Psip1 PWWP domain binds to H3K36me3.

<p>A) Diagram of p52 and p75 Psip1 isoforms showing the position of the; PWWP domain, AT hook-like domains (hatched box), C-terminal 8 a.a. unique to p52 (black box), and the p75-specific IBD. Vertical arrow indicates the site of gene trap integration in <i>Psip1^gt/gt</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002717#pgen.1002717-Sutherland1" target="_blank">[22]</a>. Horizontal lines indicate the position of epitopes recognized by antibodies A300-847 and A300-848. B) Peptide array containing 384 histone tail modification combinations incubated with GST-Psip1-PWWP and detected with αGST. Spots corresponding to unmodified H3 26–45 peptide (arrow) and H3K36me3 (arrowhead) are indicated. C) Binding specificity (calculated from the intensity of the histone peptide interaction) of Psip1-PWWP (y axis) to the top list of histone modifications arranged according to decreasing specificity (x axis). Data for all the modifications are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002717#pgen.1002717.s002" target="_blank">Table S1</a>. D) Immunoblot of biotinylated H3K36me3 peptide pull-down detecting GST-p52 with αGST antibodies. Corresponding unmodified histone H3 peptide served as control and GST-p52 was loaded as input. E) Immunoblot of A300-847 IPs with antibodies detecting; unmodified H3, H3K36me3, H3K9me2 and H3K4me3. IgG served as control and 5% of NIH3T3 nuclear extract was loaded as input.</p

Psip1/p52 interacting partners.

<p>Proteins identified by mass spectrometry of p52 IPs (200 mM KCl).</p>*<p>indicates known proteins of the ‘spliceosomal complex C’. Data on protein domains and putative protein functions were taken from <a href="http://npd.hgu.mrc.ac.uk/" target="_blank">http://npd.hgu.mrc.ac.uk/</a>.</p

Sub-cellular localization of Psip1/p52 and p75.

<p>A) Immunofluorescence and wide-field epifluorescence microscopy on human cells with; (upper row) p75-specific antibody A300-848, (lower row) A300-847 which can recognize both p52 and p75. DNA was counterstained with DAPI. B) Co-immunofluorescence of Psip1/p52 (green/A300-847) and SRSF2 (red) analyzed by confocal microscopy in untreated (upper row), or actinomycin D (ActD) treated cells. C) Co-immunofluorescence of Psip1/p75 (green/A300-848) and SRSF2 (red) in ActD treated cells and analyzed by confocal microscopy.</p

Alternative splicing in <i>Psip1</i>^gt/gt cells.

<p>RT-PCR to detect; exon inclusion (In) or skipping (Skip) of (A); <i>Ptprc</i>, <i>Ppfibp1</i>, <i>Rapgef6</i>, <i>Rasgrp3</i>, <i>Ogfrl</i>, and <i>Sorb2</i> all of which showed evidence for altered alternative splicing in array analysis, or (B) <i>Csnk1d and Alg9</i>, <i>which were unchanged in the array</i>, in RNAs prepared from wt and <i>Psip1</i>^gt/gt primary MEFs. C) Specific exon-exon junctions (constitutive-constitutive or constitutive-alternative) of <i>Vcan</i>, <i>Tpp2</i> and <i>Diap2</i> in wt and <i>Psip1</i>^gt/gt MEFs. D) Specific exon-exon junctions (constitutive-constitutive) of <i>Vcan</i> (5′ exons) and <i>Diap2</i> (3′exons) and constitutive-constitutive (5′) and constitutive-alternative of <i>Tpp2</i> alternative exon 24 in wt and <i>Psip1</i>^gt/gt MEFs. E) Specific exon-exon junctions (constitutive-constitutive or constitutive-alternative) of <i>Vcan</i>, <i>Tpp2 and Diap2</i>; exon inclusion (In) or skipping (Skip) of <i>Sorb2</i> in wt and <i>Psip1</i>^−/− MEFs, and after transfection of p52 or p75 Psip1 into <i>Psip1</i>^−/− MEFs. F) Exon inclusion (In) or skipping (Skip) of; <i>Csnk1d</i> Alg9, and constitutive-constitutive (5′) and constitutive-alternative of <i>Tpp2</i> alternative exon 24 in RNAs prepared from wt and <i>Psip1</i>^−/− MEFs, and after transfection of p52 or p75 Psip1 into <i>Psip1</i>^−/− MEFs. Sequence and position of primer pairs for each exons are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002717#pgen.1002717.s004" target="_blank">Table S3</a>. Below the gels in panels A to F, the mean ratio of alternative∶constitutive exon (+/− s.e.m.) is shown for three biological replicates. G) Immunoblots of proteins using A300-847 antibodies to detect p75 and p52 in wt and <i>Psip1</i>^−/− MEFs, also <i>Psip1</i>^−/− MEFs transfected with p52 (<i>Psip1</i>^−/− p52Res) and p75 (<i>Psip1</i>^−/− p75Res). Immunoblot with Pcna served as a loading control.</p

Genomic distribution of Psip1/p52 and H3K36me3.

<p>A) Mean log2 ChIP∶input for Psip1/p52 and H3K36me3 in MEFs for an approximately 1.2Mb genomic window from mouse chromosome 5. n = 4 (3 biological and 1 technical replicate). B) Box plots showing the distribution of log2 ChIP∶input for Psip1/p52 and H3K36me3 across exons and introns of expressed or non-expressed genes. Data are deposited in NCBI GEO (Accession no. GSM697402-GSM697411). C, D) Mean log2 ChIP∶input for Psip1/p52 and H3K36me3 in MEFs at (C) c-<i>Myc</i> and (D) <i>Xist</i> loci. H3K4me3 is also shown for XIST. Filled boxes indicate the positions of exons. n = 4 (3 biological and 1 technical replicate) for H3K36me3 and Psip1. NCBI GEO accession number for array platform is GPL13276. n = 2 biological: replicates for H3K4me3.</p

ChIP for H3K36me3, Psip1, and Srsf1 in wt and Psip1 mutant MEFs.

<p>A) Mean log2 ChIP∶input for H3K36me3, Psip1 and Srsf1 in wt MEFs across the <i>Vcan</i> (A), <i>Diap2</i> (B) and <i>Ppfibp1</i> (C) loci. Distribution of Srsf1 in chromatin from <i>Psip1</i>^−/− MEFs is also shown. Filled boxes indicate the positions of exons and the arrows indicate the position of alternatively spliced exons whose inclusion into spliced mRNAs is altered in <i>Psip1</i>^gt/gt cells. n = 2 biological replicates that also incorporate a technical (dye-swap) replicate. Array platform number is GPL14175 and the GEO accession numbers for ChIP data are; GSM782590 (Psip1), GSM782591 (H3K36me3), GSM782592 and GSM782593 (Srsf1 in wt), GSM782594 and GSM782595 (Srsf1 in <i>Psip1</i>^−/−).</p

Immunoprecipitation of Psip1/p52 and p75.

<p>A) Immunoblot of NIH 3T3 nuclear extract with antibodies; A300-848 which recognizes only the p75 isoform of Psip1, and A300-847 which detects both p52 and p75. B) IPs with IgG, A300-847 and A300-848 from NIH 3T3 nuclear extracts, immunoblotted with antibodies recognizing p75 (A300-848) or p52 (A300-847). Input is 5% total extract. C) Immunoblot of A300-847 IPs with αSRSF1. IP was also performed in the presence of RNase A. Input is 10% of total extract and IgG served as a control. D) In vitro pulldown of 293T cell expressed T7-SRSF1 using GST-p52 and Psip1-PWWP and immunoblotted with αT7. Input is 5% of T7-SRSF1 and GST alone is control. E) In vitro pulldown with GST-p52 of; T7-SRSF1 and mutants that mimic its hypo-(RG) and hyper-phosporylation (RD), T7-SRSF3 and GFP-SRSF2. Immunoblotting was with αT7 or αGFP. F) ChIP with αH3K36me3 from wild-type (wt) and <i>Psip1</i>^−/− MEFs immunoblotted with antibodies detecting Srsf1, Srsf2, Srsf3, PTB, Psip1 and H3K36me3. G) In vitro pulldown of HeLa core histones by T7-SRSF1 in the presence or absence of Psip1/p52 and immunoblotted with antibodies detecting pan H3, H3K36me3, H3K9me2 and H3K4me3.</p

<i>Hottip</i> localizes to 5′ Hoxa genes and deletion of <i>Hottip</i> reduces <i>5</i>′ Hoxa expression.

<p>(A) Schematics showing the mouse <i>Hoxa13</i> and <i>Hottip</i> loci. The CpG Island (CGI) at the <i>Hoxa13</i> promoter is shown in a grey bar. Genome co-ordinates are from the mm9 assembly of the mouse genome. Guide RNA binding sites for deletion of <i>Hottip</i> are shown as arrow heads, primers used for genotyping are shown in arrows (p1 to p4). The deletion product of <i>Hottip</i> (<i>HottipΔ</i>) is shown below. Agarose gel image showing genotyping PCR, first two lanes are amplicons of primers (p1 and p2) within the deleted <i>Hottip</i> region, second two lanes are for amplicons from primers (p3 and p4) 3’ of deleted region. (B) Mean (± s.e.m) expression, assayed by RT-qPCR and normalized to <i>Gapdh</i>, of Hoxa genes and <i>Hottip</i>, in wild-type (black bars, WT), and <i>Hottip</i> knock out (gray bars, <i>HottipΔ</i>) limb mesenchymal cells, (n = 3 biological replicates). * p < 0.05, ** p <0.01. (C) RT-qPCR showing mean (± s.e.m) ± percentage (%) enrichment over input for <i>Hottip</i>, <i>7SK</i> and <i>Gapdh</i> RNAs from <i>Hottip</i> ChIRP pulldown from two experiments. (D) qPCR showing mean (± s.e.m) percentage (%) enrichment over input of ChIRPed DNA at promoters of <i>Actb</i>, <i>Hoxa1</i>, <i>Hoxa7</i>, <i>Hoxa9</i>, <i>Hoxa10</i>, <i>Hoxa11</i>, and <i>Hoxa13</i> from Hottip ChIRP experiments in wild type (black bars, WT) and <i>Hottip</i> knock out limb mesenchymal cells (grey bars, <i>HottipΔ</i>).</p

Artificial induction of <i>Hottip</i> is sufficient to activate 5’ Hoxa genes.

<p>(A) Schematics showing UCSC genomic coordinates of <i>Hottip</i>, <i>Hoxa13</i>, CpG Islands (CGI) in the mouse (top, mm9) and human (bottom, hg19) genomes. Schematics of guide RNA mediated recruitment of dCas9-VP160 to the <i>Hottip or Hoxa13</i> promoters is also shown. Direction of transcription is indicated as arrow marks. (B) Heat map showing the log2 mean fold change in expression of Hoxa, Hoxd and pluripotency associated genes (control genes) from expression microarray experiment, upon co-transfection of guide RNAs recognizing the <i>Hottip</i> promoter (<i>Hottip</i> gRNAs + dcas9-VP160). dCas9-VP160 was also co transfected with guide-RNAs recognizing <i>Hoxa13</i> promoter (<i>Hoxa13</i> gRNAs + dcas9-VP160) (n = 3 or 4 biological replicates). (C) Similar to (B) RT-qPCR data showing mean (± s.e.m) log2 fold change in expression of <i>Hottip</i>, <i>Hoxa13</i>, <i>a11</i>, <i>a10</i>, <i>a9</i>, <i>a7</i>, <i>a1</i>, <i>Pou5f1</i> and <i>Hoxb9</i> upon guide RNA mediated recruitment of dCas9-VP160 to the <i>Hottip</i> promoter (A) in mouse ES cells. Data were normalized to those from a dcas9 control (n = 3 biological replicates). (D) Similar to (C) mean log2 fold change in <i>Hottip and Hoxa13</i> expression in wild type ES cells co-transfected with guide-RNAs recognizing the <i>Hottip</i> promoter and dCas9-VP160 (Black bars, WT). <i>Hoxa13</i> expression in <i>Hottip</i> knock out limb mesenchymal cells is also shown (grey bar, <i>HottipΔ</i>). * p < 0.05, ** p <0.01 throughout.</p

Reduced <i>Hottip</i> expression and Mll occupancy in <i>Psip1</i>^{<i>–/–</i>}.

<p>(A) Mean Log2 ChIP/input for Psip1/p75, Mll1, Menin and H3K4me3 in WT and <i>Psip1</i>^{<i>–/–</i>}MEFs over <i>Hoxa</i> clusters from custom tiling arrays[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006677#pgen.1006677.ref035" target="_blank">35</a>]. Annotated noncoding transcripts (grey, top) and Hox gene transcripts (black) are shown below. (n = 2 biological replicates). Genome co-ordinates are from the mm9 assembly of the mouse genome. Direction of transcription for <i>Hoxa13</i> and <i>Hottip</i> genes are indicated with arrow below. (B) Mean (± s.e.m) expression, assayed by RT-qPCR and normalized to <i>Gapdh</i>, of <i>Hoxa13</i> and <i>Hottip</i> in <i>WT</i> and <i>Psip1</i>^{<i>–/–</i>}MEFs, (n = 3 biological replicates). (C) Nimblegen tiling microarray data showing log2 ratio of <i>Psip1</i>^{<i>–/–</i>}/ <i>WT</i> run-on transcribed RNA (nascent RNA) over posterior Hoxa genes n = 2 technical replicates.</p