interacting proteins, DIM-5 activity can be impacted by
modification of nearby residues providing an additional layer of regulation governing constitutive heterochromatin assembly (Adhvaryu and Selker 2008;
Adhvaryu et al. 2011).
Following DIM-5 action, the resulting
H3K9me3 mark is bound by the chromodomain of HP1 (encoded by heterochromatin protein one, hpo; NCU04017). Like other
components in the DNA methylation pathway,
HP1-deficient strains completely lack DNA
methylation (i.e., these mutants have a Dim
phenotype). To date, three HP1-containing
complexes have been described. HP1 interacts
directly with DIM-2 (NCU02247), a conserved
cytosine methyltransferase enzyme that is
required for all 5
m C in vegetative tissues of N.
crassa (Kouzminova and Selker 2001; Honda
and Selker 2008). A second HP1 complex,
HCHC, is comprised of HP1, chromo-domain
protein-2 (CDP-2; NCU00738), histone deacetylase-1 (HDA-1; NCU01525), and CDP-2- and
HDA1-associating protein (CHAP; NCU01796)
(Honda et al. 2016). The HCHC complex regulates heterochromatin assembly by removing
acetyl groups from lysine residues within histone tails (Honda et al. 2012, 2016); acetyl
marks are considered active (euchromatic)
marks that loosen the DNA-histone interaction
(see Sect. IIA). The combined action of the
HP1-DIM2 complex and the HCHC complex
generates a transcriptionally repressed and
compacted chromatin structure that is characterized at the molecular level by low levels of
histone acetylation and high levels of 5
m C.
Interestingly, by using an in vivo tethering system to ectopically localize individual components of the heterochromatin machinery, it
was found that tethered HP1 can actually
induce H3K9me3 in an HCHC/HDA-1-dependent manner, suggesting deacetylated histone
tails recruit DCDC-dependent catalytic activity
and, more broadly, that some positive feedback
between HCHC and DCDC occurs (Gessaman
and Selker 2017).
A third HP1 complex, called the DNA methylation modulator complex (DMM), functions
to prevent uncontrolled spreading of heterochromatin into adjacent gene sequences
(Honda et al. 2010). The DMM complex
includes HP1, the Jumonji domain protein
DMM-1 (NCU01554) and the DNA-binding
protein DMM-2 (NCU08289) (Honda et al.
2016). Although Jumonji domain proteins are
predicted to be lysine demethylases (KDMs), an
enzymatic activity for DMM-1 has not been
described. Genetic studies highlight the important role of DMM-1 at heterochromatin/
euchromatin boundaries. Mutation of dmm-1
leads to spreading of DNA methylation and
H3 lysine 9 methylation into genes that reside
adjacent to constitutive heterochromatin
domains, which in turn leads to their aberrant
transcriptional repression and severe growth
defects. Thus, this HP1 complex functions to
restrict constitutive heterochromatin to the
correct regions of the genome.
Characterization of another dim mutant,
dim-1 (NCU06484), whose phenotype includes
the hypomethylation of constitutive heterochromatin and hypermethylation of intergenic
euchromatic regions, revealed that positioning
of nucleosomes within heterochromatic regions
is distinct from euchromatin and functionally
important (Klocko et al. 2019). The causative
mutations within the dim-1 strain were identified in the Neurospora homolog of an ATPdependent chromatin remodeler conserved
from yeast (Yta7) (Lombardi et al. 2011) to
humans (ATAD2) (Zou et al. 2007). Using
MNase-seq as described in Sect. IIA above,
nucleosome positioning in wild-type and dim1 strains was examined. Interestingly, heterochromatic nucleosomes of a wild-type Neurospora strain were found to be less well
positioned than their euchromatic counterparts
(Klocko et al. 2019). Peaks of MNase-protected
DNA were broad and short, a classic phenotype
for moving, less well-positioned nucleosomes
(Lai and Pugh 2017), in contrast to the welldefined, high-amplitude nucleosomal peaks
surrounding the transcriptional start sites of
euchromatic genes.
It can be speculated that the underlying
repetitive, AT-rich DNA in heterochromatin
may not be conducive for proper positioning
of histone octamers. Nucleosomes just outside
heterochromatic regions were often wellpositioned and stable, perhaps reflecting a
12
A. J. Courtney et al.
modification of nearby residues providing an additional layer of regulation governing constitutive heterochromatin assembly (Adhvaryu and Selker 2008;
Adhvaryu et al. 2011).
Following DIM-5 action, the resulting
H3K9me3 mark is bound by the chromodomain of HP1 (encoded by heterochromatin protein one, hpo; NCU04017). Like other
components in the DNA methylation pathway,
HP1-deficient strains completely lack DNA
methylation (i.e., these mutants have a Dim
phenotype). To date, three HP1-containing
complexes have been described. HP1 interacts
directly with DIM-2 (NCU02247), a conserved
cytosine methyltransferase enzyme that is
required for all 5
m C in vegetative tissues of N.
crassa (Kouzminova and Selker 2001; Honda
and Selker 2008). A second HP1 complex,
HCHC, is comprised of HP1, chromo-domain
protein-2 (CDP-2; NCU00738), histone deacetylase-1 (HDA-1; NCU01525), and CDP-2- and
HDA1-associating protein (CHAP; NCU01796)
(Honda et al. 2016). The HCHC complex regulates heterochromatin assembly by removing
acetyl groups from lysine residues within histone tails (Honda et al. 2012, 2016); acetyl
marks are considered active (euchromatic)
marks that loosen the DNA-histone interaction
(see Sect. IIA). The combined action of the
HP1-DIM2 complex and the HCHC complex
generates a transcriptionally repressed and
compacted chromatin structure that is characterized at the molecular level by low levels of
histone acetylation and high levels of 5
m C.
Interestingly, by using an in vivo tethering system to ectopically localize individual components of the heterochromatin machinery, it
was found that tethered HP1 can actually
induce H3K9me3 in an HCHC/HDA-1-dependent manner, suggesting deacetylated histone
tails recruit DCDC-dependent catalytic activity
and, more broadly, that some positive feedback
between HCHC and DCDC occurs (Gessaman
and Selker 2017).
A third HP1 complex, called the DNA methylation modulator complex (DMM), functions
to prevent uncontrolled spreading of heterochromatin into adjacent gene sequences
(Honda et al. 2010). The DMM complex
includes HP1, the Jumonji domain protein
DMM-1 (NCU01554) and the DNA-binding
protein DMM-2 (NCU08289) (Honda et al.
2016). Although Jumonji domain proteins are
predicted to be lysine demethylases (KDMs), an
enzymatic activity for DMM-1 has not been
described. Genetic studies highlight the important role of DMM-1 at heterochromatin/
euchromatin boundaries. Mutation of dmm-1
leads to spreading of DNA methylation and
H3 lysine 9 methylation into genes that reside
adjacent to constitutive heterochromatin
domains, which in turn leads to their aberrant
transcriptional repression and severe growth
defects. Thus, this HP1 complex functions to
restrict constitutive heterochromatin to the
correct regions of the genome.
Characterization of another dim mutant,
dim-1 (NCU06484), whose phenotype includes
the hypomethylation of constitutive heterochromatin and hypermethylation of intergenic
euchromatic regions, revealed that positioning
of nucleosomes within heterochromatic regions
is distinct from euchromatin and functionally
important (Klocko et al. 2019). The causative
mutations within the dim-1 strain were identified in the Neurospora homolog of an ATPdependent chromatin remodeler conserved
from yeast (Yta7) (Lombardi et al. 2011) to
humans (ATAD2) (Zou et al. 2007). Using
MNase-seq as described in Sect. IIA above,
nucleosome positioning in wild-type and dim1 strains was examined. Interestingly, heterochromatic nucleosomes of a wild-type Neurospora strain were found to be less well
positioned than their euchromatic counterparts
(Klocko et al. 2019). Peaks of MNase-protected
DNA were broad and short, a classic phenotype
for moving, less well-positioned nucleosomes
(Lai and Pugh 2017), in contrast to the welldefined, high-amplitude nucleosomal peaks
surrounding the transcriptional start sites of
euchromatic genes.
It can be speculated that the underlying
repetitive, AT-rich DNA in heterochromatin
may not be conducive for proper positioning
of histone octamers. Nucleosomes just outside
heterochromatic regions were often wellpositioned and stable, perhaps reflecting a
12
A. J. Courtney et al.