More recently, Assay for TransposaseAccessible Chromatin coupled to highthroughput sequencing (ATAC-seq) (Buenrostro et al. 2013) has provided even higher resolution analysis of open chromatin within N.
crassa promoters and regulatory regions (A.R.
Ferraro and Z.A. Lewis, unpublished). Curiously, a subset of N. crassa genes has promoters
that are unusually large (>2000 bp of upstream
intergenic sequence) and rich in accessible
regions. Based on these chromatin accessibility
profiles, these rare genes are likely bound by
multiple transcription factors. It is also interesting to note that this subset of genes is
enriched for regulatory proteins such as transcription factors or signal transduction proteins. One hypothesis is that these relatively
large, hyper-accessible regions seem to resemble evolutionary precursors to metazoan
enhancers.
B. Coding Sequences
In contrast to promoters, gene coding
sequences are largely inaccessible. Nucleosome
mapping by MNase-seq shows high occupancy
of well-positioned nucleosomes within gene
bodies, especially for those nucleosomes immediately adjacent to the transcriptional start site
and the NFR (Sancar et al. 2015; Seymour et al.
2016; Klocko et al. 2019), although the periodicity of nucleosome positioning degrades further from the 5
0 end of genes.
Coding sequences themselves have a
unique pattern of epigenetic marks. Trimethylation of lysine 36 on histone H3 (H3K36me3),
which is generally thought of as a “euchromatic” chromatin modification, has a critical
role in gene bodies and some promoters to
limit DNA accessibility. In S. cerevisiae, this
modification is deposited co-transcriptionally
by the enzyme SET-2, also referred to as
KMT3, using the recommended histone
methyltransferase nomenclature (Allis et al.
2007).
The SET-2 protein contains a canonical SET [Su(var)39, enhancer of zeste, trithorax] catalytic domain typical
of all histone methyltransferases, including SET-1/
KMT2, DIM-5/KMT1, and SET-7/KMT6 enzymes discussed below. SET2/KMT3 enzymes directly interact
with the C-terminal repeats of the RNA polymerase II
(RNAPII) subunit RPB1 (Kizer et al. 2005).
Co-transcriptional deposition of H3K36me3
in yeast gives rise to a characteristic pattern of
H3K36me3 in gene bodies of expressed genes,
with highest levels observed in the 3
0 end of
gene bodies (Krogan et al. 2003). It is thought
that this H3K36me3 prevents reinitiation of the
RNAPII following transcription, as deletion of
set2 in yeast causes aberrant transcription from
cryptic promoters within gene bodies (Carrozza et al. 2005; Keogh et al. 2005; Joshi and
Struhl 2005), consistent with a role for this
histone mark in limiting DNA accessibility.
Further, H3K36me3 recruits a histone deacetylase (Keogh et al. 2005; Joshi and Struhl 2005) to
further compact chromatin, thereby limiting
accessibility of underlying DNA and preventing
transcription initiation outside of promoter
regions.
These roles for methylation of H3K36 may
hold true in N. crassa as well. The N. crassa set2 gene (NCU00269) is required for H3K36me3,
like its yeast homolog, and SET-2-catalyzed
H3K36me3 is also found within expressed N.
crassa genes, suggesting that SET-2 also functions to deposit H3K36me3 during gene transcription in N. crassa (Adhvaryu et al. 2005;
Bicocca et al. 2018). In contrast to yeast, however, virtually all gene bodies in N. crassa contain high levels of H3K36me3 regardless of their
expression level (Bicocca et al. 2018). The differences in H3K36me3 localization patterns
can be explained by the fact that N. crassa
encodes a second H3K36 methyltransferase,
ASH1 (NCU01932), that dimethylates K36 in
histone H3 within the gene bodies and promoters of repressed genes (Bicocca et al. 2018).
H3K36me2 deposited by ASH1 can apparently
be converted to H3K36me3 by SET-2 through a
transcription-independent mechanism to elicit
the full H3K36me3 across all gene bodies;
these data suggest the N. crassa SET-2/KMT3
enzymes have a “roaming activity” to convert
dimethyl H3K36 to trimethyl H3K36, even at
repressed genes. Dimethylation of H3K36 by
ASH1, and the subsequent conversion to
8
A. J. Courtney et al.
crassa promoters and regulatory regions (A.R.
Ferraro and Z.A. Lewis, unpublished). Curiously, a subset of N. crassa genes has promoters
that are unusually large (>2000 bp of upstream
intergenic sequence) and rich in accessible
regions. Based on these chromatin accessibility
profiles, these rare genes are likely bound by
multiple transcription factors. It is also interesting to note that this subset of genes is
enriched for regulatory proteins such as transcription factors or signal transduction proteins. One hypothesis is that these relatively
large, hyper-accessible regions seem to resemble evolutionary precursors to metazoan
enhancers.
B. Coding Sequences
In contrast to promoters, gene coding
sequences are largely inaccessible. Nucleosome
mapping by MNase-seq shows high occupancy
of well-positioned nucleosomes within gene
bodies, especially for those nucleosomes immediately adjacent to the transcriptional start site
and the NFR (Sancar et al. 2015; Seymour et al.
2016; Klocko et al. 2019), although the periodicity of nucleosome positioning degrades further from the 5
0 end of genes.
Coding sequences themselves have a
unique pattern of epigenetic marks. Trimethylation of lysine 36 on histone H3 (H3K36me3),
which is generally thought of as a “euchromatic” chromatin modification, has a critical
role in gene bodies and some promoters to
limit DNA accessibility. In S. cerevisiae, this
modification is deposited co-transcriptionally
by the enzyme SET-2, also referred to as
KMT3, using the recommended histone
methyltransferase nomenclature (Allis et al.
2007).
The SET-2 protein contains a canonical SET [Su(var)39, enhancer of zeste, trithorax] catalytic domain typical
of all histone methyltransferases, including SET-1/
KMT2, DIM-5/KMT1, and SET-7/KMT6 enzymes discussed below. SET2/KMT3 enzymes directly interact
with the C-terminal repeats of the RNA polymerase II
(RNAPII) subunit RPB1 (Kizer et al. 2005).
Co-transcriptional deposition of H3K36me3
in yeast gives rise to a characteristic pattern of
H3K36me3 in gene bodies of expressed genes,
with highest levels observed in the 3
0 end of
gene bodies (Krogan et al. 2003). It is thought
that this H3K36me3 prevents reinitiation of the
RNAPII following transcription, as deletion of
set2 in yeast causes aberrant transcription from
cryptic promoters within gene bodies (Carrozza et al. 2005; Keogh et al. 2005; Joshi and
Struhl 2005), consistent with a role for this
histone mark in limiting DNA accessibility.
Further, H3K36me3 recruits a histone deacetylase (Keogh et al. 2005; Joshi and Struhl 2005) to
further compact chromatin, thereby limiting
accessibility of underlying DNA and preventing
transcription initiation outside of promoter
regions.
These roles for methylation of H3K36 may
hold true in N. crassa as well. The N. crassa set2 gene (NCU00269) is required for H3K36me3,
like its yeast homolog, and SET-2-catalyzed
H3K36me3 is also found within expressed N.
crassa genes, suggesting that SET-2 also functions to deposit H3K36me3 during gene transcription in N. crassa (Adhvaryu et al. 2005;
Bicocca et al. 2018). In contrast to yeast, however, virtually all gene bodies in N. crassa contain high levels of H3K36me3 regardless of their
expression level (Bicocca et al. 2018). The differences in H3K36me3 localization patterns
can be explained by the fact that N. crassa
encodes a second H3K36 methyltransferase,
ASH1 (NCU01932), that dimethylates K36 in
histone H3 within the gene bodies and promoters of repressed genes (Bicocca et al. 2018).
H3K36me2 deposited by ASH1 can apparently
be converted to H3K36me3 by SET-2 through a
transcription-independent mechanism to elicit
the full H3K36me3 across all gene bodies;
these data suggest the N. crassa SET-2/KMT3
enzymes have a “roaming activity” to convert
dimethyl H3K36 to trimethyl H3K36, even at
repressed genes. Dimethylation of H3K36 by
ASH1, and the subsequent conversion to
8
A. J. Courtney et al.