Mms 126r

Chromatin Immunoprecipitation (ChIP) experiments were performed as described in ref. 27 (link) using early embryos or L4 worms. The antibodies used are as follows: 5 μg of anti-H3 (Millipore, 05-928), 2.5 μg of anti-H3K27me3 (Diagenode, pAb-195-050), 10 μg of anti-H3K36me3 (Abcam, ab9050), 10 μg of anti-MES-4 (Novus, 29400002), 10 μg of anti-CeCENP-A (a gift from the Desai lab, validated in ref. 35 (link)), 10 μg of anti-Pol II 8WG16 (Covance, MMS-126R). Validation information for the commercial antibodies is included at the manufacturers’ websites.

ChIP was performed as described previously (28 (link)). Briefly, H358, H358 Control, or H358 Brg1i.2 cells were fixed with 1% formaldehyde in PBS for 15 minutes at room temperature, whereas H358 Brg1i.2Brmi double knockdown cells were fixed for 8 minutes. Crosslinking was quenched by adding glycine to a final concentration of 125mM for at least 10 minutes. Cells were then collected in lysis buffer (1% NP40, 0.1% SDS, 5mM EDTA, 50mM Tris, pH 8.0, 150mM NaCl, protease inhibitors cocktail tablet) and cell lysates were sonicated for 5minutes (for 1 cycle:15 sec on/45 sec off, 20 cycles) at 20% amplitude. ChIP was performed with antibodies specific to NRF2 (H300, Santa Cruz Biotechnology, Dallas, TX), BRG1 (A300–813A, Bethyl Laboratories, Inc., Montgomery, TX), RNA polymerase II (MMS-126-R, Covance, Raleigh, NC) or normal rabbit IgG (SC-2027, Santa Cruz Biotechnology, Dallas, TX). Precipitated DNA was determined by Q-PCR using gene-specific primers as listed in Table 1 and normalized against input DNA.

ChIP-seq was performed as described (Huang et al., 2016 (link)) using the antibodies for BRD4 (A301-985A, Bethyl, lot: A301-985A-1), RNAPII (MMS-126R, Covance, lot: D12LF03144) and H3K27ac (ab4729, Abcam) in K562 erythroid cells treated with DMSO (control), or 1 μM of JQ1 for 6 hours. Antibodies for NUP98 (2598, Cell Signaling Technology, lot:4) or NUP153 (906201, BioLegend, lot:B215613) were used. Cross-linked K562 chromatin was sonicated in RIPA 0 buffer (10 mM Tris-HCl, 1 mM EDTA, 0.1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 0.25% Sarkosyl, pH 8.0) to 200~500 bp. Final concentration 150 mM NaCl was added to the chromatin and antibody mixture before incubation overnight at 4°C. ChIP-seq libraries were generated using NEBNext ChIP-seq Library Prep Master Mix following the manufacturer’s protocol (New England Biolabs), and sequenced on an Illumina NextSeq500 system using the 75bp high output sequencing kit. ChIP-seq raw reads were aligned to the hg19 or mm9 genome assembly using Bowtie (Langmead et al., 2009 (link)) with the default parameters. Only tags that uniquely mapped to the genome were used for further analysis. ChIP-seq peaks were identified using MACS (Zhang et al., 2008 (link)). Gene ontology (GO) analysis was performed using GREAT (McLean et al., 2010 ).

Commercial antibodies used in this study were anti-RNA polymerase II (RNAPII ChIP 12 μg, 8WG16, MMS126R, Covance), anti-phosphorylated Ser 2 of RNAPII CTD (ChIP 12 μg, clone 3E10, 04-1571, Millipore), anti-HA (ChIP 8 μg, WB 1:2000, clone HA-11, MMS-101R, Covance), anti-H3 (ChIP 4 μg, ab1791, abcam), anti-H3K36me3 (ChIP 4 μg, ab9050, abcam). Polyclonal anti-Mex67 (WB 1:20,000) and anti-Npl3 (ChIP 1,5 μL, WB 1:10,000) antibodies were previously described (Siebel and Guthrie 1996 (link); Gwizdek et al., 2005 (link)). TAP-tagged proteins were immunoprecipitated using IgG sepharose beads (50 μL for ChIP analysis). Western blot analyses were performed using appropriate HRP-coupled secondary antibodies and chemi-luminescence protein immunoblotting reagents (Pierce).