient cohorts (Alzheimer’s disease, Fatigue); roles of lamins in DNA repair Postdoctoral Fellow 2005-Jan. 2010 National Institutes of Health, Baltimore, MD. (Vilhelm Bohr group) Summary: Oxidative DNA damage processing and mitochondrial functions in human and mouse cells including stem cells Postdoctoral Fellow 2003-2005 University of Michigan Cancer Center, Ann Arbor, MI. (Richard Miller group)
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Summary: Investigation of genetic determinants of stress resistance and delayed growth arrest in cells from long-lived Snell dwarf mice. Publications:
1. Chan JWYF, Maynard S, Goodwin PH (1998). Cloning of a two-component signal transduction system of Xanthomonas campestris pv. phaseoli var. fuscans strain BXPF65 . Can J Plant Pathol. 20: 288-295.
2. Maynard S*, Gagliardi M*, Bojovic B, Bedard PA (2001). The constitutive
activation of the CEF-4/9E3 chemokine gene depends on C/EBP in v-src transformed chicken embryo fibroblasts. Oncogene 20: 2301-2313 (*co-first authors).
3. Gagliardi M, Maynard S, Miyake T, Tjew SL, Bedard PA (2003). Opposing
roles of C/EBP and AP-1 in the control of growth-arrest specific gene expression. J Biol. Chem. 278(44): 43846-54.
4. Maynard SP, Miller RA (2006). Fibroblasts from long-lived Snell dwarf mice are resistant to oxygen-induced in vitro growth arrest. Aging Cell 5(1): 89-96.
5. Maynard S, Swistowska AM, Liu Y, Liu ST, Da Cruz AB, Lee JW, Rao M,
de Souza-Pinto N, Zeng X and Bohr VA (2008). Human embryonic stem cells have enhanced DNA repair of multiple forms of damage. Stem Cells 26 (9): 2266-2274. (91 citations)
6. Maynard S, Schurman SH, Harboe C, de Souza-Pinto NC, Bohr VA (2009). Base excision repair of oxidative DNA damage and association with cancer and aging. Review. Carcinogenesis 30 (1): 2-10. (224 citations)
7. Maynard S*, de Souza-Pinto N*, Hashiguchi K, Hu J, Muftuoglu M, Bohr VA
(2009). The recombination protein RAD52 cooperates with the base excision repair protein OGG1 for the repair of oxidative lesions in mammalian cells. Mol Cell Biol 29 (16): 4441-4454 (*co-first authors).
8. Maynard S, de Souza-Pinto NC, Scheibye-Knudsen M, Bohr VA (2010). Mitochondrial base excision repair assays. Review. Methods 51 (49) 516-425.
9. Canugovi C, Maynard S, Bayne AC, Tian J, de Souza-Pinto N, Croteau DL,
Bohr VA (2010). The mitochondrial transcription factor A functions in mitochondrial base excision repair. DNA Repair 9 (10) 1080-1089.
10. Scheibye-Knudsen M, Ramamoorthy M, Sykora P, Maynard S, Lin P-C, Minor RK, Wilson III DM, Cooper M, Spencer R, de Cabo R, Croteau DL,
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Bohr VA (2012). Cockayne syndrome group B protein prevents mitochondrial stress and promotes autophagy. J Exp Med 209(4) 855-869.
11. Maynard S*, Keijzers G*, Gram M, Desler C, Bendix L, Budtz-Jørgensen E,
Molbo D, Croteau DL, Olser M, Stevsner T, Rasmussen LJ, Dela F, Avlund K, Bohr VA (2013). Relationships between human vitality and mitochondrial respiratory parameters, reactive oxygen species production and dNTP levels in peripheral blood mononuclear cells (*co-first authors). Aging 5(11) 850-864.
12. Akbari M, Keijzers G, Maynard S, Scheibye-knudsen M, Desler C, Hickson ID, Bohr VA. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair. DNA Repair 2014 Apr; 16(4):44-53.
13. Keijzers G, Maynard S, Shamanna RA, Rasmussen LJ, Croteau DL, Bohr VA. The role of RecQ helicases in non-homologous end-joining. Review. Critical Reviews in Biochemistry and Molecular Biology. 2014. Nov-Dec; 49(6): 463-72.
14. Maynard S, Keijzers G, Hansen ÅM, Osler M, Molbo D, Bendix L, Møller P, Loft S, Moreno-Villanueva M, Bürkle A, Hvitby CP, Schurman SH, Stevnsner T, Rasmussen LJ, Avlund K, Bohr VA. Associations of subjective vitality with DNA damage, cardiovascular risk factors and physical performance. Acta Physiologica (Oxf). 2015 Jan; 213(1):156-70.
15. Maynard S*, Ghosh R*, Wu Y, Yan S, Miyake T, Gagliardi M, Rethoret K,
Bedard PA. GABARAP is a determinant of apoptosis in growth arrested embryo fibroblasts. J Cell Physiol 2015 Jul; 230(7):1475-88.
16. Maynard S, Fang EF, Scheibye-Knudsen M, Croteau DL, Bohr VA. DNA damage, DNA repair, aging and neurodegeneration. Review. The Longevity Dividend. Cold Spring Harbor Press. Editors S. Jay Olshansky, George M Martin, and James L Kirkland. Cold Spring Harbor Perspect Med. 2015 Sep 18; 5(10).
17. Maynard S, Hejl A-M. Dinh T-S T, Keijzers G, Hansen Å M, Desler C. Moreno-Villanueva M, Bürkle A, Rasmussen LJ Waldemar G, Bohr VA. Defective mitochondrial respiration, altered dNTP pools and reduced AP endonuclease 1 activity in peripheral blood mononuclear cells of Alzheimer’s disease patients. Aging. 2015 Oct; 7(10): 793-815.
18. Desler C, Frederiksen JB, Angleys M, Maynard S, Keijzers G, Stevnsner T, Bohr VA, Rasmussen LJ. dNTP imbalance in peripheral blood mononuclear cells is associated with cognitive decline in middle aged men. Mitochondrion. 2015 Sep 25; 25: 34-37.
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19. Maynard S*, Nielsen RH*, Schjerling P, Kjær M, Qvortrup K, Bohr VA
Rasmussen LJ, Jemec GB, Heidenheim M. Acquired localized cutis laxa due to increased elastin turn-over (*co-first authors). Case Reports in Dermatology (In Press).
Manuscript in preparations*
Maynard S, Keijzers G, Scheibye-Knudsen M, Akbari M, Tian J, Gonzalo S, Bartek J, Bohr VA. Base excision repair and cellular respiration are compromised in lamin A/C-depleted cells and in fibroblasts from Hutchinson Gilford progeria patients. (*see attached pdf).
Professional activities
Lecturer for Human Biology course (organized by Lotte Vogel), University of Copenhagen, 2010-2013
Project teacher for IARU CEHA summer school (organized by Ying Liu), 2011, 2012, 2013
12 conference presentations (poster seven times, speaker five times), 2002-2013
Reviewer for international peer-reviewed journals: Mutation Research, Experimental Gerontology, Mechanisms of Aging and Development, and Neuroscience, 2006-2012
Laboratory Teaching Assistant for molecular biology and genetics courses at University of Saskatchewan and York University (during MSc and PhD), 1995-2003
Committee member for York University Biology, Department Symposium, 2001 and 2002.
Graduate Representative for York University Biology Department, 2001
Computer lab supervisor for York University Biology Department (Graduate Assistantship), 2001
Awards/funding:
As a Research Assistant Professor at the University of Copenhagen (2010-2015, Vilhelm Bohr group), my research was funded by a large Nordea-fonden grant shared by members of the Center for Healthy Aging. I contributed to the grant renewal process in 2013 by assisting in writing research summaries and presenting our data to the committee.
Fellows Award for Research Excellence (FARE), 2009
Scholarship recipient for the 3 week MBL Molecular Biology of Aging course, 2007
National Institutes of Health Intramural Training Award, 2005-2010
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Scholarship recipient for the Buck Institute training Symposium on Aging, 2002
References:
1. Dr. Vilhelm Bohr (postdoctoral supervisor, 2005-present) Dept. Chief, Lab. Molecular Gerontology, NIA-IRP, NIH, Baltimore, MD, 21224 Phone: 410-558-8223; email: [email protected]
2. Dr. Lene Juel Rasmussen, Director of Center for Healthy Aging, Dept. Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark 2200 Phone: 011-45-35326717 email: [email protected]
3. Professor Alexander Bürkle,
Chair of Molecular Toxicology, Dept. of Biology, Box 628 University of Konstanz D-78457, Konstanz, Germany Tel. +49-7531-88-4035. Tel. +49-7531-88-4034(secretary) Fax +49-7531-88-4033. Email: [email protected]
4. Dr. Shepherd H. Schurman, MD
Acting Medical Director of the Clinical Research Unit PI, Environmental Polymorphisms Registry Clinical Research Program, NIEHS, NIH Research Triangle Park, NC 27709-0002 Phone: 919-541-7736 email: [email protected]
5. Dr. Richard Miller (postdoctoral supervisor, 2003-2005) Geriatrics Center, University of Michigan Ann Arbor, MI 48109 Phone: (734) 936 2122; email: [email protected]
6. Dr. Andre Bedard (PhD supervisor, 1997-2003)
Professor and PhD supervisor Department of Biology, McMaster University 1280 Main St. West, Hamilton, Ontario L8S 4K1, Canada Phone: (905) 525-9140 73; email: [email protected]
Base excision repair and cellular respiration are compromised in lamin A/C-
depleted cells and fibroblasts from Hutchinson Gilford Progeria patients
Scott Maynard1, Guido Keijzers1, Morten Scheibye-Knudsen2, Mansour Akbari1,
Jane Tian2, Susana Gonzalo3, Jiri Bartek4, and Vilhelm A. Bohr2
1Department of Cellular and Molecular Medicine, Center for Healthy Aging, University of
Copenhagen, Copenhagen, 2200, Denmark
2Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health,
Baltimore, MD 21224, USA
3Department of Biochemistry and Molecular Biology, Saint Louis University, School of Medicine,
Saint Louis, MO 63104
4Danish Cancer Society Research Center, Copenhagen, Denmark
ABSTRACT
The A-type lamins (lamin A and C encoded by the LMNA gene) are important structural components of the nuclear lamina. LMNA mutations lead to degenerative disorders termed laminopathies, including the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Altered expression levels of lamins are found in various cancers. Reports indicate the lamins play a role in DNA double strand break repair, but a role for lamins in base excision repair (BER) has not be described. In this report we provide evidence for compromised BER in lamin A/C-depleted cells (MEF LMNA knockout and siRNA knockdown in human cells), including altered oxidative stress response in microarray analysis, increased cell sensitivity to oxidative and alkylation stress, less efficient DNA repair of oxidative lesions, and impaired DNA incision activity of two base excision repair enzymes (OGG1 and APE1). These cells also displayed impaired glycolytic and mitochondrial bioenergetic fluxes, concomitant with higher levels of PARylation. Moreover, in LMNA null MEF there was a reduction in the level of several BER enzymes, and impaired recruitment of OGG1 and APE1 to damaged DNA. Many of these impairments were recapitulated in dermal fibroblasts from HGPS patients, suggesting common DNA repair and bioenergetic defects caused by both lamin depletion and the lamin mutation that leads to HGPS. These findings reveal a novel role for lamin A/C in promoting BER, with a functional link to cellular respiration through PARP1 activation. This study gives new insight into the role of A-type lamins in the DNA damage response and laminopathies, with implications for treatment.
INTRODUCTION
The nuclear lamina is located at the inner periphery of the nucleus and consists
mostly of the A-type lamins, lamin A and C (herein referred to as lamin A/C) which
arise by alternative splicing of a single gene LMNA, and of lamin B (encoded by
the LMNB gene) and associated proteins. A- and B - type lamins are type V
intermediate filament proteins and have important scaffolding roles in the nucleus,
giving it mechanical support and regulating protein transport, chromatin access,
and gene expression, ultimately regulating cellular events such as DNA replication,
transcription, and DNA damage response. Mutations in LMNA have been linked to
cancer and physiological aging, as well as degenerative disorders, broadly termed
laminopathies, including muscular dystrophy, neuropathies, lipodystrophies, and
premature aging syndromes (e.g. Hutchinson-Gilford Progeria syndrome, HGPS)
(1-7). The specific molecular mechanisms that lead to disease from defective A-
type lamins are poorly understood. However, various lines of evidence have linked
laminopathies with increased genomic instability (3;4).
In addition to the impact of LMNA mutations, non-optimal levels of wild-type lamin
A/C can also be deleterious. Accumulating evidence indicates that the nuclear
lamina composition and altered expression or steady state levels of lamins
influence tumor formation and cancer progression (8-10). Several studies indicate
that the levels of A-type lamins are either lower (or even absent) (11-18) or higher
(19-21) in various human cancers and that these changes have a significant impact
on the disease severity. These data suggest that A-type lamin expression and/or
stability is important for proper control of cell proliferation, and that improper levels
could promote tumorigenesis. Moreover, mice lacking A-type lamins develop
defects in skeletal and cardiac muscles, a phenotype characteristic of muscular
dystrophy. Cells from these mice also have deformation of the nuclear envelope,
and epigenetic alterations, concomitant with mislocalization of emerin, an inner
nuclear membrane protein linked to muscular dystrophy (22). These cells also
have a loss of chromatin integrity, thus implicating a link between lamin depletion
(absence in this case) and genomic instability in laminopathies.
Mutations in the LMNA gene as well as lamin A/C depletion has recently been
shown to lead to defects in double strand break repair, contributing to genomic
instability (3;6;23-30). Moreover, a study has shown that DNA damage results in
suppression of the GH/IGF-1 axis, which in turn leads to striking progeriod
symptoms (31). Recent studies suggests that laminopathies are associated with
altered ROS metabolism and oxidative stress and that lamins are critical in cellular
response to ROS generation (32-35). However, the underlying mechanisms by
which lamin responds to oxidative stress remain poorly understood, and there had
been no investigation into base excision repair (BER) in this regard. BER is the
major pathways for removal of oxidative and alkylated lesions and defects in this
pathway lead to accumulation of these lesions (36;37). In this pathway, a damaged
base (for example 8-oxodG) is often recognized by a DNA glycosylase (for
example OGG1) enzyme that mediates base removal before nuclease (example,
APE-1), polymerase and ligase proteins complete the repair, in processes
overlapping with those used by single strand break (SSB) repair (37). PARP1 helps
mediate BER by catalyzing the poly(ADP-ribosyl)ation of a several acceptor protein
involved in chromatin architecture. It is well established that oxidative stress
triggers assembly of BER complexes on open chromatin regions; components
include 8-oxoguanine DNA glycosylases (OGG1) and AP endonuclease-1 (APE-
1), (38). It is also well established that lamins effect chromatin organization; they
provide anchorage sites for peripheral elements of the heterochromatin, which are
involved in local regulation of gene expression (39-42). Current findings also
suggest that lamins have a role in regulating cell proliferation and longevity through
oxidative stress responses and ROS signaling pathways (43).
Relative mRNA expression data by microarray analysis of LMNA-/- versus LMNA+/+
has recently been reported (29), indicating associations between disease, such as
cancer and renal necrosis, and significant alterations of several genes classes that
included DNA repair and DNA damage response. For example, UNG and PARP1
were upregulated in the LMNA-/- cells. Both of these proteins are involved in BER.
In this current study we also perform microarray on LMNA-/- versus LMNA+/+ and
report novel data suggesting a canonical oxidative pathway alteration and that
glycolytic activity is downregulated. We then preformed cell survival and various
cellular and biochemical DNA repair assays on lamin-depleted cells to further
examine the role of A-type lamins in the repair of oxidative damage. We present a
previously unrecognized role of lamin A/C in response to oxidative stress:
specifically, promotion of repair of oxidative lesions by BER. This appears to be
mediated in part by enhanced intrinsic activity of two BER enzymes, the
glycosylase OGG1 and the endonuclease APE1. We also saw that PARP
activation (PARylation) was higher in these cells, apparently in response to
unrepaired DNA damage, and a concomitantly a reduction in cellular respiration
(glycolytic and mitochondrial respiration fluxes). BER and cellular respiration was
also found in fibroblasts from HGPS patients, thus linking these lamin-deficiency
defects not only to cancer but also to laminopathies.
MATERIALS AND METHODS
Cell culture
U2OS cells, HeLa cells (both obtained from the American Type Culture Collection;
ATCC), wild-type MEF (MEF+/+), and LMNA knockout (MEF-/-), were grown in
Dulbecco modified eagle medium (DMEM) supplemented with 10% fetal bovine
serum (Gibco BRL), 50µg/ml streptomycin and 50 U/ml penicillin at 37C with 5%
CO2.
Western blotting analysis
Proteins were separated on 12% Tris-glycine gels (456-1046; Bio-Rad), in Novex
tris-glycine SDS running buffer (LC26755; Life Technologies). 20-100 g of total
protein in whole cell extracts was loaded per lane. Transfer to PVDF membranes
(LC2002; Life Technologies) was carried out by electroblotting in transfer buffer in
Novex tris-glycine transfer buffer (LC3675; Life Technologies) containing 20%
methanol, for 1 hr at 100 V. Membranes were blocked 1 h at room temperature in
5% nonfat dry milk (232100; BD Difco) in TBST (20 mM Tris-HCl pH 7.2, 137 mM
NaCl, 0.1% Tween-20). All antibodies were diluted in fresh milk (5%)-TBST and
incubated with the membrane. Antibodies used were: anti- actin (AMB-7229;
Nordic Biosite), anti-lamin A/C (sc-20681; Santa Cruz), anti-OGG1 (S0675;
Epitomics), anti-APE1 (ab97296; Abcam), anti-ligase III (sc-135883; Santa Cruz),
anti-H2AX (07-164; Millipore), anti-PARP1 (614302; Nordic Biosite), anti-Polβ
(ab26343; Abcam). Secondary HRP-conjugated antibodies (Jackson
ImmunoResearch) and Pierce ECL Plus (32132; Thermo Scientific) were used to
visualize the protein bands on high performance chemiluminescence film
(28906837; GE Healthcare). The films were scanned and saved as 16-bit
greyscale tiff files.
Single Cell Gel Electrophoresis (Comet) Assay
Comet assays were performed on HeLa, U2OS and MEF cells, based on our
previously described method (44) and in other reports (45;46). Cells were grown
in 60 mm or 100 mm dishes and treated with 100 μM H2O2 for 30 min at 37oC.
Cells were harvested at several time points: untreated (UT), immediately after
treatment (0 h), and the indicated time points after treatment (during repair).
Harvested cells were collected and suspended in 200-600 l PBS. 6-10 l of cells
(i.e. approximately 5000 cells) were mixed with 75 l 0.5% low-melting-point
agarose in PBS, and spread on a microscope slide, pre-coated (and dried) with 1%
agarose in PBS, and allowed to cool for 5 min. The slides were then incubated
overnight in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-
100). Cells were rinsed three times, 5 min each, in neutralization buffer (0.4 M Tris-
HCl, pH 7.4). The specifically determine extent of oxidative DNA damage, the
slides were then treated with 5 U of formamidopyrimidine DNA glycosylase (FPG)
(4040-500-01; Trevigen, 5 U/l) per slide (1 l FPG in 99 l FLARE buffer I
(Trevigen) containing 0.1 mg/ml BSA) and incubated for 1 hr at 37C. Control slides
were treated with FLARE buffer/BSA. Slides were then washed in PBS three times
before incubation in cold unwinding solution (300 mM NaOH, 1 mM EDTA, pH >
13) at 4C for 30 min. Electrophoresis was carried out in the unwinding solution at
25 V, 300 mA, for 30 m. Slides were fixed in 100% ethanol for 5 m and then allowed
to dry. The slides were stained with SYBR Gold nucleic acid dye (S-11494; Life
Technologies) at 1/10,000 dilution in Tris-EDTA buffer pH 7.5. Assay results were
visualized using fluorescence microscopy (Axiovert 200M, Carl Zeiss) and
analyzed using the Komet 5 software (Andor Technology). Each data point
represents the mean of 50-100 olive tail moment (OTM) values. A larger and more
intense comet tail (OTM) indicates more DNA damage. For estimation of oxidative
damage, the OTM for buffer treated cells was subtracted from the OTM for FPG
treated cells.
DNA Incision Activity Assays
Cell extracts for OGG1 and APE1 incision assays were performed as described
previously (47) (ALSO ADD THE AD PAPER). Oligonucleotide DNA substrates for
measuring BER activities of 8-oxoG and AP incision activity have been used
extensively by our group and others: 8-oxodG. Oligonucleotides were obtained
from Midland Certified Reagent Co., and their sequences are shown in 5’-end
labeling was carried out using T4 polynucleotides kinase (New England BioLabs)
and [γ-32P] ATP. Unincorporated [32P] ATP was removed with a Sephadex G25
spin column (GE Healthcare). Incision activity of OGG1 and APE1 were measured
using 28-mer radiolabeled double-stranded DNA oligonucleotides, 8-oxodG/OG
28-comp and THF28/OG 28-comp, respectively. Reactions of OGG1 were
incubated for 16 hours at 32°C and prior to termination 10 min incubation with 10
N NaOH. Reactions of APE1 were incubated for 10 min at 37°C. Both reactions
were terminated by addition of formamide loading dye (90% formamide, 10 mmol/L
EDTA, 1 mg/mL xylene cyanol FF, and 1 mg/mL bromophenol blue) and heat
inactivated by 80°C for 10 min. Reaction products were resolved by 20%
acrylamide and 7 M visualized by phosphorimaging (Typhoon 9410, Amersham
Biosciences) and analyzed using ImageQuant 5.2 (Molecular Dynamics). Incision
activity was determined as the intensity of product bands relative to the combined
intensities of substrate and product bands. Statistical comparisons were performed
using GraphPad Prism 5.04 software (La Jolla, CA, USA).
Table 1. Oligonucleotide used as substrates in assays for DNA repair activities
Specificity Name Sequence
8-oxodG site (= O) OG 28 5’- GAA CGA CTG T(O)A CTT GAC TGC TAC TGA T
AP site (=F) THF28 5’-GAA CGA CTG T(F)A CTT GAC TGC TAC TGA T
Complementary strand OG 28-comp 3’- CTTGCTGACA CTGAACTGAC GATGACTA
Immunoprecipitation assays
For Immunoprecipitation experiments, one 150-mm plate was used per condition.
U2OS cells were grown to near confluence, treated as indicated, washed twice in
PBS and lysed at 4oC in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM
EDTA, 1% Triton X-100, and complete protease inhibitor cocktail (Roche)).
Immunoprecipitation experiments were performed using protein A-agarose beads
(Santa Cruz) and anti-lamin A/C antibody (sc-6215, goat polyclonal), according to
manufacturer’s (Santa Cruz) protocol. The immuno-precipitates were resolved on
Tris-Glycine gels (Invitrogen) followed by electro-blotting to PVDF membrane. The
immunoblots were analyzed with anti-lamin A/C (sc-20681; rabbit polyclonal), anti-
emerin (NCL-EMERIN, Leica Biosystems) as a positive control, anti-OGG1
(S0675; Epitomics), anti-APE1 (ab97296; Abcam), anti-actin (AMB-7229; Nordic
Biosite), and visualized with ECL (GE Healthcare).
Immunofluorescence
For 8-oxoG detection, lamin A-/- MEF and wild-type MEF were seeded into 4-
chamber covered slides at 200,000 cells/well. Exponentially growing cells were
treated with 100 M H2O2 in DMEM for 30 min at 37C and allowed to repair in
complete media for 10 h, or not allowed to repair (0 h); an untreated (UT) condition
was also included. Preparation of stained slides was then carried out as described
previously (48), including fixation with ice-cold 1:1 methanol-acetone for 20 minute,
and using anti-8-oxoG antibody (clone 2E2) (4354-MC-050; Trevigen). The
negative control was normal chicken IgY in place of the primary antibody. For PAR
detection, lamin-siRNA U2OS cells and control U2OS cells (scrambled-siRNA)
were seed into 4-chamber covered slides at 200,000 cells/well. Exponentially
growing cells were treated with 100 M H2O2 in DMEM for 30 min at 37C and
allowed to repair in complete media for 45 min. Cells were fixed with 4%
paraformaldehyde for 10 min and permeabilized with 0.2% triton X for 5 min. Slides
were washed with PBS twice and then blocked for 1 hr at room temperature with
10% goat serum in PBS, and then incubated with primary anti- anti-PADPR
antibody (ab14460; Abcam) overnight. The negative control was normal mouse
IgG in place of the primary antibody. In all cases, the slides were mounted with
DAPI-containing vectashield (vector laboratories) and analyzed using a Zeiss
Axiovert 200M microscope with X10 or X40 objective lens.
siRNA transfections
Lamin A/C expression in HeLa or U2OS cells was transiently reduced using ON-
TARGETplus siRNA human LMNA (J-004978-05; Thermo Scientific Dharmacon).
Control cells were those transfected with ON-TARGETplus control siRNA Non-
targeting siRNA 1 (D-001810-01-05; Thermo Scientific Dharmacon). The cells
were seeded in 100mm dishes and grown in DMEM supplemented with 10% FCS
(no antibiotic) for 24 h to reach approximately 70% confluence; 50 nM of each
siRNA were transfected into the cells using DharmaFECT 1 (T-2001-02; Thermo
Scientific Dharmacon), according to the manufacturer’s directions. The cells were
harvested for further use after 72 hr of incubation.
Cell viability assays
30,000 cells were plated in triplicate onto 96-well microtiter plates (flat bottom) in
complete growth medium. After 24 h incubation the cells were washed in PBS and
exposed to the indicated stressors over a range of doses indicated in the figure
legends. Oxidative DNA damage was induced by treatment for 4 hr with hydrogen
peroxide (H2O2) or menadione. Alkylation DNA damage was induced by methyl
methane sulfonate (MMS) treatment for 1 hr. These liquid stressors were made in
DMEM (no FCS, no antibiotic). These types of damage are typically repaired by
BER. UV-C- induced DNA damage, typically bulky DNA adducts, trigger for the
most part nucleotide excision repair (NER). At the end of the treatments, the cells
were washed with PBS; 100 l of fresh medium was added and then 10 l of WST-
1 (Roche). After 3 hr incubation in WST-1 the absorbance (450 nm minus 630 nm)
was read. This assay is based on the cleavage of tetrazolium salts to soluble
formazan dye by mitochondrial succinate-tetrazolium reductase which exists in the
mitochondrial respiratory chain and is active only in viable cells. The quantity of
formazan dye in the medium is directly proportional to the number of viable
metabolically active cells.
Measurement of 8-oxoG by HPLC
The 8-oxoG measurements were conducted, on HeLa cells transfected with lamin
A/C siRNA or scrambled siRNA, as described previously (49-51). Nucleoside
isolation was achieved by nuclease P1 and calf alkaline phosphatase treatment.
The digested material was subjected to filtration and high performance liquid
chromatography coupled to electrochemical detection using the ESA Biosciences
Inc. four-channel Coularray system (Chelmsford, MA). The experiment was
repeated three times.
Microarray
RESULTS
Lamin A/C promotes DNA repair of oxidative lesions following oxidative
stress
A role for lamin A/C in the repair of oxidative lesions has never been reported,
despite the importance of oxidative stress in laminopathies (52). We performed
microarray analysis on lamin null MEF and control MEF and found evidence that
DNA repair pathways are altered (Figure 1, Tables 1-3) including: 1. Oxidative
stress response was a top canonical pathway, 2. The gene ontology (GO term)
pathways “hydrolase activity acting on glycosylase bonds”, “damaged DNA
binding” and “DNA repair” are altered, 3. p53 is downregulated. This led us to
further pursue DNA damage response and in particular the DNA repair response
to oxidative stress, BER. Since BER deficiency is often reflected by poor cell
survival under oxidative or alkylation stress, we firstly performing cell survival
assays on cells under oxidative stress (H2O2 or menadione treatment) or during
exposure to methyl methane sulfonate (MMS; to give alkylation damage (Figure
2). These genotoxic lesions are mainly repaired by BER. In contrast, we also
measured survival after UV-C stress; this produces bulky DNA adducts that trigger
for the most part nucleotide excision repair (NER). These assays were performed
on human cells with transient knockdown of lamin A/C (lamin A/C-siRNA, referred
to in this report as “KD” for knockdown), along with control cells (human cells with
scrambled siRNA; scr-siRNA, referred to in this report as “C” for control). Lamin-
siRNA U2OS cells were more sensitive to H2O2, menadione and MMS compared
to control cells, and slightly more sensitive to UV-C (Figure 2B).
Next, we preformed the FPG-comet assay to determine if lamin A/C plays a role
specifically in DNA repair of oxidative DNA lesions (Figure 3A). This variant of the
comet assay incorporates the FPG enzyme to convert 7,8-dihydro-8-oxoguanine
(8-oxoG) and other oxidized purines to breaks. To determine comet tails values
that estimate the amount of oxidized purines (FPG-sensitive sites), the percent tail
DNA values were determined by first subtracting out all the non-FPG-treated
values (from the FPG-treated values) at each condition, then subtracting out the
values for non-H2O2 -treated cells and finally comparing to the zero hour values set
to 100. The lamin-siRNA HeLa, lamin-siRNA U2OS, and lamin A-/- MEF showed
significantly less efficient DNA repair of FPG-sensitive sites (less reduction in
comet tail size than their respective control cells during the indicated repair times).
Since the standard 20% oxygen incubation conditions in which the cells grow is
relatively toxic compared physiological 3% oxygen, we also sought to determine if
this might be reflected in oxidative lesion accumulation in the cells that were not
subjected to liquid oxidative stress (H2O2). Typically the comet tails are quite small
in untreated cells (as can be seen in the pictures of the representative comet tails
for U2OS cells), however, in the case of the MEFs, the tails were large enough to
make a sufficiently accurate determination of tails size difference between MEF+/+
and the lamin A-/- MEF. There is a more extensive accumulation of FPG-sensitive
sites in the lamin A-/- MEF. In order to support the comet assay data, we measured,
by immunofluorescence, specifically the level of 8-oxoG lesions in nuclear DNA of
lamin A-/- MEF along with control MEF, during repair after treatment with H2O2
(Figure 3B). The lamin A-/- MEF repaired significantly less efficiently than control
cells, as quantitated in the lower bar graph of the Figure 3B. In fact, even without
addition of the oxidative stressor (untreated MEFs), there was a more extensive
accumulation of 8-oxoG in the lamin A-/- MEF, relative to the MEF+/+, as illustrated
in the upper bar graph of Figure 3B. We also examined 8-oxoG lesions by HPLC,
in untreated lamin-siRNA HeLa cells and HeLa control cells (scr-siRNA) (Figure
3C). This revealed significantly more accumulation of 8-oxoG in the lamin-siRNA
HeLa.
Lamin A/C levels are higher in the chromatin fraction after oxidative stress
It is known that A-type lamins binds to chromatin to an increased extent after DSB-
inducing genotoxic stress (39-41;53); data suggests that A-type lamins might
anchor DSB repair foci to the nuclear lamin meshwork. However, no study had yet
looked for extent of lamin A/C–chromatin interaction after oxidative stress. Thus,
we decided to look for enhanced lamin A/C-chromatin interaction before and after
the oxidative damage treatments that we used for the comet assay (100 µM H2O2).
We performed western blotting on lysates from lamin-depleted cells, with and
without DNase treatment to generate chromatin and soluble fractions. This
revealed that lamin A and C bind more extensively to the chromatin after the
oxidative treatment (Figure 4). We speculate that the enhanced DNA repair that is
observed in the control cells (normal lamin A/C levels) may be due in part to the
interaction of A-type lamin with chromatin, in a similar anchorage mechanism by
which lamins assist in DSB repair.
Lamin A/C promotes the DNA excision step of base excision repair by
activation of OGG1 and APE1 activity.
Our GO term microarray data on wild-type and lamin null MEF indicated that
glycosylase activity may be altered in MEF. Also, studies suggest that OGG1
activity for removal of 8-oxoG lesions is rate limiting in BER and APE1 incision
activities are rate limiting steps in BER of strand breaks and oxidative lesions.
Thus, we investigated the effect of lamin depletion on the DNA incision step of
BER. We prepared whole cell extracts from lamin A-/- MEF and human lamin-siRNA
cells, and the corresponding control cells, and examined the DNA incision activity
of OGG1 and APE1 by using duplex oligonucleotide substrates containing an 7,8-
dihydro-8-oxoguanine (8-oxoG) lesion and AP-site (5’ tetrahydrofuran, THF) site,
respectively. Both the OGG1 and APE1 activities was shown to be reduced in lamin
A-/- MEF and lamin siRNA HeLa cells relative to control MEF (Figure 7A). This
effect does not appear to be due to the protein level of OGG1 or APE1, as they are
unchanged by lamin A/C depletion as shown by western blotting (Figure 7B). In
addition, Co-IP analysis indicated that lamin A/C does not appear to physically bind
to OGG1 or APE1. This data points to a more complex lamin-dependent
mechanism (54) by which lamin A/C enhances the activity of these BER enzymes,
such as transcription factor shuttling or nuclear import of DNA damage response
factors.
Fibroblasts from HGPS patients have reduced BER efficiency
To determine if the attenuated BER that we see in lamin deficient cells is likewise
seen in HGPS cells relative to cells from controls (cells from a non-symptomatic
direct relative), we performed FPG-comet assay on these cells. We found that the
dermal cells from the HGPS patient were deficient in BER. This indicates that there
some component of the BER response is negatively affected by both the lamin
depletion and the HGPS mutation.
DISCUSSION
Here we demonstrated for the first time that lamin A/C promotes BER and limits
the accumulation of oxidative lesions and the resultant mutations. Thus, we have
uncovered an additional manner by which lamins preserve the integrity of the
genome and restrain DNA damage-associated diseases. One mechanism appears
to involve lamin-mediated enhancement of OGG1 and APE1 DNA incision
activities, before any appreciable exogenous stress. We also found that lamin A/C
had enhanced association with chromatin after oxidative stress (during DNA
repair); the identical stress conditions (100 µM H2O2) that we had used in the comet
assays in which lamin was found to promote BER. The data warrants further
research into the lamin A/C-chromatin dynamics during repair of oxidative lesions.
This line of investigation also needs to be pursued on cells from progeria patients
(mutation in LMNA) and also cells from cancer patients in which lamin A/C
expression is found to be altered.
The strength of this study comes from the battery of complementary data
suggesting that A-type lamin participate in the following: 1. Oxidative stress
signaling (Microarray: Figure 1, Tables 1 and 3); 2. Enhanced cellular oxidative
stress resistance (Figure 2); 3. Promotion of repair of FPG-sensitive DNA sites (in
MEF and two human cell types) and prevention of 8-oxoG lesion accumulation
(Figure 3); 4. Enhanced of BER enzyme activities (OGG1 and APE1). The above
data together suggest that lamin A/C is important in preventing oxidative DNA
damage accumulation. The incorporation of FPG into the comet assay enabled
estimation of oxidative damage. FPG recognizes oxidized purines (products of
DNA oxidation) and some alkylated DNA products (55;56), and thus the FPG-
sensitive sites that we measured by comet assay includes also alkylated DNA. All
of these lesions are mainly repaired mainly by BER.
Lamin depletion led to decreased cell survival after oxidative stress and MMS
stress. Both of these stressors produces DNA lesions predominantly repaired by
BER, and have not been used in this lamin-deficiency context previously. We also
found that there as a slight decrease in survival after UV-C stress. This is
consistent with a recent report in which lamin null MEF had a slight decrease in
cell survival (compared to control MEF) under a similar range of UV stress as used
by us (29). These data imply that lamin may also play a role in nucleotide excision
repair (NER). This same group also found that loss of lamin A/C results in
decreased cyclin D1 levels. Moreover, several reports have linked lamin
expression to cell cycle progression, and so we decided to also test cell
proliferation in lamin depleted cells (supplemental Figure S1). We found that lamin
A/C knockdown in HeLa cells resulted in a small reduction in cell proliferation. This
agrees with a previous report in which lamin A/C knockdown in human primary
fibroblasts resulted in an impaired proliferation (57). However, there was no
significant reduction in lamin null MEF relative to normal MEF, unless the cells
were subjected to continuous low-level oxidative or alkylation stress. Thus lamin
deficiency combined with oxidative or alkylation stress results in a reduction in both
cell survival and cell proliferation rate (the later effect presumably due to reduced
cell cycle progression efficiency).
We found that lamin A/C promotes BER, both in cells undergoing DNA repair
(comet assay and 8-oxoG monitoring) and on damaged DNA substrates (OGG1
and APE1 activity in cellular lysates). With respect to the comet assay results, the
effect of lamin on BER in damaged cells is consistent with the role of lamins in
scaffolding and tethering functions (54). Repair enzyme activities are often
optimized by cellular processes such as tethering of chromatin and protein
complexes and by epigenetic modifications that change the enzyme activities or
chromatin structure (Gonzalo, 2013). In the case of the incision assays (Figure 5),
the biochemical activities are carried out on oligonucleotide DNA substrates with
pre-designed damage; thus, the incision activity is not influenced by chromatin
access structure during the reaction, but rather is specific for how active the OGG1
and APE1 enzymes were, in term of incision capability, at the time the cell lysates
were made. In this study, we have not determined the mechanism by which lamin
A/C promotes BER enzyme incision activity. We do know that the mechanism does
not involve altered protein levels of these repair enzymes (Figure 5B) or physical
interaction of lamin with these two enzymes (Figure 5C). In fact, a recent review of
the known protein-protein interactors of APE1 shows no evidence for a lamin
interaction (58). We speculate that these enzymes are altered in structure as a
consequence of lamin tethering the modifying proteins towards the BER enzymes.
Despite the dramatic reduction in OGG1 and APE1 incision activity in lamin-
depleted cells, we found no change in their levels on western blots and no striking
alterations in level of transcription of any BER enzyme on microarray. This is
consistent with the findings that BER enzyme activity did not correlate with BER
gene expression in PBMCs of healthy individuals (59) or in rat liver tissue (60).
In fact the latter study found the increased proliferation of rat liver tissue that occurs
during regeneration was accompanied by increased OGG1 expression, but with no
change in it activity on an 8-oxoG containing substrate; lamin-depleted cells have
been found to have slightly lower proliferation rate (…), as we also found in our
study (SUPPL). However this had no effect on either protein level or incision
activity.
Our findings are perhaps most relevant for disease known to be associated with alterations in
expression or steady-state level of A-type lamins. Certainly, cancer falls in this category.
……..(Redwood….A dual role). Studies suggest A-type lamin depletion contributes to the
genomic instability that drives malignancy, by way of defective DNA repair (in our case BER).
Going forward new cancer and laminopathy therapy strategies could can possibly target both
lamins and BER proteins simultaneously.
These data are the first to show that lamin A/C is involved in repair of oxidative damage. This
study provides more insight into the novel role and mechanism of lamin A/C in stress
response pathways and lamin-associated disease.
Similarly, silencing of LA/C expression slows cell proliferation and induces premature
senescence in human diploid fibroblasts (HDFs) (PMID:21535365). Conversely, dermal
fibroblasts from long-lived Snell dwarf mice have enhanced proliferation and cell senescence
(Maynard and Miller).
Furthermore, it has been shown that LA posttranslational processing by farnesylation is affected by oxidative
stress (Shimi and Goldman review).
Lamin expression is regulated by the tumor suppressors p53 and retinoblastoma protein (pRb) and by telomere functions; all master regulators of the cell cycle, apoptosis, replicative senescence, and autophagy. For example, LA/C expression is significantly upregulated upon the activation of p53 (Shimi and Goldman review, and PMID:17372198).
In addition, ROS-induced DNA double-strand breaks in HGPS fibroblasts are not repaired normally, and this appears to be related to their slow rate of proliferation (Shimi and Goldman review).
A-type lamins (lamin A and C) along with B type lamins and lamin-associated proteins form the nuclear lamina. This polymeric mechwork is associated with the nuclear envelope and play important roles in the temporal and spatial organization of the genome and in tethering protein complexes to the chromatin.
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Table 2. Top Molecules
Fold change up-regulated Exp. Value THY1 165.34 HOXC6 105.64 IGF2 81.96 CRABP1 66.64 Cdkn1c 59.27 H19 52.62 ADH7 43.63 THBS2 41.14 ITM2A 35.92 Klra4 (includes others) 32.74
Fold Change down-regulated IGFBP2* 373.88 LMNA* 225.12 GRB10 209.43 GDF15 76.2 CCDC68 58.78 IGFBP4* 58.56 Cbr2 47.45 ITGA7 45.78 Ppbp* 45.25 Sprr2g 41.54
Table 3. Top Upstream Regulators
Upstream regulator P-value of overlap Predicted activation state TP53 7.05E-35 Inhibited TGFB1 8.04E-33 Inhibited
TNF 2.44E-26
ERBB2 1.70E-25 HRAS 5.35E-25
Microarray – INGINUITY ANALYSIS-Lamin null MEF and control MEF
Figure 1. Significantly changed GO terms in MEF LMNA null.
Microarray-Significantly changed GO terms-Lamin null MEF and control MEF
Figure 2. Lamin A/C depletion leads to slower cell proliferation and to reduced cell survival after genotoxic stress. A. Typical siRNA lamin A/C knockdown in this project, as observed on western blot, for U2OS or HeLa cells, three days post-transfection B. Cell proliferation assays were performed on U2OS and MEF cells by seeding 25,000 cells per well in 6 well dishes, in triplicate and counting cells each day on a hemocytometer. In the case of HeLa cells, the cells were seeded in 96 well plates at 2000 cells per well in triplicate and absorbance measured each day using wst-1 cell proliferation reagent. C. Cell survival was assesses by WST-1 assay to estimate the relative number of viable cells at the indicated stressor concentrations. All data
points are the mean WST-1 measure (as percent of the value from untreated cells) from six wells (in 96 well dishes) SD. Durations of treatment for the liquid stressors were as follows: H2O2, 6h; Menadione, 4 h; MMS; 1h. KD = human cells with
transient siRNA knockdown of lamin A/C, C = control cells that were transfected with scrambled siRNA. In the case of IR stress, the cell type was HeLa; in the case of the other stressors, U2OS cells were used.
0 2 0 0 4 0 0 6 0 0 8 0 0
0
2 0
4 0
6 0
8 0
1 0 0
H y d ro g e n p e ro x id e
C o n ce n tra tio n (µ M )
% c
ell
su
rv
iva
l
C
K D
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0
0
5 0
1 0 0
M e n a d io n e
C o n ce n tra tio n (µ M )
% c
ell
su
rv
iva
l
C
K D
0 .0 0 .2 0 .4 0 .6 0 .8 1 .0
0
5 0
1 0 0
M M S
C o n c e n tra tio n (m M )
% c
ell
su
rv
iva
lC
K D
0 2 0 4 0 6 0
0
5 0
1 0 0
U V -C
U V C (J /m2)
% c
ell
su
rv
iva
l
C
K D
0 5 1 0
0
5 0
1 0 0
IR
IR (G y )
% c
ell
su
rv
iva
l
C
K D
C U2OS
GAPDH
Lamin A Lamin C
HeLa Day 6 A
C KD
0 1 2 3 4
0
1 0 0 0 0 0
2 0 0 0 0 0
3 0 0 0 0 0
4 0 0 0 0 0
D a y
Ce
ll n
um
be
r
+ /+
- / -
0 2 4 6
0 .0
0 .5
1 .0
1 .5
2 .0
D a y s a fte r s e e d in g
Ab
so
rba
nc
e (
48
0n
m-5
95
nm
)
C
K D
MEFs
HeLa cells U2OS cells
0 1 2 3 4 5
0
2 0 0 0 0 0
4 0 0 0 0 0
6 0 0 0 0 0
D a y s a fte r s e e d in g
Ce
ll n
um
be
r
S c r
la m K D
*
*
*
Lam A
Lam C
Actin
C KD C KD
Day 2 Day 5
B
A
C K D
0
2 0
4 0
6 0
8 0
1 0 0
H e L a ce lls -6 h r re p a ir
DN
A d
am
ag
e (
%)
p < 0 .0 0 0 1
C K D
0
2 0
4 0
6 0
8 0
1 0 0
U 2 O S c e lls -1 0 h r re p a ir
DN
A d
am
ag
e (
%)
p < 0 .0 0 0 1
+ /+ - /-
0
2 0
4 0
6 0
8 0
1 0 0
M E F s -1 0 h r re p a ir
DN
A d
am
ag
e (
%)
p = 0 .0 0 1 5
+ /+ -/-
0
5
1 0
1 5
2 0
A c c u m u la tio n o f F P G -s e n s itiv e s ite s in u n tre a te d M E F s
Oli
ve
ta
il m
om
en
t
p < 0 .0 0 0 1
No Fpg
No Fpg
Fpg
Fpg
C KD
0 hr
10 hr
COMET ASSAYS ON LAMIN-DEPLETED CELLS
0 hr
+/+
Untreated
10 hr
-/-
B
+ /+ -/-
0
2 0
4 0
6 0
8 0
1 0 0
% re p a ir o f 8 -o x o G
% 8
-ox
oG
p < 0 .0 0 0 1
+ /+ -/-
0
2
4
6
8
A c c u m u la tio n o f 8 -o x o G in u n tre a te d M E F
Av
era
ge
in
ten
sit
y
p < 0 .0 0 0 1
Immunofluorescence-for 8-oxoG accumulation
Figure 3. Accumulation and DNA repair of oxidative lesions in lamin A/C-depleted cells. For DNA repair determination, cells were treated with oxidative stress (100 µM H2O2) for 30 minutes and then allowed to repair in normal medium
for the indicated times. A. Fpg-comet assay. Expressed as percent of Fpg-sensitive sites (estimate of 8-oxoG lesions) relative to 0hr (immediately after treatment) after correction to the Fpg-sensitive sites in untreated cells; or expressed as olive tail moment as a measure of Fpg-sensitive site accumulation (A, at bottom right). Typical comet tails for U2OS cells are shown. B. Immunofluorescence using 8-oxoG antibody to monitor 8-oxoG accumulation (B, upper bar graph) and removal of the lesion after H2O2 treatment (B, lower bar graph) in MEFs. DAPI staining, clearly indicating
overlapping nuclear staining, not shown. Negative control using normal mouse IgG resulted in no obvious signal (data not shown). Bar represents 10 µm. C. HPLC to give estimate of 8-oxoG accumulation in untreated HeLa cells. +/+ =
Lamin A+/+
MEF, -/- = Lamin A-/-
MEF, KD = human cells with transient siRNA knockdown of lamin A/C, C = control cells that were transfected with scrambled siRNA.
C K D
0
1
2
3
4
5
8-o
xo
G/1
06
ba
se
s
p = 0 .0 2 5
C HPLC-for 8-oxoG accumulation
Will use MEF null overexpressing lam A or C to correct DNA repair defect
D
Figure 4. Incision activity of BER repair enzymes OGG1 and APE1 are reduced in lamin A/C-depleted cells. A. siRNA knockdown in human cells (HeLa) and LMNA knockout in MEF. B. siRNA knockdown in human cells (U2OS) and LMNA knockout in MEF. C. No detectable binding of lamin A/C to OGG1, APE1 or to P53 (see Table 3), as determines by co-immunoprecipitation with the lamin A/C antibody. Clear binding to known lamin A binding
protein emerin. +/+ = Lamin A+/+
MEF, -/- = Lamin A-/-
MEF, KD = human cells with transient siRNA knockdown of lamin A/C, C = control cells that were transfected with scrambled siRNA.
A
C U
T
L U
T
C 0
h
L 0
h
C 1
5m
L 1
5 m
C 1
h
L 1
h
C 4
h
L 4
h
0 .0
0 .5
1 .0
1 .5
Re
lati
ve
le
ve
ls o
f P
AR
C U
T
L U
T
C 0
h
L 0
h
C 1
5m
L 1
5 m
C 1
h
L 1
h
C 4
h
L 4
h
0 .0
0 .5
1 .0
1 .5
Re
lati
ve
le
ve
ls o
f P
AR
P1
C U
T
L U
T
C 0
h
L 0
h
C 1
5m
L 1
5 m
C 1
h
L 1
h
C 4
h
L 4
h
0 .0
0 .5
1 .0
1 .5
Re
lati
ve
le
ve
ls o
f L
IGII
I
C U
T
L U
T
C 0
h
L 0
h
C 1
5m
L 1
5 m
C 1
h
L 1
h
C 4
h
L 4
h
0 .0
0 .5
1 .0
1 .5
Re
lati
ve
le
ve
ls o
f P
olb
eta
C U
T
L U
T
C 0
h
L 0
h
C 1
5m
L 1
5 m
C 1
h
L 1
h
C 4
h
L 4
h
0 .0
0 .5
1 .0
1 .5
Re
lati
ve
le
ve
ls o
f O
GG
1
C U
T
L U
T
C 0
h
L 0
h
C 1
5m
L 1
5 m
C 1
h
L 1
h
C 4
h
L 4
h
0 .0
0 .5
1 .0
1 .5
Re
lati
ve
le
ve
ls o
f A
PE
1
B
C U
T
L U
T
C 0
h
L 0
h
C 1
5m
L 1
5 m
C 1
h
L 1
h
C 4
h
L 4
h
0 .0
0 .5
1 .0
1 .5
Re
lati
ve
le
ve
ls o
f P
AR
C U
T
L U
T
C 0
h
L 0
h
C 1
5m
L 1
5 m
C 1
h
L 1
h
C 4
h
L 4
h
0 .0
0 .5
1 .0
1 .5
Re
lati
ve
le
ve
ls o
f P
AR
P1
C U
T
L U
T
C 0
h
L 0
h
C 1
5m
L 1
5 m
C 1
h
L 1
h
C 4
h
L 4
h
0 .0
0 .5
1 .0
1 .5
Re
lati
ve
le
ve
ls o
f L
IGII
I
C U
T
L U
T
C 0
h
L 0
h
C 1
5m
L 1
5 m
C 1
h
L 1
h
C 4
h
L 4
h
0 .0
0 .5
1 .0
1 .5
Re
lati
ve
le
ve
ls o
f P
olb
eta
C U
T
L U
T
C 0
h
L 0
h
C 1
5m
L 1
5 m
C 1
h
L 1
h
C 4
h
L 4
h
0 .0
0 .5
1 .0
1 .5
Re
lati
ve
le
ve
ls o
f O
GG
1
C U
T
L U
T
C 0
h
L 0
h
C 1
5m
L 1
5 m
C 1
h
L 1
h
C 4
h
L 4
h
0 .0
0 .5
1 .0
1 .5
Re
lati
ve
le
ve
ls o
f A
PE
1
C
Figure 5. The major BER protein have reduced levels in MEF null MEF but not in U2OS siRNA lamin A/C knockdowns. Western blotting was performed on lysates prepared from untreated cells (UT) and from cells exposed to 100 µM H2O2 and left to repair for the indicated times in normal complete medium. A. U2OS cells. B. MEF cells. C. PAR was later tested in the U2OS cells under the same conditions. Abbreviations: hr = hours, m =
minutes. +/+ = Lamin A+/+
MEF, -/- = Lamin A-/-
MEF, KD = human cells with transient siRNA knockdown of lamin A/C, C = control cells that were transfected with scrambled siRNA.
Fibroblast from HGPS patients
A
B
Figure 7. The protein levels of PAR, OGG1, and APE1 mimic what the patterns seen in MEF LMNA null of Figure
5B. A. Protein levels of OGG1 and APE1 are reduced HGPS patient cells. B. PAR level (PARylation) was higher
in patient cells, after treatment with 100 µM H2O2. C = control cells (HGADFN370 from mother of patient
HGADFN371). P = patient HGADFN371.
C P C P C P C P C P
UT 0 h 15 m 1 h 4 h
Actin
OGG1
APE1
Supplemental
Figure S1. siRNA if lamin A/C in BJ cells has no effect on the protein level of APE1.
Figure S2. Demonstration that the PAR antibody used in this study was specific for activated PARP. Addition of
NAD into the media resulted in enhanced PAR; addition of PARP1 inhibitor 3AB then resulted in reduction of PAR.
Supplemental figure showing that lamin-A/C-OGG1 binding can be detected by an alternative
CO-IP strategy of over-expressing OGG1-FLAG and using FLAG beads, and also by
immunofluorescence
A B
Western blotting of soluble fraction (chromatin fraction removed) of U2OS and MEF cells
demonstrates that BER protein access to damaged DNA is reduced in lamin A/C-depleted cells
Figure S4. The effects of lamin A/C depletion on PARylation before and after oxidative stress (100 µM H2O2 for 30
minutes). A. Western blotting for PAR (estimate of PARylation) and the other major BER proteins at various time points (in complete medium) after oxidative stress in A. U2OS lamin A/C knockdown cells and B. MEF LMNA null cells.
Abbreviations: m = minutes, h = hours. +/+ = Lamin A+/+
MEF, -/- = Lamin A-/-
MEF, KD = U2OS cells with transient siRNA knockdown of lamin A/C, C = control cells that were transfected with scrambled siRNA.