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H3K9me2 genome-wide distribution in the holocentric insect Spodoptera frugiperda (Lepidoptera: Noctuidae)

TL;DR: The first genome-wide ChIP-Seq analysis conducted in S. frugiperda for H3K9me2 was performed in this article, where the authors measured an enrichment of H3kme2 at the (sub) telomeres, rDNA loci, and satellite DNA sequences, which could represent dispersed centromeric regions.
Abstract: BackgroundEukaryotic genomes are packaged by Histone proteins in a structure called chromatin. There are different chromatin types. Euchromatin is typically associated with decondensed, transcriptionally active regions and heterochromatin to more condensed regions of the chromosomes. Methylation of Lysine 9 of Histone H3 (H3K9me) is a conserved biochemical marker of heterochromatin. In many organisms, heterochromatin is usually localized at telomeric as well as pericentromeric regions but can also be found at interstitial chromosomal loci. This distribution may vary in different species depending on their general chromosomal organization. Holocentric species such as Spodoptera frugiperda (Lepidoptera: Noctuidae) possess dispersed centromeres instead of a monocentric one and thus no observable pericentromeric compartment. To identify the localization of heterochromatin in such species we performed ChIP-Seq experiments and analyzed the distribution of the heterochromatin marker H3K9me2 in the Sf9 cell line and whole 4th instar larvae (L4) in relation to RNA-Seq data. ResultsIn both samples we measured an enrichment of H3K9me2 at the (sub) telomeres, rDNA loci, and satellite DNA sequences, which could represent dispersed centromeric regions. We also observed that density of H3K9me2 is positively correlated with transposable elements and protein-coding genes. But contrary to most model organisms, H3K9me2 density is not correlated with transcriptional repression. ConclusionThis is the first genome-wide ChIP-Seq analysis conducted in S. frugiperda for H3K9me2. Compared to model organisms, this mark is found in expected chromosomal compartments such as rDNA and telomeres. However, it is also localized at numerous dispersed regions, instead of the well described large pericentromeric domains, indicating that H3K9me2 might not represent a classical heterochromatin marker in Lepidoptera.

Summary (4 min read)

Introduction

  • DNA is wrapped around histone proteins to form nucleosomes that constitute basic units of chromatin (Luger et al. 1997) .
  • Several types of heterochromatin have been described.
  • While most studies on c-Het have been performed on monocentric species, a particular case of c-Het dynamic is found in holocentric organisms.
  • A recent study proposed that in Lepidopteran, CenH3 function has been replaced by H3K27me3, a facultative heterochromatin mark (Senaratne et al. 2021) .

Material and Methods

  • Spodoptera frugiperda rearing and Sf9 cell line maintenance L4 insects have been raised in controlled laboratory conditions of 16h:8h light/dark photoperiod cycle, ~40 % mean hygrometry and ~24°c temperature.
  • The insects derived from pupae individuals collected in 2001 in Guadeloupe.
  • This laboratory population corresponds to previously published reference genome assemblies (Gouin et al. 2017; Nam et al. 2020) .
  • Immortalized Sf9 cell line derived from S. frugiperda ovarian tissues (Vaughn et al. 1977 ).
  • The cell line was acquired from ATCC (https://www.atcc.org/products/crl-1711) and has been cultured following the manufacturer protocol recommendations.

Western blot

  • Chromatin extracts were prepared from Sf9 cells and L4 insects as described below fro the ChIP procedure.
  • Cells were then diluted up to 3.105 cells/ml and 1 mL was used for each immunostaining condition for 1 well of a 12-well plate.
  • The solution was removed and staining with the primary antibody (anti-H3K9m2, Abcam 1220) diluted in PBS-BSA 0.1 % was performed.
  • Still in the dark, coverslips were rinsed once in PBS 1X, then incubated with DAPI (1mg/mL diluted 1/1000 per condition) for 5 minutes and rinsed again.
  • Coverslips were then mounted on a microscopy slide with 1 drop of ProLong Antifade Mountant (ThermoFischer Scientific) and after 30min, sealed with transparent nail polish.

ChIP-Seq and RNA-Seq

  • The authors performed ChIP-Seq following an adapted protocol by Nègre et al. 2006 on 4th instar whole larvae and Sf9 cell culture.
  • For the immunoprecipitation, sonicated chromatin is incubated for 4 h with the primary antibody at 4°C, then with Protein-A sepharose beads (CL4B) for again 4 h.
  • To reverse the cross-linking, this immuno-precipitate was incubated overnight at 65°C and then 2-3h at 50°C with proteinase K. DNA was purified from this precipitate with 500µL of phenol-chloroform and 55µL of 4M LiCl.
  • Samtools have been used for sample concatenation (Li et al. 2009 ) after Deeptools -bigwigcorrelate Pearson analysis (Ramírez et al. 2016 ).
  • Peaks were annotated using Bedtools software (--intersect) after functional annotation of genomic elements.

Genome functional element annotation

  • The authors used published S. frugiperda transcriptome (Legeai et al. 2014) for Sf9 and L4 gene annotations using Scipio software (Keller et al. 2008) .
  • Introns were annotated by subtracting CDS-exons positions (Bedtools -subtract).
  • Output in transcript per millions (TPM) was converted to log2 to determine active pool genes (log2(TPM)>1) from inactive ones (log2(TPM)<=1).
  • Major 150bp satellite regions were shuffled using Bedtools --shuffle option.
  • Finally, consensus sequence satellite of over 100 bp were annotated as satDNA.

Statistic and graphic productions

  • Barplot, scatterplot, histogram and Venn Diagram were plotted using R software.
  • Student ttest and Chi-square test were performed using R software.
  • Gene ontology enrichment has been performed using BLAST2GO annotations followed by Fisher exact test analysis (Conesa et al. 2005) .

1. H3K9me2 genome-wide distribution in Sf9 and L4 genomes

  • While their global analysis gives clues about the general distribution of H3K9me2 in S.frugiperda, it failed to detect its expected association with telomeres or rDNA locus, with no enrichment found compared to broader regions such as repeated sequences and genes (Supplementary Fig. 6 ).
  • As this result could be due to a very small fraction of telomeric and rDNA loci in the genome (~80 kb over 400 MB; Supplementary Table 3 & 4 ), the authors specifically analyzed the H3K9me2 distribution around the telomeric regions and rDNA locus (see Results section below) that they annotated in both reference sequences.

2. H3K9me2 signal in telomeres repetitions

  • In Noctuidae, the Lepidoptera family to which belongs the S.frugiperda species, chromosome pairs number is stably equal to 31 (Robinson 1971) .
  • Hence, consensual [TTAGG]n motif constituting telomeres is expected to be annotated at least 62 times in haploid genome assemblies (two tips per chromosome) (Gong et al. 2015; Vershinina, Anokhin, et Lukhtanov 2015) .
  • For each presumptive telomeric region, the authors checked whether it was associated with any H3K9me2 peak.

3. H3K9me2 signal in rDNA locus

  • Previous FISH cytology experiments conducted in Noctuidae showed the conservation of one rDNA locus located interstitially in an autosome (Nguyen et al. 2010) .
  • This polycistronic-like cluster is made of repeated 18S, 5.8S and 28S genes with additional 5S RNA being anti-sense included or extra located (Shaw et McKeown 2011) .
  • Like the authors did for telomere sequences, they compared H3K9me2 enrichment and transcription at rDNA loci.
  • The authors searched both reference genomes for rDNA sequences and found one major locus in both, even though relatively shorter rDNA sequences and even larger ones can be detected at various places in the genome (Supplementary Table 4 ).
  • Thus, if only one or few portions are H3K9me2 enriched in biological reality, every identical DNA sequence would be predicted as associated with the mark.

4. H3K9me2 enrichment around satellite DNA regions

  • The authors wondered whether heterochromatin in holocentric species is also associated with pericentric regions.
  • This was confirmed for the majority of monocentric organisms but contested for holocentric ones (Melters et al. 2013 ) with only Rynchospora pubera plant sharing this characteristic (Marques et al. 2015) .
  • Interestingly, H3K9me2 enrichment profile around satDNA was found similar in the two references with a systematic decrease of the mark inside candidate sequences opposing a broader adjacent signal.
  • In both references, highest adjacent H3K9me2 peaks are stably found around 1000bp from major 150bp satellites (Fig. 4D ).
  • While this result might indicate an association of heterochromatin with centromeric regions in holocentric species, additional studies would be needed to confirm that satDNA corresponds to bona fide centromeres in S. frugiperda.

5. H3K9me2 enrichment in repeat elements families

  • The authors annotated in both reference genomes the different categories of repeat sequences, whether they correspond to tandem repeats, such as micro-and mini-satellite sequences, or transposable elements (Fig. 5A ).
  • The authors then calculated the proportion of each category associated with H3K9me2 (Fig. 5A ).
  • A higher proportion of satellite sequences and transposable elements is found associated with H3K9me2 compared to micro-and mini-satellite, which agrees with their expectation of a role of heterochromatin associated with repeat-rich regions of the genome.
  • Indeed, because of their potential deleterious effects on the genome when they are mobilized, transposable elements are often transcriptionally repressed by small RNA targeted H3K9me (Klenov et al.
  • In order to determine if a similar role of H3K9me2 exists in S. frugiperda, the authors analyzed the RNA-Seq data from L4 tissues and Sf9 cells to classify transcribed and non-transcribed transposable elements and observe the association of each category with H3K9me2 signal (Fig. 5B ).

6. H3K9me2 signal enrichment in genes

  • Due to the classical repressive nature of heterochromatin, the authors expected H3K9me2 to be associated with mainly inactive genes.
  • The authors analyzed if some gene regions like promoters, UTRs or gene bodies were more enriched in H3K9me2 than others (Fig. 6A ).
  • The authors also noticed the presence of genes involved in heterochromatin maintenance and formation, suggesting possible molecular feedback.

Discussion

  • The authors report the first H3K9me2 genome-wide ChIP-Seq analysis conducted in S. frugiperda, a Lepidopteran species.
  • The authors detected H3K9me2 at expected chromosomal compartments such as telomeres and rDNA locus but no major centromeric locus.
  • Instead, the authors observed a scattered distribution of H3K9me2 along the chromosomes colocalizing with genes and repeat elements, independent of their transcriptional status.

Localization of H3K9me2 at expected heterochromatin compartments

  • In many monocentric organisms, c-Het is found in large regions surrounding the centromere.
  • As mentioned in the introduction, H3K9me2 surrounding the major 150bp satDNA repetition is a conserved pattern for pericentric regions in monocentric species.
  • The distribution of H3K9me2 was not assessed in their study and the authors don't know whether it colocalizes with H3K27me3 or is excluded.
  • The authors did not observe a strong complete association of the mark with repeat elements with only 24% and 38% of transposable elements intersecting H3K9me2 peaks (Fig. 5A ).

H3K9me2 association with genes bodies

  • This histone mark has been described to cover coding sequences in two different situations.
  • Their function reflects constitutive roles such as general transcription factors, ribosomal genes or mitochondrial genes, which fits with heterochromatic genes hypothesis.
  • In order to confirm a splicing role for this mark in S.frugiperda, it would be necessary to annotate exons nature (internal vs. external, constitutive vs. alternative, etc.) and check if alternative mRNA transcripts are produced given presence or absence of exonic H3K9me2 signal.
  • The authors search for a similar motif in S.frugiperda using two dedicated softwares failed to give the same results.
  • In addition, the correspondence of classical HP1 proteins with homologs in Lepidoptera is not clear.

Conclusion / Article summary

  • The authors produced the first genome-wide analysis of holocentric Lepidoptera S. frugiperda heterochromatin distribution by analyzing H3K9me2 ChIP-Seq data in two cell models.
  • In contrast to studies suggesting unusual behavior of modified histones, their results supported a conserved pattern with invariant classic c-Het domains such as (sub)telomeres, rDNA locus and even peripheral major 150bp satDNA that could be associated with centromeric functions.
  • SN produced the bioinformatic and statistical analysis with the help of KWN.
  • SN and NN wrote the manuscript with the input of all co-authors.
  • Mean log2(H3K9me2/Input) signal (y-axis) in all exons (left) and introns (right, x-axis), also known as For upper graphs.

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1
H3K9me2 genome-wide distribution in
1
the holocentric insect Spodoptera
2
frugiperda (Lepidoptera: Noctuidae)
3
Sandra Nhim
1
, Sylvie Gimenez
1
, Rima Nait-Saidi
1
, Dany Severac
2
, Kiwoong Nam
1
,
4
Emmanuelle d’Alençon
1
& Nicolas Nègre
1#
5
6
1
DGIMI, Univ Montpellier, INRAE, Montpellier, France
7
2
MGX, Univ Montpellier, CNRS, INSERM, Montpellier, France
8
9
#
to whom correspondence should be addressed: nicolas.negre@umontpellier.fr
10
Abstract
11
Background: Eukaryotic genomes are packaged by Histone proteins in a structure called
12
chromatin. There are different chromatin types. Euchromatin is typically associated with
13
decondensed, transcriptionally active regions and heterochromatin to more condensed regions
14
of the chromosomes. Methylation of Lysine 9 of Histone H3 (H3K9me) is a conserved
15
biochemical marker of heterochromatin. In many organisms, heterochromatin is usually
16
localized at telomeric as well as pericentromeric regions but can also be found at interstitial
17
chromosomal loci. This distribution may vary in different species depending on their general
18
chromosomal organization. Holocentric species such as Spodoptera frugiperda (Lepidoptera:
19
Noctuidae) possess dispersed centromeres instead of a monocentric one and thus no
20
observable pericentromeric compartment. To identify the localization of heterochromatin in
21
such species we performed ChIP-Seq experiments and analyzed the distribution of the
22
heterochromatin marker H3K9me2 in the Sf9 cell line and whole 4
th
instar larvae (L4) in relation
23
to RNA-Seq data.
24
25
Results: In both samples we measured an enrichment of H3K9me2 at the (sub) telomeres,
26
rDNA loci, and satellite DNA sequences, which could represent dispersed centromeric regions.
27
We also observed that density of H3K9me2 is positively correlated with transposable elements
28
and protein-coding genes. But contrary to most model organisms, H3K9me2 density is not
29
correlated with transcriptional repression.
30
31
Conclusion: This is the first genome-wide ChIP-Seq analysis conducted in S. frugiperda for
32
H3K9me2. Compared to model organisms, this mark is found in expected chromosomal
33
compartments such as rDNA and telomeres. However, it is also localized at numerous
34
.CC-BY-NC-ND 4.0 International licenseavailable under a
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

2
dispersed regions, instead of the well described large pericentromeric domains, indicating that
35
H3K9me2 might not represent a classical heterochromatin marker in Lepidoptera.
36
(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
42
(Luger et al. 1997). Two types of chromatin are classically described based on the compaction
43
of nucleosomes along the genome. The euchromatin represents “open” and less compacted
44
chromatin structures and is usually associated with active gene transcription. On the other
45
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.
48
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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
57
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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.CC-BY-NC-ND 4.0 International licenseavailable under a
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

3
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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.CC-BY-NC-ND 4.0 International licenseavailable under a
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

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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
124
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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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

5
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
135
cycle, ~40 % mean hygrometry and ~24°c temperature. The insects derived from pupae
136
individuals collected in 2001 in Guadeloupe. This laboratory population corresponds to
137
previously published reference genome assemblies (Gouin et al. 2017; Nam et al. 2020).
138
Immortalized Sf9 cell line derived from S. frugiperda ovarian tissues (Vaughn et al. 1977). The
139
cell line was acquired from ATCC (https://www.atcc.org/products/crl-1711) and has been
140
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

Citations
More filters
Journal ArticleDOI
01 Aug 2022-Cell
TL;DR: The authors compare genome organization and evolution as a function of centromere type by assembling chromosome-scale holocentric genomes with repeat-based holocentromeres from three beak-sedge (Rhynchospora pubera, R. breviuscula and R. tenuis) and their closest monocentric relative, Juncus effusus.

22 citations

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More filters
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21 Nov 2012-Cell
TL;DR: It is shown that the HMG protein Maelstrom is essential for Piwi-mediated silencing in Drosophila and shows that Piwi is required to establish heterochromatic H3K9me3 marks on transposons and their genomic surroundings.

559 citations

Journal ArticleDOI
TL;DR: The spectrum of histone modifications present in human and Drosophila melanogaster CEN chromatin is distinct from that of both euchromatin and flanking heterochromatin, and it is speculated that this distinct modification pattern contributes to the unique domain organization and three-dimensional structure of centromeric regions.
Abstract: Post-translational histone modifications regulate epigenetic switching between different chromatin states. Distinct histone modifications, such as acetylation, methylation and phosphorylation, define different functional chromatin domains, and often do so in a combinatorial fashion. The centromere is a unique chromosomal locus that mediates multiple segregation functions, including kinetochore formation, spindle-mediated movements, sister cohesion and a mitotic checkpoint. Centromeric (CEN) chromatin is embedded in heterochromatin and contains blocks of histone H3 nucleosomes interspersed with blocks of CENP-A nucleosomes, the histone H3 variant that provides a structural and functional foundation for the kinetochore. Here, we demonstrate that the spectrum of histone modifications present in human and Drosophila melanogaster CEN chromatin is distinct from that of both euchromatin and flanking heterochromatin. We speculate that this distinct modification pattern contributes to the unique domain organization and three-dimensional structure of centromeric regions, and/or to the epigenetic information that determines centromere identity.

557 citations

Journal ArticleDOI
TL;DR: RepeatExplorer as mentioned in this paper is a collection of software tools for characterization of repetitive elements which is accessible via web interface and uses graph-based sequence clustering algorithm to facilitate de novo repeat identification without the need for reference databases of known elements.
Abstract: Motivation: Repetitive DNA makes up large portions of plant and animal nuclear genomes, yet it remains the least characterized genome component in most species studied so far. Although the recent availability of high throughput sequencing data provides necessary resources for in-depth investigation of genomic repeats, its utility is hampered by the lack of specialized bioinformatics tools and appropriate computational resources that would enable large-scale repeat analysis to be run by biologically-oriented researchers. Results: Here we present RepeatExplorer, a collection of software tools for characterization of repetitive elements which is accessible via web interface. A key component of the server is the computational pipeline employing a graph-based sequence clustering algorithm to facilitate de novo repeat identification without the need for reference databases of known elements. Since the algorithm uses short sequences randomly sampled from the genome as input, it is ideal for analyzing next generation sequence reads. Additional tools are provided to aid in classification of identified repeats, investigate phylogenetic relationships of retroelements and perform comparative analysis of repeat composition between multiple species. The server allows to analyze several million sequence reads which typically results in identification of most high and medium copy repeats in higher plant genomes. Implementation and availability: RepeatExplorer was implemented within the Galaxy environment and set up on a public server at http://repeatexplorer.umbr.cas.cz/. Source code and instructions for local installation are available at http://w3lamc.umbr.cas.cz/lamc/ resources.php.

549 citations

Journal ArticleDOI
TL;DR: Ch Chromatin structure analysis revealed that the variegating inserts showed a reduction in accessibility to restriction enzyme digestion in the hsp26 regulatory region in isolated nuclei, suggesting that altered chromatin packaging plays a role in PEV.
Abstract: A euchromatic gene placed in the vicinity of heterochromatin by a chromosomal rearrangement generally exhibits position effect variegation (PEV), a clonally inherited pattern showing gene expression in some somatic cells but not in others The mechanism responsible for this loss of gene expression is investigated here using fly lines carrying a P element containing the Drosophila melanogaster white and hsp26 genes Following mobilization of the P element, a screen for variegation of white expression recovered inserts at pericentric, telomeric, and fourth chromosome regions Previously identified suppressors of PEV suppressed white variegation of pericentric and fourth chromosome inserts but not telomeric inserts on the second and third chromosomes This implies a difference in the mechanism for gene repression at telomeres Heat shock-induced hsp26 expression was reduced from pericentric and fourth chromosome inserts but not from telomeric inserts Chromatin structure analysis revealed that the variegating inserts showed a reduction in accessibility to restriction enzyme digestion in the hsp26 regulatory region in isolated nuclei Micrococcal nuclease digests showed that pericentric inserts were packaged in a more regular nucleosome array than that observed for euchromatic inserts These data suggest that altered chromatin packaging plays a role in PEV

496 citations

Journal ArticleDOI
TL;DR: In this paper, the authors discuss conserved principles of heterochromatin formation and function using selected examples from studies of a range of eukaryotes, from yeast to human, with an emphasis on insights obtained from unicellular model organisms.
Abstract: Heterochromatin is a key architectural feature of eukaryotic chromosomes, which endows particular genomic domains with specific functional properties. The capacity of heterochromatin to restrain the activity of mobile elements, isolate DNA repair in repetitive regions and ensure accurate chromosome segregation is crucial for maintaining genomic stability. Nucleosomes at heterochromatin regions display histone post-translational modifications that contribute to developmental regulation by restricting lineage-specific gene expression. The mechanisms of heterochromatin establishment and of heterochromatin maintenance are separable and involve the ability of sequence-specific factors bound to nascent transcripts to recruit chromatin-modifying enzymes. Heterochromatin can spread along the chromatin from nucleation sites. The propensity of heterochromatin to promote its own spreading and inheritance is counteracted by inhibitory factors. Because of its importance for chromosome function, heterochromatin has key roles in the pathogenesis of various human diseases. In this Review, we discuss conserved principles of heterochromatin formation and function using selected examples from studies of a range of eukaryotes, from yeast to human, with an emphasis on insights obtained from unicellular model organisms.

476 citations

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Q1. What have the authors contributed in "H3k9me2 genome-wide distribution in the holocentric insect spodoptera frugiperda (lepidoptera: noctuidae)" ?

To identify the localization of heterochromatin in 21 such species the authors performed ChIP-Seq experiments and analyzed the distribution of the 22 heterochromatin marker H3K9me2 in the Sf9 cell line and whole 4th instar larvae ( L4 ) in relation 23 to RNA-Seq data. In both samples the authors measured an enrichment of H3K9me2 at the ( sub ) telomeres, 26 rDNA loci, and satellite DNA sequences, which could represent dispersed centromeric regions. CC-BY-NC-ND 4. 0 International license available under a was not certified by peer review ) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.