Figure 1: A) ​Venn diagram showing overlap between euchromatic TEs and heterochromatic TEs

defined in Papareddy et al (2020) with TEs that are CHH hypomethylated in ​drm1​/​drm2 ​or cmt2
​ troud et al. 2013)​. ​P value < 0.0001 obtained from Fisher's exact
mutant leafs respectively (S
test by comparing overlap of TEs classifications relative to genomic background was
repressednted by ****. ​B) ​Boxplots of CHH, CHG and CG methylation levels in leaves (lf),
floral buds (fb), microspore (msp)​(Calarco et al. 2012)​, meiocyte (mio)​(Walker et al. 2018)​,
 sperm (sp)​(Ibarra et al. 2012) and late torpedo-mature green embryos (lt-mg) (​Ibarra et al. 2012)​.
C) Bar plot representing enrichment of heterochromatic vs euromatic TE methylation difference
between sperm and leaf. Thick horizontal white bars indicate medians, and the top and bottom
edges of the box indicate the 75th and 25th percentiles, respectively. ​D) Boxplot representing
difference in CHH methylation between sperm and leaf in 1kb bins divided into percentiles and
ordered based on their distance to the centromere. ​E) ​Enrichment of H1.1 ​(Choi et al. 2019) ​F)
H3K9me1 and ​G) H3K9me2 ​(Stroud et al. 2014)​. Colored bars represent a range between 75th
and 25th percentiles of distribution. Grey horizontal bars with black dotted ends represent 1.5 x
inter quartile range.

Result 1: Heterochromatic siRNA independent regions are liable to mCHH

reconfiguration during male sexula lineage formation.

● In this section, the idea is to identify regions of genome in which DNA methylation
reprogramming happens under wild type condition.This is important because, if stress or any
other induced DMRs (appears to occur mostly at CHH context) occur in regions of genome that
undergo reprogramming they may not be able to inherit into next generation. But if they happen
to be occurring in regions that are not being reprogrammed then they have a good chance to be
inherited. Because of the lack of base-pair resolution methylome of the egg cell we confined our
analysis to male germline only.

● In the arabidopsis genome, transposable elements (TEs) are kept silenced near
centromeric-pericentromeric regions with high DNA methylation and other repressive chromatin
marks. Symmetric methylation CG and CHG are largely mediated by METHYLTRANSFERASE1
(MET1) and CHROME METHYLTRANSFERASE 3 (CMT3) irrespective of genomic location.
Euchromatic TEs are mainly methylated in RNA directed DNA methylation fashion [REF] , where
RNA polymerase IV (Pol IV)-RNA dependent RNA polymerase 2 (RDR2) co- transcribed
siRNAs.Precursor siRNA diced into 24 mers by DICER LIKE 3 (DCL 3) are loaded in
ARGONAUTE 4 (AGO4) and guide ​DOMAINS REARRANGED METHYLTRANSFERASE 1/2
(DRM1/2) to methylate their target regions. Although DRM1/2 can methylate cytosines
irrespective of their context, CHH (where H ≠ G) is a hallmark for the RdDM pathway. Whereas
Heterochromatic TEs in CHH context are methylated by CMT2 in a Histone 3 Lysine 9
 di-methylation (H3K9me2) dependent pathway and moreover RdDM is inhibited on
heterochromatic TEs somatic tissue.
● T​he process of erasure and reestablishment of epigenetic marks such as Histone modifications
and DNA methylation towards faithful transmission of epigenome through generations is
described as epigenetic reprogramming[REF].In animals, two waves of global erasure and re
establishment of methylation occurs[REF]. In plants, during male gametogenesis whilst
maintaining symmetric mCG and mCHG are robustly, mCHH is hypomethylated methylation.
● Studies from various stress induced DNA methylation changes presented substantial numbers of
differentially methylated regions (DMRs) near TE rich genomic regions at mCHH context.
Therefore we hypothesised that for an induced DMR (particularly hyper CHH) in soma to be
inherited to the next generation it should not undergo reprogramming. Towards this end, we first
sought to investigate methylation dynamics over TEs methylation changes in leaf (somatic
control), floral bud (composite of somatic and reproductive), microspore, meiocytes, sperm and
late embryos.
● Based on genomic location and siRNA dynamics we previously classified TEs into euchromatic
and heterochromatic types (papareddy et al 2020) . Euchromatic TEs that are enriched for
siRNAs and hypomethylated in Pol IV mutant (REF) showed almost 80% significant overlap with
drm1/2 targeted TEs (Fig 1A). Whereas, heterochromatic TEs that are unaffected in Pol IV
mutants showed 87% overlapped (Fig .1A). ​###I will explain in detail later but it’s better to use
the TEs classification I made for my previous paper based on PolIV-siRNA dynamics. Because
transcription, abundance and effects of siRNA has been defined from ten biological stages.
Whereas regularly used TE classification as RdDM and CMT2 is simply based on static leaf
drm1/2 and cmt2 mutants (stroude et al). While extensive analysis on similarities between
stroude et al and my classification was presented in (papareddy et al 2020), a simple
comparison like in the venn diagram(Fig 1A) required to grow readers confidence before we dig
deep#####. ​When next used these euchromatic and heterochromatic TEs to calculated the
weighted cytosine methylation rate.

● CHH methylation loss in sperm compared to somatic leaf tissue is more pronounced over
heterochromatic TEs (Fig 1B and C). In contrast symmetric CG and CHG methylation is gained in
nearly equal proportions on TEs irrespective of their targeting preference (Fig 1B and C).However
the loss of CHH observed in sperm is already reestablished by torpedo stage (REF) and shows
hypermethylation in late torpedo-mature green stage (Fig 1B). CG methylation on both TE types
and CHG methylation on euchromatic TEs are resetted to levels observed in leafs by late
torpedo-mature green stage (Fig 1B).
● Contrary to popular belief that loss of CHH methylation during male secular linea is reestablished
in embryos by siRNA we saw major loss of CHH methylation in heterochromatic TEs relative to
heterochromatic TEs (Fig 1C). Because Heterochromatic TEs are swarmed near centromeric
regions and euchromatic TEs are relative further away we ask whether distance from centromeric
regions alone can explain targeted reprogramming of CHH methylation. Accordingly, we divided
the genome into 1kb bins and ranked them in percentiles based on distance to centromeric
regions (centromeric regions as defined in Qiu, Mei et al 2019 ) . Decrease in sperm CHH
methylation compared to leaf was linear until 2.69 Mb to the centromeric region genomewide and
largely unchanged there after (Fig 1D) . Moreover the reprogrammable CHH methylation during
male gametogenesis is enriched for H3K9me2,H3k9me1 and H1 (Fig 1E-G) further suggesting
that RdDM targeted euchromatic regions largely remains unchanged at least in male germline .
Majority of genes are spread across euchromatic-chromosomal arm regions and RdDM can
regulate gene expression by targeting cis regulators or promotor proximal regions. Therefore
stress or artificially induced methylation gain in euchromatic regions of soma is more likely to be
inherited than those which are around 2.6 MB of deep heterochromatic region.

● Result 2: Are DMRs that didn't make it to the next generation in the
heterochromatic region while that were inherited to the next generation far away
from deep heterochromatin?