Cell culture and reagents
Mouse embryonic fibroblasts (MEFs), wild-type HEK293T cells and HEK293T cells constitutively expressing the V5-tagged NM1 constructs V5-wtNM1, V5-RK605AA NM1 or V5-ΔC NM1 were grown in DMEM medium (Gibco, Life Technology, Carlsbad, CA, USA), supplemented with 10 % fetal bovine serum (Gibco) and a 1 % penicillin/streptomycin cocktail (Gibco) as previously performed [25, 26]. The antibodies against WSTF (ab50850), SNF2h (ab3749), H3K4m1 (ab8895), H3k4me3 (ab8580), H3K9Ac (ab10812) and H3k27ac (ab4729), Set1/Ash2 (ab70378), Rpb8 (ab104802), as well as 8WG16 (ab817) and 4H8 (ab5408) respectively targeting non-phosphorylated and hypophosphorylated (phospho-S5) heptapeptide repeats from the CTD of the largest Pol II subunit were all purchased from Abcam, Cambridge, UK. The antibodies against PCAF (sc13124) and Rpb6 (sc28711) were from Santa Cruz Biotechnology, Inc. Dallas, TX, USA. The antibodies to actin are specific for the β-isoform and were purchased from Sigma-Aldrich, St. Louis, MO, USA (clone AC74). The antibody against the V5 epitope (A190-120A) was purchased from Bethyl, Montgomery, TX, USA Laboratories. The non-specific rabbit IgGs (ab46540) were from Abcam. The antibody against NM1 has previously been characterized . The anti-pan-myosin-Ic monoclonal antibody that recognizes an epitope in the tail region of myosin Ic has been previously characterized  and was provided by Dr. W. Hofmann (University at Buffalo-SUNY). RNAi duplexes against the target sequence GCACACGGCUUGGCACAGA in the mouse NM1 (NM1 RNAi) or control scrambled versions (scrRNAi) were purchased from Dharmacon, Lafayette, CO, USA; GE Healthcare and applied by transfection with Lipofectamine RNAi Max (Invitrogen, Waltham, Massachusetts, USA) at a final concentration of 30 nM. RNAi duplexes against human NM1 or control scrambled versions were previously described and they were applied by transfection with Lipofectamine RNAi Max (Invitrogen) at a final concentration of 30 nM as previously described . The HEK293T cells stably expressing V5-wt NM1, V5-RK605AA NM1 and V5-ΔC NM1 were a kind gift of Ingrid Grummt, German Cancer Research Center, Heidelberg, Germany .
MEFs transfected with NM1 RNAi duplexes or control scrRNAi duplexes were grown in 10 cm dishes for 24 h at 37 °C. Total RNA was extracted with the TRI reagent as specified by the manufacturer’s instruction manual (Sigma). For analysis of transcripts, polyA mRNA was isolated from NM1-silenced or control cells using the Oligotex mRNA Mini Kit according to the manufacturer’s protocol (Qiagen, Venlo, Limburg, Netherlands) and treated with DNase1. cDNA was then synthesized using Superscript II reverse transcriptase (Invitrogen) using oligo dT primers according to the manufacturer’s instructions. The concentration of cDNA was determined by nanodrop. Semi-quantitative RT-qPCR was performed using the cDNA templates prepared from MEFs treated with control scrRNAi duplexes or from MEFs treated with NM1 RNAi duplexes, a Power SYBR Green PCR kit (Life Technology, Carlsbad, CA, USA), specific primers amplifying the mouse class II genes Rplp0, Rpl13a, Rpl19, Junb, Rad9a, Wtap, Ddx46 as well as Bad and the human class II genes RAD9A and RPL19 (see Additional file 8: Table S3) and a 7300 Real Time PCR System (Applied Biosystems, Waltham, Massachusetts, USA). For all primers the annealing temperature was 60 °C. All samples were run in triplicate. Relative changes in RNA levels were calculated against the reference β-actin gene using the delta-delta Ct method as previously described .
Cells were lysed in RIPA buffer (50 mM Tris‐HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 % NP‐40, 0.5 % sodium deoxycholate, 0.1 % SDS) supplemented with protease inhibitors (cOmplete cocktail, Roche, Basel, Switzerland). For denaturation, protein extracts were incubated in Laemmli buffer at 95 °C for 10 min, separated by SDS–PAGE under reducing conditions and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, Massachusetts, USA) by semidry blotting (Biorad, Hercules, CA, USA). Primary antibodies and dilutions used were NM1 (1:1000), β-actin (Sigma, 1:50000), WSTF (Abcam, 1:2000), SNF2h (Abcam, 1:500), PCAF (Abcam, 1:500), Set1/Ash2 (Abcam, 1:500). Immunoreactive bands were visualized by chemiluminescence (Amersham, GE Healthcare Life Sciences, Pittsburgh, USA).
High-resolution MNase assay
These experiments were essentially performed as described [25, 34]. Briefly, HEK293T cells subjected to control or NM1 gene knockdown by RNAi , and HEK293T stably expressing V5-wtNM1, V5-RK605AA NM1 or V5-ΔC NM1 were crosslinked with 1 % formaldehyde for 20 min. Chromatin was prepared as for ChIP (see below), but washed with Buffer D containing 25 % glycerol, 5 mM magnesium acetate, 50 mM Tris (pH 8.0), 0.1 mM EDTA, 5 mM DTT. Before digestion with MNase the chromatin was lightly sonicated in MNase buffer (60 mM KCl, 15 mM NaCl, 15 mM Tris at pH 7.4, 0.5 mM DTT, 0.25 M sucrose, 1.0 mM CaCl2), 8 times for 30 s. The equivalent of 0.46 × 106 cells was used in each reaction, and the level of DNA was first adjusted to be in the same range in the samples from all different treatments. Several MNase concentrations were used such that the reaction occurred in the linear range of digestion. Two samples from each treatment were used for the calculations: 10 U MNase and one concentration between 10 U and 20 U MNase. The reactions were performed at 37 °C for 30 min and then stopped by adding 12.5 mM EDTA/0.5 % SDS. After three hours of proteinase K treatment, the cross-linking was reversed at 65 °C for five hours. DNA was extracted  and the digest was evaluated by qPCR with primers amplifying around the transcription start site of the mouse Rpl19 gene (primers available upon request) and human RPL19 gene (for the mouse primers sequences see Additional file 9: Table S4), giving a product of approximately 100 bp. The results were analyzed by calculating ΔCt between the reactions performed with and without MNase. The values are presented as 2ΔCt. Chromatin from cells transfected with control siRNA oligonucleotides and chromatin from untransfected cells gave the same MNase digestion pattern. P-values (significances) were obtained by Student’s t-test as previously described .
ChIP and qPCR analysis
ChIP on growing MEFs was performed as previously described . Briefly, formaldehyde cross-linked chromatin was obtained from in vivo cross-linked MEFs and subjected to immunoprecipitations with antibodies to Pol II (8WG16 and 4H8), Rbp6, Rbp8, NM1, actin, WSTF, SNF2h, H3K9Ac, H3K27Ac, H3K4me1, H3K4me3, PCAF, Set1/Ash2, HDAC1 and non-specific rabbit IgGs. DNA-protein complexes were analyzed by qPCR with specific primers amplifying class II promoters (see Additional file 10: Table S5 for the primers sequences). qPCR was performed using SYBR-green from Applied Biosystems according to the manufacturer’s instructions. The primer concentration was 2.5 mM and the samples analyzed by qPCR (7300 Real Time PCR System, Applied Biosystem). The PCR conditions were: hold 50 °C for 2 min, 95 °C for 10 min, 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s. The results were analyzed using an average of Ct of no antibody as background. The 2ΔCt of each sample in triplicate was related to the 2ΔCt of the input sample. P-values (significances) were obtained by Student’s t-test as previously described .
ChIP assays were also performed on formaldehyde crosslinked chromatin isolated from wild-type HEK293T and HEK293T cells expressing V5-wtNM1, V5-RK605AA NM1 and V5-ΔC NM1 mutants using antibodies against the V5 epitope, NM1 and histone H3 as well as non-specific rabbit IgGs. Precipitated chromatin was analyzed by PCR with primers to the EP300 gene promoter and exonic sequences and the PCR products visualized by agarose gel electrophoresis. For the primers sequences see .
ChIP-Seq, sequencing, data alignment and analysis
For ChIP-Seq analysis, crosslinked chromatin from MEFs was subjected to immunoprecipitation with antibodies to NM1. A total of 5 ng of precipitated DNA was used to prepare sequencing libraries at the Bejing Genome Institute (Hong Kong) using the Illumina HiSeq 2000 platform. The analysis procedure involved the use of the SOAP2 program to map the reads to the mouse reference genome. Sequences with more than two mismatches were discarded from further analysis. The resulting individual sequences were remapped back to the annotated UCSC MM9 reference sequence which allows for the identification of peaks corresponding to the levels of association of the ChIP target with those loci. The ChIP-Seq data sets are available for download in the Gene Expression Omnibus (GEO) database (accession number GSE66542). The ChIPseek program was used to analyze the genomic distribution of NM1 across coding and non-coding elements and around the transcription start site (TSS) . For this all genomic locations with a score of 10 sequences per 50 bp or above were selected. The ChIPseek program was also used for pairwise comparison of the NM1 ChIPSeq data with the SNF2h ChIPSeq data .