Supplementary Materials and Methods
Plasmids. The table below contains information about all the plasmids utilized in our studies.
Plasmid
Previously described
Gal4-RelA
Yes (Schmitz and Baeuerle 1991)
His-HA-Ub
Yes, gift from Richard Baer
His-RelA
No
HA-RelA
No
His-K63 only Ubiquitin
Yes, gift from Richard Baer
His-K63R only Ubiquitin
Yes, gift from Richard Baer
Cloning strategy
Fig 1
His tag was fused to the C-terminus of
RelA using the pEF vector (Schmitz lab)
RelA with C-terminally located HA-tag
was introduced in pcDNA3 (Schmitz lab)
Mutagenesis of K28,37, 56, 62, 79, 83,
122, 123, 195, 218, 221 to R (Schmitz
lab)
Mutagenesis of K 301, 303, 310, 314,
315, 343, 427 to R (Schmitz lab)
Mutagenesis of K28,37, 56, 62, 79, 83,
122, 123, 195, 218, 221, 301, 303, 310,
314, 315, 343, 427 to R (Schmitz lab)
HA-RelA K/R RHD
HA-RelA K/R TAD
HA-RelA K/R All
Fig 2
pEBB
pEBB-HA-RelA
Yes (Mizushima and Nagata
1990)
Yes (Burstein et al 2005)
pcDNA-Flag-p300
pEBB-HA-RelA K4RNonAc
Yes, gift from Colin Duckett
pEBB-HA-RelA K4R
No
pEBB-HA-RelA K5R
No
pEBB-HA-RelA K310R
No
pEBB-HA-RelA K8R
No
His-RelA K5R
No
pEBB-RelA-TB
pEBB-RGS-His6-human
ubiquitin
pEBG
Yes (Maine et al 2010)
pEBG-RelA
Yes (Burstein et al 2005)
pCW7-His6-Myc-Ub
Yes (Yu and Kopito 1999)
No
Mutagenesis of K56,62,79,195 to R
(Burstein lab)
Mutagenesis of K122,123,314,315 to R
(Burstein lab)
Mutagenesis of K122,123,310,314,315 to
R (Burstein lab)
Mutagenesis of K310 to R (Burstein lab)
Fig 3
Fig 4
Yes (Maine et al 2010)
Yes (Sanchez et al 1994)
Mutagenesis of
K56,62,79,122,123,195,310,314,315 to R
(Burstein lab)
RelA K5R was cut out of the pEBB-HA
vector and ligated into the pEF-His vector
(Schmitz lab)
pEBB-Flag-COMMD1
Yes (Burstein et al 2004)
pEBB-Flag-Cul2
Yes (Maine et al 2007)
pEBB-Flag GCN5 E575Q
Yes (Mao et al 2009)
YFP-CBP
Yes (Tini et al 2002)
FG9
Yes (Wright and Duckett 2009)
FG9-HA-mRelA WT
No
FG9-HA-mRelA K310R
No
FG9-HA-mRelA K4R
No
FG9-HA-mRelA K5R
No
FG9-HA-mRelA K310Q
No
FG9-HA-mRelA K4Q
No
FG9-HA-mRelA K5Q
No
Fig 5
PCR from Image clone 3489295 (Burstein
lab)
Mutagenesis of K310 to R (Burstein lab)
Mutagenesis of K122,123,314,315 to R
(Burstein lab)
Mutagenesis of K122,123,310,314,315 to
R (Burstein lab)
Mutagenesis of K310 to Q (Burstein lab)
Mutagenesis of K122,123,314,315 to Q
(Burstein lab)
Mutagenesis of K122,123,310,314,315 to
Q (Burstein lab)
Antibodies. The following antibodies were utilized for immunoblotting in our studies.
Antibody
Supplier
Cat. No.
Ubiquitin (P4D1)
Cell Signaling
3936
RelA (C-Term, C-20)
Santa Cruz Biotechnology
sc-372
RelA (N-Term, F-6)
Santa Cruz Biotechnology
sc-8008
Tubulin
Sigma
T4026
K63-only Ubiquitin
Biomol
PW0600
Acetylated lysine
Cell Signaling
9441
FLAG
Sigma
A8592
HA
CoVance
MMS101R
Ubiquitin (P4D1)
Cell Signaling
3936
HA
Covance
MMS101R
RelA (C-Term, C-20)
Santa Cruz Biotechnology
sc-372
K63-only Ubiquitin
Biomol
PW0600
Tubulin
Sigma
T4026
Streptavidin-HRP
BioLegend
405210
Acetylated lysine
Cell Signaling
9441
FLAG
Sigma
A8592
GST
Santa Cruz Biotechnology
sc-459
HA
Covance
MMS101R
Ubiquitin (P4D1)
Cell Signaling
3936
GFP
Roche
11814460001.00
HA
Covance
MMS101R
RelA
Santa Cruz Biotechnology
sc-8008
IB-
Upstate Biotechnologies
06-494
IB-
Santa Cruz Biotechnology
sc-9130
IB-
Santa Cruz Biotechnology
sc-7155
p50
Santa Cruz Biotechnology
sc-8414
p52
Santa Cruz Biotechnology
sc-848
RelB
Santa Cruz Biotechnology
sc-226
c-Rel
Santa Cruz Biotechnology
sc-6955
-Actin
Sigma
A5441
RelA(C-Term, C-20)
Santa Cruz Biotechnology
sc-372
Fig 1
Fig 2
Fig 3
Fig 4
Fig 5
Fig S1
Tubulin
Sigma
T4026
HDAC1 (H11)
Santa Cruz
G1008
Primers. The following table provides information regarding the qRT-PCR and ChIP primers
utilized in our studies.
qRT-PCR
Gene target
(mouse)
Nfkbia
Sense primer
Antisense primer
Reference
CGCAGACCTGCACACCCCAG
GGAGGGCTGTCCGGCCATTG
This study
Vcam1
AGTTGGGGATTCGGTTGTTCT
CCCCTCATTCCTTACCACCC
(Moreno et al 2010)
Icam1
GGAGACGCAGAGGACCTTAAC
CGCTCAGAAGAACCACCTTC
(Geng et al 2009)
Mmp13
ACCTCCACAGTTGACAGGCT
AGGCACTCCACATCTTGGTTT
(Moreno et al 2010)
Saa3
CTGTTCAGAAGTTCACGGGAC
AGCAGGTCGGAAGTGGTT
(Moreno et al 2010)
Il6
TGGATGCTACCAAACTGGAT
GGACTCTGGCTTTGTCTTTC
Lamb3
GGCTGCCTCGAAATTACAACA
ACCCTCCATGTCTTGCCAAAG
Cxcl10
AGTGAACTGCGCTGTCAATG
CTTCAGGGTCAAGGCAAACT
(Moreno et al 2010)
(Spandidos et al 2010)
Primerbank ID: 6678660a1
(Moreno et al 2010)
Mmp3
ACCTATTCCTGGTTGCTG
GCCTTGGCTGAGTGGTAG
(Moreno et al 2010)
Mmp9
CGAACTTCGACACTGACAAGAAGT
GCACGCTGGAATGATCTAAGC
(Wells et al 2003)
Expi
GGAGATGGATCGTGCTCTGG
GGCTAGCCATCAGTCCTGC
(Korsten et al 2009)
Ccl9
CCAGTGGTGGGTGTACCAG
CTCCGATCACTGGGGTTG
(van Erp et al 2006)
ChIP
Gene promoter
(species)
Vcam1 (mouse)
Sense primer
Antisense primer
Reference
TCAGCCCAGAAAGCAGC
AAAGTGTTCAGCCTCCA
(Moreno et al 2010)
Icam1 (mouse)
AGGGGACTAGGCAGTAGTCAATCAG
GAACGAGGGCTTCGGTATTT
(Geng et al 2009)
ICAM1 (human)
CCCGATTGCTTTAGCTTGGAA
CCGGAACAAATGCTGCAGTTAT
(Horion et al 2007)
Mmp13 (mouse)
GCAGACATTTTCCTTA
ATGTTTGTGACTTGGA
(Moreno et al 2010)
Microarray analysis
RNA extraction and quality control. RNA was extracted using the Trizol method according to
the manufacturer’s instructions (Invitrogen; Carlsbad, California, USA). RNA integrity was
validated using an Agilent Bioanalyzer. All RNA preparations had RIN values above 8.8 except
for samples from rela-/- fibroblasts reconstituted with vector control and treated for 8h with TNF.
The latter showed substantial degradation of total RNA as expected from TNF-dependent
apoptosis in the absence of protection by NF-B/RelA dependent gene expression. However,
these samples could also be subjected to the microarray analysis protocol.
Microarray-based mRNA expression analysis (Single Color Mode).
The “Whole Mouse
Genome Oligo Microarray V2” (G4846A, ID 026655, Agilent Technologies; Santa Clara,
California, USA) used in this study contains 44397 oligonucleotide probes covering the entire
murine transcriptome. Synthesis of Cy3-labeled cRNA was performed with the “Quick Amp
Labeling kit, one color” (#5190-0442, Agilent Technologies) according to the manufacturer’s
recommendations. cRNA fragmentation, hybridization and washing steps were also carried-out
exactly as recommended: “One-Color Microarray-Based Gene Expression Analysis Protocol
V5.7” (see http://www.agilent.com for details). Slides were scanned on the Agilent Micro Array
Scanner G2565CA (pixel resolution 5 µm, bit depth 20). Data extraction was performed with the
“Feature Extraction Software V10.7.3.1” by using the recommended default extraction protocol
file: GE1_107_Sep09.xml.
Normalization. The single channel data generated by the Feature Extraction software were
normalized and analyzed in Genespring GX software, version 11.5.1 (Agilent Technologies).
Expression values were log2 transformed, normalized to 75th percentile of each array and inter
array median centered according to the standard procedure in Genespring GX.
Identification of RelA- and K5-dependent gene sets.
According to their annotation in
EntrezGene, out of 44397 probes 35843 probes corresponding to 28299 genes were selected. Of
these, 24902 probes corresponding to 19833 genes showed hybridization signals above the 20%
percentile in at least 4 of the 24 microarray hybridizations and were marked as measurable
fluorescence intensity values in at least 16 out of 24 hybridizations. In this data set 419 probes
corresponding to 398 genes were regulated by 1h or 8h TNF stimulation by at least two-fold in
both independent experiments in rela-deficient fibroblasts stably reconstituted with wild type
mRelA. In addition to the regulation by TNF, these genes showed a mean raw expression value
above or equal to 50 fluorescence units across all 24 hybridizations. Thereof a total of 197 probe
values corresponding 185 genes indicated differential regulation by two-fold compared to
averaged values from wild type-reconstituted cells in at least one of the experiments using K5
mutants at least one time point. For these 185 genes, average ratios values of the wild-type
reconstituted cells, and, individual ratio values for either K5 reconstituted cell line compared to
the WT-reconstituted cells, were analyzed by hierarchical cluster analysis using MeV
MultiExperimentViewer, version 4.6.2, 2011, (www.tm4.org) with cluster settings “complete” as
linkage method and “Pearson correlation” for distance measure (Saeed et al 2003). A distance
threshold of 2.065 resulted in 10 gene clusters, marked by blue triangles as shown in Fig. 5E.
Ratio values for rela-/- cells were not included in the cluster analysis but are displayed along the
gene list to facilitate identification of RelA-dependent genes.
Chromatin immunoprecipitation.
Cells were cross-linked with 1% (v/v) formaldehyde, collected and then lysed in RIPA buffer [10
mM Tris pH 7.5, 150 mM NaCl, 1% (v/v) NP-40, 1% (w/v) Na-Deoxycholate, 0.1% (w/v) SDS,
1 mM EDTA and 10 µg/ml aprotinin]. DNA was sheared by sonification using a Branson
sonifier 250. After removal of cellular debris by centrifugation, equal amounts of DNA were
incubated with 2 μg of anti-RelA, anti-HA, anti-CBP, anti-Acetyl H3 or control IgG antibodies
previously bound to ChIP-grade protein G-coupled magnetic beads (Cell Signaling; Danvers,
Massachusetts, USA). After rotating for 2 h at 4°C on a spinning wheel, the magnetic beads
were washed five times using the following buffers: RIPA buffer (twice), RIPA high-salt buffer
[2 M NaCl, 10 mM Tris pH 7.5, 1% (v/v) NP-40, 0.5% (v/v) Na-Deoxycholate and 1 mM
EDTA], RIPA and TE buffer (10 mM Tris pH 7.5 and 1 mM EDTA).
The precipitated
fragments were eluted with TE buffer containing 1% SDS. For re-ChIP assays, the eluted
material was diluted 1:10 with RIPA buffer lacking SDS and precipitated again with the antiRelA antibody. The eluted DNA was quantified by real-time PCR using specific primer sets
flanking the expected RelA binding site. The signal was normalized by the background IgG
precipitation and expressed as fold of the WT unstimulated sample. The following antibodies
were used for immunoprecipitations: ChIP-grade HA (Abcam ab 9110), ChIP grade RelA (C-20,
Santa Cruz sc-372x), CBP (C-20, Santa Cruz sc-583), acetyl-Histone H3 (Milipore 06-599).
References
Burstein E, Ganesh L, Dick RD, van De Sluis B, Wilkinson JC, Klomp LW et al (2004). A novel
role for XIAP in copper homeostasis through regulation of MURR1. EMBO Journal 23: 244254.
Burstein E, Hoberg JE, Wilkinson AS, Rumble JM, Csomos RA, Komarck CM et al (2005).
COMMD proteins: A novel family of structural and functional homologs of MURR1. J Biol
Chem 280: 22222-22232.
Geng H, Wittwer T, Dittrich-Breiholz O, Kracht M, Schmitz ML (2009). Phosphorylation of NFB p65 at Ser468 controls its COMMD1-dependent ubiquitination and target gene-specific
proteasomal elimination. EMBO Rep 10: 381-386.
Horion J, Gloire G, El Mjiyad N, Quivy V, Vermeulen L, Vanden Berghe W et al (2007).
Histone deacetylase inhibitor trichostatin A sustains sodium pervanadate-induced NF-B
activation by delaying IBmRNA resynthesis: comparison with tumor necrosis factor . J Biol
Chem 282: 15383-15393.
Korsten H, Ziel-van der Made A, Ma X, van der Kwast T, Trapman J (2009). Accumulating
progenitor cells in the luminal epithelial cell layer are candidate tumor initiating cells in a Pten
knockout mouse prostate cancer model. PLoS One 4: e5662.
Maine GN, Mao X, Komarck CM, Burstein E (2007). COMMD1 promotes the ubiquitination of
NF-B subunits through a Cullin-containing ubiquitin ligase. EMBO Journal 26: 436-447.
Maine GN, Li H, Zaidi IW, Basrur V, Elenitoba-Johnson KS, Burstein E (2010). A bimolecular
affinity purification method under denaturing conditions for rapid isolation of a ubiquitinated
protein for mass spectrometry analysis. Nat Protoc 5: 1447-1459.
Mao X, Gluck N, Li D, Maine GN, Li H, Zaidi IW et al (2009). GCN5 is a required cofactor for
a ubiquitin ligase that targets NF-B/RelA. Genes Dev 23: 849-861.
Mizushima S, Nagata S (1990). pEF-BOS, a powerful mammalian expression vector. Nucleic
Acids Res 18: 5322.
Moreno R, Sobotzik JM, Schultz C, Schmitz ML (2010). Specification of the NF-B
transcriptional response by p65 phosphorylation and TNF-induced nuclear translocation of
IKK. Nucleic Acids Res 38: 6029-6044.
Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N et al (2003). TM4: a free, open-source
system for microarray data management and analysis. Biotechniques 34: 374-378.
Sanchez I, Hughes RT, Mayer BJ, Yee K, Woodgett JR, Avruch J et al (1994). Role of
SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun. Nature
372: 794-798.
Schmitz ML, Baeuerle PA (1991). The p65 subunit is responsible for the strong transcription
activating potential of NF-B. EMBO J 10: 3805-3817.
Spandidos A, Wang X, Wang H, Seed B (2010). PrimerBank: a resource of human and mouse
PCR primer pairs for gene expression detection and quantification. Nucleic Acids Res 38: D792D799.
Tini M, Benecke A, Um SJ, Torchia J, Evans RM, Chambon P (2002). Association of CBP/p300
acetylase and thymine DNA glycosylase links DNA repair and transcription. Mol Cell 9: 265277.
van Erp K, Dach K, Koch I, Heesemann J, Hoffmann R (2006). Role of strain differences on host
resistance and the transcriptional response of macrophages to infection with Yersinia
enterocolitica. Physiol Genomics 25: 75-84.
Wells JE, Rice TK, Nuttall RK, Edwards DR, Zekki H, Rivest S et al (2003). An adverse role for
matrix metalloproteinase 12 after spinal cord injury in mice. J Neurosci 23: 10107-10115.
Wright CW, Duckett CS (2009). The aryl hydrocarbon nuclear translocator alters CD30mediated NF-B-dependent transcription. Science 323: 251-255.
Yu H, Kopito RR (1999). The role of multiubiquitination in dislocation and degradation of the
alpha subunit of the T cell antigen receptor. J Biol Chem 274: 36852-36858.