Proper assembly of constitutive heterochromatin domains is also needed for genome
stability. Deletion mutants that lack HP1 or the
H3K9 methyltransferase DIM-5 displays defects
in centromere structure (Smith et al. 2011),
which leads to defects in chromosome segregation, as DCDC mutants have increased segregation defects including chromosome bridges
(Lewis et al. 2010a). These heterochromatindefective mutants display other signs of
genome instability as well, including elevated
levels of ɣH2A and high sensitivity to DNAdamaging agents (Sasaki et al. 2014; Basenko
et al. 2015). It is not entirely clear why hpo and
dim-5 strains display increased sensitivity to
DNA-damaging agents, but this is at least partially caused by mis-localization of the repressive chromatin modifications dimethyl and
trimethyl H3K27 (H3K27me2/3). In the absence
of DIM-5 or HP1, H3K27me2/3 aberrantly
accumulates in genomic regions that are typically assembled into constitutive heterochromatin (see Sect. IIIC). Why mis-localization of
H3K27me2/3 leads to genotoxic stress is
unknown, but one reasonable hypothesis is
that heterochromatin structure is improperly
regulated during S-phase. It may also be related
to misregulation of catalase-3 expression
(Wang et al. 2016).
Recent work suggests that constitutive heterochromatin may play a key role in genome
organization within the three-dimensional
space of the nucleus. The Neurospora genome
is non-stochastically organized within the
nucleus; this organization is similar to other
eukaryotes but has properties specific to the
unique composition of Neurospora DNA. The
first evidence of the 3D architecture of Neurospora was gleaned from electron microscopy of
Neurospora nuclei, which showed contrasting
light and dark stained material, which we
now realize comprise active euchromatic DNA
and silent heterochromatic DNA, respectively
(Shatkin and Tatum 1959). Much of the heterochromatin was observed to associate with the
nuclear membrane. Further cytological experiments using fluorescently tagged centromeric
and telomeric proteins showed clustering of
these genomic structures. GFP fusions to a centromeric protein showed that all seven centromeres cluster into a single focus (Smith et al.
2011), while GFP fusions with proteins that
form the telomere protection complex shelterin
showed that the 14 telomeres cluster into two to
four foci (Galazka et al. 2016). Interestingly,
both centromeric and telomeric foci are in
close proximity to the nuclear membrane,
which speaks to their heterochromatic nature.
This chromosome organization is consistent
with what had been observed by Rabl (Rabl
1885) and what has now become known as the
Rabl chromosome configuration: clusters of
centromeres and telomeres at the nuclear membrane are essential for organizing eukaryotic
nuclear DNA. Despite the characterization of
chromosomal structure, little information has
been gleaned about the interactions of individual loci from cytological studies.
Recent technological advances have
enabled the genome-wide analysis of chromosomal contacts across the Neurospora genome,
revealing important aspects of threedimensional (3D) chromosome structure
within the nucleus. The extremely powerful
protocol chromosome conformation capture
(3C) monitors the interactions between genomic loci: in vivo chromatin is nonspecifically
cross-linked, digested with a restriction
enzyme, and ligated, thus covalently linking
interacting genomic loci into a single DNA
molecule (Dekker et al. 2002). When 3C is coupled with high-throughput sequencing (Hi-C)
(Lieberman-Aiden et al. 2009), it became possible to monitor the long-range contacts of every
locus in the genome, with the only limitations
being the density of restriction enzyme sites
and sequencing depth. Two noteworthy publications detailed the genome organization of
wild-type N. crassa and initially characterized
the epigenetic factors that contribute to proper
3D genome architecture (Galazka et al. 2016;
Klocko et al. 2016). Importantly, due to the
action of RIP, the degenerate nature of
heterochromatin-associated repeats in N.
crassa allows for contacts across the whole
genome to be analyzed, including sequences
found in silent heterochromatin. This is not
possible in other organisms.
The seven chromosomes of the Neurospora
genome form both strong intrachromosomal
14
A. J. Courtney et al.
stability. Deletion mutants that lack HP1 or the
H3K9 methyltransferase DIM-5 displays defects
in centromere structure (Smith et al. 2011),
which leads to defects in chromosome segregation, as DCDC mutants have increased segregation defects including chromosome bridges
(Lewis et al. 2010a). These heterochromatindefective mutants display other signs of
genome instability as well, including elevated
levels of ɣH2A and high sensitivity to DNAdamaging agents (Sasaki et al. 2014; Basenko
et al. 2015). It is not entirely clear why hpo and
dim-5 strains display increased sensitivity to
DNA-damaging agents, but this is at least partially caused by mis-localization of the repressive chromatin modifications dimethyl and
trimethyl H3K27 (H3K27me2/3). In the absence
of DIM-5 or HP1, H3K27me2/3 aberrantly
accumulates in genomic regions that are typically assembled into constitutive heterochromatin (see Sect. IIIC). Why mis-localization of
H3K27me2/3 leads to genotoxic stress is
unknown, but one reasonable hypothesis is
that heterochromatin structure is improperly
regulated during S-phase. It may also be related
to misregulation of catalase-3 expression
(Wang et al. 2016).
Recent work suggests that constitutive heterochromatin may play a key role in genome
organization within the three-dimensional
space of the nucleus. The Neurospora genome
is non-stochastically organized within the
nucleus; this organization is similar to other
eukaryotes but has properties specific to the
unique composition of Neurospora DNA. The
first evidence of the 3D architecture of Neurospora was gleaned from electron microscopy of
Neurospora nuclei, which showed contrasting
light and dark stained material, which we
now realize comprise active euchromatic DNA
and silent heterochromatic DNA, respectively
(Shatkin and Tatum 1959). Much of the heterochromatin was observed to associate with the
nuclear membrane. Further cytological experiments using fluorescently tagged centromeric
and telomeric proteins showed clustering of
these genomic structures. GFP fusions to a centromeric protein showed that all seven centromeres cluster into a single focus (Smith et al.
2011), while GFP fusions with proteins that
form the telomere protection complex shelterin
showed that the 14 telomeres cluster into two to
four foci (Galazka et al. 2016). Interestingly,
both centromeric and telomeric foci are in
close proximity to the nuclear membrane,
which speaks to their heterochromatic nature.
This chromosome organization is consistent
with what had been observed by Rabl (Rabl
1885) and what has now become known as the
Rabl chromosome configuration: clusters of
centromeres and telomeres at the nuclear membrane are essential for organizing eukaryotic
nuclear DNA. Despite the characterization of
chromosomal structure, little information has
been gleaned about the interactions of individual loci from cytological studies.
Recent technological advances have
enabled the genome-wide analysis of chromosomal contacts across the Neurospora genome,
revealing important aspects of threedimensional (3D) chromosome structure
within the nucleus. The extremely powerful
protocol chromosome conformation capture
(3C) monitors the interactions between genomic loci: in vivo chromatin is nonspecifically
cross-linked, digested with a restriction
enzyme, and ligated, thus covalently linking
interacting genomic loci into a single DNA
molecule (Dekker et al. 2002). When 3C is coupled with high-throughput sequencing (Hi-C)
(Lieberman-Aiden et al. 2009), it became possible to monitor the long-range contacts of every
locus in the genome, with the only limitations
being the density of restriction enzyme sites
and sequencing depth. Two noteworthy publications detailed the genome organization of
wild-type N. crassa and initially characterized
the epigenetic factors that contribute to proper
3D genome architecture (Galazka et al. 2016;
Klocko et al. 2016). Importantly, due to the
action of RIP, the degenerate nature of
heterochromatin-associated repeats in N.
crassa allows for contacts across the whole
genome to be analyzed, including sequences
found in silent heterochromatin. This is not
possible in other organisms.
The seven chromosomes of the Neurospora
genome form both strong intrachromosomal
14
A. J. Courtney et al.