
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

39229016
10.1101/2024.06.07.597955
preprint
1
Article
A Constitutive Heterochromatic Region Shapes Genome Organization and Impacts Gene Expression in Neurospora crassa
http://orcid.org/0000-0003-2686-0183
Reckard Andrew T. 1
http://orcid.org/0009-0008-7759-9361
Pandeya Abhishek 2*
http://orcid.org/0009-0004-7930-4337
Voris Jacob M. 1*
http://orcid.org/0009-0008-1457-0537
Gonzalez Cruz Carlos G. 1
http://orcid.org/0000-0002-5264-2342
Oluwadare Oluwatosin 2
http://orcid.org/0000-0002-2236-672X
Klocko Andrew D. 1‡
1 Department of Chemistry & Biochemistry, University of Colorado Colorado Springs, Colorado Springs, CO 80918, USA
2 Department of Computer Science, University of Colorado Colorado Springs, Colorado Springs, CO 80918, USA
* Equal Contribution

Author Contributions: A.T.R. and A.D.K. designed research; A.T.R., A.P., J.V., C.G.C, O.O. and A.D.K. performed research; A.T.R., A.P., J.V., C.G.C., O.O. and A.D.K. analyzed data; and A.T.R., A.P., J.V., C.G.C., O.O. and A.D.K. wrote the paper.

‡ Corresponding Author: Andrew D. Klocko, aklocko@uccs.edu
09 6 2024
2024.06.07.597955https://creativecommons.org/licenses/by-nd/4.0/ This work is licensed under a Creative Commons Attribution-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, and only so long as attribution is given to the creator. The license allows for commercial use.
nihpp-2024.06.07.597955.pdf
Genome organization is essential for proper function, including gene expression. In metazoan genome organization, chromatin loops and Topologically Associated Domains (TADs) facilitate local gene clustering, while chromosomes form distinct nuclear territories characterized by compartmentalization of silent heterochromatin at the nuclear periphery and active euchromatin in the nucleus center. A similar hierarchical organization occurs in the fungus Neurospora crassa where its seven chromosomes form a Rabl conformation, where heterochromatic centromeres and telomeres independently cluster at the nuclear membrane, while interspersed heterochromatic loci in Neurospora aggregate across megabases of linear genomic distance for forming TAD-like structures. However, the role of individual heterochromatic loci in normal genome organization and function is unknown. Here, we examined the genome organization of a Neurospora strain harboring a ~47.4 kilobase facultative (temporarily silent) heterochromatic region deletion, as well as the genome organization of a strain deleted of a 110.6 kilobase permanently silent constitutive heterochromatic region. While the facultative heterochromatin deletion had little effect on local chromatin structure, the constitutive heterochromatin deletion altered local TAD-like structures, gene expression, and the predicted 3D genome structure by qualitatively repositioning genes into the nucleus center. Our work elucidates the role of individual heterochromatic regions for genome organization and function.

genome organization
chromosome conformation
heterochromatin
Neurospora crassa
gene expression
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pmcIntroduction

The correct organization of a genome, including proper chromosome folding, is necessary for the function of DNA-templated processes, including gene expression regulation (1–5). Indeed, improper DNA folding altering transcriptional patterns occurs in human cancer cells (6–8). Eukaryotic genomes are hierarchically folded in nuclei, where DNA molecules are wrapped about histone octamers to form chromatin fibers that are extruded into globules (“loops”) by cohesin and compacted into Topologically Associated Domains (TADs) (9–17). TADs of similar transcriptional activity are then compartmentalized into the nucleus (10, 11, 17). Specifically, the more open, gene-rich, and actively transcribed euchromatin primarily localizes to the nucleus center, while the silent, often gene-poor, and compacted heterochromatin is confined to the nuclear periphery (18–20). Heterochromatin segregation may drive chromatin compartmentalization, although transcriptional activity might force silent genomic loci to the nuclear periphery (19). In metazoans, the formation of individual chromosome territories typified by reduced inter-chromosomal contacts leads to the clustering of intra-chromosomal heterochromatic regions (21, 22). In contrast, the independent aggregation of the heterochromatic centromeres and telomeres into the Rabl conformation promotes extensive intra- and inter-chromosomal heterochromatic contacts across fungal chromosomes (9, 23–25). All told, heterochromatin bundling is a conserved feature of eukaryote genome organization.

Fungi are excellent models for studying genome organization. Resembling metazoans, fungal genomes are partitioned into euchromatin and heterochromatin, and the heterochromatin of many filamentous fungi, including Neurospora crassa, is subdivided into permanently silent constitutive heterochromatin and temporarily silent facultative heterochromatin (26–28). The adenine and thymine (AT)-rich, gene-poor, and repetitive constitutive heterochromatic regions, comprising ~16% of the Neurospora genome, are marked by the tri-methylation of lysine 9 on histone H3 (H3K9me3) that is catalyzed by the histone methyltransferase DIM-5 (KMT1) (27, 29–31). Specific H3K9me3-marked regions found within the seven Neurospora chromosomes (“Linkage Groups” [LGs]) include the regional centromeres, the 14 telomeres, and ~200 interspersed heterochromatic regions (27, 31). Neurospora facultative heterochromatin is delineated by the subtelomeric di- or tri-methylation of lysine 27 on histone H3 (H3K27me2/3), which is catalyzed by the SET-7 (KMT6) histone methyltransferase of the Polycomb Repressive Complex 2 (PRC2) (32–34). In the Neurospora three-dimensional (3D) genome organization, as assessed by in nucleus chromosome conformation capture coupled with high-throughput sequencing (in situ Hi-C), robust intra- and inter-chromosomal heterochromatic interactions are readily observed across megabases of linear genomic distance with few heterochromatin-euchromatin contacts, highlighting the compartmentalization of fungal chromatin (9, 23, 24). The clustering of heterochromatic regions forms the anchors of TAD-like structures; similar structures are observed in other ascomycete fungi, suggesting the use of heterochromatin to organize fungal genomic DNA may be conserved (9, 23–25, 35). Further, subtelomeric facultative heterochromatin enriched with H3K27me2/3 mediates the interaction between chromosome ends and the nuclear periphery (36). Together, both heterochromatin types are necessary to organize the Neurospora genome into the Rabl chromosome conformation.

To understand the importance of individual facultative and constitutive heterochromatic regions on 3D genome organization in Neurospora, we performed chromatin-specific in situ Hi-C on a strain harboring a deletion of a ~47.4 kilobase (kb) H3K27me2/3 domain on the LG VI left arm (33), as well as a strain in which we deleted a ~110 kb H3K9me3 domain on the LG II right arm. While the H3K27me2/3 deletion minimally impacts genome organization, the H3K9me3 deletion (“ΔLGIIK9het25”) reorganizes the heterochromatin bundle and alters regional TADs across LG II without affecting deposition of H3K9me3 on nearby AT-rich loci. The 3D structure prediction of individual ΔLGIIK9het25 strain chromosomes is qualitatively different than those of its wild type (WT) counterpart, and numerous genes became upregulated in the ΔLGIIK9het25 strain, possibly due to altered gene positioning within the 3D genome. We conclude that a constitutive heterochromatic region organizes the Neurospora crassa genome for proper fungal genome function.

Results

Loss of a H3K27me2/3-enriched region minimally affects the local chromatin landscape.

Since interactions between regions of similar heterochromatin type comprise many of the strongest intra-chromosomal contacts in the Neurospora genome (23, 24), we examined how deletions of either type, facultative or constitutive heterochromatin, could impact fungal heterochromatin clustering. Starting with facultative heterochromatin, we examined a previously constructed strain, N4933, originally used to assess position-dependent signals for H3K27me2/3 deposition in Neurospora genome (33). N4933 harbors a 47.4 kb deletion of LG VIL, from basepairs 175,121 to 222,557, which removes fourteen H3K27me2/3-enriched genes (“ΔK27”). To assess how ΔK27 impacts genome organization, we performed two in situ Hi-C replicates with DpnII to capture gene-rich chromatin interactions within DNA of higher guanine and cytosine basepair percentages, including those in facultative heterochromatic regions, in the nucleus. Our replicates show highly similar Hi-C contact matrices across LG VI and are highly correlated (Figure S1), allowing replicate merging into a single Hi-C dataset with 9.2 million (M) valid reads. For comparison, we processed an identical number of valid Hi-C reads from our WT in situ DpnII Hi-C dataset (23). Our initial observation is that, despite having nearly identical valid reads, the ΔK27 dataset has a slight bias in capturing more local contacts, most likely due to Hi-C experiment variability, which concomitantly reduces inter-chromosomal contacts. Indeed, decreased inter-centromeric contacts are observed in raw datasets due to fewer centromeric DpnII sites (Figures 1, S2-S3). Inter-centromeric interactions are present upon Knight-Ruiz (KR) (37) correction (Figure S3), indicating capture of biologically relevant structures. Upon directly comparing WT and ΔK27 contact matrices, the decrease in moderate and long-range contacts is evident (Figure S3). Therefore, we focused our analyses on the local LG VIL changes that manifest with the H3K27me2/3 region deletion.

On WT LG VI, we observe consistent off-diagonal contacts that inversely decrease with genomic distance, in addition to strong inter-telomeric contacts and the insulation of centromeric DNA from local euchromatin, in both raw and KR corrected heatmaps (Figure 1A). A higher resolution (5kb) heatmap of the 500 kb surrounding the region deleted in N4933 shows a gradual decrease of contacts from the diagonal (Figure 1A). In contrast, the ΔK27 deletion Hi-C, mapped to the Neurospora reference genome version 14 (“nc14”) (23), appears as a white cross emanating from the deletion locus indicating a gap in mappable Hi-C reads (Figure 1B), since the DNA in this region was not present for nuclear ligation capture. This gap occurs in both the raw and KR corrected contact probability heatmaps, although the base of the diagonal on the KR corrected heatmap has some signal (Figures 1B-C), perhaps due to correctional bias. Importantly, stronger off diagonal signal at the boundary intersections of the facultative heterochromatin deletion appear (Figures 1B-C, black arrowheads), showing how new interactions occur between the H3K27me2/3-enriched chromatin flanking the ΔK27 deletion. Interestingly, another N4933 locus devoid of Hi-C signal is observed over the sly1-1 transposase gene NCU09969 (Figure 1C, gray arrowhead), suggesting the ΔK27 strain had a transposition event. Regarding telomere clustering, the ΔK27 strain is still capable of forming intra- and inter-chromosomal subtelomeric interactions (Figures 1D-E), with strong contacts between chromosome ends originating from the terminal ~100kb chromatin enriched with H3K27me2/3. We conclude a deletion of a H3K27me2/3-enriched region minimally impacts the organization of the remaining LGVIL facultative heterochromatin.

Deletion of a ~110 kb constitutive heterochromatic region alters silent region clustering.

In Neurospora nuclei, gene-poor AT-rich constitutive heterochromatin is compartmentalized from gene-rich euchromatin or facultative heterochromatin (23, 38). To ask if altered constitutive heterochromatin composition could have consequences on genome organization and function, we deleted a 110,609 bp H3K9me3-enriched region on LG IIR, aptly called “LGIIK9het25” as this region is the 25th constitutive heterochromatic region from the LG II left telomere. We chose LGIIK9het25 for deletion for a high probability of observing changes to genome organization by deleting a large AT-rich region without compromising chromosome function by removing the centromere. Using split-marker homologous recombination in a Dmus-52 strain (39), we replaced the 110.6 kb LGIIK9het25 with a hygromycin resistance gene (hygromycin phosphotransferase; hph) controlled by a strong constitutive trpC promoter (a PtrpC::hph construct; Figure S4A). We note a similar-sized DNA replacement could have more accurately represented the LG II chromosome length, but we wanted to avoid cryptic promoters, repetitive sequences for RIP (27, 40, 41), or AT-rich DNA sequences forming novel constitutive heterochromatic regions within the inserted DNA. We successfully obtained hygromycin-resistant primary transformants containing the LGIIK9het25 deletion (DLGIIK9het25); in sum, we removed 109,208 bp from the Neurospora genome, given that PtrpC::hph inserts 1,401 bp. A hygromycin-resistant progeny from a sexual cross, xKL9AR-17, was characterized by PCR analysis as harboring both the DLGIIK9het25 allele (Figure S4B) and a wild type mus-52+ allele. The final strain, NKL2 (genotype: mat A; DLGIIK9het25::hph), had no noticeable growth defect from a WT strain (Figure S4C-D).

We performed chromatin-specific Hi-C (23) using the restriction enzyme DpnII to monitor the contacts of gene-rich euchromatin, while MseI Hi-C captures interactions between AT-rich, gene-poor constitutive heterochromatic regions. Our DpnII and MseI DLGIIK9het25 Hi-C replicates show highly reproducible and correlated interactions (Figure S5), allowing us to merge replicates into enzyme-specific datasets, with the NKL2 DpnII dataset containing 20.6M valid reads and the NKL2 MseI dataset comprising 12.2M valid reads. All Hi-C datasets display the inter-chromosomal centromeric interactions independent of telomere clusters, indicating our Hi-C captured biologically relevant structures (Figures S6-S7). As before, we selected nearly identical valid read numbers from previously published WT DpnII and MseI datasets for comparisons (23).

Compared to a WT DpnII Hi-C dataset (Figure 2A), the deleted LGIIK9het25 region appears as a large white “cross” devoid of Hi-C interactions on LGII in the NKL2 DpnII contact probability matrix (Figure 2B). Looking specifically at a 500 kb region surrounding LGIIK9het25, the WT dataset has extensive interactions within and around LGIIK9het25, while the NKL2 has contact probability loss, as no interactions are observed in the raw interaction heatmap and sparse contacts at the diagonal in the KR corrected dataset (Figure 2A-B), which may be due to a few reads mapping to repetitive DNA sequences at this locus. The Hi-C contacts surrounding DLGIIK9het25 appear slightly enhanced, as the flanking euchromatin moderately gains contacts in the NKL2 DpnII dataset, relative to a WT dataset (Figure 2A-B). Similar contact probability changes occur in WT and NKL2 MseI datasets, where in the WT dataset the LGIIK9het25 region extensively contacts internal heterochromatin yet is isolated from surrounding euchromatin, but the NKL2 dataset completely loses heterochromatic contacts from the LGIIK9het25 region yet gains flanking euchromatin interactions (Figure 2C-D). Direct comparison of the WT and NKL2 DpnII and MseI datasets surrounding LGIIK9het25 shows the contact probability gain between flanking euchromatic regions when a separating heterochromatic region is lost (Figure 2E-F, arrowheads). This surrounding euchromatin may gain local, on-diagonal interactions, as shown in the MseI comparison (Figure 2F), possibly due to either reduced heterochromatic interactions or a local contact capture bias in the NKL2 dataset. Between LG I and LG II, the LGIIK9het25 deletion causes a loss of inter-heterochromatic region interactions, including from the largest non-centromeric heterochromatic region on LG I (Figure 2G, white arrowhead), while the LG II centromere loses inter-centromeric contacts but modestly gains interactions with interspersed heterochromatic regions (Figure 2G, black arrowhead). Similar gains in inter-heterochromatic region interactions are evident across the genome in WT vs. NKL2 MseI Hi-C dataset comparisons (Figure S8), possibly from the absence of LGIIK9het25 contacts influencing euchromatin interactions. As evident in WT vs. NKL2 DpnII comparisons, the inter-chromosomal contact strength between centromere-proximal euchromatin and more-distant euchromatin on chromosome arms is increased, yet chromosome-internal euchromatic contacts are reduced (Figure S9), possibly due to enhanced centromere-interspersed heterochromatin bundling. Similarly, both DLGIIK9het25 Hi-C datasets have depleted inter-centromeric contacts; KR correction reduces DpnII contact biases (Figures S8-S9). All told, the LGIIK9het25 deletion moderately impacts the organization of the entire Neurospora genome.

The LGIIK9het25 deletion minimally alters H3K9me3 deposition but alters TAD-like structures.

Upon deleting LGIIK9het25, the possibility exists that genome function, specifically H3K9me3 deposition on nearby AT-rich loci, may be impacted. To assess if constitutive heterochromatic region loss limits histone post-translational modification enrichment, we performed H3K9me3 Chromatin Immunoprecipitation-sequencing (ChIP-seq) on the NKL2 strain. Our two H3K9me3 ChIP-seq replicates were highly reproducible (Figure S10) and were merged into a single Reads Per Kilobase per Million (RPKM) normalized file for direct comparison to previously published WT H3K9me3 ChIP-seq datasets, although using spike-in DNA controls may be best practice for assessing strain differences (42, 43). We mapped the ChIP-seq reads to either the nc14 WT genome or an “NKL2 reference genome” which replaced the AT-rich DNA of LGIIK9het25 with the PtrpC::hph sequence; enhanced images show low levels of background signal across the PtrpC::hph sequence in an NKL2 H3K9me3 ChIP-seq dataset that are absent in WT H3K9me3 data (Figure S4E).

H3K9me3 ChIP-seq enrichment exhibits similar patterns across LG II, independent of the reference genome used for mapping reads. For ChIP-seq data mapped to the WT nc14 genome, identical H3K9me3-enriched regions are observed, except peaks to the right of LGIIK9het25 are shifted by ~110kb from the deletion site (Figure 3A), given that genomic elements positions within bedgraph/bigwig enrichment count files are shifted based on input bam file read mapping position. Qualitatively, the NKL2 H3K9me3 peaks are higher (Figure 3A), possibly reflecting increased H3K9me3 purification from the remaining constitutive heterochromatic regions or enhanced sequencing depth limiting background signal. When H3K9me3 ChIP-seq data from both strains was mapped to the NKL2 reference genome, the loss of LGIIK9het25 signal from the WT dataset is apparent, while the remaining constitutive heterochromatic regions have identical LG II genomic positions (Figure 3B).

To further assess local chromatin changes with the LGIIK9het25 deletion, we examined Topologically Associated Domain (TAD)-like structure positions on LG IIR. The TAD-like structures in WT and NKL2 DpnII datasets, mapped to the nc14 (WT) genome, show striking differences across euchromatin, with larger TAD-like structures predicted with the ΔLGIIK9het25 allele, as opposed to the smaller TAD-like structures in WT datasets (Figure 4A), although TAD structure prediction can vary (44). To more clearly understand the strain-specific chromatin folding changes without the ΔLGIIK9het25 signal gap, we predicted TAD-like structures WT and NKL2 DpnII and MseI datasets mapped to the NKL2 reference genome. Here, clear changes to TAD-like structures are observed at the deletion site, with the euchromatin in the DpnII dataset having larger TAD-like structures in the NKL2 strain, while the NKL2 ΔLGIIK9het25 MseI strain has smaller TAD-like structures on the diagonal (Figure 4B, black arrowheads), with this difference likely stemming from the chromatin monitored – and the density of restriction sites – in each Hi-C experiment. The flanking TAD-like structures are nearly identical in both datasets, except for two larger TADs-like structures in the NKL2 MseI data (Figure 4B, open arrowheads). Interestingly, at the PtrpC::hph insertion, two novel yet small TAD-like structures are observed in both NKL2 datasets, combined with additional stronger Hi-C interactions in MseI data (Figure 4B, purple line), suggesting the constitutive PtrpC promoter could influence local contacts – and possibly regulate expression – of nearby genes. Overall, the deletion of the constitutive heterochromatic region LGIIK9het25 alters the local folding of the euchromatin surrounding the deletion site.

The LGIIK9het25 deletion alters the three-dimensional folding of Neurospora chromosomes.

It remained possible that a large constitutive heterochromatic region deletion would alter chromosome folding. To assess the 3D folding of LG II, we computationally predicted the LG II 3D structure using WT and NKL2 ΔLGIIK9het25 Hi-C datasets. We chose to merge all DpnII and MseI data together from each strain to maximize the possible Hi-C contacts across their genomes; we did not combine DpnII and MseI data at ratios equivalent to the Neurospora chromatin distribution, as previously performed (23). With the maximal contact probability merged matrices, we iteratively generated LG II 3D structures using 3DMax (45) customized for biologically relevant structures in fungal genomes.

In the WT strain, LG II has an elongated structure with condensed chromosome arms, a more isolated centromere, telomeres localized to a single “side”, and extensive intervening chromatin loops (Figure 5A). Similar 3D structure prediction changes are observed on the other six WT chromosomes (Figure S11). The LGIIK9het25 region forms a loop and associates with nearby chromatin loops (Figure 5A), consistent with the presence of nine, moderately sized heterochromatic regions in the terminal 800 kb of the LG II right arm (Figure 3A). In contrast, the chromosome arms within the LG II 3D structure when LGIIK9het25 is deleted are more compact and form a wider structure, despite similar centromere isolation, telomere locations, and looping of intervening chromatin (Figure 5B). In fact, the terminal right arm of NKL2 LG II does not form any loops in this 3D structure prediction, suggesting that the requirement for telomere clustering impacts the chromosome folding prediction in the NKL2 strain (Figure 5B). Similar folding differences occur on each LG in NKL2, with some chromosomes - specifically LG I and LG IV - having drastically altered 3D structures relative to WT LG folding (Figures S11, S12). Together, the deletion of a ~110kb constitutive heterochromatic region modifies the predicted 3D structures of Neurospora LGs.

The LGIIK9het25 deletion increases gene expression due to altered 3D genome folding.

To understand if constitutive heterochromatin could influence genome function through gene expression, we assessed changes in polyadenylation messenger RNA sequencing (polyA mRNA-seq) in WT and NKL2 ΔLGIIK9het25 strains. As a control for complete loss of constitutive heterochromatin function, we examined gene expression changes in a Δdim-5 strain, which is devoid of H3K9me3 genome-wide (29). Volcano plots that display the significant gene expression changes (log2 > 3.0 or < −3.0; adjusted p-value < 0.001) in the NKL2 strain relative to the WT strain show 84 genes are significantly upregulated and no genes significantly downregulated, (Figure 6A, left; Supplementary File S1), consistent with the removal of a repressive heterochromatic region; additional genes could be considered differentially regulated with less stringent cutoff values. In contrast, the Δdim-5 strain has 175 differentially regulated genes compared to WT, with genes having both increased and decreased transcription (Figure 6A, right; Supplementary File S2), including the strong reduction of the gene NCU04402 encoding DIM-5 (Figure 6B), highlighting our polyA mRNA-seq dataset accuracy. Many Δdim-5 differentially expressed genes associate with altered peaks of H3K27me2/3 (Figure S13), as this facultative heterochromatin mark relocates to constitutive heterochromatic regions in a Δdim-5 strain (46, 47). Unique genes are found in the ΔLGIIK9het25 and Δdim-5 datasets, with only 25 overlapping differentially regulated genes. Notably, ΔLGIIK9het25 causes less drastic expression changes relative to the Δdim-5 polyA mRNA-seq (Figure 6A-B). Further, there are no differentially expressed genes in the ΔLGIIK9het25 strain wtih putative functions in chromatin or transcriptional regulation (Supplementary File S3), although numerous hypothetical genes without defined functions are present, preventing a complete assessment of possible genes involved in regulating transcription. Interestingly, gene expression changes in the ΔLGIIK9het25 strain occur on each chromosome (Figures 6B, S14), with many genes near H3K9me3-marked heterochromatic regions. Specifically, the gene NCU08696, directly adjacent to LGIIK9het25, is downregulated in the Δdim-5 strain but upregulated in ΔLGIIK9het25 (Figure 6B, zoomed region), the latter possibly due to its proximity to the strong PtrpC promoter. While longer chromosomes would be expected to have more differentially expressed genes, the short ~4.3 Mb LG VII has more genes with changed expression (n = 24 differentially expressed genes [DEGs]) than either the ~10 Mb LG I (n = 20 DEGs) or the 6.0 Mb LG IV (n =11 DEGs) (Figures 6B and S14, Supplementary File S2). We conclude that a constitutive heterochromatic region controls gene expression across the fungal genome.

To assess if altered 3D chromosome folding could explain the transcriptional changes in a ΔLGIIK9het25 strain, we colored bins harboring differentially expressed genes on models of NKL2 LGs. Qualitatively, the positions of the bins containing the differentially expressed genes are modified on the 3D chromosome structures in the ΔLGIIK9het25 strain relative to the WT strain (Figures 6C-D, S15, S16). Specifically, on LG II, the bins with differentially expressed genes appear more “enclosed” within the WT structure, while these same bins are repositioned to another “face” of the NKL2 LG II structure (Figure 6C-D). Similar qualitative bin movements altering gene positions are readily observed on other LGs (Figure S16).

Finally, we examined the positions of ΔLGIIK9het25 differentially expressed genes relative to the 3D folding predicted for the WT and NKL2 genomes. Our 3D genome models maintain the biologically relevant centromere cluster independent of the bundling of telomeres (Figure S17). The WT genome model forms an elongated structure with the LGIIK9het25 region close to the structure bottom (Figure 7A), consistent with the “stretched” conformation of individual chromosomes (Figures 5, S15-S16) and a previous report (48), despite different programs predicting each 3D structure. The NKL2 genome deleted of LGIIK9het25 is wider and less elongated (Figure 7B), similar to the individual NKL2 chromosomes. Intriguingly, the 20 kb bins containing the ΔLGIIK9het25 differentially expressed genes are qualitatively oriented towards the WT genome periphery, while the same bins are more centrally localized in the NKL2 genome, consistent with the increased gene expression that occurs with the LGIIK9het25 deletion. We conclude that the loss of a single heterochromatic region alters the 3D conformation of the genome and disrupts normal genome function in the Neurospora nucleus.

Discussion

Here, we examined if facultative and constitutive heterochromatic regions organize the fungal genome by performing Hi-C on strains harboring silent region deletions. Our results suggest that gene-rich facultative heterochromatic loci are plastic in nature, as a facultative heterochromatin deletion simply fuses nearby H3K27me2/3-enriched regions within the linear chromosome, which minimally impacts facultative heterochromatin clustering. Specifically, the subtelomere bundling at the terminal ~150 kb of each LG is maintained in the facultative heterochromatin deletion strain. Subtelomere clustering may be compromised by larger deletions or only deletions targeting the terminal ~150 kb of a chromosome. Consistent with these hypotheses, genome-wide loss of H3K27me2/3 in a Neurospora Δset-7 (Δkmt-6) strain causes subtelomere cluster dissociation from the nuclear periphery (36), suggesting at least some subtelomeric H3K27me2/3 is needed for nuclear membrane association. While larger or telomere-proximal deletions of facultative heterochromatin could assess subtelomere clustering requirements, Neurospora may endure alterations to gene-rich genomic loci, assuming no essential genes are deleted, given the absence of wide-ranging topological or physiological consequences. Since most genes enriched with H3K27me2/3 are novel genes with a hypothetical function (32), Neurospora may tolerate subtelomeric structural variants if facultative heterochromatin is deposited. In contrast, some Fusarium species require H3K27me2/3 for normal growth (49), meaning facultative heterochromatic structural variants may be selected against in Fusarium.

We also assessed the role of a constitutive heterochromatic region for organizing the Neurospora genome. The most recent Neurospora reference genome (nc14) (23) has ~200 interspersed H3K9me3-enriched regions distributed across its seven chromosomes, and apart from the regional centromeres and telomeres of each LG required for chromosome function, the remaining interspersed heterochromatic regions have often been dismissed as “junk DNA”. However, when we delete the largest, non-centromeric heterochromatic region on LG II, genome organization and function are impacted. Specifically, the remaining heterochromatic regions have altered clustering when LGIIK9het25 is deleted, as increases in intra- and inter-chromosomal heterochromatic region contacts are observed, including between interspersed heterochromatic regions and centromeres. These data suggest the Neurospora heterochromatin bundle is dynamic, with no “defined” interactions mediated by specific DNA binding motifs in each heterochromatic region. Thus, heterochromatic region aggregation mediated by Liquid Liquid Phase Separation or a chromatin binding protein, such as Heterochromatin Protein-1 (HP1) (50–52) may allow shifting contacts between constitutive heterochromatic regions. Since HP1 loss does not radically disrupt the Neurospora heterochromatin bundle (24), perhaps a combination of redundant factors drive silent chromatin bundling in fungi. Similarly, HP1-independent pericentromere clustering is observed in Drosophila (53). Future experiments may elucidate additional mechanisms necessary for heterochromatin clustering in eukaryotes.

The LGIIK9het25 deletion also showed changes in the local chromatin structure and the folding of the 3D genome. In a manner synonymous to the deletion of the H3K27me2/3 region, the loss of LGIIK9het25 caused the flanking chromatin to simply “fuse”, as shown by interaction gains between neighboring euchromatin bins. However, removing the intervening permanently silent region causes the predicted TADs within this newly fused chromatin to change – forming a larger TAD-like structure comprised of unique chromatin loops, suggesting the loss of LGIIK9het25 alters the regional chromatin looping. Since interspersed heterochromatic regions in Neurospora and other filamentous fungi act as “anchors” at the bases of TAD-like structures (9, 23–25, 35), in a mechanism tantamount to CTCF forming metazoan loops (10), removing a heterochromatic “boundary element” would allow distant euchromatic regions to interact within a single TAD-like structure. Complicating matters is how fungal TAD-like structures have extensive inter-TAD interactions (23–25), meaning that the non-uniform TAD-like structures in Neurospora may minimize the functional consequences of altering loop structures. Further, it would be expected that TAD-like structures would differentially form on fungal chromosomes with different patterns of interspersed heterochromatic regions. While fungi and metazoans employ different chromatin folding mechanisms, it is possible that a deletion of a metazoan constitutive heterochromatic region would also disrupt TADs by changing the highly isolated TAD-internal interactions, in a similar manner to our observations in fungi. Here, loss of a H3K9me3-enriched region from a metazoan genome could alter TAD compartmentalization or misregulate gene expression if several smaller TADs are merged into a larger TAD. Unfortunately, the consequences to metazoan genome function would be more dire, given how TAD boundary dysfunction compromises proper gene expression (6–8). In fact, our chromosome and genome structure predictions suggest local TAD changes could manifest into a completely unique folding pattern for the overall genome. Here, the ΔLGIIK9het25 deletion caused each chromosome, and the entire genome, to form a conformation qualitatively distinct from a wild type genome structure. While these models are merely predictions and should be interpreted lightly, it is impactful that the repositioning of a single locus has the potential to alter the 3D folding of the whole genome. Thus, despite the species-specific differences in TADs or loops, it is hypothetically possible that syntenic changes of linear chromosomal DNA could alter a species’ genome organization and function.

Importantly, we show a novel function for a constitutive heterochromatic region, as the ΔLGIIK9het25 strain is important for downregulating gene expression in a WT straiin, consistent with the repressive nature of H3K9me3. The gene expression changes we observe when a single constitutive heterochromatic region is deleted are not at the same extent as if all H3K9me3 is removed in a Δdim-5 strain, with numerous genes both strongly up- and down-regulated (36), suggesting the LGIIK9het25 deletion may minorly affect genome function. Overall, this single constitutive heterochromatic region may subtly repress gene expression in the Neurospora genome, which was hypothesized when strong gene – constitutive heterochromatin contacts were observed in a high-resolution Neurospora Hi-C dataset (23). However, it is difficult to imagine that the single LGIIK9het25 region deletion directly causes these gene expression changes genome wide. More likely, the ΔLGIIK9het25 allele alters the positioning of the heterochromatin bundle – and the nearby euchromatin – at the nuclear periphery, which repositions some gene promoters for increased transcription machinery accessibility. This hypothesis is supported by modeling the gene expression changes on the ΔLGIIK9het25 chromosomes and across the genome and reasonably explains gene activation in the NKL2 strain. Of course, altered promoters can also drive gene expression changes, as observed by the insertion of the strong PtrpC promoter on LG II caused a local increase in Hi-C contacts and the production of mRNA from gene NCU08696 proximal to the LGIIK9het25 region. In fact, NCU08696, and others close to constitutive heterochromatic regions, are downregulated in a Δdim-5 strain devoid of H3K9me3. Further, the proper expression of the Neurospora met-8 gene requires the immediately adjacent interspersed constitutive heterochromatic region (54). Together, these data support a hypothetical role for constitutive heterochromatin in regulating gene expression. In fact, the local deposition of H3K9me3 at a gene promoter can repress gene expression in metazoans, as the control of mouse embryonic stem cell identity requires the co-deposition of H3K9me3 and H3K36me3 for transcriptional repression (55). Thus, in addition to the canonical repression of transposable elements and repetitive sequences in eukaryotic genomes (56–59), H3K9me3 directly regulates gene transcription. We propose heterochromatin-mediated gene regulation also occurs through 3D organization, where genes interact with H3K9me3-enriched regions. Specifically, genes would associate with constitutive heterochromatic regions to localize to the more repressive periphery for reducing transcription, and should these genes move to the more active nuclear center, transcription would increase. Currently, it is unknown how the constitutive heterochromatin – gene interaction occurs, but aggregation of HP1 bound to H3K9me3 at promoters with the constitutive heterochromatin bundle is an attractive model. Fungi may be more apt to employ this transcriptional regulation mechanism, as opposed to their metazoan counterparts, given how fungal TAD-like structures have more promiscuous contacts and how the Rabl chromosome conformation has extensive inter-chromosomal contacts (9, 23–25, 35). Recently, the histone methyltransferase SETDB1, which catalyzes H3K9me3 across metazoan genomes, was shown to maintain gene expression in addition to driving chromatin compartmentalization (60), suggesting that constitutive heterochromatic regions have additional, underappreciated roles in both genome organization and function across diverse species. Further research in multiple species should help elucidate novel functional roles for this “junk DNA” that is pervasive yet evolutionarily retained in genomes from fungi to metazoans.

Materials and Methods

Strains and growth conditions

Neurospora crassa strains WT N150 (derived from the strain 74-OR23-IVA [Fungal Genetics Stock Center #2489]), N2930 (mat A his-3; Δmus52::bar+), N3944 (mat x; Δdim-5::bar+), and N4933 (mat a; Δ47.4 kb::hph+) were gifts from Eric U. Selker (University of Oregon). All strains were grown with 1x Vogels minimal medium + 1.5% sucrose and supplements at 32°C (61). All strains are available upon request.

The ΔLGIIK9het25 (NKL2) strain replacing 110,609 basepairs (bp) of AT-rich DNA from H3K9me3-enriched region #25 on LG II (between bp 3,602,033 and 3,712,857 of Neurospora reference genome version 14 [nc14]) (23) with 1,401 bp of the PtrpC::hph hygromycin resistance cassette (deleting a total of 109,208 bp of DNA from the Neurospora genome) was created by split marker gene replacement. Briefly, the ~1,000 bp upstream of LGIIK9het25 was PCR amplified with oligonucleotides (“oligos”) oKL14 and oKL15, while the ~1,000 bp downstream of LGIIK9het25 was amplified with oligos oKL18 and oKL19, by Phusion DNA polymerase (cat# F530-L; ThermoFisher Scientific) using the manufacturer’s conditions; oligos oKL15 and oKL18 had ten 5’ complementary nucleotides specific to the PtrpC promoter and hph gene 3’ end, respectively. Oligo sequences are provided in Table S1. DNA fragments were gel purified using the GenCatch Gel Extraction Kit (cat# 2260250; Epoch Life Sciences), and split marker fragments fusing the LGIIK9het25 upstream to the PtrpC promoter (“UP”; oKL14 and 2955) or fusing the hph gene to the LGIIK9het25 downstream (“DOWN”; oligos 2954 and oKL19) were PCR amplified with the upstream and downstream fragments, and an hph gene containing plasmid (p3xFLAG::hph::LoxP) (62) using LA Taq (cat# RR002M; Takara), and gel purified. The UP and DOWN split marker fragments were transformed into N2930 by electroporation (BioRad MicroPulser Electroporator, cat# 1652100EDU). Hygromycin resistant colonies were selected on 1x Vogels+FGS media with 100 µg/mL hygromycin (cat# ant-hg-5; Invivogen). Genomic DNA was isolated from resultant colonies, using standard protocols (63), and proper hygromycin cassette integration at LGIIK9het25 borders was performed by PCR using oKL49 and 2955 for the left border, 2954 and oKL50 for the right border, and oKL49 and oKL50 for the entire integrated PtrpC::hph gene. Positive integrant strain tKL1A-2 was crossed to N150 (xKL9AR) and genomic DNA was isolated from hygromycin resistant progeny; similar PCR reactions were performed to confirm the presence of the ΔLGIIK9het25 allele in individual cross progeny. PCR with oligos oKL55 and oKL56 determined Δmus-52::bar allele absence; strain mating type was established by crosses with strains of known mating types. The final homokaryotic strain NKL2 from cross progeny xKL9AR-17 contains only the ΔLGIIK9het25 deletion (NKL2 genotype: mat A; ΔLGIIK9het25::hph).

Hi-C library construction and bioinformatic analyses

In situ Hi-C was performed as previously described (23, 24). Hi-C libraries were Illumina sequenced at the Genomics and Cell Characterization Core Facility (GC3F; University of Oregon), either on a Hi-Seq 2500 as PE100 reads or a NovaSeq 6000 as PE59 reads. For comparison to mutant Hi-C datasets, WT DpnII and MseI in situ Hi-C datasets (NCBI Gene Expresssion Omnibus (GEO) accession number GSE173593) (23) were generated by extracting the number of total reads from WT datasets using the sed command to provide approximately equal numbers of valid reads as mutant datasets. Reads were mapped with bowtie2 (64) to the Neurospora crassa version 14 (nc14) reference genome (23), the output sam files were used to build the initial Hi-C contact matrix at a high [1 kb] resolution with hicExplorer software package (65), and the output contact matrix was used to analyze replicate dataset similarity (hicCorrelate), generate lower resolution contact matrices (hicMergeMatrixBins), compare WT and mutant datasets (hicCompareMatrices), KR correct matrices (hicCorrectMatrix), assess TADs (hicFindTADs [using --correctForMultipleTesting false discovery rate, --thresholdComparisons 0.3, and --delta 0.15 values to make TAD bed files] and hicPlotTADs), and generate images (hicPlotMatrix). Individual chromosome 3D structure prediction was performed with 3DMax (https://github.com/BDM-Lab/3DMax)(45) while entire Neurospora 3D genome structure prediction was performed by LorDG (66), both using custom scripts that are available on the Oluwadare lab GitHub website (https://github.com/OluwadareLab/Fungi_3DGenome), tailored to fungal chromosome biology. Input contact matrices for structure prediction were made by merging the individual DpnII and MseI matrices (hicSumMatrices) and converting the .h5 output format to NxN text files with a custom script (23) (https://github.com/Klocko-Lab). Output PDB files were displayed on ChimeraX (67), individual bins with differentially expressed genes or chromosomal features were manually colored, and high-resolution images were used for figure construction. PDB files are available on Zenodo (10.5281/zenodo.11245552).

Chromatin Immunoprecipitation-sequencing (ChIP-seq) library construction and bioinformatic analyses

ChIP-seq was performed as previously described (38), using anti-H3K9me3 (Active Motif; cat# 39161, lot# 30220003) and anti-H3K27me2/3 (Active Motif; cat# 39535, lot# 23120014) antibodies. All ChIP-seq libraries were Illumina sequenced at the GC3F on a NovaSeq 6000 as SR118 reads. Fastq dataset files were mapped to nc14 with bowtie2 (64), converted to bam files, sorted, and indexed with samtools (68), made into bigwig or bedgraph files with Deeptools (69), and displayed on Integrative Genomics Viewer (IGV) (70), with exported png images from IGV used for figure creation. Previously published NCBI GEO or the NCBI Sequence Read Archive (SRA) datasets for WT H3K9me3 (GEO accession numbers GSE68897 and GSE98911), WT H3K27me2/3 (GEO accession numbers GSE68897 and GSE100770), and Δdim-5 H3K27me2/3 (GEO accession number GSE68897 and SRA accession number SRP058573)(31, 46, 47, 71), were mapped to the nc14 genome (23) and used for bigwig/bedgraph file creation for image generation.

Total RNA isolation, polyadenylated RNA-seq library construction, and bioinformatic analyses

Total RNA was isolated from overnight liquid cultures of Neurospora strains grown in 1x Vogels, 1.5% sucrose plus supplements (32, 36). Cultures were harvested by vacuum filtration, placed in 2.0mL screw-top tubes, and 300µL of acid washed glass beads suspended in dH2O (150–212µm, Sigma Aldrich cat# G1145–10G), 350µL Acid Phenol:chloroform (5:1; ThermoFisher cat# AM9720), and 350µL NETS (300mM NaCl, 1mM EDTA, 10mM Tris pH 7.5, 0.2% SDS) were added. Samples were vortexed for 10 minutes at maximum speed and centrifuged at 4°C for 5 minutes at 13k rpm. The aqueous layer was transferred to two tubes (~250µL each), 650µL of cold 100% EtOH was added, and RNA was precipitated on ice for 10 minutes and pelleted by microcentrifugation at 4°C for 10 minutes at 13k rpm. The pellet was washed with cold 70% EtOH (made with DECP-treated dH2O), the supernatant was removed, and pellets were allowed to air dry for 15 minutes and resuspended in 50µL DEPC-treated dH2O. Insoluble material was removed by centrifugation, and the initial RNA concentration was measured by Nanodrop. Total RNA (10µg) was resuspended in 50µL of DEPC-treated dH2O. Total RNA cleanup was performed by the Zymo RNA clean and concentrator kit (Zymo Research cat# R1013), per the manufacturer’s protocol; the 15µL elution was divided into 4µL for Qubit concentration (RNA HS assay kit; ThermoFisher Scientific, cat# Q32852) and TapeStation (Agilent) quality control analysis, while the remaining 11µL was stored at −80oC. Only total RNA samples with high ribosomal RNA to messenger RNA (mRNA) ratios were used for library construction. Polyadenylated mRNA (polyA mRNA) was selected with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs [NEB], cat# E7490L) and polyA mRNA sequencing libraries were generated with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, cat# E7760L and E7765L) per the manufacturer’s protocols; quadruplicate polyA mRNA-seq replicate libraries were constructed for each strain. All polyA mRNA-seq libraries were Illumina sequenced at the GC3F on a NovaSeq 6000 as SR118 reads. Raw fastq files were mapped to the nc14 genome using HiSat2 (72), and gene count files were generated with htseq-count (73). Differential Expression analysis between strains was performed with DESeq2 (74) in R/Rstudio (75, 76), selecting significantly changed genes as those with expression log2 > 3.0 or log2 < −3.0 and an adjusted p-value < 0.001; bed files for display on IGV were generated from the DESeq2 output gene lists in Microsoft Excel, and volcano plots of log2 fold change vs. adjusted p-value were generated in R/R studio.

Supplementary Material

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Acknowledgements

The authors thank Sara Rodriguez, Yulia Shtanko, and Ashley Scadden for Hi-C library generation help; Sara Hanson (Colorado College) for TapeStation use and RNA-seq protocol advice; the University of Oregon GC3F for Illumina library sequencing service, and Klocko lab members and UCCS Department of Chemistry & Biochemistry colleagues for helpful comments and discussions. Funding was provided by a UCCS Undergraduate Research Academy award to A.T.R., start-up funds from the UCCS College of Engineering and Applied Science to O.O., a Maximizing Investigators Research Award grant (1R35GM150402–01) to O.O., start-up funds from the UCCS College of Letters, Arts, and Sciences to A.D.K., and a NIH Academic Research Enhancement Award grant (1R15GM140396–01) to A.D.K.

Data Availability

All genomic datasets have been deposited to the NCBI GEO under the SuperSeries accession number GSE269235. Individual datasets have the subseries numbers GSE269228 (ChIP-seq), GSE269229 (Hi-C), and GSE269230 (polyA mRNA-seq).

Figure 1. A strain harboring a 47.4 kilobase deletion of a H3K27me2/3-enriched domain has minimal genome organization change.

(A-B) Contact probability heatmaps of DpnII (euchromatin-specific) in situ Hi-C datasets at 20 kb resolution across Linkage Group VI (LG VI) of the (A) wild type (WT) or (B) N4933 strains, the latter deleted of a Δ47.4kb H3K27me2/3-enriched region (“ΔK27”). Enhanced heatmaps show the terminal 500 kb of LG VI, harboring the deleted H3K27me2/3 region, at 5 kb resolution. Each Hi-C image displayed has the raw contact probability heatmap shown above the diagonal and the Knight Ruiz (KR) corrected contact probability heatmap, for reducing inherent contact probability biases (37), displayed below the diagonal. Contact probability scale bars shown below the images. Images of wild type H3K9me3 (green) and H3K27me2/3 (purple) ChIP-seq tracks, displayed on IGV (70), above and to the right. The purple line in (A) shows the WT location of the H3K27me2/3-enriched region that was deleted in the ΔK27 strain, while the black arrowhead in (B) shows the deletion site in the N4933 strain. (C) KR corrected contact probability heatmaps of the DpnII in situ Hi-C datasets of WT and ΔK27 strains primarily showing on-diagonal contacts at 5 kb bin resolution. Wild type H3K9me3 ChIP-seq (green), H3K27me2/3 ChIP-seq (purple), and gene (gray) tracks shown below. Contact probability scalebar to the left. The black arrowhead shows the gain in contacts between fused euchromatic DNA flanking the ΔK27 deletion. The gray arrowhead shows a loss of contact probability at a putative transposable element (gene NCU09969; sly-1). (D-E) Contact probability heatmaps of telomere interactions within WT and ΔK27 strains, as (D) shows intra-chromosomal (e.g., the contacts between the left and right telomeres of LG VI), while (E) shows inter-chromosomal (e.g., the contacts between the left telomere of LG VI and the right telomere of LG V) telomere contacts. Telomere schematic, with green circle showing the position of the telomere repeats, H3K27me2/3 ChIP-seq (purple), and gene (gray) tracks shown above and to the right. The red “X” shows the H3K27me2/3 domain that is deleted, while the black arrowhead shows the depletion of contacts at that deletion site.

Figure 2. Deletion of a ~110 kb H3K9me3-enriched domain alters constitutive heterochromatic region clustering.

(A-D) Contact probability heatmaps of (A-B) DpnII (euchromatin-specific) and (C-D) MseI (heterochromatin specific) in situ Hi-C datasets at 20 kb resolution across LG II, as in Figure 1A-B, of the (A, C) wild type (WT) or (B, D) NKL2 strains, the latter deleted of the 25th H3K9me3-enriched region from the LG II left telomere (“ΔLGIIK9het25”). The purple line on panel A shows the deleted LGIIK9het25 region. Enhanced heatmaps at 5 kb resolution show a 500 kb region of LG II surrounding the ΔLGIIK9het25 allele, as in Figure 1A-B. (E-F) Images showing the log2 change in Hi-C contact probability between WT and ΔLGIIK9het25 (E) DpnII (euchromatin-specific) or (F) MseI (heterochromatin-specific) datasets at 5 kb resolution across a 500 kb region of LG II surrounding the ΔLGIIK9het25 allele. Scalebar of contact probability changes is shown below. (G) Images showing the change in inter-chromosomal MseI (heterochromatin-specific) Hi-C contact probability between the entire LG I chromosome and the (top) LG II centromere or (bottom) LGIIK9het25 interspersed heterochromatic region.

Figure 3. The LGIIK9het25 deletion does not compromise the deposition of H3K9me3 at other gene-poor constitutive heterochromatic regions in the NKL2 strain.

(A-B) Images of WT (dark green) or NKL2 ΔLGIIK9het25 (light green) H3K9me3 ChIP-seq and gene (gray) tracks, displayed on IGV, of the entire LG II chromosome or enhanced regions. H3K9me3 ChIP-seq reads were mapped to the (A) WT nc14 reference genome or (B) the NKL2 ΔLGIIK9het25 reference genome. Numbers in the enhanced panel in Figure 3A count the heterochromatic regions from the LG II left telomere, while the black arrowheads show the position at which the DNA sequence has been deleted in the NKL2 ΔLGIIK9het25 strain, and the purple line shows the ~110 kb of DNA that is missing from the NKL2 genome (causing all peaks to be shifted by ~110 kb starting at the black arrowhead). The red arrow in panel B shows the position of the inserted PtrpC::hph gene in the NKL2 reference genome.

Figure 4. The LGIIK9het25 deletion alters the predicted TAD-like structures in the surrounding chromatin.

(A-B) Contact probability heatmaps of DpnII (KR corrected) or MseI (raw) in situ Hi-C datasets of WT and ΔLGIIK9het25 strains primarily showing on-diagonal contacts at 5 kb bin resolution, mapped to either the (A) nc14 WT reference genome or the (B) NKL2 ΔLGIIK9het25 reference genome. Wild type H3K9me3 ChIP-seq (green) and gene (gray) tracks shown below. The contact probability scalebar is shown to the left of each heatmap. Black triangular lines show predicted TAD-like structures, as calculated by the program hicFindTADs, in the defined regions. In panel B, the black arrowheads indicate predicted TAD-like structures surrounding the LGIIK9het25 constitutive heterochromatic region deletion that change, while the open arrowheads show altered TAD-like structures in more distant, flanking euchromatin. The red arrow shows the position of the PtrpC::hph gene in the NKL2 reference genome, while the purple line shows increased chromatin contacts surrounding the PtrpC promoter.

Figure 5. The predicted 3D folding of a single chromosome is changed with the LGIIK9het25 deletion.

(A-B) Images depicting the predicted 3D modeling of LG II from (A) WT or (B) NKL2 ΔLGIIK9het25 strains at 20 kb resolution. The green highlighted region shows the 20 kb bins covering the LGIIK9het25 region in the WT structure. The enhanced image shows the folding of the chromatin at the right arm of LG II surrounding the LGIIK9het25 region in a WT strain. The arrow shows the LGIIK9het25 deletion location.

Figure 6. The LGIIK9het25 region deletion causes increased gene expression genome wide.

(A) Volcano plots showing the log2 fold change from WT expression level of all Neurospora genes verses adjusted p-values from (left) the NKL2 ΔLGIIK9het25 strain or (right) the Δdim-5 strain. Points colored red are genes that have significant expression changes, with log2 fold changes > 3.0 or < −3.0 and adjusted p-values < 0.001. The number of significantly changed genes in each strain indicated in red at the top of the plot. (B) Images of IGV tracks showing WT H3K9me3 ChIP-seq (green), genes (gray), or the positions of differentially expressed genes with log2 fold changes > 3.0 (“up” genes) or < −3.0 (“down” genes) and adjusted p-values < 0.001. The purple “X” symbols show the LGIIK9het25 constitutive heterochromatic region deleted in the NKL2 strain, while the black arrows show the positions of genes NCU08696 on LG II or dim-5 (NCU04402) on LG IV. (C-D) Images depicting the predicted 3D modeling of LG II from (C) WT or (D) NKL2 ΔLGIIK9het25 strains at 20 kb resolution. The green highlighted region shows the 20 kb bins encompassing the LGIIK9het25 region in the WT structure, while red highlights show the 20 kb bins containing differentially expressed genes in the NKL2 strain, relative to a WT strain; the same bins are marked on both structures. The arrow in panel D shows the LGIIK9het25 deletion location.

Figure 7. The predicted 3D structure of the entire genome, as well as the positions of differentially expressed genes, are altered when the LGIIK9het25 region is deleted.

(A-B) Images depicting the predicted 3D modeling of the entire genome of (A) WT or (B) NKL2 ΔLGIIK9het25 strains. The model on the left shows the side view of the genome structure, which is rotated up 90° to obtain the right model, which shows the bottom of the structure from the centromere cluster. The green highlighted region shows the 20 kb bins covering the LGIIK9het25 region in the WT structure, while the red regions highlight the 20 kb bins containing differentially expressed genes in the NKL2 ΔLGIIK9het25 strain.

Declaration of Interest Statement

The authors report there are no competing interests to declare.
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