local binding site for complexes that manage
heterochromatin borders. Interestingly, the
periodicity of nucleosomes in heterochromatin
was antiparallel to cytosine methylation enrichment (Klocko et al. 2019), suggesting regions of
DNA not protected by nucleosomes (i.e., linker
DNA) were more apt to contain DNA methylation. In support, a deletion strain lacking histone H1, which is thought to bind linker DNA,
exhibited increased methylation at typical constitutive heterochromatin domains (Seymour
et al. 2016). The loss of cytosine methylation
within a dim-1 strain could be explained by
the nucleosome disorder that was observed.
Heterochromatic nucleosome positioning was
exacerbated upon inactivation of the DIM-1
chromatin remodeler—any positioning of
nucleosomes was essentially abolished and
linker DNA availability for methylation was
thus reduced (Klocko et al. 2019). This same
nucleosome disorder was observed at intergenic euchromatic regions gaining cytosine
methylation in a Ddim-1 strain, leading to speculation that disordered nucleosomes may be a
signal for heterochromatin formation.
Heterochromatin domains in N. crassa are
also enriched for a phosphorylated form of
H2A, referred to as ɣH2A (Sasaki et al. 2014).
In both Saccharomyces cerevisiae and humans,
phosphorylation of a C-terminal serine on H2A
or H2A.X, respectively, to yield ɣH2A occurs
typically at sites of DNA damage, where it is
contributes to proper DNA repair (Chambers
and Downs 2007; Dickey et al. 2009). Thus,
one can speculate the role of ɣH2A in Neurospora
heterochromatin is to maintain the integrity of the
underlying A/T-rich DNA. In S. cerevisiae, ɣH2A
is deposited at sites of DNA damage by the Tel1
and Mec1 kinases (homologs of human ATM and
ATR) (Downs et al. 2000), but the enzyme that
phosphorylates H2A to yield ɣH2A in N. crassa
heterochromatin domains is unknown.
Despite several decades of work, the
mechanisms that control assembly of constitutive heterochromatin remain poorly understood. In the fission yeast Schizosaccharomyces
pombe, small interfering RNAs are important
for directing H3K9 methylation to the appropriate regions of the genome (Martienssen and
Moazed 2015). In N. crassa, however, the RNA
interference pathway is not required (Freitag
et al. 2004b). In contrast, the ectopic introduction of A/T-rich DNA is sufficient to direct 5
m C
and presumably heterochromatin formation
(Miao et al. 2000; Tamaru and Selker 2003),
but how this occurs is unknown. In addition,
repeated DNA (i.e., paired repeated sequences)
may play an important role in directing
assembly of constitutive heterochromatin. A
tandem array of the euchromatic albino-1
gene generated by transformation was associated with H3K9me3, and a synthetic
sequence duplication was subjected to DIM2-dependent RIP during the sexual cycle
(Chicas et al. 2005; Gladyshev and Kleckner
2017). In both cases, the DNA sequences
could recruit components of the constitutive
heterochromatin machinery even though
they were not A/T-rich. Together, these
observations support the idea that multiple,
redundant, or partially redundant mechanisms regulate assembly of heterochromatin
domains. Future studies are needed to uncover
these regulatory mechanisms.
2. Functions of Constitutive Heterochromatin
In N. crassa, heterochromatin domains are
devoid of genes, yet it is clear that proper
assembly of heterochromatin is important for
genome function. Indeed, mutants that lack key
components of the constitutive heterochromatin pathway exhibit severe growth defects (e.g.,
dim-5, hpo, and others). The most obvious
function for constitutive heterochromatin is to
defend the genome against transposable elements. The presence of cytosine methylation
in DNA was shown to inhibit transcription
elongation by RNA polymerase II in vivo leading to transcriptional repression of the methylated sequence (Rountree and Selker 1997). Due
to the action of RIP, N. crassa lab strains contain only a single active DNA transposon typically present as a single copy (Wang et al. 2015).
Functional retrotransposons are present in
some wild isolates, however, and the DNA
methyltransferase DIM-2 is required to suppress mobilization of active Tad elements
(Zhou et al. 2001).
1 Chromatin Structure and Function in Neurospora crassa
13
heterochromatin borders. Interestingly, the
periodicity of nucleosomes in heterochromatin
was antiparallel to cytosine methylation enrichment (Klocko et al. 2019), suggesting regions of
DNA not protected by nucleosomes (i.e., linker
DNA) were more apt to contain DNA methylation. In support, a deletion strain lacking histone H1, which is thought to bind linker DNA,
exhibited increased methylation at typical constitutive heterochromatin domains (Seymour
et al. 2016). The loss of cytosine methylation
within a dim-1 strain could be explained by
the nucleosome disorder that was observed.
Heterochromatic nucleosome positioning was
exacerbated upon inactivation of the DIM-1
chromatin remodeler—any positioning of
nucleosomes was essentially abolished and
linker DNA availability for methylation was
thus reduced (Klocko et al. 2019). This same
nucleosome disorder was observed at intergenic euchromatic regions gaining cytosine
methylation in a Ddim-1 strain, leading to speculation that disordered nucleosomes may be a
signal for heterochromatin formation.
Heterochromatin domains in N. crassa are
also enriched for a phosphorylated form of
H2A, referred to as ɣH2A (Sasaki et al. 2014).
In both Saccharomyces cerevisiae and humans,
phosphorylation of a C-terminal serine on H2A
or H2A.X, respectively, to yield ɣH2A occurs
typically at sites of DNA damage, where it is
contributes to proper DNA repair (Chambers
and Downs 2007; Dickey et al. 2009). Thus,
one can speculate the role of ɣH2A in Neurospora
heterochromatin is to maintain the integrity of the
underlying A/T-rich DNA. In S. cerevisiae, ɣH2A
is deposited at sites of DNA damage by the Tel1
and Mec1 kinases (homologs of human ATM and
ATR) (Downs et al. 2000), but the enzyme that
phosphorylates H2A to yield ɣH2A in N. crassa
heterochromatin domains is unknown.
Despite several decades of work, the
mechanisms that control assembly of constitutive heterochromatin remain poorly understood. In the fission yeast Schizosaccharomyces
pombe, small interfering RNAs are important
for directing H3K9 methylation to the appropriate regions of the genome (Martienssen and
Moazed 2015). In N. crassa, however, the RNA
interference pathway is not required (Freitag
et al. 2004b). In contrast, the ectopic introduction of A/T-rich DNA is sufficient to direct 5
m C
and presumably heterochromatin formation
(Miao et al. 2000; Tamaru and Selker 2003),
but how this occurs is unknown. In addition,
repeated DNA (i.e., paired repeated sequences)
may play an important role in directing
assembly of constitutive heterochromatin. A
tandem array of the euchromatic albino-1
gene generated by transformation was associated with H3K9me3, and a synthetic
sequence duplication was subjected to DIM2-dependent RIP during the sexual cycle
(Chicas et al. 2005; Gladyshev and Kleckner
2017). In both cases, the DNA sequences
could recruit components of the constitutive
heterochromatin machinery even though
they were not A/T-rich. Together, these
observations support the idea that multiple,
redundant, or partially redundant mechanisms regulate assembly of heterochromatin
domains. Future studies are needed to uncover
these regulatory mechanisms.
2. Functions of Constitutive Heterochromatin
In N. crassa, heterochromatin domains are
devoid of genes, yet it is clear that proper
assembly of heterochromatin is important for
genome function. Indeed, mutants that lack key
components of the constitutive heterochromatin pathway exhibit severe growth defects (e.g.,
dim-5, hpo, and others). The most obvious
function for constitutive heterochromatin is to
defend the genome against transposable elements. The presence of cytosine methylation
in DNA was shown to inhibit transcription
elongation by RNA polymerase II in vivo leading to transcriptional repression of the methylated sequence (Rountree and Selker 1997). Due
to the action of RIP, N. crassa lab strains contain only a single active DNA transposon typically present as a single copy (Wang et al. 2015).
Functional retrotransposons are present in
some wild isolates, however, and the DNA
methyltransferase DIM-2 is required to suppress mobilization of active Tad elements
(Zhou et al. 2001).
1 Chromatin Structure and Function in Neurospora crassa
13