Identification of chemicals that mimic transcriptional changes associated with autism, brain aging and neurodegeneration

Environmental factors, including pesticides, have been linked to autism and neurodegeneration risk using retrospective epidemiological studies. Here we sought to prospectively identify chemicals that share transcriptomic signatures with neurological disorders, by exposing mouse cortical neuron-enriched cultures to hundreds of chemicals commonly found in the environment and on food. We find that rotenone, a pesticide associated with Parkinson's disease risk, and certain fungicides, including pyraclostrobin, trifloxystrobin, famoxadone and fenamidone, produce transcriptional changes in vitro that are similar to those seen in brain samples from humans with autism, advanced age and neurodegeneration (Alzheimer's disease and Huntington's disease). These chemicals stimulate free radical production and disrupt microtubules in neurons, effects that can be reduced by pretreating with a microtubule stabilizer, an antioxidant, or with sulforaphane. Our study provides an approach to prospectively identify environmental chemicals that transcriptionally mimic autism and other brain disorders

Polycomb proteins control proliferation and transformation independently of cell cycle checkpoints by regulating DNA replication

The ability of PRC1 and PRC2 to promote proliferation is a main feature that links polycomb (PcG) activity to cancer. PcGs silence the expression of the tumour suppressor locus Ink4a/Arf, whose products positively regulate pRb and p53 functions. Enhanced PcG activity is a frequent feature of human tumours, and PcG inhibition has been proposed as a strategy for cancer treatment. However, the recurrent inactivation of pRb/p53 responses in human cancers raises a question regarding the ability of PcG proteins to affect cellular proliferation independently from this checkpoint. Here we demonstrate that PRCs regulate cellular proliferation and transformation independently of the Ink4a/Arf-pRb-p53 pathway. We provide evidence that PRCs localize at replication forks, and that loss of their function directly affects the progression and symmetry of DNA replication forks. Thus, we have identified a novel activity by which PcGs can regulate cell proliferation independently of major cell cycle restriction checkpoints. \ua92014 Macmillan Publishers Limited. All rights reserved

Effect of PRC2 inactivation on established <i>Ezh2^ΔSET/ΔSET</i> iPSC clones and TF–induced reprogramming.

<p>A. Western blot analysis of EED protein levels in two <i>Ezh2^ΔSET/ΔSET</i> iPSC clones infected with control virus (empty) or lentiviruses expressing independent short hairpin (sh) RNAs targeting <i>Eed</i> (#19 and #21)(left). Quantification of EED protein levels in infected cells after normalization based on Vinculin levels (right). B. H3K27me3 status (left panel) and expression levels (right panel) measured respectively by ChIP-qPCR and qRT-PCR, of 4 representative genes up regulated in MEF relative to iPSC, in two <i>Ezh2^ΔSET/ΔSET</i> iPSC clones. <i>Ezh2</i>-mutant iPSC were infected with viruses expressing two independent hairpins for <i>Eed</i> or with a control virus. Status of H3K27me3 (±SEM) is represented as enrichment relative to input, after normalization for H3 density within the same amplicon. Expression levels are shown as fold change relative to iPSC infected with the empty vector. Error bars refer to qPCR triplicates. C. Western blot analysis of EED and H3K27me3 protein levels at day-6 of puromycin selection on <i>Cdkn2a^−/− Ezh2</i>-proficient TTF expressing three independent <i>Eed</i> hairpins (lines 1, 2 and 3) or infected with a control virus (line 4). Quantification of protein levels relative to Vinculin or Histone H3 are shown in the right panel. D. AP staining of primary iPSC colonies obtained upon reprogramming of 1×10³ (upper panel) <i>Cdkn2a^−/− Ezh2</i>-proficient TTF expressing three independent <i>Eed</i> hairpins (lines 1, 2 and 3) or infected with an empty lentiviral vector (line 4) used as control. E. Quantification of AP⁺ iPSC colonies. Column height represents number of AP⁺ iPSC colonies obtained from 1×10³ TTF expressing either one of the three independent <i>Eed</i> hairpins (lines 1, 2 and 3) or infected with an empty lentivirus vector (line 4) as control. Data are representative of two independent experiments performed using three different shRNAs.</p

Mass spectrometry analysis: H3K27me3 levels below the limit of detection in <i>Ezh2^ΔSET/ΔSET</i> iPSCs.

*<p>Perkins et al 1999.</p>**<p>Cox and Mann 2010.</p><p>Mascot and PTM scores attributed in control (<i>Ezh2^+/+</i>) and mutant (<i>Ezh2^ΔSET/ΔSET</i>) iPSC clones to each combination of amino acid modifications on the H3^27–40 tri-, tetra- and penta-methylated peptide.</p

Characterization of pluripotency in iPSC clones reprogrammed upon <i>Ezh2</i> inactivation.

<p>A. Images of phase contrast (left), alkaline phosphatase staining (middle) and <i>Oct4</i>-driven GFP fluorescence (right) of representative control (<i>Ezh2^+/ΔSET</i>, upper row) and mutant (<i>Ezh2^ΔSET/ΔSET</i>, lower row) iPSC colonies. B. Flow cytometric analysis of SSEA-1 and endogenous OCT4 (OCT4-GFP) levels in representative control (<i>Ezh2^+/ΔSET</i>, upper row) and mutant (<i>Ezh2^ΔSET/ΔSET</i>, lower row) iPSC clones (cl.1 and cl.2). Embryonic stem cells (ESC) were used as positive control for SSEA1 expression (upper left). C. Growth curve of representative control (<i>Ezh^+/+</i>, grey) and mutant (<i>Ezh2^ΔSET/ΔSET</i>, purple) iPSC clones cultured in 2i/LIF medium for the indicated hours. Column height represents cell number. D. Hematoxylin & eosin staining and immunohistochemical analysis of representative sections of teratomas generated from either <i>Ezh2</i> control (<i>Ezh2^+/+</i>) or mutant (<i>Ezh2^ΔSET/ΔSET</i>) iPSC cells of two representative clones. Stainings for mesoderm (upper row), endoderm (middle row) and ectoderm (lower row) markers are displayed. Data displayed in A, B, C and D are representative of at least four independent experiments, using two iPSC clones per genotype. E. Scatter plots showing global gene expression correlation analyses between <i>Ezh2^+/+</i> and <i>Ezh2^ΔSET/ΔSET</i> iPSC (left panel), <i>Ezh2^+/+</i> iPSCs and MEF (central panel) and <i>Ezh2^ΔSET/ΔSET</i> iPSC and MEF (right panel). Correlation coefficients (r) reveal the degree of similarity for each comparison. Genes within red lines differ less than 1.5-fold. F. Heat map representation of expression levels of genes involved in pluripotency, stemness and differentiation in two control (<i>Ezh2^+/+</i>, first and second column) and two mutant (<i>Ezh2^ΔSET/ΔSET</i>, third and fourth column) iPSC clones. ESC (fifth column) and MEF (last column) were used for comparison. Colors range from yellow (low dCt, higher expression) to black (high dCt, lower expression). Hierarchical clustering of samples is also shown.</p

Genome-wide distribution of H3K27me3 in <i>Ezh2^ΔSET/ΔSET</i> iPSC revealed through ChIP–seq.

<p>A. Pie chart showing partition of Polycomb targets based on H3K27 methylation status in <i>Ezh2^ΔSET/ΔSET</i> iPSCs. B. Venn diagram displaying overlap between H3K27me3⁺ genes in <i>Ezh2</i>^+/+ (grey) and <i>Ezh2^ΔSET/ΔSET</i> (purple) iPSC. C. Venn diagram showing overlap between H3K27me3⁺ genes in <i>Ezh2</i>^+/+ iPSC (grey), H3K27me2⁺/H3K27me3⁻ genes in <i>Ezh2^ΔSET/ΔSET</i> iPSC (orange) and H3K27me2⁺/H3K27me3⁻ in <i>Ezh2</i>^+/+ iPSC (light blue). D. Analysis of transcript levels measured by qRT-PCR, and status of SUZ12 binding, H3K27me2 and H3K27me1 enrichment revealed by ChIP q-PCR at promoters of 7 genes overexpressed in MEF relative to iPSC and representative of groups of genes marked by either H3K27me2⁺/H3K27me3⁺ or H3K27me2⁺/H3K27me3⁻ in <i>Ezh2^ΔSET/ΔSET</i> iPSC. For all analyses, two <i>Ezh2</i> control (+/+; grey) iPSC clones were compared to two mutant (<i>Δ</i>SET/<i>Δ</i>SET; purple) counterparts. Levels of expression are shown as ddCt (log₂ scale) relative to MEF. Status of a particular histone modification (±SEM) is represented as fold change of enrichment relative to input, after normalization for H3 density within the same amplicon. SUZ12 enrichment at promoters of indicated genes was determined comparing it to that of an unrelated IgG. Error bars refer to qPCR triplicates. E. Average reads distribution of H3K27me3 around the TSS in <i>Ezh2</i>^+/+ iPSC. Genes were divided in two classes based on H3K27me3 status in <i>Ezh2^ΔSET/ΔSET</i> iPSC (H3K27me3⁺ genes in green, H3K27me3⁻ genes in red) and compared to all genes (black) F. Gene ontology analysis of genes belonging to the three main groups based on H3K27 methylation status in <i>Ezh2^ΔSET/ΔSET</i> iPSC depicted in panel A. Bars represent <i>P</i>-values in –Log₂ scale of the corresponding biological processes. Dashed lines identify the significance threshold. G. Pie chart of H3K27 methylation status in <i>Ezh2^ΔSET/ΔSET</i> iPSC of MEF specific genes marked by H3K27me3 in <i>Ezh2</i>^+/+ iPSC. Color code is shown in panel A.</p

Establishment of iPSC clones upon genome-wide erasure of H3K27me3 at the onset of reprogramming.

<p>A. Western blot analysis of EZH2 and H3K27me3 protein levels respectively at onset or 48 hr after reprogramming in <i>Ezh2^+/ΔSET</i> and <i>Ezh2^ΔSET/ΔSET</i> MEFs. Data are representative of two experiments. Quantification of protein levels at the indicated time points is shown in the right panel (controls in grey, mutants in purple). B. Strategy to induce reprogramming of tail tip fibroblasts (TTFs) lacking H3K27me3 at the onset of reprogramming. C. Western blot analysis of EZH2 and H3K27me3 protein levels in <i>Cdkn2a^−/−</i> TTF carrying either one (+/<i>Δ</i>SET) or both (<i>Δ</i>SET/<i>Δ</i>SET) <i>Ezh2</i> mutant alleles. As comparison, representative <i>Ezh2</i>-proficient (+/+) and -deficient (<i>Δ</i>SET/<i>Δ</i>SET) iPSC clones were analyzed. The band corresponding to H3K27me3 in mutant TTF has the same intensity of that from a mutant iPSC clone for which mass-spectrometry did not detect the presence of H3K27me3. D. Quantification of AP-positive primary iPSC colonies obtained from infection of 2.5×10³<i>Cdkn2a^−/−</i> TTF, respectively proficient (grey bar) or deficient (purple) for <i>Ezh2</i>, assessed in three independent experiments.</p

Derivation and biochemical analysis of iPSC upon conditional <i>Ezh2</i> inactivation.

<p>A. Diagram of the reprogramming protocol of MEF. B. Alkaline phosphatase staining of control (lower row) and mutant (upper row) primary iPSC colonies one week following doxycycline withdrawal. C. Number of AP-positive primary iPSC colonies obtained upon infection of, respectively, 2×10³, 5×10³, 1×10⁴ or 6×10⁴ MEF in two experiments performed with two biological replicates per genotype. D. EZH2, H3K27me1, H3K27me2 and H3K27me3 protein levels assessed by Western blot in two representative <i>Ezh2</i> control (<i>+/+</i>) and mutant <i>(ΔSET/ΔSET</i>) iPSC clones. Vinculin and Histone H3 were used as loading controls for, respectively, EZH2 and methylated forms of H3K27. E. Relative abundance in control (upper row) and mutant (lower row) iPSC clones of the six possible methylation isoforms of the Histone H3 peptide spanning amino acids 27–40, as determined by mass spectrometry.</p