E2f4 regulates fetal erythropoiesis through the promotion of cellular proliferation
Blood Kinross et al.
108: 886
Supplemental materials for: Kinross et al, Vol 108, Issue 3, 886-895
SUPPLEMENTARY MATERIALS AND METHODS
Differential expression analysis using cDNA Microarrays
Total RNA was extracted using TRIzol (Life Technologies, Bethesda, MD) and RNeasy minicolumns (QIAGEN, Valencia, CA) according to the manufacturer’s specifications. RNA from 2 to 3 E2f4−/− or E2f4+/+ E15.5 whole FLs was pooled and labeled indirectly via reverse transcription using superscript II reverse transcriptase (Life Technologies), oligo dT, and amino-allyl dUTP (Sigma, St Louis, MO). Subsequently, cDNA was purified with QIAquick columns (QIAGEN) and coupled to Cy3- or Cy5-monofunctional NHS ester (Amersham Pharmacia, Piscataway, NJ). Labeled cDNAs were cohybridized to the NIA 15K mouse cDNA array1 printed at the Peter MacCallum Cancer Centre Microarray Facility (www.ccgpm.org/microarray/), with subsequent slide washing and scanning as previously described.2 Expression intensities were determined using Quantarray (GSI Lumonics, Bedford, MA) and data analysis was performed using Genespring (Silicon Genetics, Redwood City, CA) following LOWESS normalization.3 Clones with differential expression of greater than 1.66/0.6-fold between E2f4+/+ and E2f4−/− samples in 3 or more of 7 independent arrays were considered differentially expressed.
Real-time PCR
cDNA was created using random primers and M-MLV reverse transcriptase RNase H− (InVitrogen, Frederick, MD) and amplified by PCR in triplicate with SYBR Green (Applied Biosystems, Foster City, CA) as per the manufacturer’s specifications on the ABI PRISM 7700 system (Applied Biosystems).
Oligonucleotide primers used for expression analysis were:
H66∼6¹
fwd, 5´-AACGCCGATGAAGTTGGTG-3´
rev, 5´-AAGGGTAGACAACCAGCAGCC-3´
Slc4a1
fwd, 5´-CCTGTGACCGAGATGCAGG-3´
rev, 5´-GGCTGTTTGCTCTGTGGGAA-3´
β2M
fwd, 5´-TTCACCCCCACTGAGACTGAT-3´
rev, 5´-GTCTTGGGCTCGGCCATA-3´
Ccna2
fwd, 5´-AGCAAGAAAACCACTGACACCTCT-3´
rev, 5´-TCAAAACTGCCATCCATTGGA-3´
Cbx5
fwd, 5´-CCCACCCCTTTCCATCCTAA-3´
rev, 5´-CAGTGATGAGCGCAGTGGTT-3´
H19
fwd, 5´-GCAGTCATCCAGCCTTCTTGA-3´
rev: CTTCTACGACAAGGCGCCA-3´
Ccnd2
fwd, 5´-GCTCTGTGCGCTACCGACTT-3´
rev, 5´-AATCATCGACGGCGGGTAC-3´
Gapdh
fwd, 5´-GTATGACTCCACTCACGGCAAA-3´
rev, 5´-GGTCTCGCTCCTGGAAGATG-3´
Chromatin Immunoprecipitation (ChIP) Analysis
ChIPs were carried out essentially as described, with some modifications.4 Cells (20 × 106/IP) were pooled and fixed in 0.4% formaldehyde at 5 × 105 cells/mL for 10 minutes at room temperature. Cross-linking was terminated by addition of 0.125 M glycine for 5 minutes, and followed by 2 washes in ice-cold PBS. Nuclei were isolated in cell lysis buffer (10 mM Tris pH 8.0; 10 mM NaCl; 0.2% NP40; proteinase inhibitors) for 10 minutes. Pelleted nuclei were lysed in 0.6 mL SDS lysis buffer for 10 minutes on ice (50 mM Tris pH 8.1, 10 mM EDTA; 1% SDS; proteinase inhibitors). Samples were diluted in 0.4 mL ChIP dilution buffer (20 mM Tris pH 8.1; 2 mM EDTA; 150 mM NaCl; 1% Triton X-100; 0.01% SDS; protease inhibitors) and sonicated to shear chromatin (300 to 600 bp). Soluble chromatin was collected and diluted 1:4 with ChIP dilution buffer. Protein A-Sepharose CL-4B (PAS; Amersham Biosciences) was prepared as per the manufacturer’s specifications, blocked with 200 µg/µL salmon sperm DNA and 1 µg/µL bovine serum albumin (BSA), and made to a 50:50 slurry with ChIP dilution buffer. Chromatin was precleared with preimmune rabbit serum and PAS overnight at 4°C. The sample was separated equally into 3 IP tubes and incubated with the immunoprecipitating antibody (rabbit 
-E2F4 sc866), rabbit -acetylated histone H3 (Upstate Biotech), rabbit -GAL4 (DNA-binding domain; Upstate Biotech), or 25 µL preimmune rabbit serum for 2 to 3 hours. An input control containing 20% chromatin amount and a no-chromatin negative control were also prepared. Immune complexes were retrieved with 60 µL blocked PAS for 1 hour, washed for 3 minutes at 4°C 3 times in ChIP wash 1 (20 mM Tris pH 8.1; 2 mM EDTA; 50 mM NaCl, 1% Triton X-100; 0.01% SDS), twice in LiCl immune complex wash buffer (Upstate Biotech), once with TE (10 mM Tris pH 8.0, and 0.1 mM EDTA), and were then eluted in elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature. Protein/DNA cross-links were reversed by overnight incubation at 65°C with 200 mM NaCl. Proteins and RNA were degraded in Rnase and proteinase K, and DNA was isolated by a phenol-chloroform and chloroform extraction followed by ethanol precipitation with the inclusion of glycogen. DNA pellets were washed with 70% ethanol and resuspended in 200 µL TE buffer and stored at −20°C. Quantification of IP-enriched DNA sequences was conducted using real-time PCR as above. Data are represented as percentage of input, calculated by 2deltaCT × 20, where deltaCT is determined by CT(Input) — CT(IP sample), and 20 refers to input being 20% of chromatin amount exposed to IP.5
Oligonucleotide primers used for ChIP were:
Ccna2.E
fwd, 5´-CTCCGGACGCCCTCATT-3´
rev, 5´-TTGTAGTTCAAGTAGCCCGCG-3´
Ccna2.U
fwd, 5´-GAAAGATAAACAAGGTAGGCCAACA-3´
rev, 5´-TCAGGCTCTGCTTGCTTCTTC-3´
Mcm2.E
fwd, 5´-TCACGTGGTCTAGTTTTCGCG-3´
rev, 5´-CTCCACCGCACCAGCTG-3´
Mcm2.U
fwd, 5´-TACCCCTCTCCGTTGTCACC-3´
rev, 5´-GAGTCCAAGTAGCCTGGGCC-3´
Ccnd2.E
fwd, 5´-GGACAACAGCTTGAAAGTTATCAGG-3´
rev, 5´-AAAGGTTATGCCCCTCAAGCTT-3´
Ccnd2.U
fwd, 5´-CCTTTCTTTACATGCAGTAGATTGGAT-3´
rev, 5´-CTATCAATGGCAGCGGGAAT.
References
1. Tanaka TS, Jaradat SA, Lim MK, et al. Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray. Proc Natl Acad Sci U S A. 2000;97:9127-9132.
2. Boussioutas A, Li H, Liu J, et al. Distinctive patterns of gene expression in premalignant gastric mucosa and gastric cancer. Cancer Res. 2003;63:2569-2577.
3. Yang YH, Dudoit S, Luu P, et al. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 2002;30:e15.
4. White J, Stead E, Faast R, Conn S, Cartwright P, Dalton S. Developmental activation of the Rb-E2F pathway and establishment of cell cycle-regulated cyclin-dependent kinase activity during embryonic stem cell differentiation. Mol Biol Cell. 2005;16:2018-2027.
5. Frank SR, Schroeder M, Fernandez P, Taubert S, Amati B. Binding of c-Myc to chromatin mediates mitogen-induced acetylation of histone H4 and gene activation. Genes Dev. 2001;15:2069-2082.
