Histone sample preparation for quantitative mass spectrometry (qMS)
Cell pellets were lysed, nuclei isolated, and histones extracted as previously described16
. For each sample, approximately 100 μg of extracted histones were re-suspended in 30μL of 100mM ammonium bicarbonate, pH 8.0. Chemical propionylation derivatization, digestion and desalting of histones was performed as described25
, except that histones were digested for 6 hours. We performed both label free and isotopically labeled peptide relative quantification. For isotopically labeled peptide comparative MS analysis, d0
- and d10
-propionic anhydride were used as previously described15
. All proteomics data are available at the Stem Cell Omics Repository at http://scor.chem.wisc.edu/
Mass spectrometry and data analysis
Samples were analyzed by LC-MS and MS/MS as described15
. In brief, digested samples were loaded by an Eksigent AS2 autosampler onto 75 μm ID fused silica capillary columns packed with 12 cm of C18-reversed phase resin (Magic C18, 5 μm particles, Michrom BioResources), constructed with an electrospray ionization tip. Peptides were separated by nanoflowLC and introduced into a hybrid linear quadrupole ion trap-Orbitrap mass spectrometer (ThermoElectron, San Jose, CA), and resolved with a gradient from 5 to 35% Buffer B in a 110-min gradient (Buffer A: 0.1 M acetic acid, Buffer B: 70% acetonitrile in 0.1 M acetic acid) with a flow rate of 150 nl/min on an Agilent 1200 binary HPLC system. The Orbitrap was operated in data-dependent mode essentially as previously described15
. Relative abundances of peptide species were calculated by chromatographic peak integration of full MS scans using an in-house developed computer program. Peptide identity and modifications were verified by manual inspection of MS/MS spectra. Cluster 3.0 was used to create hierarchical clustering of ratio data and Java Treeview for visualization of the output.
Cell lines used for analysis of histone PTMs
The following cell lines were used for histone PTM qMS analysis in and : a female iPSC (2D4) line generated by retroviral expression of Oct4, Sox2, c-Myc, and Klf413
; a male iPSC line (C3) obtained upon retroviral expression of Oct4, Sox2, and Klf4 (i.e. in the absence of cMyc); a female pre-iPSC line (1A2)13
and a male pre-iPSC line (12-1) both obtained upon retroviral expression of Oct4, Sox2, Klf4, and cMyc in Nanog-GFP reporter MEFs. In addition, we used the male ESC line V6.5, and male and female wild-type MEFs from d13.5 embryos. ESCs, iPSCs, and pre-iPSCs were grown in standard mouse ESC media and MEFs in the same media lacking LIF.
Reprogramming experiments were carried out from Oct4
reporter MEFs using pMX retroviruses encoding Oct4, Sox2, and Klf4 as described previously27
and conducted in media containing 15% serum (FBS). MEFs containing a single polycistronic, tet-inducible cassette carrying the four reprogramming factors Oct4, Sox2, Klf4, and cMyc in the Col1A locus, the tet-transactivator M2rtTA in the R26 locus, and the Oct4
-GFP reporter, were generated as described50
, and induced to reprogram with 2ug/ml doxycycline. Reprogramming was scored by counting the number of GFP-positive ESCs-like colonies at indicated days. All reprogramming experiments from fibroblasts and pre-iPSC experiments were done in biological triplicates, and for each figure, error bars represent standard deviation from two technical replicates of a representative experiment. For pre-iPSC experiments, reprogramming to iPSCs upon siRNA knockdown was assessed by counting Nanog-GFP-positive colonies or quantifying GFP-positive cells by FACS at indicated days. For FACS analysis, 12-1 pre-iPSCs were harvested with trypsin, passed through a 40um cell strainer to obtain single-cell suspensions and analyzed on a LSR cytometer (BD Biosciences). Data were analyzed using the FlowJo software (TreeStar). Fuw-tetO-loxp-mNANOG was created by ligation-independent cloning (Infusion, Clontech Mountain View, CA) by digesting the vector Fuw-tetO-loxp-hKLF4 (Addgene 20727) with EcoRI and PCR-amplifying mouse Nanog from pMX-mNANOG (Addgene 13354). The vector was cotransfected with pCMV-delta8.9 and pCAGS-VSVg (Generous gift from Dr. Donald Kohn, UCLA) into 293T cells and viral conditioned media harvested 48 hours post transfection in serum free media (Ultraculture, Lonza). Expression was confirmed by immunostaining. For over expression, Jmjd2c was cloned from a PCR product using gene specific primers (Forward: ATGCGAATTCATGGAGGTGGTGGAGGTG, Reverse: ATGCGCGGCCGCCTACTGTCTCTTCTGACA) into the EcoRI and NotI sites of pMX vector. Expression was confirmed by qRT-PCR performed with gene specific primers RNA three days after transduction (For-GGCCATGGAAGTAACCTTGA, Rev-GAGGCTTACCAAGTGGATGG).
Sets of four different siRNAs were purchased from Dharmacon and transfected using lipofectamine–RNAi max (Life technologies) according to manufacturers instructions. Of the set of four siRNAs, the one producing the most efficient knockdown was used in reprogramming experiments at a final concentration of 20uM: Cbx1- MU-060281-01 #2, Cbx3 - MU-044218-01 #2, Cbx5 - MU-040799-01 #2, Setdb1 - MU-040815-01 #4, Ehmt1 - LU-059041-01- #3, Ehmt2 - MU053728-00- #3. For control siRNA treatments, we used the non-targeting Luciferase control- D-001210-02. The timing of siRNA transfections is indicated in each figure. For pre-iPSCs reprogramming experiments, reverse transfection of siRNAs was performed once, on 200,000 cells of the 12-1 pre-iPSC line, plated on gelatin. To test knockdown efficiency during reprogramming, RNA was harvested three days after the first transfection and on day 22, i.e. 3 days after the last transfection, and qRT-PCR was performed with gene-specific primers listed below.
Expression Analysis and Data Processing
To determine the transcriptional changes upon 3XHMT and Cbx3
knockdown in pre-iPSCs, total RNA was extracted from the pre-iPSC line 12-1 three days after the cells were subjected to transfection with control siRNAs (in biological triplicates), si-Cbx3
(in biological triplicates), or a pool of si-Ehmt1
(in biological duplicates), and analyzed on an Affymetrix GeneChip Mouse Genome 430 2.0 array at the UCLA Clinical Microarray core facility. Quantile normalization was performed using the Affymetrix package (affy) from Bioconductor. To convert probe data into gene expression data, probes ending in “_at” and “_a_at” were averaged for each gene. Data at the probe level were normalized with previously published data for ESCs, iPSCs, and MEFs27
. Data are deposited in the GEO database under GSE44084.
RNA-seq was performed using 4 ug of mRNA as starting material from ESCs and pre-IPSCs, using standard illumina RNA-seq library construction protocols. Briefly polyadenylated RNA was purified by two rounds of oligo-dT bead selection followed by divalent cation fragmentation under elevated temperature. Following cDNA synthesis with random hexamers, the double-stranded products were end repaired, a single “A” base was added, and Illumina adaptors were ligated onto the cDNA products. Ligation products with an average size of 300 bp were purified by means of agarose gel electrophoresis. The adaptor ligated single-stranded cDNA was then amplified with 10 cycles of PCR. RNA-Seq libraries were sequenced on Illumina HiSeq 2000. The RPKM (reads per kilobase of exon per million) was then computed for each gene.
Med29 Immunoprecipitation and PIC capture assay
ESCs expressing FLAG-Med29 were obtained by targeting the 3xFlag-Med29 under control of a tet-inducible promoter into the ColA1 locus51
in V6.5 ESCs carrying the M2rtTA in the R26 locus. Targeting was confirmed by Southern Blotting. Neural precursors were differentiated from these cells after suspension culture of embryoid bodies for four days, and selection in ITSF media for six days52
. Nuclear extract of ESCs and neural precursors were prepared and the purification of protein complexes containing Med29 was performed as in previously described53
. PIC assembly was performed from HeLa nuclear extract using the immobilized G5E4T and analyzed by Western blotting as described54
. All primary antibodies were used at 1:1000 dilution and secondary antibodies at 1:10000 dilution- anti-FLAG from Sigma (F-1804), anti-Med6 (sc-9434), anti-Cbx3 from Millipore (05690), from SantaCruz anti-Med1 (sc-8998), anti-RBBP5 (Bethyl-A300-109), anti-Cdk8 (sc-1521).
ChIP-Seq and ChIP-chip analysis
12-1 pre-iPSCs or V6.5 ESCs were chemically cross-linked by the addition of formaldehyde to 1% final concentration for 10 minutes at room temperature, and quenched with 0.125 M final concentration glycine. Cells were washed twice in PBS, re-suspended in sonication buffer (50mM Hepes, 140mM NaCl, 1mM EDTA, 1% TritonX-100, 0.1% Na-deoxycholate, 0.1% SDS), and sonicated with a Diagenode Bioruptor. Cell extracts were incubated with an antibody against Cbx3 (Millipore, 05-690; clone 42s2) or Med1 (sc-8998) overnight at 4°C and immunoprecipitates collected with magnetic beads.
Beads were washed twice with RIPA buffer, low salt buffer (20mM Tris pH 8.1, 150mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), high salt buffer (20mM Tris pH 8.1, 500mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), LiCl buffer (10mM Tris pH 8.1, 250mM LiCl, 1mM EDTA, 1% deoxycholate, 1% NP-40), and with 1xTE. Reverse cross-linking occurred overnight at 65°C with 1% SDS and proteinase K. Illumina/Solexa sequence preparation, sequencing, and quality control were performed according to Illumina protocols, with the minor modification of limiting the PCR amplification step to 10 cycles.
Reads were mapped to mm9 genome using the Bowtie software and only those reads that aligned to a unique position with no more than two sequence mismatches were retained for further analysis. Significant binding events were called as peaks using MACS2.0 using an FDR of 0.05 and the –broadpeaks setting that allows calling of broader domains. Location analysis of called peaks was performed using the Sole-search tool. Visualization of the ChIP-seq signal around the TSS is provided by heatmaps generated using Java Treeview. Briefly, enrichment is displayed after normalization to 1 million reads and subtraction of normalized input values per 100bp window. Data are deposited in the GEO database under GSE44242.
For ChIP-chip of H3K18me1 and H3K79me2, chromatin fragments (500ug) from V6.5 ESCs were enriched with specific antibodies, labeled and hybridized, along with corresponding input fragments, to an Agilent promoter microarray (Agilent-G4490) that contains the promoter regions of 18,300 annotated mouse genes, encompassing regions 5.5kb upstream to 2.5 kb downstream of the respective transcription start sites (TSS) as described27
. The H3K18me1 antibody was generated by N. Mishra, and the H3K79me2 antibody was kindly provided by Michael Grunstein at UCLA. The ChIP-chip data sets for H3K4me3 and RNA PolII data have been previously published5,27
. Hybridization onto the arrays, washing, and scanning were done according to manufacturer’s protocols. Average probe signals were extracted in a 500bp window-step-wise manner as described previously27