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H3K9me2 genome-wide distribution in
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the holocentric insect Spodoptera
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frugiperda (Lepidoptera: Noctuidae)
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Sandra Nhim
1
, Sylvie Gimenez
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, Rima Nait-Saidi
1
, Dany Severac
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, Kiwoong Nam
1
,
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Emmanuelle d’Alençon
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& Nicolas Nègre
1#
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DGIMI, Univ Montpellier, INRAE, Montpellier, France
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MGX, Univ Montpellier, CNRS, INSERM, Montpellier, France
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9
#
to whom correspondence should be addressed: nicolas.negre@umontpellier.fr
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Abstract
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Background: Eukaryotic genomes are packaged by Histone proteins in a structure called
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chromatin. There are different chromatin types. Euchromatin is typically associated with
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decondensed, transcriptionally active regions and heterochromatin to more condensed regions
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of the chromosomes. Methylation of Lysine 9 of Histone H3 (H3K9me) is a conserved
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biochemical marker of heterochromatin. In many organisms, heterochromatin is usually
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localized at telomeric as well as pericentromeric regions but can also be found at interstitial
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chromosomal loci. This distribution may vary in different species depending on their general
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chromosomal organization. Holocentric species such as Spodoptera frugiperda (Lepidoptera:
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Noctuidae) possess dispersed centromeres instead of a monocentric one and thus no
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observable pericentromeric compartment. To identify the localization of heterochromatin in
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such species we performed ChIP-Seq experiments and analyzed the distribution of the
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heterochromatin marker H3K9me2 in the Sf9 cell line and whole 4
th
instar larvae (L4) in relation
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to RNA-Seq data.
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Results: In both samples we measured an enrichment of H3K9me2 at the (sub) telomeres,
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rDNA loci, and satellite DNA sequences, which could represent dispersed centromeric regions.
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We also observed that density of H3K9me2 is positively correlated with transposable elements
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and protein-coding genes. But contrary to most model organisms, H3K9me2 density is not
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correlated with transcriptional repression.
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Conclusion: This is the first genome-wide ChIP-Seq analysis conducted in S. frugiperda for
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H3K9me2. Compared to model organisms, this mark is found in expected chromosomal
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compartments such as rDNA and telomeres. However, it is also localized at numerous
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dispersed regions, instead of the well described large pericentromeric domains, indicating that
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H3K9me2 might not represent a classical heterochromatin marker in Lepidoptera.
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(242 words)
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Keywords: heterochromatin, H3K9me2, Spodoptera frugiperda, holocentrism, ChIP-Seq
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Introduction
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The nuclear organization of the genome into chromatin is a hallmark of eukaryotes. DNA is
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wrapped around histone proteins to form nucleosomes that constitute basic units of chromatin
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(Luger et al. 1997). Two types of chromatin are classically described based on the compaction
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of nucleosomes along the genome. The euchromatin represents “open” and less compacted
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chromatin structures and is usually associated with active gene transcription. On the other
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hand, heterochromatin designates regions of the chromosomes that are more compact, with a
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higher nucleosome density (Heitz, 1928). Genes within heterochromatin are regarded to be
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transcriptionally repressed.
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Several types of heterochromatin have been described. Constitutive heterochromatin (c-Het),
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contrary to facultative heterochromatin, remains persistently compacted despite cell cycle and
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developmental stages, environmental states or even studied species (Dillon 2004; Allshire et
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Madhani 2018; Janssen, Colmenares, et Karpen 2018). It is usually located at important
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chromosomal features such as telomeres, rDNA loci and pericentromeric regions (Riddle et al.
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2011). Those regions are usually gene poor and transcriptionally silenced (Dillon 2004; Allshire
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et Madhani 2018). Understood as a genomic safety guard from transposons (Janssen,
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Colmenares, et Karpen 2018), c-Het associated regions are often enriched in repeated
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sequences such as satellite DNA and transposable elements. The transcriptional silencing by
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c-Het is due to its compaction which is achieved by chemical modifications of histones. The
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classical marker of c-Het in all eukaryotes is the post translational methylation of the lysine 9
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of Histone H3 (H3K9me) (Kharchenko et al. 2011; Liu et al. 2011; Ho et al. 2014). This
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methylation mark deposited by SET domain proteins such as Su(var)3-9 (Rea et al. 2000) and
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G9a (M. Tachibana et al. 2001; Makoto Tachibana et al. 2002; Kondo et al. 2008) is recognized
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by chromo-domain containing proteins belonging to HP1s family (Minc et al. 1999; Lachner et
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al. 2001; Maison et Almouzni 2004; Lomberk, Wallrath, et Urrutia 2006). HP1 proteins
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assemble as homodimers to form the ultrastructural 3D compaction detected by cytology
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(Verschure et al. 2005). This compaction impairs the binding of other DNA associated proteins
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such as transcription factors and RNA polymerases, which explains the repressive effect of
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heterochromatin. These properties of c-Het have been well described in model organisms,
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from yeast to mammals and thus are thought to be conserved.
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With developments of sequencing, biochemical methods and growing interest for non-model
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organisms (Tagu, Colbourne, et Nègre 2014), the classical c-Het features are being
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reconsidered (Grewal et Jia 2007). Underlying DNA sequences can show rapid turnover, a fact
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particularly true for centromeres (Henikoff, Ahmad, et Malik 2001). Depending on cell cycle
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phases, nascent non coding RNAs from telomeres and pericentromeres contribute to the
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regulation of their biology (Bierhoff, Postepska-Igielska, et Grummt 2014; Allshire et Madhani
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2018). H3K9me distribution has been shown to vary and dynamic apposition of histone marks
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has also been reported outside of primary c-Het regions (Wen et al. 2009) in heterochromatin
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“islands” (Riddle et al. 2011; Lee et al. 2020). In human and mouse, interstitial domains called
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LOCKS, spanning several Mb, dynamically switch to heterochromatin state, marked by Lysine
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9 methylation, in specialized cells, supposedly to limit pluripotency (Wen et al. 2009; Madani
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Tonekaboni, Haibe-Kains, et Lupien 2021). Beside these controlled variations, c-Het can
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unpredictably change in terms of associated proteins or even DNA sequences. This causes
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several defects in development or represent molecular basis of hybrid incompatibilities
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between close species (Ferree et Barbash 2009; Hughes et Hawley 2009; Johnson 2010;
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Crespi et Nosil 2013; Satyaki et al. 2014).
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While most studies on c-Het have been performed on monocentric species, a particular case
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of c-Het dynamic is found in holocentric organisms. Their chromosomes have no cytological
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hypercompacted regions and possess dispersed centromeres instead of unique ones per
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chromosome (Schrader 1935). Classical heterochromatin compartmentation and properties
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are thought to be absent. Holocentrism has been described in several plants, in some
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nematodes and some insect orders (Wolf 1996; Drinnenberg et al. 2014). Phylogenetic
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analysis indicates that holocentric species might have derived several times from monocentric
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species by convergent evolution (Melters et al. 2012; Escudero, Márquez-Corro & Hipp 2016).
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This is supported by different centromeric molecular signatures between holocentric species.
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In monocentric species, major 150bp satellite forming centromeres are packaged in CenH3
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rich nucleosomes that are encompassed by peripheral H3K9me2/3 regions (Melters et al.
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2013; Drinnenberg et al. 2014). But, except for the plant Rhynchospora pubera (Marques et al.
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2015), described holocentric species have lost those centromeric repeated sequences. CenH3
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is present in nematodes (Gassmann et al. 2012; Steiner et Henikoff 2014) and in plants (Zedek
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et Bureš 2016; Oliveira et al. 2020) but has been lost in other holocentric insects (Drinnenberg
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et al. 2014). A recent study proposed that in Lepidopteran, CenH3 function has been replaced
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by H3K27me3, a facultative heterochromatin mark (Senaratne et al. 2021). More importantly
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H3K9me2 signal surrounding centromeres is thought to be lost in these organisms, unlike
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holocentric C.elegans (Steiner et Henikoff 2014). Previous studies conducted on Lepidopteran
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showed nonetheless that H3K9me2 is still associated to repeated DNA at rDNA loci and sexual
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chromosomes (Stanojcic et al. 2011; Borsatti et Mandrioli 2013).
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In this paper, we aim to clarify H3K9me2 heterochromatin distribution in Spodoptera frugiperda
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(Lepidoptera: Noctuidae), a crop pest causing severe damages to plants at larval stage. Since
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the S. frugjperda distribution area has recently been extended from the American continent to
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a worldwide invasion (Goergen et al. 2016), there is an urge to understand its adaptive potential
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when confronted with new ecosystems. In particular, chromatin properties could influence
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phenotypic plasticity in response to environmental conditions (Simola et al. 2016; Gibert et al.
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2016). S.frugiperda constitutes also an emergent epigenetic model organism with published
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4
reference genomes for different strains and cell lines (Kakumani et al. 2014; Nandakumar, Ma,
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et Khan 2017; Gouin et al. 2017; Nam et al. 2020; L. Zhang et al. 2020), histone modifications
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and non-coding RNAs being previously characterized (Stanojcic et al. 2011; d’Alençon et al.
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2011; Moné et al. 2018) as well as a growing body of RNA-Seq data (Orsucci et al. 2020).
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Another advantage lies in the well-established Sf9 cell line, derived from S. frugiperda ovarian
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tissues, providing non limiting material for biochemical assays (Vaughn et al. 1977). We
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performed H3K9me2 ChIP-Seq and RNA-Seq on two different samples: Sf9 cell lines and
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whole 4th instar larvae (L4). In both samples, we confirmed the association of H3K9me2 at c-
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Het domains such as telomeres, rDNA locus and satellite sequences that might represent
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vestigial centromeres. We found a strong association of H3K9me2 with repeat elements as
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well as gene bodies. And we show that H3K9me2 enrichment at these elements is not
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associated with transcriptional repression or activation, raising the question of its role in
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chromosomal organization in holocentric lepidopteran.
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Material and Methods
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Spodoptera frugiperda rearing and Sf9 cell line maintenance
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L4 insects have been raised in controlled laboratory conditions of 16h:8h light/dark photoperiod
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cycle, ~40 % mean hygrometry and ~24°c temperature. The insects derived from pupae
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individuals collected in 2001 in Guadeloupe. This laboratory population corresponds to
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previously published reference genome assemblies (Gouin et al. 2017; Nam et al. 2020).
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Immortalized Sf9 cell line derived from S. frugiperda ovarian tissues (Vaughn et al. 1977). The
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cell line was acquired from ATCC (https://www.atcc.org/products/crl-1711) and has been
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cultured following the manufacturer protocol recommendations.
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Western blot
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Chromatin extracts were prepared from Sf9 cells and L4 insects as described below fro the
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ChIP procedure. Total proteins have been first quantified by colorimetric Bradford method and
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equivalent quantities were used for a 15% SDS/PAGE. After migration, proteins from the gel
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were transferred onto a PVDF membrane. The membrane was incubated overnight with
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mouse monoclonal H3K9me2 antibody (Abcam 1220) and revealed with an ECL kit.
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Immunofluorescence on Sf9 cell lines
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Sf9 cells were grown to confluence with standard Schneider medium, then scraped and
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collected in 50 mL Falcon tubes. Cells were then diluted up to 3.105 cells/ml and 1 mL was
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used for each immunostaining condition for 1 well of a 12-well plate. Plates with round glass
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coverslips and 1 mL of cell dilution were then placed at 28 °C for 4 h, allowing the cells to
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sediment on the coverslip. The culture medium was then removed, plates washed with 1X PBS
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and fixed with paraformaldehyde 4%, 20 min at room temperature. Coverslips were rinsed
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twice with PBS before processing with immunostaining.
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For immunostaining, coverslips in the plates were permeabilized with 1 mL of PBS 1X + Triton
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1% per well for 30 min at room temperature. The solution was removed, and the cells were
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blocked with PBS 1X + BSA 1% for 30 min at room temperature. The solution was removed
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and staining with the primary antibody (anti-H3K9m2, Abcam 1220) diluted in PBS-BSA 0.1 %
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was performed. We used 1/100 and 1/200 dilutions (50 µL / well) and we kept a control non-
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treated well. Incubation was done 1 h at room temperature. Primary antibody was rinsed twice
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in PBS 1X before adding the secondary antibody (anti-mouse Alexa568) diluted 1/500 in PBS
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1X – BSA 0.1% (~100 µL per well) 30-45 min at room temperature in the dark. Still in the dark,
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coverslips were rinsed once in PBS 1X, then incubated with DAPI (1mg/mL diluted 1/1000 per
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condition) for 5 minutes and rinsed again. Coverslips were then mounted on a microscopy slide
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with 1 drop of ProLong Antifade Mountant (ThermoFischer Scientific) and after 30min, sealed
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with transparent nail polish. Slides were kept in the dark at 4°C until observation with an
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Apotome microscope (Zeiss).
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ChIP-Seq and RNA-Seq
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 8, 2021. ; https://doi.org/10.1101/2021.07.07.451438doi: bioRxiv preprint