(a) Mouse RPB1 CTD is composed of 52 heptapeptide repeats with consensus amino-acid sequence YSPTSPS, which is represented 21 times at the most proximal CTD region. Non-consensus amino acids are …
(a) Outline of strategy used to generate mouse cell lines bearing K7-to-S7 mutations and study CTD-K7 methylation. Red bars represent CTD repeats with K7 residues. The nomenclature of the cell lines …
Sections shown in Figure 2a are highlighted with dashed boxes. High and low exposure blots are shown for YFP, S5p, and S2p. For total RPB1, YFP, S5p and S2p, two different cell lines (a and b) …
(a) Amino-acid sequence of CTD-K7-methyl peptides used for immunization, designed based on the sequence of mouse CTD repeats 35 and 36. (b) Schematic representation of strategy used for production …
(a) Complete western blot used in Figure 3d. Sections shown in Figure 3d are highlighted with dashed boxes. In the case of 8WG16, two different cell lines (a and b) that stably express each of the …
(a) CTD K7me1 and K7me2 mark hypophosphorylated and intermediately phosphorylated Rpb1 forms, but not the hyperphosphorylated (II0) form. Western blotting using the indicated antibodies was …
(a) Complete western blot used in Figure 4a. Used sections are highlighted with dashed boxes and low and higher exposure blot versions are displayed. Sections of the membrane treated with alkaline …
(a) K7me1 and K7me2 are enriched at promoters of active genes. ChIP-seq profiles for K7me1, K7me2, K7ac, 8WG16, S5p, S7p and S2p, and mRNA-seq profiles are represented for the inactive gene Myf5, …
(a) Abundance and distribution of K7me1, K7me2, and S5p was assessed by ChIP followed by qPCR at promoters and coding regions of active (β-Actin, Oct4, Nanog and Polr2a) and inactive (Gata1 and Myf5)…
(a) Percentage of genes positive for K7me1, K7me2 and K7ac in three different classes of genes: active, polycomb repressed (PRCr) and inactive. Active genes are positive for S5p, S7p, S2p and 8WG16, …
(a) Matrix of Spearman’s correlation coefficients between the levels of K7me1, K7me2, K7ac, 8WG16, S5p, S7p, S2p (2 kb window after TES), mRNA and mock ChIP control ordered according to increasing …
(a) Matrix of Spearman’s correlation coefficients between the levels of several histone and CTD modifications, mRNA, CpG and mock control. The correlations present in Figure 6a were expanded here to …
Typical LASSO regression models to predict S2p levels select K7me1 or K7me2 as negative predictor variables at the optimum penalty (blue line, top graph). LASSO regression employs the L1 norm to …
(a) K7me2/K7ac ratio correlates negatively with mRNA expression. (b) Distribution of K7me2/K7ac ratios, and their division into three quantiles: low (green), medium (yellow) and high (blue) ratios. …
(a) CTD K7 methylation / acetylation ratio negatively correlates with S2p (TES) and mRNA. Matrix of Spearman’s correlation coefficients between ratios K7me1/K7ac and K7me2/K7ac and K7me1, K7me2, …
(a) Heatmaps of ChIP-seq density using a ± 500 bp window centered around the TSS (unless stated otherwise) of active genes defined as positive for S5p, S7p and S2p, as negative for H3K27me3 and …
(a) Range of K7me2/K7ac ratios for genes used in expression analysis after Trichostatin A (TSA) treatment. (b) Total RNA levels were analysed by quantitative RT-PCR after treatment of ES cells with …
Sections shown in Figure 7g are highlighted with dashed boxes. High and low exposure blots are shown for K7me2. In the H3K9ac western blot, the sections shown with increasing exposure time …
Mouse ES cells were treated with P300 inhibitor C646 (30 μM, 3h), before western blotting with antibodies specific for total RPB1, K7me1, K7me2 and S7p. Hypo- (IIa) and hyperphosphorylated (II0) …
(A) Range of K7me2/K7ac ratios for genes used in expression analysis after C646 treatment. (B) Total RNA levels were measured by quantitative RT-PCR after treatment of ES cells with C646 (30 μM, 3h) …
(A) Western blotting was performed using protein extracts from mouse ES cells with conditional knockout for H3K4 methylase Setd1a or Setd1b for total RPB1 and K7me1 in 2 biological replicates. Hypo- …
RPB1 CTD peptides. CTD peptides with unmodified, mono-, di-, tri-methyl and acetyl K7 residues were used in ELISA assays to characterize the specificity of the CTD methyl antibodies produced in this …
Peptide sequence | Modification |
---|---|
SYSPTSPKYTPTSPSC | Unmodified |
SYSPTSPKme1YTPTSPSC | K7 monomethyl |
SYSPTSPKme2YTPTSPSC | K7 dimethyl |
SYSPTSPKme3YTPTSPSC | K7 trimethyl |
SYSPTSPKacYTPTSPSC | K7 acetyl |
CTD, C-terminal domain; ELISA, enzyme-linked immunosorbent assay.
List of Antibodies used in this study. Full description of the antibodies and the amounts or concentrations used in this study for WB, ChIP or IF.
Amount/dilution | |||||||
---|---|---|---|---|---|---|---|
Antibody | Raised in (isotype) | Clone | Stock | WB | ChIP | IF | Source |
S5p | Mouse (IgG) | CTD4H8 (MMS-128P) | 1 mg/ml | 1/200,000 | 10 µl (10 µg) | 1/3000 | Covance |
S7p | Rat (IgG) | 4E12 | - | 1/10 | - | - | Kind gift from Dirk Eick |
S2p | Mouse (IgM) | H5 (MMS-129R) | 1–3 mg/ml | 1/500 | - | - | Covance |
Unphospho-S2 | Mouse (IgG) | 8WG16 (MMS-126R) | 1–3 mg/ml | 1/200 | - | - | Covance |
N-terminus (Total RPB1) | Rabbit (IgG) | H224 (sc-9001x) | 200 µg/ml | 1/200 | - | - | Santa Cruz Biotechnology |
K7me1 | Mouse (IgG) | CMA611 | 10 mg/ml | 1/1000 | 5 µl (50 µg) | 1/200 | This study |
K7me2 | Mouse (IgG) | CMA612 | 10 mg/ml | 1/1000 | 5 µl (50 µg) | 1/200 | This study |
H3K9ac | Rabbit serum | 39585 | - | 1/1000 | - | - | Active Motif |
Lamin B | Goat (IgG) | C-20 Sc-6216 | 200 µg/ml | 1/500 | - | Santa Cruz Biotechnology | |
α-tubulin | Mouse (IgG) | T6074 | 2 mg/ml | 1/10,000 | - | - | Sigma |
GFP | Rabbit (IgG) | A11122 | 2 mg/ml | 1/1000 | - | - | Life Technologies |
Digoxigenin | Mouse (IgG) | 200–002-156 | 1.2 mg/ml | - | 10 µl (12 µg) | - | Jackson ImmunoResearch |
ChIP, chromatin immunoprecipitation; IF, immunofluorescence; WB, western blotting.
List of PCR primers used in this study. Primer sequences (F, forward; R, reverse) are represented (5´to 3´orientation) for promoter and coding regions of active and inactive genes. Primers designed …
Gene | Primer sequence | Gene | Primer sequence |
---|---|---|---|
ChIP primers β-actin (promoter) F | GCAGGCCTAGTAACCGAGACA | Gene expression primers β-actin 5´F | CCACCCGCGAGCACA |
β-actin (promoter) R | AGTTTTGGCGATGGGTGCT | β-actin 5´R | CCGGCGTCCCTGCTTAC |
β-actin (coding) F | TCCTGGCCTCACTGTCCAC | β-actin Exon1 F | TCTTTGCAGCTCCTTCGTTG |
β-actin (coding) R | GTCCGCCTAGAAGCACTTGC | β-actin Exon2 R | ACGATGGAGGGGAATACAGC |
Oct4 (promoter) F | GGCTCTCCAGAGGATGGCTGAG | Oct4 5´F | TGAGCCGTCTTTCCACCA |
Oct4 (promoter) R | TCGGATGCCCCATCGCA | Oct4 5´R | TGAGCCTGGTCCGATTCC |
Oct4 (coding) F | CCTGCAGAAGGAGCTAGAACA | Sox2 5´F | AGGGCTGGGAGAAAGAAGAG |
Oct4 (coding) R | TGTGGAGAAGCAGCTCCTAAG | Sox2 5´R | ATCTGGCGGAGAATAGTTGG |
Myf5 (promoter) F | GGAGATCCGTGCGTTAAGAATCC | Serpine1 5´F | CCCCGAGAGCTTTGTGAAG |
Myf5 (promoter) R | CGGTAGCAAGACATTAAAGTTCCGTA | Serpine1 5´R | AAGGTGCCTTGTGATTGGCT |
Myf5 (coding) F | GATTGCTTGTCCAGCATTGT | Dusp1 5´F | CGGTGAAGCCAGATTAGGAG |
Myf5 (coding) R | AGTGATCATCGGGAGAGAGTT | Dusp1 5´R | AGGAGCGACAATCCAACAAC |
Gata1 (promoter) F | AGAGGAGGGAGAAGGTGAGTG | Ctgf 5´F | GACTCAGCCAGATCCACTCC |
Gata1 (promoter) R | AGCCACCTTAGTGGTATGACG | Ctgf 5´R | GTGCAGAGGCGACGAGAG |
Gata1 (coding) F | TGGATTTTCCTGGTCTAGGG | ||
Gata1 (coding) R | GTAGGCCTCAGCTTCTCTGTAGTA | ||
Polr2a (-1 kb) F | CCGTAAAGCTATTAGAGCACAGG | ||
Polr2a (-1 kb) R | ATGCATAAGGCAGGCAAGAT | ||
Polr2a (-0.5 kb) F | GTAACCTCTGCCGTTCAGGA | ||
Polr2a (-0.5 kb) R | TTTCTCCCTTTCCGGAGATT | ||
Polr2a (E1) F | CAGGCTTTTTGTAGCGAGGT | ||
Polr2a (E1) R | GACTCAGGACTCCGAACTGC | ||
Polr2a (I1) F | CAGAGGGCTCTTTGAATTGG | ||
Polr2a (I1) R | GCATCAGATCCCCTTCATGT | ||
Polr2a (I2/E3) F | GCCCTCTTCTGGAGTGTCTG | ||
Polr2a (I2/E3) R | AGGAAGCCCACATGAAACAC | ||
Polr2a (E19/I19) F | CCAAGTTCAACCAAGCCATT | ||
Polr2a (E19/I19) R | TCTTAACCGCTGAGCCATCT | ||
Polr2a (E28) F | TCTCCCACTTCTCCTGGCTA | ||
Polr2a (E28) R | CCGAGGTTGTCTGACCCTAA | ||
Polr2a (+2 kb TES) F | GAGGGGCAGACACTACCAAA | ||
Polr2a (+2 kb TES) R | AAAAGGCCAAAGGCAAAGAT |
Description of ChIP-seq and messenger RNA datasets used in this study. Full description of the ChIP-seq datasets produced or re-analysed in this study. NCBI Gene Expression Omnibus (GEO) Sample …
ChIP-seq dataset | Dataset origin | Antibody clone | Mapped reads (millions) | ES cell line |
---|---|---|---|---|
RPB1-K7me1 (GSM1874007) | This study | CMA611 (this study) | 64 | ESC OS25 |
RPB1-K7me2 (GSM1874008) | This study | CMA612 (this study) | 69 | ESC OS25 |
RPB1-K7ac (SRR1028808) | Schröder et al. (2013) | AcRPB1 (Schröder et al., 2013) | 85 | ESC |
Input (SRR1028807) | Schröder et al. (2013) | - | 21 | ESC |
RPB1-S5p (GSM850467) | Brookes et al. (2012) | CTD4H8 (MMS-128P, Covance) | 22 | ESC OS25 |
RPB1-S7p (GSM850468) | Brookes et al. (2012) | 4E12 (Chapman et al., 2007) | 11 | ESC OS25 |
RPB1-S2p (GSM850470) | Brookes et al. (2012) | H5 (MMS-129R, Covance) | 33 | ESC OS25 |
Unphospho-S2 (8WG16) (GSM850469) | Brookes et al. (2012) | 8WG16 (MMS-126R, Covance) | 24 | ESC OS25 |
Mock IP (GSM850473) | Brookes et al. (2012) | - | 12 | ESC OS25 |
H3K4me3 (GSM307618) | Mikkelsen et al. (2007) | ab8580 (Abcam) | 9 | ESC V6.5 |
H3K36me3 (GSM850472) | Brookes et al. (2012) | 13C9 (Rechtsteiner et al., 2010) | 23 | ESC OS25 |
H2Aub1 (GSM850471) | Brookes et al. (2012) | E6C5 (Upstate) | 18 | ESC OS25 |
H3K27me3 (GSM307619) | Mikkelsen et al. (2007) | 07–449 (Upstate) | 8 | ESC V6.5 |
RNA datasets | Dataset origin | Mapped reads (millions) | Cell line | |
mRNA-seq (GSM850476) | Brookes et al. (2012) | 74 | ESC OS25 | |
GRO-seq (GSE48895) | Jonkers et al. (2014) | 25 | ESC V6.5 (“untreated”) |
ChIP-seq, chromatin immunoprecipitation with sequencing; ES, embryonic stem; GRO-seq, global run-on sequencing.