1. Volume 52, Issue 5, Pages 746-757 (December 2013)
Regulation of Heterochromatin Transcription by Snail1/LOXL2 during Epithelial-to- Mesenchymal Transition 
Alba Millanes-Romero, Nicolás Herranz, Valentina Perrera, Ane Iturbide, Jordina Loubat-Casanovas, Jesús Gil, Thomas Jenuwein, Antonio García de Herreros, Sandra Peiró 
Molecular Cell 
Volume 52, Issue 5, Pages (December 2013)
DOI: /j.molcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2013 52, 746-757DOI: (10.1016/j.molcel.2013.10.015)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1
Heterochromatin Organization Is Compromised in the Absence of Snail1
(A) Western blot for Snail1 in pMEFs Snai1F/F/Cre+ and Snai1F/F/Cre− treated with tamoxifen.
(B) A representative image of pMEFs Snai1F/F/Cre− (CT) Snai1F/F/Cre+(KO) stained with DAPI and immunolabeled with anti-HP1α and anti-H3K9me3 antibodies after tamoxifen treatment.
(C) The graph depicts the average of foci number per nucleus in pMEFs Snai1F/F/Cre+ and Snai1F/F/Cre− detected with DAPI, H3K9me3, and HP1α. Error bars indicate SD for at least three independent experiments; in all of them, at least 100 nuclei were analyzed. Scale bar, 10 μm. ∗∗p < 0.01.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Snail1 Regulates Major Satellite Transcription and Binds to Pericentromeric Regions in Mouse and Human Cells
(A) Left: real-time quantitative RT-PCR (qRT-PCR) shows the changes in expression of the major satellite and other repetitive sequences in control and KO MEFs. The levels of major satellite transcription were also analyzed by qRT-PCR after Snail1 transfection in Snai1-depleted MEFs (KO + Sna). Right panel: qRT-PCR shows the re-expression of Snai1 gene in KO MEFs. Gene expression was normalized against an endogenous control (HPRT or Pumilio) and presented as RNA levels over those obtained in KO MEFs, which was set as 1.
(B) Snail1 binding to major satellite region was determined by ChIP in control and KO MEFs. The α-globin gene was used as a negative control in the ChIP assay. Data from real-time quantitative PCR (qPCR) amplifications of the major satellite region and α-globin promoter in control MEFs were normalized to the input and expressed as fold enrichment over the data obtained in KO MEFs, which was set as 1. This value was not significantly different to that obtained in control MEFs using an irrelevant immunoglobulin G (IgG).
(C) Whole human chromosome view of Snail1 mapping in pericentromeric regions. Rectangles mark the detected binding site in pericentromeric regions.
(D) Snail1 ChIPs in the human colorectal cancer cell line Sw620 in pericentromeric regions corresponding to chromosomes 4, 7, and 20. HP1α was used as positive control in the same regions. qPCR data of the heterochromatin regions immunoprecipitated with antibodies against Snail1 were normalized to the input and expressed as fold change over the data obtained when an irrelevant IgG was used (dark bars). The value given for the IgG sample was set as 1.
Error bars indicate SD in at least three independent experiments. ∗p < 0.05, ∗∗p < 0.01.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Oxidized Levels of Histone H3 in Major Satellite Controlled by LOXL2 Regulates Pericentromeric Transcription
(A) H3 oxidation in major satellite region was determined by sequential ChIP (re-ChIP) in control and KO MEFs. The lysate was incubated with biotin-hydrazide (an activated biotin that reacts with oxidized H3) before re-ChIP. Extracts were sequentially immunoprecipitated with anti-H3 and streptavidin beads. DNA binding was quantified by qPCR. Data were normalized to the total amount of H3 immunoprecipitated and to the input and expressed as fold enrichment over the data obtained when an irrelevant IgG was used. The value given for the IgG sample was set as 1.
(B) LOXL2 overexpression restored major satellite downregulation in Snail1 KO MEFs. LOXL2 was ectopically expressed after retrovirus infection in Snail1 KO MEFs. Major satellite expression levels (left panel) and LOXL2 mRNA levels (right panel) were determined by qRT-PCR. Expression levels were normalized to an endogenous control and are shown relative to the MEFs KO values (arbitrarily set as 1).
(C) qRT-PCR results revealed the specific upregulation of major satellite transcription in wild-type MEFs infected with small hairpin RNA (shRNA) controls or against shLOXL2. Expression levels were normalized to an endogenous control and expressed relative to the shRNA-control-infected cells (arbitrarily set as 1). The lower panel shows a representative western blot for LOXL2 from shControl or shLOXL2 MEFs.
(D) LOXL2 and H3K4me3 ChIP and re-ChIP for oxidized H3 in MEFs infected with an shRNA control or an shRNA against LOXL2. ChIP was conducted as in (A). qPCR data from the major satellite region in MEFs treated with either shRNA control or shRNA against LOXL2 were normalized to the input and expressed as fold enrichment relative to the data obtained in shRNA control, which was set as 1.
Error bars indicate SD in at least three independent experiments. ∗p < 0.05, ∗∗p < 0.01.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
HP1α Distribution Changes during EMT in a Snail1-Dependent Manner
(A) NMuMG cells were treated with TGF-β and immunolabeled with HP1α antibody or stained with DAPI. Scale bar, 10 μm.
(B) The graph depicts the average of foci number per nucleus of HP1α and DAPI-positive cells at different times of TGF-β treatment.
(C) Purified nuclei from NMuMG cells treated with TGF-β were extracted with NaCl (0.5 M), and the extracted fractions or the input were analyzed by western blotting (left panel). Quantification is shown at the bottom. Right panel: the release of HP1α from the chromatin fraction of NMuMG cells treated with TGF-β was monitored by western blot. HP1α levels were standardized using H3 levels. The quantification shows the increase or decrease in the amount of HP1α from treated as compared to untreated cells (time point 0), which was set as 1.
(D) NMuMG cells were transfected with an siRNA control and an siRNA against Snail1. After 24 hr, cells were treated with TGF-β. The average of HP1α-foci number per cell was quantified prior or 8 hr after TGF-β addition.
Error bars indicate SD in at least three independent experiments. ∗∗p < 0.01.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Snail1 Regulates Pericentromeric Transcription during EMT through LOXL2
(A) Snail1 induction upon TGF-β treatment is shown by western blot in NMuMG cells at different time points. qRT-PCR shows major satellite transcript levels after normalization versus an endogenous control in NMuMG cells treated with TGF-β.
(B) Snail1 ChIP (left) and re-ChIP for oxidized H3 (right) in major satellite regions in NMuMG cells treated with TGF-β.
(C) qRT-PCR shows major satellite transcription in NMuMG cells transfected with a siRNA against Snail1 upon EMT induction by TGF-β.
(D) qRT-PCR shows major satellite transcription in NMuMG cells infected with an shRNA against LOXL2 upon EMT induction by TGF-β (left panel). The right panel shows a ChIP analysis of oxidized H3 under the same conditions.
Error bars indicate SD for at least three independent experiments. ∗p < 0.05, ∗∗p < 0.01.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 Ectopic Major Satellite Expression Alters the EMT
(A) Control or NMuMG cells ectopically expressing major satellites were treated with TGF-β and immunolabeled for HP1α. Scale bar, 10 μm. Inset shows magnified representative nucleus, scale bar 5 μm.
(B) The percentage of cells positive for HP1α-foci is shown in the graph.
(C) Gene ontology analysis of differentially expressed genes in major satellite-NMuMG cells compared with control cells 8 hr after TGF-β treatment. List of the selected mesenchymal and epithelial genes that are differentially expressed in major-NMuMG cells compared with control-NMuMG cells upon TGF-β treatment. LogFC indicates log2 fold changes.
(D) Validation by qRT-PCR of selected genes.
(E) Major satellite-NMuMG and control NMuMG cells were treated with TGF-β; after 24 hr, cells were reseeded on transwell chambers and incubated for 10 hr (left panel, migration) or placed in Matrigel-coated transwells and incubated for 24 hr (right panel, invasion). Nonmigrating and noninvading cells were removed from the upper surface of the membrane, while cells present at the lower surface were fixed and stained with DAPI. The DAPI-stained nuclei were counted in four different fields per filter by ImageJ software.
Error bars indicate the SD for at least four independent experiments. ∗p < 0.05, ∗∗p < 0.01.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7 Depletion of HP1α or Major Satellites Does Not Affect EMT
(A and B) NMuMG cells were transfected with an siRNA control or an siRNA against HP1α (A) or with LNA-DNA control gapmers or with a mix of LNA-DNA gapmer1 and LNA-DNA gapmer2 to deplete major satellite expression (B). After transfection, cells were treated with TGF-β for 24 hr when indicated. Levels of FN1, Zeb1/Zeb2, Slug (Snail2), CD44, HP1α, and major satellites were determined by qRT-PCR. Error bars indicate SD in at least three independent experiments.
(C) Working model. Upon TGF-β induction of EMT, Snail1 is rapidly upregulated, binds to pericentromeric regions, and recruits LOXL2 to oxidize H3 and repress major satellite transcription. As a consequence, HP1α is released from heterochromatin, enabling chromatin reorganization and acquisition of mesenchymal traits. This Snail1 action is transient, and in 24 hr major satellite levels and HP1α binding to heterochromatin have been recovered. Therefore, for the conversion of an epithelial cell to a mesenchymal cell during TGF-β-induced EMT, there is a window of time during which major satellite transcription is downregulated due to the actions of Snail1 and LOXL2 and the oxidation of H3. HP1α is released from heterochromatin during this time frame. Altogether, this allows chromatin reorganization and mesenchymal traits to be acquired.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions