Supplementary Materials and Methods
Promoter Enrichment-Quantitative Mass Spectrometry (PE-QMS)
The promoter region of Abca1 was used to enrich for associated gene regulatory
complexes as described below. The 1X reaction ratio for promoter enrichment was 10µL
NeutrAvidin-agarose slurry (Thermo Fisher Scientific) for 500fmol DNA, 300µg nuclear
extract, and 250ng HaeIII-digested E. coli DNA per reaction condition tested. Binding
reactions were scaled up 20-30 times.
Promoter Synthesis
The biotinylated double stranded promoter region of
Mus musculus Abca1
(chr4:53,172,714-53,173,035; NCBI Build 37; mm9) used for enrichment was generated
by PCR amplification from mouse genomic DNA using a biotinylated forward primer
(Supplementary Figure S5, Supplementary Table S3). The LXRE DR4 mutant was
generated by overlapping PCR using the above construct as a template (Supplementary
Figure S5). All constructs were sequenced prior to use.
Promoter Immobilization
To immobilize biotinylated promoters, NeutrAvidin-agarose beads (Thermo Fisher
Scientific) were washed 2X with Binding Buffer (10mM Hepes, pH 7.4, 1mM EDTA, 1M
NaCl) and incubated with biotinylated DNA with constant agitation for 30 min at room
temperature. Promoters immobilized on NeutrAvidin-agarose beads were washed with
Binding Buffer and non-specific interactions were blocked with 20mM Hepes, pH 7.4,
100mM NaCl, 5mM MgCl2, 1mM EDTA, 2mM DTT, 5mg/ml Polyvinylpyrrolidone,
40mg/ml Casein for 30 min at room temperature. Blocked promoters were further
washed 3X with 25mM Hepes, pH 7.4, 5mM MgCl2, 1mM EDTA, 2mM DTT.
Preparation of Nuclear Extracts
Nuclear extracts were prepared essentially as described (Dignam et al, 1983). Briefly,
cells were swollen in Hypotonic Buffer (10mM Hepes, pH 7.4, 10mM KCl, 0.5mM DTT)
and lysed 5X with a 27 gauge needle. Intact nuclei were collected by centrifugation and
their contents extracted in 15mM Hepes, pH 7.4, 420mM NaCl, 25% Glycerol, 0.1%
NP40 (IGEPAL CA-630; CAS 9036-19-5), 2mM DTT. Extracts were passed 5X through
a 28 gauge insulin syringe, then agitated for 60 min at 4C. Nuclear extracts were
clarified by centrifugation and dialyzed to reduce concentrations of NaCl and Glycerol to
150mM and 20%, respectively.
Promoter Enrichment of Protein Complexes
Nuclear extracts were diluted to achieve a final buffer concentration of 20mM Hepes, pH
7.4, 10% Glycerol, 60mM NaCl, 1mM EDTA, 5mM MgCl2, 2mM DTT. NP40 was added
to 0.05% and samples were incubated for 10 min at room temperature and clarified by
centrifugation to remove insoluble material. NeutrAvidin-agarose-bound biotinylated
promoters were used to enrich for protein complexes from nuclear extracts for 1.5 h at
room temperature. HaeIII-digested E. coli DNA was also added to the reaction. DNAprotein complexes were washed 3X with Wash Buffer (20mM Hepes, pH 7.4, 60mM
NaCl, 5mM MgCl2, 1mM EDTA, 2mM DTT, 0.05% NP40), then 2X with Wash Buffer
lacking NP40. Protein complexes were eluted twice with 15mM Tris, pH 8.3, 600mM
NaCl, 5.5M Urea, 1mM EDTA for 30 min at 37C each.
Mass Spectrometry Sample Preparation
Eluted proteins were reduced with 5mM DTT, alkylated with 25mM Iodoacetamide, and
digested with Lys-C (Roche Applied Science) and Trypsin (Promega) essentially as
described (Mirzaei et al, 2013). Samples were acidified with Formic Acid (Thermo
Fisher
Scientific)
to
stop
digestion
and
purified
using
C18
reversed-phase
chromatography (SUM SS18V; Nest Group) according to manufacturers’ protocol.
Quantitative Mass Spectrometry
Orthogonal label free and labeled strategies were separately employed for protein
quantification. Label free MS1 intensity analysis quantifies proteins based on the
intensity of peptides in one mass spec run relative to another (Bensimon et al, 2012).
Labeled approaches involved labeling peptides post-digest with either light or heavy
isotopic versions of the primary amine-reactive mTRAQ (Life Technologies) or N-isotag
(Smolka et al, 2005) reagents. Protein quantification for these labeled samples is based
on relative peptide intensities within the same run (Bensimon et al, 2012).
Label Free Approach
C18 purified peptides were further purified by hydrophilic interaction chromatography
(HILIC; SEM HIL; Nest Group) according to manufacturers’ protocol. Purified peptides
were then separated by online nanoscale HPLC (NanoLC Ultra 2D Plus; Eksigent) with
a C18 reversed-phase column packed 15 cm in house (ReproSil-Pur C18-AQ 3um; Dr.
Maisch GmbH) over an increasing gradient of 2-35% Buffer B (100% Acetonitrile, 0.1%
Formic Acid) over 100 min at a flow rate of 280nl/min. Eluted peptides were analyzed
with an LTQ-Orbitrap Velos (Thermo Scientific) operated in data dependent mode to
acquire the top 8 most intense peptides per MS1 survey scan. MS1 survey scans were
performed in the Orbitrap at a resolution of 30,000 at m/z 400, with a lock mass of
445.120025 and charge state rejection enabled, while fragmentation was by collisioninduced dissociation (CID) in the LTQ, with a minimum signal of 3000. Dynamic
exclusion was set to 3 min to prevent repeat identifications. Raw output files were
processed using Genedata Expressionist Refiner MS to reduce noise, align spectra,
detect peaks and isotope clusters, determine intensities, as well as initiate searches
using Mascot (Matrix Science) (Perkins et al, 1999) against the IPI murine database
(v3.69) (Guryca et al, 2012; Ueda et al, 2011). A reverse database was used to assess
FDR. Data was subsequently normalized by central tendency and analyzed using
Genedata Expressionist Analyst (Askovich et al, 2013).
Isotopic Labeling Approach
C18-purified peptides were labeled with mTRAQ (Life Technologies) according to
manufacturers’ protocol, or labeled with N-isotag as described (Smolka et al, 2005).
Labeled peptides were fractionated with Partisphere strong cation exchange (SCX)
resin using in house packed microbore guard columns (Upchurch Scientific) and a
syringe pump (30µl/min; Harvard Apparatus). Peptides were eluted with a stepwise
gradient of 25mM, 40mM, 50mM, 60mM, 75mM, 100mM, and 750mM NH4-Formate in
25% Acetonitrile. Eluted peptide fractions were evaporated, then separated by online
HPLC (1100; Agilent Technologies) with a C18-reversed phase column packed 10 cm in
house (Magic C18 AQ 5um; Bruker) over an increasing gradient of 2-35% Buffer B over
125 min at a flow rate of 350nl/min. Eluted peptides were analyzed with an LTQOrbitrap XL mass spectrometer operated in data dependent mode to acquire the top 3
most intense peptides per MS1 survey scan. All other run details were similar to those
listed above, except MS2 minimum signal was 100 and dynamic exclusion was set to 60
sec. Raw files were searched against the IPI murine database (v3.66) using X!Tandem
(Thermo Scientific) to identify peptides. A reverse database was used to assess false
discovery rate (FDR). Probabilities were determined by PeptideProphet and
ProteinProphet (Keller et al, 2002; Nesvizhskii et al, 2003), and quantification was
performed by ASAP Ratio (Li et al, 2003), as part of the Trans-Proteomic Pipeline.
Data Filtering
Data was manually inspected to ensure accuracy of identifications and quantifications.
Data was filtered to remove single peptide identifications, along with proteins not
satisfying a 1% FDR cutoff. In addition, proteins corresponding to genes not expressed
in primary BMMs were removed (Gold et al, 2012; Ramsey et al, 2008). Proteins were
required to be present in both the labeled and label free ligand-stimulated experiments.
From this, we generated a list of 188 promoter-associated proteins. We used
quantitative information to further restrict this list to 79 based on the criteria that proteins
must have correlated binding intensities (i.e. either both positive or both negative
values) in the ligand-stimulated experiments. We used 1.5-fold cutoffs as described in
the Results to subdivide proteins by their ligand dependency and their LXR
dependency. Heatmaps were generated using Genedata Expressionist Analyst.