MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing

Origin recognition complex (ORC)-dependent loading of the replicative helicase MCM2-7 onto replication origins in G1-phase forms the basis of replication fork establishment in S-phase. However, how ORC and MCM2-7 facilitate genome-wide DNA licensing is not fully understood. Mapping the molecular footprints of budding yeast ORC and MCM2-7 genome-wide, we discovered that MCM2-7 loading is associated with ORC release from origins and redistribution to non-origin sites. Our bioinformatic analysis revealed that origins are compact units, where a single MCM2-7 double hexamer blocks repetitive loading through steric ORC binding site occlusion. Analyses of A-elements and an improved B2-element consensus motif uncovered that DNA shape, DNA flexibility, and the correct, face-to-face spacing of the two DNA elements are hallmarks of ORC-binding and efficient helicase loading sites. Thus, our work identified fundamental principles for MCM2-7 helicase loading that explain how origin licensing is realised across the genome

LINT, a Novel dL(3)mbt-Containing Complex, Represses Malignant Brain Tumour Signature Genes

Mutations in the l(3)mbt tumour suppressor result in overproliferation of Drosophila larval brains. Recently, the derepression of different gene classes in l(3)mbt mutants was shown to be causal for transformation. However, the molecular mechanisms of dL(3)mbt-mediated gene repression are not understood. Here, we identify LINT, the major dL(3)mbt complex of Drosophila. LINT has three core subunits—dL(3)mbt, dCoREST, and dLint-1—and is expressed in cell lines, embryos, and larval brain. Using genome-wide ChIP–Seq analysis, we show that dLint-1 binds close to the TSS of tumour-relevant target genes. Depletion of the LINT core subunits results in derepression of these genes. By contrast, histone deacetylase, histone methylase, and histone demethylase activities are not required to maintain repression. Our results support a direct role of LINT in the repression of brain tumour-relevant target genes by restricting promoter access

Promoter recruitment of LINT subunits results in transcriptional repression.

<p>(A) A <i>Firefly</i> luciferase reporter construct (schematic representation on top) was transiently cotransfected into RNAi treated Kc cells along with a <i>Renilla</i> luciferase reporter and varying amounts of expression vectors for LexA or dL(3)mbt-/dLint-1-LexA fusion proteins as indicated. Repressor activities of dL(3)mbt-LexA and dLint-1-LexA are presented as -fold repression normalized against activities measured for LexA expression alone. (B) Kc cells were treated with no dsRNA (mock) or dsRNA against EGFP, dL(3)mbt, dLint-1, dCoREST, dLsd1, G9a and Pc. Cells were then cotransfected with reporter and expression vectors as in (A).</p

L(3)mbt and LSD1/CoREST complexes.

<p>Schematic representation of complex composition of mammalian L3MBTL1 (left), <i>Drosophila</i> LINT (middle) and mammalian LSD1/CoREST (right) complexes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002676#pgen.1002676-Trojer1" target="_blank">[5]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002676#pgen.1002676-Lee1" target="_blank">[21]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002676#pgen.1002676-Shi1" target="_blank">[22]</a>. Only core subunits are shown. Shared homologous subunits are indicated by color (red: L(3)mbt, blue: CoREST). Proposed repression mechanisms for each complex are indicated below.</p

dLint-1 is a novel dL(3)mbt–interacting protein.

<p>(A) Nuclear extract from Kc cells was fractionated over a Superose 6 column. Fractions were analyzed by Western blot using the antibodies indicated. Fraction numbers and molecular mass standards are denoted on top. Input: 5% of extract loaded. (B) Nuclear extracts from control S2 cells (mock, lanes 1 and 3) and S2 cells stably expressing FLAG-dL(3)mbt (lanes 2 and 4) were subjected to FLAG affinity purification, elution with FLAG peptide, SDS-PAGE and silver staining (lanes 3 and 4). Input: 2 µg of nuclear extracts (lanes 1 and 2). The positions of FLAG-dL(3)mbt and dLint-1 are indicated on the right. (C) <i>In vitro</i> translated, ³⁵S-labeled dLint-1 (upper panel) or luciferase (lower panel) were incubated with FLAG beads (beads, lane 3) or beads loaded with FLAG-dL(3)mbt (lane 2). Bound proteins were separated by SDS-PAGE and detected by autoradiography. Lane 1: 1% input. (D) Sf9 cells were infected with baculoviruses expressing dL(3)mbt-FLAG or dLint-1 as indicated on top. Extracts were immunoprecipitated and subjected to Western blot using FLAG and dLint-1 #2 antibodies (lanes 2, 4 and 6). Lanes 1, 3 and 5: 5% input. (E) Nuclear extracts from control S2 cells (mock, lanes 1 and 3) and S2 cells stably expressing dLint-1-FLAG (lanes 2 and 4) were subjected to FLAG affinity purification, elution with FLAG peptide, SDS-PAGE and silver staining (lanes 3 and 4). Input: 2 µg of nuclear extracts (lanes 1 and 2). The position of dLint-1-FLAG and copurifying proteins are indicated on the right. * denotes that eIF-4B was also recovered from the control and is considered to be a contaminant. Note that dCoREST and dLsd1 have the same molecular weight and comigrate. (F) Nuclear extracts from control S2 cells (mock, lanes 1 and 2) and S2 cells stably expressing FLAG-dLint-1 (lanes 3 and 4) were precipitated with FLAG antibody and analyzed by Western blot as indicated (lanes 2 and 4). dPR-Set7 served as a negative control. Lanes 1 and 3: 5% input.</p

Purification of the LINT complex.

<p>(A) Nuclear extracts of Kc cells treated with dsRNA directed against EGFP (control, lanes 1 and 3) and dLint-1 (lanes 2 and 4) were subjected to Western blot using dLint-1 antibody #1 (left upper panel), dLint-1 antibody #2 (right upper panel) and tubulin antibody (lower panels). (B) Nuclear extracts from Kc cells were precipitated with protein G beads (beads control, lane 3) and beads loaded with dLint-1 #1 antibody (lane 2) and analyzed by Western blot as indicated (lanes 2 and 3). dMi-2 served as a negative control. Lane 1: 5% input; lane 4 contains dLint-1 antibody (antibody control). * denotes a polypeptide that unspecifically crossreacts with the dLsd1 antibody (compare lanes 2 and 4). (C) Nuclear extract from Kc cells was fractionated over a Superose 6 column. Fractions were analyzed by Western blot. Fraction numbers and molecular mass standards are denoted on top. Input: 5% of extract loaded. (D) Kc nuclear extract was separated by sequential ion exchange chromatography over Q-Sepharose and MonoQ columns. MonoQ fractions were analyzed by Western blot as indicated. Fraction numbers are denoted on top. (E) Extract from third instar larval brains was precipitated with protein G beads (beads control, lane 3) and beads loaded with dLint-1 antibody (lane 2) and analyzed by Western blot as indicated (lanes 2 and 3). Lane 1: 5% input; lane 4 contains dLint-1 antibody (antibody control). * denotes a polypeptide that unspecifically crossreacts with the dLsd1 antibody (compare lanes 2 and 4).</p

LINT represses MBTS genes with germline function.

<p>(A) Gene expression analysis upon RNAi mediated depletion of dL(3)mbt and dLint-1 in Kc cells. Kc cells were treated with double-stranded RNA (dsRNA) directed against EGFP, dL(3)mbt or dLint-1. Nuclear extracts were analyzed by Western blot as indicated. (B) Venn diagrams of dL(3)mbt and dLint-1 regulated genes (fold change ≥1.5, adj. p≤0.05). (C) Target gene expression upon depletion of dL(3)mbt and dLint-1 in flies. RNAi depletion was achieved by crossing the <i>da-GAL4</i> driver strain to <i>w¹¹¹⁸</i> (control) and strains carrying dL(3)mbt or dLint-1 RNAi transgenes under UAS control, respectively. RNA was isolated from 3rd instar larvae and transcription was determined by RT-qPCR. Transcription levels in control crosses were set to 1. Tudor serves as a negative control.</p

dLsd1, dRpd3, and dPR-Set7 are not essential for MBTS gene repression.

<p>(A) Kc cells were treated with dsRNA directed against EGFP, dL(3)mbt and dLsd1. Nuclear extracts of RNAi treated Kc cells were subjected to Western blot and analyzed using antibodies as indicated. (B) Chromatin of RNAi treated cells was precipitated with H3K4me2 and H3 antibodies as indicated. The ratio of H3K4me2 and H3 ChIP signals is shown for <i>swa</i> and <i>nos</i> promoter and ORF regions and an unrelated intergenic region (interg.). Genes analyzed are denoted below the panel. Amplified regions are indicated by boxed letters and have the following distances from the transcriptional start site as illustrated on the right: c, 0–0.15 kb (promoter); d, 1.5 kb downstream. (C) Kc cells were treated with dsRNA directed against EGFP, dL(3)mbt, dLint-1, dRpd3, dCoREST and dLsd1 as indicated and transcription was determined by RT-qPCR. Transcription levels in EGFP RNAi treated cells were set to 1. Tudor serves as a negative control. (D) Kc cells were treated with dsRNA directed against EGFP and dPR-Set7 as indicated. Nuclear extracts and acid extracted histones were analyzed by Western blot as indicated. (E) Transcription levels of target genes were determined by RT-qPCR. Transcription levels in cells treated with dsRNA against EGFP were set to 1. (F) Chromatin from cells treated with RNAi against dPR-Set7 or EGFP (control) was precipitated with H4K20me1 and IgG (left panel) or H4K20me2 and IgG (right panel) antibodies as indicated. ChIP signals are shown for <i>swa</i> and <i>nos</i> promoter regions, an intergenic region and the <i>actin</i> gene as denoted below the panel.</p