and interchromosomal contacts: the chromosomal territories of near-exclusive intrachromosomal interactions that dominate the
human Hi-C heatmaps are less prevalent in
Neurospora (Galazka et al. 2016). Strong longrange interchromosomal contacts between the
seven centromeres or between the 14 telomeres
are readily observed, confirming microscopic
observations of centromere or telomere clustering and the Rabl organization pattern of Neurospora chromosomes (Galazka et al. 2016).
Interestingly, strong interactions between heterochromatin dominate the organizational
landscape. Silent DNA marked by H3K9me3,
or to a lesser extent H3K27me2/3, strongly
interacts to form a “heterochromatic bundle”
of intrachromosomal and interchromosomal
interactions; few paired interactions between
heterochromatin and euchromatin are
observed (Galazka et al. 2016). On a single
chromosome, interactions between interspersed heterochromatic regions marked by
H3K9me3 often form the base of euchromatin
loops (Galazka et al. 2016). These loops of
active DNA, which average < 100 kilobases in
size, may play a structural role for genome
compaction or may be essential for optimal
transcription induction. Surprisingly, deletion
of the genes encoding the H3K9 methyltransferase DIM-5 or its cognate binding partner
HP1 has little impact on the genome organization, as the heterochromatin bundle still forms
in their absence. Hi-C datasets of Ddim-5 and
Dhpo strains only have decreased centromereflank interactions and reduced packaging of
interspersed heterochromatic regions (Galazka
et al. 2016). Only a dim-3 strain encoding a
neomorphic importin-a allele that compromises, but does not abolish, H3K9me3 and
cytosine methylation levels (Klocko et al.
2015) shows any drastic differences in genome
organization. As the average volume of nuclei is
significantly larger with defective importin a,
sub-telomeres have a greater tendency to dissociate from the nuclear membrane, compromising the normal folding of chromosomes, as HiC of a dim-3 strain shows increased centromere—euchromatin
interactions
and
decreased intra- and inter-telomeric contacts
(Galazka et al. 2016). Thus, noncanonical proteins can have a profound yet potentially indirect impact on the Neurospora genome
architecture.
B. ERI1-Directed Facultative Heterochromatin
In contrast to constitutive heterochromatin,
facultative heterochromatin assembles on gene
sequences under certain conditions and may
contribute to gene regulation (Trojer and Reinberg 2007). Based on genome-wide analysis of
DNA methylation and H3K9me3 by microarray
and
high-throughput
sequencing-based
approaches, it was initially concluded that
both modifications were restricted to repeatrich constitutive heterochromatin domains
and therefore were not associated with facultative heterochromatin (Lewis et al. 2009). However, subsequent studies have identified low
levels of DNA methylation and H3K9me3 at
certain euchromatic sequences in the N. crassa
genome. This was first observed at the frq locus
of N. crassa, when DNA methylation was
detected in the promoter by Southern blotting
(Belden et al. 2011). Interestingly, inspection of
the data revealed that most copies of frq are
unmethylated, while a fraction of nuclei exhibit
heavy DNA methylation in which most Cs are
methylated. High-throughput sequencing of
small RNAs revealed a second unusual feature
of the frq locus. High levels of small RNAs were
produced from the frq promoter region on both
sense and antisense strands and were processed
via a Dicer-independent mechanism (Lee et al.
2010). Together, these observations raised the
intriguing possibility that small RNAs could
direct DNA methylation, H3K9 methylation,
or both modifications to these regions. This
idea was particularly attractive given the wellestablished links between small RNAs and
H3K9 methylation in Schizosaccharomyces
pombe and the existence of the extensively
studied RNA-directed DNA methylation pathway in Arabidopsis thaliana (Martienssen and
Moazed 2015; Wendte and Pikaard 2017).
In addition to frq, production of Dicerindependent small-interfering RNAs (disiRNAs) from other loci is reportedly correlated
with low levels of DNA methylation (Dang et al.
1 Chromatin Structure and Function in Neurospora crassa
15
human Hi-C heatmaps are less prevalent in
Neurospora (Galazka et al. 2016). Strong longrange interchromosomal contacts between the
seven centromeres or between the 14 telomeres
are readily observed, confirming microscopic
observations of centromere or telomere clustering and the Rabl organization pattern of Neurospora chromosomes (Galazka et al. 2016).
Interestingly, strong interactions between heterochromatin dominate the organizational
landscape. Silent DNA marked by H3K9me3,
or to a lesser extent H3K27me2/3, strongly
interacts to form a “heterochromatic bundle”
of intrachromosomal and interchromosomal
interactions; few paired interactions between
heterochromatin and euchromatin are
observed (Galazka et al. 2016). On a single
chromosome, interactions between interspersed heterochromatic regions marked by
H3K9me3 often form the base of euchromatin
loops (Galazka et al. 2016). These loops of
active DNA, which average < 100 kilobases in
size, may play a structural role for genome
compaction or may be essential for optimal
transcription induction. Surprisingly, deletion
of the genes encoding the H3K9 methyltransferase DIM-5 or its cognate binding partner
HP1 has little impact on the genome organization, as the heterochromatin bundle still forms
in their absence. Hi-C datasets of Ddim-5 and
Dhpo strains only have decreased centromereflank interactions and reduced packaging of
interspersed heterochromatic regions (Galazka
et al. 2016). Only a dim-3 strain encoding a
neomorphic importin-a allele that compromises, but does not abolish, H3K9me3 and
cytosine methylation levels (Klocko et al.
2015) shows any drastic differences in genome
organization. As the average volume of nuclei is
significantly larger with defective importin a,
sub-telomeres have a greater tendency to dissociate from the nuclear membrane, compromising the normal folding of chromosomes, as HiC of a dim-3 strain shows increased centromere—euchromatin
interactions
and
decreased intra- and inter-telomeric contacts
(Galazka et al. 2016). Thus, noncanonical proteins can have a profound yet potentially indirect impact on the Neurospora genome
architecture.
B. ERI1-Directed Facultative Heterochromatin
In contrast to constitutive heterochromatin,
facultative heterochromatin assembles on gene
sequences under certain conditions and may
contribute to gene regulation (Trojer and Reinberg 2007). Based on genome-wide analysis of
DNA methylation and H3K9me3 by microarray
and
high-throughput
sequencing-based
approaches, it was initially concluded that
both modifications were restricted to repeatrich constitutive heterochromatin domains
and therefore were not associated with facultative heterochromatin (Lewis et al. 2009). However, subsequent studies have identified low
levels of DNA methylation and H3K9me3 at
certain euchromatic sequences in the N. crassa
genome. This was first observed at the frq locus
of N. crassa, when DNA methylation was
detected in the promoter by Southern blotting
(Belden et al. 2011). Interestingly, inspection of
the data revealed that most copies of frq are
unmethylated, while a fraction of nuclei exhibit
heavy DNA methylation in which most Cs are
methylated. High-throughput sequencing of
small RNAs revealed a second unusual feature
of the frq locus. High levels of small RNAs were
produced from the frq promoter region on both
sense and antisense strands and were processed
via a Dicer-independent mechanism (Lee et al.
2010). Together, these observations raised the
intriguing possibility that small RNAs could
direct DNA methylation, H3K9 methylation,
or both modifications to these regions. This
idea was particularly attractive given the wellestablished links between small RNAs and
H3K9 methylation in Schizosaccharomyces
pombe and the existence of the extensively
studied RNA-directed DNA methylation pathway in Arabidopsis thaliana (Martienssen and
Moazed 2015; Wendte and Pikaard 2017).
In addition to frq, production of Dicerindependent small-interfering RNAs (disiRNAs) from other loci is reportedly correlated
with low levels of DNA methylation (Dang et al.
1 Chromatin Structure and Function in Neurospora crassa
15