Vectors and plasmids
full length cDNA was isolated from mouse testis tissue, amplified by PCR and then sequenced. Full length Taf7l
, and Pparγ
cDNAs were inserted into pCMV 3×FLAG-10 vector to construct pCMV 3×FLAG-TAF7L/TAF7/PPARγ. Taf7l
, and Pparγ
full length cDNAs were cloned into the pCS2+ vector with either HA or FLAG tag at their N-terminus. pLKO.1 shGFP and pLKO.1 shTAF7L vectors were purchased from Open Biosystems. shTAF7L-resistant pCMV 3×Flag-TAF7LmA was made by site-directed mutagenesis to introduce two silent mutations in shTAF7L targeted TAF7L cDNA sequence (Ding et al., 2008
Antibody production and purification, antibodies
A fragment of the Taf7l cDNA corresponding to residues 400–600 was cloned into the pGEX-4T-1 vector carrying a GST tag. GST-TAF7L (a. a. 400–600) was expressed and purified from E.coli and injected into rabbits by Covance (Covance Research Products Inc.,Denver, PA). Bleeds were collected after three boosts and TAF7L antibodies were tested and confirmed by in vitro transcribed and translated TAF7L protein and whole protein extracts from mouse testis of WT and Taf7l KO mice (data not shown). The antisera obtained were affinity-purified using antigen immobilized on Affigel 10/15 resin (Bio-rad, Hercules, CA). For Pol II antibody, monoclonal anti-Pol II (8WG16) was concentrated from hybridoma supernatant with Protein A Sepharose Beads (GE Healthcare, Piscataway, NJ).
Antibody information: anti-TAF4 (BD 612054), anti-TBP (abcam 62126), anti-FLAG (Sigma, F3165), anti-HA (abcam 9110), anti-β-actin (Sigma, A2228), anti-TAF7 (abnova H00006879-M01), anti-FABP4 (abcam 66682), Pol II (monoclonal 8GW16, protein-A purified), PPARγ (sc-7196), mouse and rabbit IgG (prepared in-house and concentrated with Protein A Sepharose Beads).
Cells culture, stable cell line establishment
C3H10T1/2, 3T3-L1, C2C12, HeLa, and 293T cells were cultured in high glucose DMEM with 10% fetal bovine serum at 10% CO2.
C3H10T1/2 shGFP and shTAF7L lentiviral shRNA knockdown stable cell lines were established by transfecting pLKO.1 shGFP or pLKO.1 shTAF7L into C3H10T1/2 cells and then subjecting to puromycin selection for 3 weeks.
C3H10T1/2 shTAF7L cells were transfected with pCMV 3×Flag vector, pCMV 3×Flag-TAF7 or pCMV 3×Flag-TAF7LmA and then subjected to G418 and puromycin double selection for 3 weeks to establish shTAF7L+vector, shTAF7L+TAF7 or shTAF7L+TAF7LmA cell lines used in ‘rescue’ experiments in .
C2C12.CNTL and C2C12.TAF7L were established by transfecting pCMV 3×Flag vector or pCMV 3×Flag-TAF7L vector into C2C12 cells and then underwent G418 selection for 3 weeks.
Adipocyte differentiation, Oil red O staining and C2C12 myogenesis
For adipogenesis, 3T3-L1 and C3H10T1/2 cells were grown in high glucose DMEM supplemented with 10% fetal bovine serum. At confluence, cells were exposed to induction medium containing dexamethasone (1 μM), isobutylmethylxanthine (IBMX, 0.1 mM), insulin (5 μg/ml), rosiglitazone (1 μM), and 10% FBS. 3 days later, cells were further cultured in high glucose DMEM containing insulin (5 μg/ml) and rosiglitazone (1 μM) until they were ready for harvest. C3H10T1/2 cells form mature adipocytes 5 days post induction; 3T3-L1 cells require 7–8 days to form adipocytes.
For Oil red O staining, pre- and post-differentiated C3H10T1/2 and 3T3-L1 cells, WT and Taf7l
KO adipose-derived primary fibroblasts, shGFP and shTAF7L-treated C3H10T1/2 cells, C2C12.CNTL and C2C12.TAF7L cells were washed once in PBS and fixed with freshly prepared 4% formaldehyde in 1×PBS for 30 min, followed by standard Oil red O staining method described previously (Tang et al., 2004
For C2C12 myogenesis, C2C12 cells are cultured in maintenance media until confluence was reached. 2 days post confluence, cells were switched to differentiation media comprised of low glucose DMEM, 2% horse serum, and 5 μg/ml insulin, 3 days later, change fresh differentiation media and culture cells for additional 2 days, differentiated myotubes were harvested and purified by collecting the suspended cells after splitting and reseeding the cells for 1 hr.
RNA isolation and real-time PCR analysis
Total RNA from cultured cells or mouse tissues was isolated using QIAGEN RNeasy Plus mini columns according to the manufacturer's instructions (Qiagen Inc., Germantown, MD). For RT-qPCR analysis, 1 μg total RNA was reverse transcribed using cDNA reverse transcription kit (Invitrogen, Carlsbad,, CA). SYBR green reactions using the SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) were performed according to the manufacturer's instruction using an ABI 7300 real time PCR machine (Applied Biosystems, Foster City, CA). Relative expression of mRNA was determined after normalization to total RNA amount. Student's t-test was used to evaluate statistical significance.
Western blot analysis, immunoprecipitation
Whole cell extracts were prepared from cells by homogenization in lysis buffer containing 50 mM Tris–Cl, pH 8.0, 500 mM NaCl, and 0.1% Triton X-100, 10% glycerol and 1 mM EDTA, supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN) and phenylmethylsulphonyl fluoride (PMSF). Fifteen micrograms (μg) of whole-cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membrane. For immunoblotting, membranes were blocked in 10% milk, 0.1% Tween-20 in TBS for 30 min, and then incubated with TAF7L, TAF4, TAF7, FLAG, β-actin, PPARγ and TBP antibodies for 2 hr at room temperature; detailed Western blotting procedure was performed as previously described (Zhou et al., 2006
500 μg whole-cell extracts from 293T cells transfected with FLAG-TAF7L and HA-PPARγ, FLAG-PPARγ and HA-TBP, or FLAG-TAF7L and HA-TBP were immunoprecipitated with FLAG or HA antibodies at 4°C for overnight under the conditions of 0.3 M NaCl and 0.2% NP-40, 30 μl protein A/G beads were added and incubated for additional 2 hr at 4°C, after extensive washing with buffer containing 0.15 M NaCl and 0.1% NP-40, remaining beads were subjected to 10% SDS-PAGE and followed by western blotting analysis with FLAG and HA antibodies to detect tagged-proteins in the inputs and IPs as previously described (Ding et al., 2008
Animals and genotype analysis
The derivation of Taf7l
-knockout mice has been previously described (Cheng et al., 2007
). All animal experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved animal use protocols (#R007) by Animal Care and Use Committee (ACUC) of the University of California, Berkeley. Mice were maintained on a standard rodent chow diet with 12 hr light and dark cycles. Taf7l
KO mouse line was maintained on a C57/Bl6 background. Genotyping was performed by PCR as previously described (Cheng et al., 2007
Preparation of primary fibroblast and induction of adipogenesis
Fresh inguinal adipose tissues were removed from 3 week old euthanized WT and Taf7l KO mice and finely minced, digested with 0.25% trypsin for 30 min at 37°C, and centrifuged for 5 min at 2,000g. The pellet was resuspended in culture media before plated on gelatin coated plates. Cells were cultured at 37°C in high glucose DMEM supplemented with 20% FBS. Adipocyte differentiation and staining were followed the same procedure as C3H10T1/2 cells.
For histological analysis on interscapular tissue of E18.5 embryos from WT and Taf7l
KO mice, freshly-harvested mouse embryos were genotyped and then interscapular regions of embryos were transversally dissected and then fixed in 10% formaldehyde for 24 hr at 4°C; tissue was embedded in paraffin using the microwave method and then sectioned into 8–10 μm sections to mount on slides. This method and the following immunohistochemistry by haematoxylin and eosin (H&E) staining were performed using the method described previously by Steven Ruzin (Schichnes et al., 1998
), and FABP4 immunostaining method was modified from the one described previously (Kajimura et al., 2008
) (Gupta et al., 2010
ChIP, ChIP libraries preparation, and deep sequencing (ChIP-seq)
Fix C3H10T1/2 cells and differentiated adipocytes with 1% formaldehyde for 10 min at room temperature then use 0.125 M glycine to stop the crosslinking for an additional 5 min. Collect cells and extract nuclei with extraction buffer. The chromatin obtained from C3H10T1/2 cells and adipocytes was fragmented to sizes ranging from 175 to 225 bp using a Covaris-S2 sonicator (Covaris, Inc., Woburn, MA) for a total processing time of 40 min (20 s on, 20 s off). 900 µg of the sonicated chromatin was used in each immunoprecipitation reaction as previously described (Liu et al., 2011
) with the Pol II, TBP, PPARγ, and TAF7L antibodies, mouse and rabbit IgG were used as negative controls respectively. Preparation of the sequencing libraries on the DNA samples of the immunoprecipitation from antibodies and IgGs precisely followed the instructions from Illumina (Illumina Inc., San Diego, CA), qualities of the libraries were assessed by 2100 Bioanalyzer (Functional Genomics Laboratory, Berkeley,CA) and then subjected to ultra-high throughput sequencing on an Illumina HiSeq 2000 sequencer (GSL core facility, Berkeley, CA) as previously described (Fong et al., 2011
; Liu et al., 2011
), each sample yielded ~50–200 million reads.
Mapping sequencing reads to the genome
Sequenced reads were mapped to the July 2007 assembly of the mouse genome (UCSC version mm9, NCBI37) using Bowtie (Langmead et al., 2009
) with the command-line options ‘-c -q -n 2 -l 48 -m 1’, thereby keeping for further analyses only reads that mapped uniquely to the genome with at most two mismatches at the first 48 bases.
Peak calling methods
To accurately identify significant binding positions across the genome, we incorporated two peak calling methods, as described previously (May et al., 2012
). First, we used MACS (Zhang et al., 2008
) (version 1.4, with default settings except: --nomodel --shiftsize = 110 --pvalue=1e-2 --mfold = 10,10,000 --slocal = 2000 --llocal = 20,000), with an FDR significant threshold of 0.05 (using IgG ChIP as a control). Second, we applied the Grizzly peak fitting algorithm (Harrison et al., 2011
), which uses a model-based iterative approach to accurately identify multiple binding loci at every enriched region. Peaks were then associated with the nearest TSS (using gene annotation from UCSC, version mm9), and classified as promoter peaks (up to 500 bp from start site), proximal enhancer peaks (larger than 500 bp and less than 5 kb), distal enhancer peaks (less than 50 kb from TSS), or none (further away than any start site).
The DNA sequence associated with each of the peaks is a 250 bp fragment that is centered on the highest point peak within the TAF7L binding region as defined by Grizzly Peak. Weeder (version 1.4.2) (Pavesi et al., 2004
) searches the inputted sequences for enriched motifs. The Weeder output is then passed through STAMP (Mahony and Benos, 2007
) to compare the identified motif's frequency matrix against the JASPAR v2010 database and identify which transcription factors have that DNA binding motif.
mRNA-seq libraries preparation and deep sequencing
Total RNAs were extracted from C3H10T1/2 cells and adipocytes, differentiated shTAF7L-C3H10T1/2 cells, differentiated C2C12.CNTL and C2C12.TAF7L cells by RNeasy Plus Mini Kit (Qiagen), 8 μg of each sample was used to purify mRNA and subsequently converted into to mRNA-seq library using mRNA-Seq Sample Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2000 sequencer. 50 bp paired-end were used for the C3H10T1/2 samples and 100 bp single-end were used for the C2C12 samples, both resulting in over 175 million reads yield per sample.
Digital gene expression of mRNA-seq
The reads were than mapped to the mouse transcriptome (created using UCSC table browser, version mm9, on February 2012), using TopHat (Trapnell et al., 2009
), version v1.4.0., using default parameters. We then applied cufflinks (Trapnell et al., 2010
), version v1.3.0, using the default parameters except: --max-mle-iterations 1, to estimate the digital expression levels at each transcript. Due to the high number of sequence reads, these steps were done in seven batches of 25 million reads each, per sample.