regulatory roles in nearly all nuclear DNAbased processes, including transcription, DNA
repair, DNA replication, and chromosome segregation (Allis and Jenuwein 2016). For the
purposes of this chapter, we will use the terms
euchromatin and heterochromatin to describe
active and silent chromatin compartments,
respectively, but it should be clear that a still
undetermined number of chromatin states exist
within each of these nuclear compartments.
For over three decades, the fungus Neurospora crassa has been an important gene and
concept discovery platform for many aspects
of chromatin structure and function. Work
with N. crassa continues to yield discoveries in
this field with broad relevance to fungi and to
eukaryotes in general. Identifying the structural
and functional relationships within chromatin
domains remains an active area of research in
N. crassa with many open questions that warrant further study.
II. Euchromatin
Most genes in N. crassa are arranged closely
together on the chromosome, with adjacent
genes typically separated by only 500–2000
base pairs of intergenic DNA sequences (Galagan et al. 2003). The vast majority of genes in N.
crassa can be classified as “euchromatic” based
on the absence of conserved heterochromatinassociated modifications (see Sect. III, “Heterochromatin”) (Lewis et al. 2009; Jamieson et al.
2013; Bicocca et al. 2018). Chromatin structure
within the euchromatin compartment of the N.
crassa genome has not been as extensively studied as heterochromatin, but important structural and regulatory information has been
generated from genetic and genomic studies
by a number of research laboratories. For
example, genome-wide approaches have been
applied to examine chromatin accessibility,
nucleosome occupancy, nucleosome positioning, the presence of the conserved histone variant H2A.Z, and several conserved chromatin
modifications. These analyses revealed that
gene promoters and gene coding sequences
reside in strikingly different chromatin environments. Promoters and regulatory regions are
highly accessible and often depleted for nucleosomes. On the other hand, the coding
sequences and most intergenic sequences are
characterized by low DNA accessibility and
high histone occupancy. General features of
chromatin structure in regulatory regions and
gene bodies are discussed separately below.
A. Promoters and Regulatory Regions
A key structural feature of euchromatin is the
presence of “open” or “accessible” regions
corresponding to gene promoters and/or distal
gene regulatory regions (Fig. 1.1a). These open
regions have been identified by mapping nucleosome occupancy using an approach called
MNase-seqMicrococcal nuclease (MNase) (Lai
and Pugh 2017). MNase-seq has been carried
out with N. crassa by several groups (Sancar
et al. 2015; Seymour et al. 2016; Liu et al. 2017;
Klocko et al. 2019). This method first uses
micrococcal nuclease (MNase) to partially
digest chromatin to yield mono-nucleosomes
(a histone octamer and approximately 146 bp
of DNA). The DNA bound by histones is protected from MNase digestion, whereas “free”
linker DNA is degraded. Protein is then
removed and the remaining DNA fragments
are analyzed by high-throughput sequencing.
Subsequent bioinformatic analysis is performed to determine nucleosome occupancy
across the entire genome. Inaccessible regions
with high nucleosome occupancy are identified
by relatively high sequence coverage, whereas
regions that are depleted for nucleosomes (i.e.,
accessible) can be identified based on low
sequence coverage. In addition to occupancy,
this approach can provide information about
the positioning of nucleosomes at specific loci.
Nucleosomes that have a stable position within
a population of nuclei give rise to a clear alternating pattern of high and low sequence coverage corresponding to bound nucleosomes and
linker DNA, respectively (Lai and Pugh 2017).
Inspection of MNase-seq data from N. crassa
reveals a characteristic Nucleosome Free
Region (NFR) near the transcriptional start
site of many genes (Sancar et al. 2015; Seymour
4
A. J. Courtney et al.
repair, DNA replication, and chromosome segregation (Allis and Jenuwein 2016). For the
purposes of this chapter, we will use the terms
euchromatin and heterochromatin to describe
active and silent chromatin compartments,
respectively, but it should be clear that a still
undetermined number of chromatin states exist
within each of these nuclear compartments.
For over three decades, the fungus Neurospora crassa has been an important gene and
concept discovery platform for many aspects
of chromatin structure and function. Work
with N. crassa continues to yield discoveries in
this field with broad relevance to fungi and to
eukaryotes in general. Identifying the structural
and functional relationships within chromatin
domains remains an active area of research in
N. crassa with many open questions that warrant further study.
II. Euchromatin
Most genes in N. crassa are arranged closely
together on the chromosome, with adjacent
genes typically separated by only 500–2000
base pairs of intergenic DNA sequences (Galagan et al. 2003). The vast majority of genes in N.
crassa can be classified as “euchromatic” based
on the absence of conserved heterochromatinassociated modifications (see Sect. III, “Heterochromatin”) (Lewis et al. 2009; Jamieson et al.
2013; Bicocca et al. 2018). Chromatin structure
within the euchromatin compartment of the N.
crassa genome has not been as extensively studied as heterochromatin, but important structural and regulatory information has been
generated from genetic and genomic studies
by a number of research laboratories. For
example, genome-wide approaches have been
applied to examine chromatin accessibility,
nucleosome occupancy, nucleosome positioning, the presence of the conserved histone variant H2A.Z, and several conserved chromatin
modifications. These analyses revealed that
gene promoters and gene coding sequences
reside in strikingly different chromatin environments. Promoters and regulatory regions are
highly accessible and often depleted for nucleosomes. On the other hand, the coding
sequences and most intergenic sequences are
characterized by low DNA accessibility and
high histone occupancy. General features of
chromatin structure in regulatory regions and
gene bodies are discussed separately below.
A. Promoters and Regulatory Regions
A key structural feature of euchromatin is the
presence of “open” or “accessible” regions
corresponding to gene promoters and/or distal
gene regulatory regions (Fig. 1.1a). These open
regions have been identified by mapping nucleosome occupancy using an approach called
MNase-seqMicrococcal nuclease (MNase) (Lai
and Pugh 2017). MNase-seq has been carried
out with N. crassa by several groups (Sancar
et al. 2015; Seymour et al. 2016; Liu et al. 2017;
Klocko et al. 2019). This method first uses
micrococcal nuclease (MNase) to partially
digest chromatin to yield mono-nucleosomes
(a histone octamer and approximately 146 bp
of DNA). The DNA bound by histones is protected from MNase digestion, whereas “free”
linker DNA is degraded. Protein is then
removed and the remaining DNA fragments
are analyzed by high-throughput sequencing.
Subsequent bioinformatic analysis is performed to determine nucleosome occupancy
across the entire genome. Inaccessible regions
with high nucleosome occupancy are identified
by relatively high sequence coverage, whereas
regions that are depleted for nucleosomes (i.e.,
accessible) can be identified based on low
sequence coverage. In addition to occupancy,
this approach can provide information about
the positioning of nucleosomes at specific loci.
Nucleosomes that have a stable position within
a population of nuclei give rise to a clear alternating pattern of high and low sequence coverage corresponding to bound nucleosomes and
linker DNA, respectively (Lai and Pugh 2017).
Inspection of MNase-seq data from N. crassa
reveals a characteristic Nucleosome Free
Region (NFR) near the transcriptional start
site of many genes (Sancar et al. 2015; Seymour
4
A. J. Courtney et al.