tures are absent from major yeast models such
as Saccharomyces cerevisiae and Schizosaccharomyces pombe. Application of powerful genetic
approaches in N. crassa has thus led to several
important discoveries regarding heterochromatin.
A. Constitutive Heterochromatin (RepeatAssociated Heterochromatin)
Constitutive heterochromatin in N. crassa
shares many features with constitutive heterochromatin found in multicellular eukaryotes.
Like plants and animals, N. crassa heterochromatin domains are rich in repeated DNA
sequences, but repeats in N. crassa are degenerate due to the action of repeat-induced point
mutation (RIP), highlighting a key difference
between N. crassa and other systems (Selker
et al. 1987; Selker 2002; Galagan and Selker
2004). RIP is a genome defense system that
introduces C to T mutations in duplicated
DNA sequences during the sexual phase of the
N. crassa life cycle (Selker et al. 1987; Selker
2002; reviewed in Gladyshev 2017) and requires
a DNA methyltransferase homolog RID (RIPdefective, NCU02034) (Freitag et al. 2002).
Approximately 20% of the N. crassa genome
exhibits sequence hallmarks of RIP, and most
of these DNA sequences appear to be nonfunctional transposon sequences (Galagan et al.
2003). These regions make up the constitutive
heterochromatin component of the N. crassa
genome and are easily identified by their high
content of A/T nucleotides relative to gene-rich
euchromatin (Lewis et al. 2009). In addition,
these genomic regions are enriched for chromatin features that are associated with constitutive heterochromatin domains in plants and
animals including 5-methylcytosine (5
m C), histone H3 trimethylated at lysine 9 (H3K9me3),
and heterochromatin protein 1 (HP1;
NCU04017), a conserved chromatin-binding
protein that interacts specifically and
directly with H3K9me3 (Nielsen et al. 2002;
Freitag et al. 2004a; Lewis et al. 2009). Within
the N. crassa genome, heterochromatin
domains can be relatively large, ranging from
~300 bp to over 300 kilobases (kb) in the centromeres. In the case of Linkage Group VII, the
heterochromatic centromere domain is
enriched for both H3K9me3 and the
centromere-specific H3 variant CenH3 and
makes up to approximately 10% of the entire
chromosome (Lewis et al. 2009; Smith et al.
2011).
1. Assembly of Constitutive Heterochromatin
Domains
A great deal of information regarding assembly
of constitutive heterochromatin in N. crassa has
come from genetic studies designed to elucidate
control of cytosine methylation in DNA, which
was first identified in N. crassa within duplicated sequences that were products of RIP
(Selker et al. 1993; Margolin et al. 1998). Isolation of mutants with abolished or reduced
levels of 5
m C, the so-called defective in methylation (dim) mutants, uncovered key genes
whose protein products are needed to assemble
and regulate constitutive heterochromatin in N.
crassa. To date, nine dim mutants have been
identified, although some of these have yet to be
described. Additional information obtained
from proteomics and molecular studies has
led to a detailed working model for assembly
of constitutive heterochromatin domains.
A/T-rich DNA can recruit the H3K9
methyltransferase complex, DCDC (DIM-5,7,9,
CULLIN4, DNA damage-binding protein 1
Complex, named after its confirmed components, gene IDs NCU04402, NCU04152,
NCU01656, NCU00272, and NCU06605)
(Tamaru and Selker 2001; Tamaru et al. 2003;
Zhao et al. 2010; Lewis et al. 2010b, a; Xu et al.
2010). Once recruited, DCDC catalyzes the
covalent addition of three methyl groups to
lysine 9 of H3 (Fig. 1.2a) (Tamaru et al. 2003).
The catalytic subunit of the complex is DIM-5, a SET
domain methyltransferase that belongs to the KMT1
family of enzymes. DIM-5 is homologous with KMT1
family enzymes including Drosophila SuVar3-9 (suppressor of variegation 3–9) and its mammalian homologs SUV39H1 and SUV39H2, as well as the plant
enzyme KRYPTONITE. All of these homologs methylate H3K9 in constitutive heterochromatin domains
(Rea et al. 2000; Jackson et al. 2002; Allis et al. 2007).
DIM-7 is required for proper recruitment of DCDC to
10
A. J. Courtney et al.
as Saccharomyces cerevisiae and Schizosaccharomyces pombe. Application of powerful genetic
approaches in N. crassa has thus led to several
important discoveries regarding heterochromatin.
A. Constitutive Heterochromatin (RepeatAssociated Heterochromatin)
Constitutive heterochromatin in N. crassa
shares many features with constitutive heterochromatin found in multicellular eukaryotes.
Like plants and animals, N. crassa heterochromatin domains are rich in repeated DNA
sequences, but repeats in N. crassa are degenerate due to the action of repeat-induced point
mutation (RIP), highlighting a key difference
between N. crassa and other systems (Selker
et al. 1987; Selker 2002; Galagan and Selker
2004). RIP is a genome defense system that
introduces C to T mutations in duplicated
DNA sequences during the sexual phase of the
N. crassa life cycle (Selker et al. 1987; Selker
2002; reviewed in Gladyshev 2017) and requires
a DNA methyltransferase homolog RID (RIPdefective, NCU02034) (Freitag et al. 2002).
Approximately 20% of the N. crassa genome
exhibits sequence hallmarks of RIP, and most
of these DNA sequences appear to be nonfunctional transposon sequences (Galagan et al.
2003). These regions make up the constitutive
heterochromatin component of the N. crassa
genome and are easily identified by their high
content of A/T nucleotides relative to gene-rich
euchromatin (Lewis et al. 2009). In addition,
these genomic regions are enriched for chromatin features that are associated with constitutive heterochromatin domains in plants and
animals including 5-methylcytosine (5
m C), histone H3 trimethylated at lysine 9 (H3K9me3),
and heterochromatin protein 1 (HP1;
NCU04017), a conserved chromatin-binding
protein that interacts specifically and
directly with H3K9me3 (Nielsen et al. 2002;
Freitag et al. 2004a; Lewis et al. 2009). Within
the N. crassa genome, heterochromatin
domains can be relatively large, ranging from
~300 bp to over 300 kilobases (kb) in the centromeres. In the case of Linkage Group VII, the
heterochromatic centromere domain is
enriched for both H3K9me3 and the
centromere-specific H3 variant CenH3 and
makes up to approximately 10% of the entire
chromosome (Lewis et al. 2009; Smith et al.
2011).
1. Assembly of Constitutive Heterochromatin
Domains
A great deal of information regarding assembly
of constitutive heterochromatin in N. crassa has
come from genetic studies designed to elucidate
control of cytosine methylation in DNA, which
was first identified in N. crassa within duplicated sequences that were products of RIP
(Selker et al. 1993; Margolin et al. 1998). Isolation of mutants with abolished or reduced
levels of 5
m C, the so-called defective in methylation (dim) mutants, uncovered key genes
whose protein products are needed to assemble
and regulate constitutive heterochromatin in N.
crassa. To date, nine dim mutants have been
identified, although some of these have yet to be
described. Additional information obtained
from proteomics and molecular studies has
led to a detailed working model for assembly
of constitutive heterochromatin domains.
A/T-rich DNA can recruit the H3K9
methyltransferase complex, DCDC (DIM-5,7,9,
CULLIN4, DNA damage-binding protein 1
Complex, named after its confirmed components, gene IDs NCU04402, NCU04152,
NCU01656, NCU00272, and NCU06605)
(Tamaru and Selker 2001; Tamaru et al. 2003;
Zhao et al. 2010; Lewis et al. 2010b, a; Xu et al.
2010). Once recruited, DCDC catalyzes the
covalent addition of three methyl groups to
lysine 9 of H3 (Fig. 1.2a) (Tamaru et al. 2003).
The catalytic subunit of the complex is DIM-5, a SET
domain methyltransferase that belongs to the KMT1
family of enzymes. DIM-5 is homologous with KMT1
family enzymes including Drosophila SuVar3-9 (suppressor of variegation 3–9) and its mammalian homologs SUV39H1 and SUV39H2, as well as the plant
enzyme KRYPTONITE. All of these homologs methylate H3K9 in constitutive heterochromatin domains
(Rea et al. 2000; Jackson et al. 2002; Allis et al. 2007).
DIM-7 is required for proper recruitment of DCDC to
10
A. J. Courtney et al.