Supplemental Information The Bcl6-SMRT/NCoR Cistrome

Cell Metabolism, Volume 15
Supplemental Information
The Bcl6-SMRT/NCoR Cistrome
Represses Inflammation
to Attenuate Atherosclerosis
Grant D. Barish, Ruth T. Yu, Malith S. Karunasiri, Diana Becerra, Jason Kim,
Tiffany W. Tseng, Li-Jung Tai, Matthias LeBlanc, Cody Diehl, Leandro Cerchietti,
Yury I. Miller, Joseph L. Witztum, Ari M. Melnick, Alexander L. Dent,
Rajendra K. Tangirala, and Ronald M. Evans
Inventory of Supplemental Information
1.
2.
3.
4.
Four Supplemental Figures (S1 – S4)
Supplemental Figure Legends
Supplemental Experimental Procedures
Supplemental References
Supplemental Information
Supplemental Figures
Supplemental Figure Legends
Figure S1 Ldlr-/- mice transplanted with Bcl6-/- bone marrow do not develop myocarditis or
pulmonary vasculitis but develop atherosclerosis, xanthomatous tendonitis, and diminished
body weights on atherogenic diet. (A) Representative H & E stained sections of myocardium in
BMT-WT versus BMT-KO mice at low (25x) and high (100x) power magnifications. (B)
Representative H & E stained sections of lung in BMT-WT versus BMT-KO mice at low (50x)
and high power (200x) magnifications. (C) Representative Sudan IV-stained aortas from BMTWT (left) versus BMT-KO (right) mice after 8 weeks of atherogenic diet. Lesions (red) are
evident at intercostal branch points in BMT-KO mice. (D) Low power representative sagittal
sections comparing BMT-WT and BMT-KO hind limbs, H & E staining. Arrows indicate lesions
in BMT-KO mice. (E) High power section of BMT-KO mouse shows inflammatory infiltration of a
tendon (left). Higher power view of infiltrating cells in BMT-KO tendon (right). (F) Masson
trichrome staining shows connective tissue deposition anterior to the tibio-talar joint (black
arrow) in BMT-KO mouse. (G) Moma-2 macrophage marker immunostaining (red) with
hematoxylin counter-stain of BMT-WT (left) and BMT-KO (right) forepaws after 11 weeks of
atherogenic diet. Insets are high power views of regions marked in yellow. Strong Moma-2
staining is observed along the tendon of BMT-KO forepaw (right inset). (H) Representative
sagittal sections (top) comparing BMT-WT and BMT-KO hind limbs after 8 weeks of exposure to
atherogenic diet, H & E staining. Yellow boxed area (magnified below) indicates tendon
xanthoma in representative BMT-KO mouse and normal corresponding area in BMT-WT
control. (I) Table of terminal body, liver, and inguinal white adipose tissue (WAT) weights, total
body fat (based on MRI), plasma total cholesterol and triglycerides in BMT-WT and BMT-KO
mice used for atherosclerotic lesion analyses. Measurements in some cohorts were not
determined (n.d). (J, K) FPLC pooled plasma lipoprotein fraction analysis of cholesterol and
triglycerides, respectively, from BMT-WT and BMT-KO mice before (week 0) or after 8 weeks
(week 8) of exposure to atherogenic diet. (L, M) Serial analysis of body weight (L) and total
body fat based on MRI (M), respectively, during 16 week exposure of a representative cohort of
BMT-WT and BMT-KO mice to atherogenic diet. (N) Relative gene expression data in inguinal
white adipose tissue from BMT-WT versus BMT-KO mice (n = 6 per group) after 2 weeks of
atherogenic diet. Values are expressed as means + SD. Statistical significance using 2-tailed ttests: +p < 0.05, *p < 0.001.
Figure S2 Non-hypercholesterolemic C57 mice deficient in bone marrow Bcl6 do not develop
atherosclerosis or xanthomatous tendonitis on a standard or atherogenic diet. (A) Terminal
body weights in C57 BMT-WT and C57 BMT-KO mice (n = 6 for each cohort) after
transplantation and exposure to standard chow diet for 30 weeks. (B, C) Terminal plasma
cholesterol and triglyceride levels in C57 BMT-WT versus C57 BMT-KO mice on standard chow
diet (n = 6 for each cohort). (D, E) FPLC pooled plasma lipoprotein fraction analysis of
cholesterol and triglycerides, respectively, from C57 BMT-WT and C57 BMT-KO mice exposed
to a standard chow diet. (F) Representative Sudan IV-stained aortas of C57 BMT-WT and C57
BMT-KO mice after transplantation and exposure to standard chow diet for 30 weeks, revealing
no evident lesions. (G) Quantification of distal hind limb volumes in C57 BMT-WT versus C57
BMT-KO mice (n = 6 for C57 BMT-WT, n = 6 for C57 BMT-KO). (H) Expression analysis of
flushed bone marrow from C57 BMT-WT (n = 6) and C57 BMT-KO (n = 6) mice after 30 weeks
of standard chow diet. (I) Gene expression in whole aortas from C57 BMT-WT (n = 5) and C57
BMT-KO (n= 5) mice after two weeks of standard chow diet normalized to expression of the
F4/80 macrophage marker gene. (J, K) Serial analysis of body weight (J) and total body fat
based on MRI (K), respectively, in C57 BMT-WT and C57 BMT-KO mice over a 14 week
exposure to atherogenic diet. (L, M) Terminal plasma cholesterol and triglyceride levels in C57
BMT-WT versus C57 BMT-KO mice (n = 11 and 13, respectively) exposed to atherogenic diet
for 14 weeks. (N, O) FPLC pooled plasma lipoprotein fraction analysis of cholesterol and
triglycerides, respectively, from C57 BMT-WT and C57 BMT-KO mice exposed to atherogenic
diet for 14 weeks. (P) Representative Sudan IV-stained aortas of C57 BMT-WT and C57 BMTKO mice after transplantation and exposure to atherogenic diet for 14 weeks, revealing no
evident lesions. (Q) Quantification of distal hind limb volumes in C57 BMT-WT versus C57
BMT-KO mice after 14 weeks of atherogenic diet (n = 11 for C57 BMT-WT, n = 13 for C57 BMTKO). (R) Representative sagittal sections comparing C57 BMT-WT and C57 BMT-KO hind
limbs, H & E staining, after 14 week exposure to atherogenic diet revealing no xanthomas. (S)
Expression analysis of flushed bone marrow from C57 BMT-WT (n = 5) and C57 BMT-KO (n =
5) mice after 2 weeks of atherogenic diet. (T) Gene expression in whole aortas from C57 BMTWT (n = 5) and C57 BMT-KO (n= 5) mice after two weeks of atherogenic diet normalized to the
F4/80 macrophage marker gene. Values are expressed as means + SD and statistical
significance determined using 2-tailed t-tests: +p < 0.05, #p < 0.01, *p < 0.001.
Figure S3 Analysis of inflammation and lipid homeostasis in wild type versus Bcl6-deficient
macrophages. (A) Quantitative PCR assessment of atherogenic gene expression in Bcl6+/+
(WT), Bcl6+/- (HET), and Bcl6-/- (KO) macrophages with or without exposure to mmLDL. Values
are expressed as means + SD. Statistical testing to compare the genotypes for each treatment
condition performed with one-way ANOVA and Tukey’s multiple comparison tests: +p < 0.05, #p
< 0.01, *p < 0.001. (B) Multiplex suspension array quantification of Ccl2 and Ccl7 protein in cell
culture supernatants from WT, HET, and KO macrophages with or without 6-hour mmLDL
stimulation. Values are expressed as means + SD. Statistical testing to compare the
genotypes for unstimulated or stimulated treatment conditions performed with one-way ANOVA
and Tukey’s multiple comparison tests: #p < 0.01, *p < 0.001. (C) In vitro chemotaxis assay
comparing migration of WT and KO macrophages to gradients of Ccl2, Ccl3, or
unsupplemented media (control). (D) In vivo migration assay comparing peritoneal leukocyte
counts in C57 BMT-WT and C57 BMT-KO mice 3 days after intraperitoneal thioglycollate
injection. (E) Western blot comparing Plau protein levels in WT, HET, and KO macrophages
versus a -actin control. (F) Quantitative PCR assessment of atherogenic gene expression in
Bcl6+/+ (WT) and Bcl6-/- (KO) macrophages with or without 6-hour exposure to TLR2 agonist
(Pam3CSK4, 100 ng/ml). (G) Assay of diI-acetylated and diI-oxidized LDL uptake in WT versus
KO macrophages. Fluorescence was normalized to cellular protein concentration. (H) 3Hcholesterol efflux assay comparing WT versus KO macrophages. Data is presented as percent
efflux of 3H-cholesterol to control media, HDL, or ApoA1 relative to total 3H-cholesterol. (I - J)
Quantification of esterified and total cholesterol levels from WT and KO foam cells, either from
bone marrow-differentiated macrophages exposed in vitro to ac-LDL (100g/ml) for 24 hours (I),
or from thioglycollate-elicited peritoneal macrophages from hypercholesterolemic BMT-WT and
BMT-KO mice exposed to atherogenic diet (J). Data is normalized to cellular protein
concentration. For C – D and F - J, values are expressed as means + SD and statistical
significance determined using 2-tailed t-tests: +p < 0.05, #p < 0.01, *p < 0.001.
Figure S4 Bcl6 mediates atherogenic repression through SMRT and NCoR. (A) ChIP qPCR
interrogation of SMRT and NCoR at Bcl6 binding sites in Bcl6 WT and KO macrophages using
IgG, SMRT, or NCoR antibodies. SMRT and NCoR enrichment is lost in KO cells specifically at
Bcl6 binding sites. Values are expressed as means + SD and statistical significance
determined using 2-tailed t-tests: +p < 0.05, #p < 0.01, *p < 0.001. (B) Distribution of binding
sites relative to gene positions for SMRT (left) or NCoR (right) based on ChIP-seq in wild type
macrophages. (C) Distribution of binding sites relative to gene positions for Bcl6-SMRT (left) or
Bcl6-NCoR (right) sub-cistromes. (D) Outline of ChIP-seq method for determining binding sites
at which Bcl6 was directly associated with SMRT or NCoR. (E) ChIP qPCR interrogation of
additionally identified Bcl6 binding sites from Bowtie alignment of Bcl6 ChIP-sequencing data
(uncalled peaks in prior Eland alignment) using IgG and Bcl6 antibodies. Values are expressed
as means + SD and statistical significance determined using 2-tailed t-tests: +p < 0.05, #p <
0.01, *p < 0.001. (F, G) ChIP-sequencing tracks for SMRT and NCoR in wild type and Bcl6 KO
macrophages compared to Bcl6 tracks along the Slain2 (F) and Nos2 (G) genes. A previously
reported binding site for NCoR is observed on the Nos2 promoter (*).
Supplemental Experimental Procedures
Animal experiments
Mice were fed standard chow or atherogenic diet (TD94059 – Harlan Teklad) as indicated.
Distal hind limbs were quantified using volumetric displacement. Serial analysis of body
composition was performed using EchoMRI (Echo Medical Systems). For sterile peritonitis
studies, transplanted mice were given 2 ml intraperitoneal injections of 3% thioglycollate, and
peritoneal exudates were collected 72 hours later by saline lavage. Cells were counted with an
automated cell counter (Invitrogen). Fasting plasma cholesterol and triglycerides were
determined by enzymatic assays (Wako Chemicals and Thermo) and particle size distribution of
the lipoproteins by FPLC of pooled plasma samples as described elsewhere (Li et al., 2004).
Quantification of Atherosclerosis and Histology
Mice were euthanized and perfused with 7.5% sucrose in paraformaldehyde. Aortas were
dissected, pinned, and stained with Sudan IV. Images were captured with a Sony 3-CCD video
camera and analyzed using ImagePro software. Lesion development is expressed as
percentage of total covering the aortic surface (Tangirala et al., 1995). Aortic root lesion
histology was obtained from 15-m sections cut from OCT-embedded hearts using a cryostat.
Aortic root lesion quantification was performed as described previously (Tangirala et al., 1995).
Necrotic core areas were determined as acellular (non-staining) regions of H & E stained aortic
root sections as described elsewhere (Han et al., 2006). Representative sections were stained
with Masson’s trichrome or immunostained using antibodies to Moma-2 (Accurate Chemical),
Ccl2 (Santa Cruz), F3 (AbCam), -smooth muscle actin (Sigma), Plau (Abcam), and Il-1
(AbCam). Limbs were fixed in paraformaldehyde, decalcified in 10% EDTA, paraffin-embedded,
sectioned, and stained as indicated. For immunostaining, forelimbs were embedded in OCT,
cut into 30-m sections, fixed, and stained. Slides were reviewed by a pathologist in a blind
study.
Ligand and inhibitor studies in bone marrow derived macrophages
For studies with mmLDL, media was changed to DMEM (unstimulated) or DMEM with mmLDL
50 g/ml prepared as described previously (Miller et al., 2005). For TLR2 ligand studies, cells
were incubated in MSF media (Invitrogen) and treated with or without Pam3CSK4 100 ng/ml
(Invivogen). For Bcl6 inhibitor studies, cells were exposed to either 5 M control or RI-BPI
peptide in MSF media.
Cholesterol uptake, efflux and foam cell formation
Cholesterol uptake was assessed by exposing bone marrow derived macrophages to 10 g/ml
diI-acetylated LDL or diI-oxidized LDL (Biomedical Technologies, Inc.) for four hours in serumfree media. Cells were washed twice in PBS + 0.4% BSA, then three times in PBS, and lysed in
a solution of 0.1% SDS and and 0.1 M NaOH. Fluorescence was quantified on a fluorometer
(Tecan) with 520 nm excitation / 580 nm emission settings and fluorescence values were
normalized to protein concentration using the BCA protein assay (Pierce)(Teupser et al., 1996).
Cholesterol efflux was performed as previously described (Chawla et al., 2001) with minor
modifications. Bone marrow derived macrophages were incubated in DMEM with 10% lipidreduced serum, 50 g/ml acetylated LDL (Biomedical Technologies, Inc.) and 2 Ci/ml 3Hcholesterol (MP Biomedical) for 16 hours. Cells were then washed, equilibrated in DMEM with
0.2% fatty acid free BSA for 2 hours, and then media was replaced with fresh medium
containing 20 g/ml human ApoA1, 50 g/ml human HDL (both from Biomedical Technologies,
Inc.), or negative control media with neither acceptor. Cells were then incubated for 4 hours.
An aliquot of medium was removed, centrifuged to remove debris, and radioactivity was
determined by liquid scintillation counting. Total cell-associated radioactivity was determined by
dissolving the cells in isopropanol. Results are expressed as the percentage of 3H cholesterol in
the medium divided by the total 3H cholesterol in the medium and cells and performed in
triplicate. In vitro foam cell formation was performed by exposing bone marrow derived
macrophages to 100 g/ml acetylated LDL for 24 hours, followed by quantification of total and
free cholesterol using the Amplex Red Cholesterol Assay Kit (Invitrogen) as described
elsewhere (McLaren et al., 2010). In vivo foam cell quantification of total and free cholesterol
was performed in the same manner, using peritoneal macrophages from thioglycollate-injected
Ldlr-/- transplanted mice exposed to 4 weeks of standard or atherogenic diet.
In Vitro Chemotaxis
Assays were performed using FluoroBlok plates (BD Biosciences) per manufacturer’s
instructions. Briefly, macrophages were labeled with 5 M Vybrant CFDA-SE dye (Invitrogen),
counted and applied to 8.0 M porous inserts. Inserts were then added above wells containing
20 ng/ml recombinant Ccl2 or Ccl3 (Peprotech). Fluorescence emitted from cells that migrated
to the bottom surface of the insert was measured at 492/517 nm (ex/em) 14 hours later.
Gene Expression & Pathway Analysis
Microarrays were performed using triplicates for each experimental condition and analyzed
using VAMPIRE (Hsiao et al., 2005). Lists of altered genes were mapped to KEGG pathways
using GeneCodis (Nogales-Cadenas et al., 2009).
Chromatin immunoprecipitation (ChIP) and ChIP-sequencing
For ChIP-seq, chromatin-antibody complexes were precipitated with anti-IgG paramagnetic
beads (Invitrogen). Sequencing libraries were constructed using ChIP-Seq library kits
(Illumina). Libraries were size-selected to include fragments ~200 – 300 base pairs in length
and sequenced using an Illumina Genome Analyzer II. DNA reads were aligned against the
mouse mm9 reference genome using the Bowtie aligner using standard parameters that allow
up to 2 mismatches in the first 28 bases of the read.
Immunoblotting & Protein Quantification
For immunoblots, lysates were separated on 10% SDS-PAGE gel (Invitrogen), transferred, and
interrogated with primary antibody to urokinase (AbCam) or beta-actin (Sigma). Cytokines and
chemokines from mouse plasma or cell culture supernatants were quantified using a Bioplex
200 Suspension Array and assay kits (Panomics).
Statistical Analysis
Data was interpreted with 2-tailed t-tests or one-way ANOVA with Tukey’s multiple comparison
tests as indicated. Gene expression microarray and ChIP-seq were analyzed as described
above.
Supplemental References
Chawla, A., Boisvert, W.A., Lee, C.H., Laffitte, B.A., Barak, Y., Joseph, S.B., Liao, D., Nagy, L.,
Edwards, P.A., Curtiss, L.K., Evans, R.M., and Tontonoz, P. (2001). A PPAR gamma-LXRABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. Mol Cell 7,
161-171.
Han, S., Liang, C.P., DeVries-Seimon, T., Ranalletta, M., Welch, C.L., Collins-Fletcher, K.,
Accili, D., Tabas, I., and Tall, A.R. (2006). Macrophage insulin receptor deficiency increases ER
stress-induced apoptosis and necrotic core formation in advanced atherosclerotic lesions. Cell
Metab 3, 257-266.
Hsiao, A., Ideker, T., Olefsky, J.M., and Subramaniam, S. (2005). VAMPIRE microarray suite: a
web-based platform for the interpretation of gene expression data. Nucleic Acids Res 33, W627632.
Li, A.C., Binder, C.J., Gutierrez, A., Brown, K.K., Plotkin, C.R., Pattison, J.W., Valledor, A.F.,
Davis, R.A., Willson, T.M., Witztum, J.L., Palinski, W., and Glass, C.K. (2004). Differential
inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPARalpha,
beta/delta, and gamma. J Clin Invest 114, 1564-1576.
McLaren, J.E., Calder, C.J., McSharry, B.P., Sexton, K., Salter, R.C., Singh, N.N., Wilkinson,
G.W., Wang, E.C., and Ramji, D.P. (2010). The TNF-like protein 1A-death receptor 3 pathway
promotes macrophage foam cell formation in vitro. J Immunol 184, 5827-5834.
Miller, Y., Viriyakosol, S., Worrall, D., Boullier, A., Butler, S., and Witztum, J. (2005). Toll-like
receptor 4-dependent and -independent cytokine secretion induced by minimally oxidized lowdensity lipoprotein in macrophages. Arterioscler Thromb Vasc Biol 25, 1213-1219.
Nogales-Cadenas, R., Carmona-Saez, P., Vazquez, M., Vicente, C., Yang, X., Tirado, F.,
Carazo, J.M., and Pascual-Montano, A. (2009). GeneCodis: interpreting gene lists through
enrichment analysis and integration of diverse biological information. Nucleic Acids Res 37,
W317-322.
Tangirala, R.K., Rubin, E.M., and Palinski, W. (1995). Quantitation of atherosclerosis in murine
models: correlation between lesions in the aortic origin and in the entire aorta, and differences in
the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-deficient
mice. J Lipid Res 36, 2320-2328.
Teupser, D., Thiery, J., Walli, A.K., and Seidel, D. (1996). Determination of LDL- and
scavenger-receptor activity in adherent and non-adherent cultured cells with a new single-step
fluorometric assay. Biochim Biophys Acta 1303, 193-198.

Download Report