Additional file 1 of ddClone: joint statistical inference of clonal populations from single cell and bulk tumour sequencing data

Supplementary information. A PDF file that contains supplementary figures, details of the mathematical derivation of the ddClone model, and the description and algorithms to simulate data from the Generalized Dollo model. (PDF 2200 kb

MLL5_PHD binding to H3K4 peptides as detected by fluorescence polarization.

<p>MLL5_PHD binding to H3K4 peptides as detected by fluorescence polarization.</p

Discovery of Novel 1,4-Diacylpiperazines as Selective and Cell-Active eIF4A3 Inhibitors

Eukaryotic initiation factor 4A3 (eIF4A3), a member of the DEAD-box RNA helicase family, is one of the core components of the exon junction complex (EJC). The EJC is known to be involved in a variety of RNA metabolic processes typified by nonsense-mediated RNA decay (NMD). In order to identify molecular probes to investigate the functions and therapeutic relevance of eIF4A3, a search for selective eIF4A3 inhibitors was conducted. Through the chemical optimization of 1,4-diacylpiperazine derivatives identified via high-throughput screening (HTS), we discovered the first reported selective eIF4A3 inhibitor <b>53a</b> exhibiting cellular NMD inhibitory activity. A surface plasmon resonance (SPR) biosensing assay ascertained the direct binding of <b>53a</b> and its analog <b>52a</b> to eIF4A3 and revealed that the binding occurs at a non-ATP binding site. Compounds <b>52a</b> and <b>53a</b> represent novel molecular probes for further study of eIF4A3, the EJC, and NMD

Solution NMR Structure and Histone Binding of the PHD Domain of Human MLL5

<div><p>Mixed Lineage Leukemia 5 (MLL5) is a histone methyltransferase that plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. In addition to its catalytic domain, MLL5 contains a PHD finger domain, a protein module that is often involved in binding to the N-terminus of histone H3. Here we report the NMR solution structure of the MLL5 PHD domain showing a variant of the canonical PHD fold that combines conserved H3 binding features from several classes of other PHD domains (including an aromatic cage) along with a novel C-terminal α-helix, not previously seen. We further demonstrate that the PHD domain binds with similar affinity to histone H3 tail peptides di- and tri-methylated at lysine 4 (H3K4me2 and H3K4me3), the former being the putative product of the MLL5 catalytic reaction. This work establishes the PHD domain of MLL5 as a bone fide ‘reader’ domain of H3K4 methyl marks suggesting that it may guide the spreading or further methylation of this site on chromatin.</p></div

Multiple sequence alignment of PHD domains of MLL5 in various organisms.

<p>Conserved residues are marked with red circles at the bottom <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077020#pone.0077020-Celniker1" target="_blank">[29]</a>. Organism of origin is shown on the left-hand-side of each sequence. Secondary structure elements of the PHD domain are shown above its sequence for clarity (α-helix as cylinder and β-strands as arrows). The residues coordinating Zn1 and Zn2 atoms are marked with blue and black dots at the top, respectively. Homologous domains are identified using protein blast against non-redundant protein database (<a href="http://blast.ncbi.nlm.nih.gov/blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/blast.cgi</a>). multiple sequence alignment is performed using clustalw2 (<a href="http://www.ebi.ac.uk/tools/clustalw2" target="_blank">www.ebi.ac.uk/tools/clustalw2</a>).</p

MLL5_iso1 and histone H3 peptides complexes.

<p>Purified MLL5_iso1 incubated with biotinylated H3 peptides and complex was pulled down using streptavidin agarose beads and detected using anti-MLL5 antibody. (A) Pull down assay using H3 peptides with methylation at different lysine sites. (B) Pull down assay using H3 peptides with varying degrees of methylation at the K4 site.</p

Comparison of PHD domains of MLL5 and PHF13 proteins.

<p>Ribbon representation of the domains with superimposed backbone, MLL5_PHD in blue and unbound PHF13_PHD (PDB ID 3O70) in orange.</p

Surface comparison between PHD domains of PHF13_PHD, [PDB ID: 3O7A] (A, AIRE_PHD, [PDB ID 2KFT] (B) and that of MLL5_PHD (C).

<p>Peptide K4 is in orange stick, R2 in green stick. (A) PHF13's Trp255, Phe241, Met246, Thr234 are highlighted in yellow. (B) AIRE's Cys310, Asp312, Thr334, Trp335 are highlighted in magenta (C) MLL5's Trp141, Met132, His127, Thr119 are highlighted in yellow and Cys134, Asp136, Thr157, Tyr158 are highlighted in magenta. Residues for which chemical shifts have changed by more than 0.3 ppm are indicated.</p

NMR data and refinement statistics.

a<p>Values calculated for the ordered regions, as reported by PSVS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077020#pone.0077020-Bhattacharya1" target="_blank">[26]</a>: residues 118–183.</p>b<p>r.m.s.d calculated for residues 118–183.</p>c<p>Calculated by PSVS.</p>d<p>RPF scores <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077020#pone.0077020-Huang1" target="_blank">[27]</a> reflecting the goodness-of-fit of the structural ensemble to the NMR data.</p

Solution structure of PHD domain of human MLL5 (A) The backbone trace of the 20 structures comprising the lowest energy NMR ensemble is shown in blue (B) Ribbon diagram of a representative structure of the NMR ensemble. Zinc atoms are shown as green spheres.

<p>Solution structure of PHD domain of human MLL5 (A) The backbone trace of the 20 structures comprising the lowest energy NMR ensemble is shown in blue (B) Ribbon diagram of a representative structure of the NMR ensemble. Zinc atoms are shown as green spheres.</p