Supplement for the Materials and Method section:
Primers
ChIP DNA was amplified by PCR using the following primers: Forward FGF9 primer 5’AAGTCGGGGACAGAGAAGGT-3’ and reverse FGF9 primer 5’CACACACACACACGCAGATG-3’; forward FHX primer 5’CTCAGCTGTAGGGGGTGCT-3’ and reverse FHX primer 5’GTGCTGATGAGGGTGGTAGC-3’; forward H2BFC primer 5’TGAAAAGAGCCTTTGGTTCC-3’ and reverse H2BFC primer 5’AAAGTCACCATCGCACAGG-3’; forward RYBP primer 5’ACCACCCCCTAAAAAGGAGA-3’ and reverse RYBP primer 5’GGCCTTGAGGTGTGATTTGT-3’; forward ARHG primer 5’GCCCTGTAACTGGAAGTGGA-3’ and reverse ARHG primer 5’AGGTGTGGAGGGAGACGAC-3’; forward FEN1 primer 5’AATAATCCAGGGATGGACCTG-3’ and reverse FEN1 primer 5’CATGTTCTGGTTCCCAACCT-3’; forward GADD45G primer 5’AAAGCCAGGCGAGATGAAAT-3’ and reverse GADD45G primer 5’GCACCCGCTTTCTGATGTAA-3’; forward APEX primer 5’GGGTGTTTGTCATTCCCTTG-3’ and reverse APEX primer
5’CGGCCGTCTTACTCTTCTTG-3’; forward ORC4L primer 5’TCCCTGGTATTCTGGAGTGG-3’ and reverse ORC4L primer 5’TTTGATCACGTCCCTTCTCC-3’; forward SST primer 5’GGAGGAAATAAAGAGGGCTCA-3’ and reverse SST primer 5’-
TGCACACAAATGTACCCAGA-3’; forward ESR2 primer 5’TCACTGAGCTGGTGTGAGGA-3’ and reverse ESR2 primer 5’CTGGAAATGGAAACCGTCAT-3’; forward SREBF2 primer 5’CTGGTCCCATTGACAACAAA-3’ and reverse SREBF2 primer 5’CCATGACACCCGACAACC-3’; forward OAS1 primer 5’TCCAAGCTCAGTCAGCAGAA-3’ and reverse OAS1 primer 5’TGTCAATGGCATGGTTGATT-3’; forward GCN5L2 primer 5’AACTCCTGCAGGGCTCAAG-3’ and reverse GCN5L2 primer 5’GGTGGTGACTTGGGTGTGTT-3’; forward pS2 primer 5’GTGAGCCACTGTTGTCACG-3’ and reverse pS2 primer 5’CGAGCCCCGGATTTTATAG-3’.
Peptide synthesis
All peptides were prepared on a PAL-PEG-polystyrene resin by continuous flow solid
phase synthesis on a PerSeptive Biosystems Pioneer synthesizer (Framingham, MA)
using HBTU-activated Fmoc (N-(9-fluorenyl)methoxycarbonyl) amino acids. Peptides
were purified using conventional reversed phase HPLC on Vydac C18 (Hesperia, CA)
with an overall yield of 25-30%, based on starting resins. The purity of the peptides was
confirmed by analytical reversed phase HPLC, capillary zone electrophoresis, and
MALDI-TOF mass spectrometry. The quantity of each peptide was determined by BioRad protein assay as well as running small aliquots on 4-20% or 15% SDS/PAGE
followed by silver staining (Silver Stain Plus, Bio-Rad). Sequences of the purified
peptides were verified using an ABI PRISM automated sequencer (Applied Biosystems,
Foster City, CA). The peptides were lyophilized and stored at 4°C prior to use.
In Vitro Binding Assays
35
S-labeled HA-cyclin D1 was produced using the TNT Coupled Reticulocyte Lysate
System (Promega, Madison, WI). pcDNA3-HA cyclin D1 [a kind gift from Dr. Doris
Germain (Russell et al., 1999)] was used to produce the 35S-labeled protein. Binding
assays included GST-BRCA1 constructs (1 g) or GST (5 g) and 5 l of TNT reactions
and were carried out at 4C overnight. The following day, complexes were washed with
TNE150 + 1% NP-40 and TNE50 + 0.1% NP-40. Complexes were separated on a 4-20%
SDS-PAGE, dried, and exposed to a PhosphorImager cassette.
siRNA Transfections
Specific cdk2, cdk4 and cdk9 siRNAs sequences have previously been described
(Ammosova T, Berro R, Kashanchi F, Nekhai S. (2005). RNA interference directed to
CDK2 inhibits HIV-1 transcription. Virology 341: 171-178; Liang WS, Maddukuri A,
Teslovich TM, de la Fuente C, Agbottah E, Dadgar S et al. (2005). Therapeutic targets
for HIV-1 infection in the host proteome. Retrovirology 2: 20). The siRNAs were
transfected at final concentration of 500 nM using Lipofectamin reagent (Invitrogen,
Carlsbad, CA) according to the manufacturer's recommendations. The siRNAs were
incubated with cells for 48 h before cells were lysed for Western blotting or ChIP
analysis.