
==== Front
Nucleic Acids Res
Nucleic Acids Res
nar
Nucleic Acids Research
0305-1048
1362-4962
Oxford University Press

39106166
10.1093/nar/gkae664
gkae664
AcademicSubjects/SCI00010
Gene regulation, Chromatin and Epigenetics
H3T11 phosphorylation by CKII is required for heterochromatin formation in Neurospora
Tian Yuan MOA Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100193, China

Zhang Chengcheng MOA Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100193, China

Tian Xiang MOA Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100193, China

Zhang Lu State Key Laboratory for Conservation and Utilization of Bio-Resources and Center for Life Science, School of Life Sciences, Yunnan University, Kunming, Yunnan 650091, China

Yin Tong State Key Laboratory for Conservation and Utilization of Bio-Resources and Center for Life Science, School of Life Sciences, Yunnan University, Kunming, Yunnan 650091, China

https://orcid.org/0000-0002-8206-0271
Dang Yunkun State Key Laboratory for Conservation and Utilization of Bio-Resources and Center for Life Science, School of Life Sciences, Yunnan University, Kunming, Yunnan 650091, China

https://orcid.org/0000-0002-8801-9317
Liu Yi Department of Physiology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA

https://orcid.org/0000-0003-4465-6186
Lou Huiqiang MOA Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100193, China

https://orcid.org/0000-0002-2629-1673
He Qun MOA Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100193, China

To whom correspondence should be addressed. Tel: +86 10 62731206; Fax: +86 10 62731206; Email: qunhe@cau.edu.cn
09 9 2024
06 8 2024
06 8 2024
52 16 95369550
22 7 2024
19 6 2024
30 1 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.
2024
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Abstract

Heterochromatin is a key feature of eukaryotic genomes and is crucial for maintaining genomic stability. In fission yeast, heterochromatin nucleation is mainly mediated by DNA-binding proteins or the RNA interference (RNAi) pathway. In the filamentous fungus Neurospora crassa, however, the mechanism that causes the initiation of heterochromatin at the relics of repeat-induced point mutation is unknown and independent of the classical RNAi pathway. Here, we show that casein kinase II (CKII) and its kinase activity are required for heterochromatin formation at the well-defined 5-kb heterochromatin of the 5H-cat-3 region and transcriptional repression of its adjacent cat-3 gene. Similarly, mutation of the histone H3 phosphorylation site T11 also impairs heterochromatin formation at the same locus. The catalytic subunit CKA colocalizes with H3T11 phosphorylation (H3pT11) within the 5H-cat-3 domain and the deletion of cka results in a significant decrease in H3T11 phosphorylation. Furthermore, the loss of kinase activity of CKII results in a significant reduction of H3pT11, H3K9me3 (histone H3 lysine 9 trimethylation) and DNA methylation levels, suggesting that CKII regulates heterochromatin formation by promoting H3T11 phosphorylation. Together, our results establish that histone H3 phosphorylation by CKII is a critical event required for heterochromatin formation.

Graphical Abstract

Graphical Abstract

National Natural Science Foundation of China 10.13039/501100001809 32370638 32171262 31871254 National Key Research and Development Program of China 10.13039/501100012166 2018YFA0900500 National Institutes of Health 10.13039/100000002 R35GM118118 Welch Foundation 10.13039/100000928 I-1560
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pmcIntroduction

In eukaryotes, genomic DNA wraps around histones to form chromatin, which is divided into gene-rich, less condensed euchromatin and gene-poor, highly condensed heterochromatin (1). Constitutive heterochromatin is mainly located in the highly repetitive DNA sequences of chromosomal centromeres and telomeres, which can maintain the stability and integrity of the genome by inhibiting the activity of transposable elements (2). Constitutive heterochromatin structure is characterized by histone hypoacetylation, histone H3 lysine 9 methylation (H3K9me) and the enrichment of heterochromatin protein 1 (HP1). The assembly of heterochromatin domain requires the concerted actions of chromatin-modifying enzymes and consists of three steps: initiation, spreading and maintenance (2–5). In fission yeast, heterochromatin is initiated at the nucleation centers by DNA-binding proteins or RNA interference (RNAi) machinery, both of which recruit histone deacetylases and histone H3K9 methyltransferases, leading to histone hypoacetylation and H3K9me at the nucleation site (6–9). Once nucleated, H3K9me and HP1 gradually spread outward from the initial nucleation site, thus allowing them to exert control over adjacent sequences (3,5,10,11).

The filamentous fungus Neurospora crassa possesses heterochromatic features found in higher eukaryotes, such as C5 cytosine methylation (5mC), trimethylation of H3K9 (H3K9me3) and HP1, providing an excellent model for studying heterochromatin formation (12). Constitutive heterochromatin is mainly assembled in AT-rich DNA sequences that are relics of the repeat-induced point mutation (RIP) (13). RIP is a genome defense system that can detect duplicated DNA sequences and introduce C:G to T:A mutations to both copies during premeiosis (14). The AT-rich DNA sequences in RIP relics recruit the histone methyltransferase DIM-5 by unknown mechanisms, leading to H3K9me3 modification (15). HP1 recognizes and binds H3K9me3 through its chromodomain (16), which further recruits histone deacetylase complex HCHC and DNA methyltransferase DIM-2 to promote histone deacetylation and cytosine methylation in this region, eventually forming a highly condensed heterochromatin structure (17–19). Proteins containing AT-hook motifs were supposed to recognize AT-rich sequences and trigger heterochromatin formation. However, a previous study found that none of the strains with disrupted genes encoding proteins containing two or more AT-hook motifs showed a dramatic reduction in DNA methylation (20). Importantly, the establishment of heterochromatin is independent of the RNAi machinery in N. crassa (21,22). Moreover, unlike the H3K9 methyltransferases in fission yeast and mammals, the N. crassa DIM-5 protein does not possess the chromodomain that is responsible for recognizing and binding H3K9me3 modification (15). Therefore, the mechanism by which DIM-5 is recruited to RIP’d relics and initiates heterochromatin formation remains unclear.

Phosphorylation of histone H3 (H3T3/S10/T11/S28) was discovered to be linked to chromosome condensation (23,24). Interestingly, the substitution of H3T11 residue with alanine (A) greatly reduces DIM-5 activity (25). Structural studies indicate that the H3T11 residue can directly interact with the DIM-5 protein and may provide critical information for substrate recognition (26). Moreover, the exchange of H3T11 residue with glutamate (E) significantly reduced H3K9me3 in vitro, suggesting that the phosphorylation of H3T11 may regulate the activity of DIM-5 (27). In budding yeast, H3T11 phosphorylation (H3pT11) is required for telomeric silencing by maintaining SIR (silent information regulator) complex occupancy (28). Given the high conservation of H3T11 phosphorylation, it is attractive to resolve the role of H3T11 phosphorylation in heterochromatin formation in organisms that assemble heterochromatin through the H3K9me/HP1 system.

Several different kinases are responsible for H3T11 phosphorylation (29). Notably, the casein kinase II (CKII) complex is required for the regulation of H3T11 phosphorylation in low-glucose conditions in budding yeast (30,31). CKII is a constitutively active serine/threonine kinase with a large number of substrates and is involved in many fundamental biological processes, including apoptosis, cell growth and proliferation, transcription, translation, circadian rhythm and DNA damage response (32). CKII holoenzyme generally exists as a tetramer consisting of two regulatory subunits and two catalytic subunits (32,33). In N. crassa, the CKII complex contains two identical catalytic subunits, CKA, and two distinct regulatory subunits, CKB1 and CKB2 (34,35). Interestingly, loss of CKII-dependent Swi6/HP1 phosphorylation alleviates heterochromatic transcriptional gene silencing without affecting heterochromatin structure in fission yeast (36). However, the role of CKII in heterochromatin regulation in N. crassa remains unknown.

In this study, we demonstrated that the CKII-dependent H3T11 phosphorylation is localized in the 5H-cat-3 domain and required for the 5-kb heterochromatin formation. Importantly, genome-wide analyses further confirmed the general role of H3T11 phosphorylation in regulating H3K9me3 and DNA methylation. Thus, our results uncovered a critical chromatin regulatory process that mediates heterochromatin formation.

Materials and methods

Strains and culture conditions

The 87-3 (bd, a) and 4200 (nbd, a) strains were used as the N. crassa wild-type (WT) strains in this study (37). The strains used for screening are all in the nonband (nbd) background and contain a WT ras-1 gene (37). The ku70RIP strain, in which the endogenous ku70 open reading frame was mutated with multiple premature stop codons (38), was used as the recipient strain for constructing the knockout strains (ckaKO, ckb1KO, ckb2KO and ckb1KOckb2KO) and the knock-in strains (ckaK61A, ckaD149A, H3T11A and H3T11E). The previously constructed cat-3KO, hpoKO, dim-5KO, cul4KO, ddb1KO, dcaf26KO, H3K9L, H3K9R, met-8KO, H3K9Q and H3S10A strains were also used in this study (39–42). The strains expressing green fluorescent protein (GFP)-tagged CKA and red fluorescent protein (RFP)-tagged HP1 were obtained by transforming the pcfp-5Myc-6His-CKA-GFP and pcfp-5Myc-6His-HP1-RFP plasmids into the 301-6 (bd, his-3−) host strain. For complementary strains, the plasmids pcfp-5Myc-6His-CKA, pcfp-5Myc-6His-CKB1 and pcfp-5Myc-6His-CKB2 were introduced into ckaKO, ckb1KO and ckb2KO, respectively.

The medium used for plate assays contained 1× Vogel’s salts, 3% sucrose and 1.5% (w/v) agar with or without H2O2. Liquid cultures were grown in minimal medium (1× Vogel’s and 2% glucose) under continuous light oscillation at 25°C for 18 h.

Generation of antiserum against CKA

The GST–CKA (containing CKA amino acids Met1–Asn336) fusion protein was expressed in Escherichia coli BL21 cells and the purified soluble recombinant protein was used as the antigen to immunize rabbits to obtain polyclonal antiserums, as described previously (42).

Race tube assay

We inoculated the mycelial mats of the strains in race tubes containing 0, 10 or 20 mM H2O2 medium, respectively, and incubated them under the conditions of 25°C and continuous light for ∼8 days, during which the hyphal growth frontiers were labeled every 24 h. Subsequently, the relative growth rate of each strain was measured by calculating the average growth rate of each strain on the medium containing H2O2 relative to that on the medium without H2O2.

Protein analysis

Protein extraction, nuclei isolation, quantification and western blot analyses were performed as described previously (43,44). Equal amounts of protein (40 μg) were loaded into each lane of the gel. After electrophoresis, proteins were transferred onto a polyvinylidine difluoride membrane, and western blot analysis was performed using antibodies against the target protein. The following antibodies were used: anti-H3 (Bioeasy, BE3017), anti-H3pT11 (EASYBIO, BE3216), anti-H3K9me3 (Abcam, ab8898), anti-H3pS10 (Abcam, ab5176), anti-CKA (self-prepared) and anti-CAT-3 (self-prepared).

In-gel assay for activity of catalases

Sample preparation, protein extraction and quantification for the in-gel assay were performed as described previously (39). Equal amounts of total protein (30 μg) were loaded into a 7.5% native polyacrylamide slab gel. After 3 h of electrophoresis at 4°C, the gel was gently incubated in 10 mM H2O2 for 10 min. After the incubation, the gel was immediately transferred into the mixed solution [1% potassium hexacyanoferrate(III) and 1% iron(III) chloride hexahydrate; use it right after it is ready]. Catalytic activities were visualized as the bright bands where H2O2 was decomposed by catalases.

Fluorescence microscopy

The conidial suspension was observed with Zeiss LSM880 confocal microscopes. GFP and RFP were visualized at 488 and 543 nm, respectively. A line sequential scanning mode was used for image capture at a 1024 × 1024 pixel resolution. The value of gains was set to 600–900. Images were processed with ImageJ (NIH, USA) software.

RNA analysis

Neurospora crassa total RNA was extracted with the TRIzol agent and treated with DNase I to digest genomic DNA, as described previously (45): reverse transcription (RT) with Maxima H Minus reverse transcriptase (Thermo Fisher Scientific, #M1682) after quantification of 5 μg per RNA sample, and then amplification by real-time polymerase chain reaction (PCR) (7500; ABI). The primers used for quantitative PCR (qPCR) are shown in Supplementary Table S1. Analysis of relative values of gene expression was performed using the 2−△△CT method by comparing the cycle number for each sample with that for the untreated control (46). The calculation results were normalized to the expression level of β-tubulin gene.

ChIP analysis

Chromatin immunoprecipitation (ChIP) assays were performed as described previously (41). The mycelial mats were grown in 300 ml of liquid medium at 25°C for 18 h. The cross-linking was performed in 1% formaldehyde and quenched by adding 10 ml of 2.5 M glycine. Harvested cells were ground to powder and lysed in lysis buffer containing proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 25 mM NaF, 10 mM Na4P2O7·10H2O, 2 mM Na3O4V and 1 mM ethylenediaminetetraacetic acid). Chromatin was sheared by sonication into ∼500–1000-bp fragments. The ChIP assay was carried out with anti-H3K9me3 (2 μl; Abcam, ab8898), anti-H3pT11 (4 μl; Abcam, ab5168), anti-HP1 (10 μl; self-prepared) or anti-CKA (15 μl; self-prepared). Immunoprecipitated DNA was quantified using real-time PCR (7500; ABI) with primer pairs listed in Supplementary Table S2. ChIP-qPCR data are presented as a percentage of input DNA.

Bisulfite sequencing and data analysis

Bisulfite sequencing sample preparation was performed using the MGIEasy WGBS Library Prep Kit (1000005251; MGI) for the DNBSEQ™ platform (MGI) and the EZ DNA Methylation-Gold Kit (Zymo Research). Data acquisition and processing were performed as previously described (45). The position and density of 5mC were visualized on the Integrative Genomics Viewer (47).

Results

CKII deficiency leads to activation of cat-3 gene expression by disrupting its upstream 5-kb heterochromatin

The association of histone phosphorylation with chromosome condensation prompted us to explore the role of histone kinases in heterochromatin formation (24,48,49). By conducting a homologous comparison, we initially focused on several histone kinases (24,29,50) (Supplementary Figure S1A). We selected a previously identified 5-kb heterochromatin domain (5H-cat-3 domain) located between the NCU00354 and NCU00355 (cat-3) genes as a target for screening, which negatively regulates the expression of these genes. The strains with disruption of the 5H-cat-3 domain resulted in significantly increased cat-3 expression and H2O2-insensitive phenotypes (39,45). To identify histone kinases that contribute to heterochromatin assembly, we examined the H2O2 sensitivity of single gene knockout mutants of N. crassa. Through screening, we found that the ckaKO (NCU03124) mutant strain exhibited a significant H2O2-insensitive phenotype compared to the WT strain, with its mycelial growth rate mostly unaffected by H2O2 (Supplementary Figure S1B and C). As previously reported (35), the ckaKO mutant showed severe conidial defects in slants and a significantly reduced mycelial growth rate on plates (Figure 1A and B). CKA is the catalytic subunit of CKII, which has two regulatory subunits, CKB1 (NCU05485) and CKB2 (NCU02754) (34,35). Interestingly, the ckb1KO, ckb2KO and ckb1KOckb2KO double mutant strains showed modest H2O2-insensitive phenotypes compared with the WT strain (Supplementary Figure S2A and B). Moreover, the CKB mutants exhibited moderately slow hyphal growth and nearly normal conidial development (Figure 1A and B). These results indicated that the CKII complex plays a crucial role in maintaining normal growth and coping with H2O2-induced oxidative stress. To further confirm the functions of the CKII complex in H2O2 resistance and vegetative development, three constructs carrying sequences encoding Myc-tagged CKA, CKB1 or CKB2 were transferred into each corresponding knockout strain. As expected, ectopic expression of Myc-tagged CKA, CKB1 or CKB2 fully complemented the growth deficiency and H2O2 sensitivity of each mutant (Figure 1A and B, and Supplementary Figure S2A and B), respectively, indicating that the observed growth defects and H2O2 insensitivity of mutants were due to the loss of these CKII subunits.

Figure 1. CKII deficiency leads to activation of cat-3 gene expression by disrupting its upstream 5-kb heterochromatin. Growth phenotypes of WT, CKII mutants and CKII complementary transformants on slants (A) and plates (B), respectively. (C) In-gel analysis of catalase activity in extracts from WT, CKII mutants, CKII complementary transformants and cat-3KO strains. (D) Western blot analysis of CAT-3 protein in WT, CKII mutants, CKII complementary transformants and cat-3KO strains. The membrane stained by Coomassie blue served as the loading control. (E) RT-qPCR assay analyzing the levels of cat-3 messenger RNA (mRNA) in WT, ckaKO, ckb1KO, ckb2KO and ckb1KOckb2KO strains. Error bars indicate standard deviation (SD; n = 3). ***P < 0.001. Unpaired Student’s t-test was used. (F) Schematic diagram displaying a 5-kb heterochromatin domain (5H-cat-3 domain) located between cat-3 (NCU00355) and NCU00354 genes on linkage group III of the N. crassa genome. Primer pairs from 1 to 8 under the schematic diagram indicate regions tested by ChIP-qPCR. ChIP assays with H3K9me3 and HP1 antibodies revealing the enrichment profiles of H3K9me3 (G) and HP1 (H) at the 5H-cat-3 domain in WT, ckaKO and ckb1KOckb2KO strains. The H3K9L and hpoKO strains were used as the negative controls in panels (G) and (H), respectively. Error bars indicate SD (n = 3). ***P < 0.001; n.s., not significant. Unpaired Student’s t-test was used.

Since catalases are responsible for converting H2O2 into water and oxygen, these results suggest that the CKII complex may regulate the expression of the catalases in response to H2O2-induced oxidative stress. To test this possibility, we detected the zymogram of catalases using an in-gel experiment. As shown in Figure 1C, the bands corresponding to CAT-3 activity were significantly brighter in the ckaKO strain, while only moderately enhanced in the ckb1KO, ckb2KO and ckb1KOckb2KO strains compared to the WT strain. Consistently, the levels of CAT-3 protein (Figure 1D) and cat-3 mRNA (Figure 1E) were also significantly increased in the ckaKO strain than those in the WT, ckb1KO, ckb2KO and ckb1KOckb2KO strains. Moreover, ectopic expression of Myc-tagged CKA, CKB1 or CKB2 in the corresponding knockout mutant reduced CAT-3 activity (Figure 1C) and CAT-3 protein (Figure 1D) to WT levels. Together, these results demonstrate that CKII plays a key role in the repression of cat-3 gene transcription.

To determine whether the transcriptional activation of cat-3 in CKII mutants is due to the disruption of the 5-kb heterochromatin, we examined the H3K9me3 levels at the 5H-cat-3 domain by ChIP in the WT, ckaKO and ckb1KOckb2KO strains. Chromatin samples were immunoprecipitated with the antibody against H3K9me3 and analyzed by qPCR with primer pairs targeting the 5H-cat-3 domain (Figure 1F). As shown in Figure 1G, H3K9me3 was specifically enriched in the 5H-cat-3 domain (primer pairs 2 and 4) in the WT strain, while the enrichment was dramatically decreased in the ckaKO strain and slightly reduced in the ckb1KOckb2KO strain. Similarly, ChIP assays with an HP1-specific antibody revealed that the specific enrichment of HP1 within the 5H-cat-3 domain was significantly reduced in the ckaKO strain but not in the ckb1KOckb2KO strain (Figure 1H). Interestingly, in the ckb1KOckb2KO strain, we observed a discrepancy between the alterations in H3K9me3 and HP1 levels (Figure 1G and H) and the degree of cat-3 derepression (Figure 1C–E). These findings indicate that CKII is capable of modulating cat-3 transcription independently of the 5H-cat-3 domain. Together, these results indicate that CKII deficiency leads to increased cat-3 gene expression by disrupting its upstream 5-kb heterochromatin.

The kinase activity of CKA is required for the proper formation of the 5-kb heterochromatin

CKA proteins are evolutionarily conserved in eukaryotes (33). The K61 (K68 in Homo sapiens) and the D149 (D156 in H. sapiens) residues of the N. crassa CKA protein are identified as its ATP binding site and catalytic site, respectively (51,52) (Supplementary Figure S3A). To determine whether the kinase activity of CKA is involved in the regulation of the 5H-cat-3 domain, we constructed the ckaK61A or ckaD149A mutant by introducing K61A or D149A point mutation at the original cka gene locus, respectively. The ckaD149A strain is similar to the ckaKO strain, exhibiting obvious growth defects and a strong H2O2-insensitive phenotype, and the ckaK61A strain also showed mild growth defects and a moderate H2O2-insensitive phenotype (Figure 2A and B, and Supplementary Figure S3B and C). Together, these results indicate that the kinase activity of CKA is required for maintaining normal growth and coping with H2O2-induced oxidative stress.

Figure 2. The kinase activity of CKA is required for the proper formation of the 5-kb heterochromatin. Growth phenotypes of WT, ckaKO, ckaK61A and ckaD149A strains on slants (A) and plates (B), respectively. (C) In-gel analysis of catalase activity in extracts from WT, ckaKO, ckaK61A, ckaD149A and cat-3KO strains. (D) Western blot analysis of CAT-3 protein in WT, ckaKO, ckaK61A, ckaD149A and cat-3KO strains. The membrane stained by Coomassie blue served as the loading control. (E) RT-qPCR assay analyzing the levels of cat-3 mRNA in WT, ckaKO, ckaK61A and ckaD149A strains. Error bars indicate SD (n = 3). ***P < 0.001. Unpaired Student’s t-test was used. ChIP assays showing the binding patterns of H3K9me3 (F) and HP1 (G) at the 5H-cat-3 domain in WT, ckaKO, ckaK61A and ckaD149A strains. The H3K9L and hpoKO strains were used as the negative controls in panels (F) and (G), respectively. Error bars indicate SD (n = 3). ***P < 0.001. Unpaired Student’s t-test was used.

We then detected the cat-3 expression in the WT, ckaK61A and ckaD149A strains. As expected, compared with the WT strain, the CAT-3 activity (Figure 2C), CAT-3 protein (Figure 2D) and cat-3 mRNA (Figure 2E) levels were remarkably increased in the ckaD149A and ckaKO strains and moderately increased in the ckaK61A strain, indicating that the kinase activity of CKA is required for the repression of cat-3 transcription. Consistently, ChIP analyses revealed that the depositions of H3K9me3 (Figure 2F) and HP1 (Figure 2G) at the 5H-cat-3 domain were significantly decreased in the ckaD149A, ckaKO and ckaK61A strains compared to those in the WT strain, indicating that the loss of CKA kinase activity disrupted the 5H-cat-3 heterochromatin domain.

CKII regulates the 5H-cat-3 domain through H3T11 phosphorylation

Previous studies have shown that CKII participates in the phosphorylation of histone H3T11, H4S1 or H2AY57 residues to affect cell metabolic regulation, DNA damage repair and transcriptional elongation, respectively (31,53,54). In budding yeast, H3T11 phosphorylation affects telomeric silencing by promoting the recruitment of the SIR complex (28). Given the disruption of the 5H-cat-3 domain in the ckaKO and ckaD149A strains, we hypothesized that CKII may affect the stability of heterochromatin through H3T11 phosphorylation. To test this possibility, we generated H3T11A and H3T11E mutant strains in which the T11 to A mutation abolishes H3T11 phosphorylation and the mutation of T11 to E mimics H3T11 phosphorylation (55,56). Neurospora genome contains a single histone H3 gene (57), which simplifies the testing of mutations in this gene. Previous studies have found that the strain with a deletion of the dim-5 gene, which encodes the histone H3K9 methyltransferase, exhibits growth defects and an H2O2-insensitive phenotype (15,39). Phenotypic examination revealed that the H3T11A mutant resembled the ckaKO strain, showing severe growth defects and a strong H2O2-insensitive phenotype, whereas the H3T11E mutant is similar to the dim-5KO strain, showing modest development defects and a moderate H2O2-insensitive phenotype (Figure 3A and B, and Supplementary Figure S4A and B). Consistently, the zymogram in-gel assay revealed that the stained bands corresponding to the CAT-3 activity in the ckaKO and H3T11A strains were much brighter and wider than those of the WT strain (Figure 3C). However, in the H3T11E and dim-5KO strains, both bands corresponding to CAT-3 activity exhibited increased brightness and downward migration (Figure 3C), suggesting that the expression and modification of CAT-3 protein were similar in these mutants. Moreover, the levels of CAT-3 protein (Figure 3D) and cat-3 mRNA (Figure 3E) were notably increased in both ckaKO and H3T11A strains compared to those in H3T11E, dim-5KO and WT strains, suggesting that CKA mediates its function in suppressing cat-3 expression by promoting H3T11 phosphorylation.

Figure 3. CKII participates in the regulation of the 5H-cat-3 domain through H3T11 phosphorylation. Growth phenotypes of the WT, ckaKO, H3T11A, H3T11E and dim-5KO strains on slants (A) and plates (B), respectively. (C) In-gel analysis of catalase activity in extracts from WT, ckaKO, H3T11A, H3T11E, dim-5KO and cat-3KO strains. (D) Western blot analysis of CAT-3 protein levels in WT, ckaKO, H3T11A, H3T11E, dim-5KO and cat-3KO strains. The membrane stained by Coomassie blue served as the loading control. (E) RT-qPCR assay analyzing the levels of cat-3 mRNA in WT, ckaKO, H3T11A, H3T11E and dim-5KO strains. Error bars indicate SD (n = 3). ***P < 0.001. Unpaired Student’s t-test was used. ChIP assays showing the enrichment profiles of H3K9me3 (F) and HP1 (G) at the 5H-cat-3 domain in WT, H3T11A and H3T11E strains. The H3K9L and hpoKO strains were used as the negative controls in panels (F) and (G), respectively. Error bars indicate SD (n = 3). ***P < 0.001. Unpaired Student’s t-test was used. (H) ChIP assay with phosphorylated H3T11 (H3pT11) antibody showing the enrichment of H3pT11 at the 5H-cat-3 domain and its adjacent cat-3 gene in WT and ckaKO strains. The H3T11A strain was used as the negative control. Error bars indicate SD (n = 3). ***P < 0.001. Unpaired Student’s t-test was used. (I) ChIP assay with the CKA antibody displaying the binding profiles of CKA protein at the 5H-cat-3 domain and its adjacent cat-3 gene in the WT, ckb1KOckb2KO, ckaK61A, ckaD149A and H3T11A mutant strains; the ckaKO mutant was used as the negative control. Error bars indicate SD (n = 3). **P < 0.01 and ***P < 0.001. Unpaired Student’s t-test was used.

To further investigate whether H3T11 substitution leads to the deficiency of the 5H-cat-3 domain, we detected the enrichment profiles of H3K9me3 and HP1 in the WT, H3T11A and H3T11E strains. ChIP results revealed that the depositions of H3K9me3 and HP1 at the 5H-cat-3 domain were significantly reduced in the H3T11A and H3T11E strains compared to those in the WT strain (Figure 3F and G), indicating that the substitutions of H3T11 residue result in a severe deficiency of the 5-kb heterochromatin. Although H3T11A and H3T11E strains mimic different phosphorylation states, the substitution of H3T11 to A or E may affect the recognition and activity of histone methyltransferase DIM-5 in vivo as those in vitro (27). In cases of extensive damage to heterochromatin (Figure 3F and G), the levels of cat-3 expression in ckaKO and H3T11A strains are significantly higher than those in dim-5KO and H3T11E strains (Figure 3C–E), indicating that H3T11 phosphorylation by CKII can regulate cat-3 transcription independently of the 5-kb heterochromatin. Collectively, these results suggest that the phosphorylation of H3T11 residue is necessary for the establishment of the 5-kb heterochromatin.

To determine whether CKA specifically regulates the H3T11 phosphorylation state, we used a commercial H3pT11-specific antibody to detect the levels of H3pT11 at the 5-kb heterochromatin and its adjacent cat-3 gene. As shown in Figure 3H, H3pT11 was highly enriched in the 5H-cat-3 domain (primer pairs 2–4) in the WT strain, which was colocalized with H3K9me3 and HP1, whereas the enrichment was significantly decreased in the ckaKO strain, suggesting that CKA regulates H3T11 phosphorylation within the 5-kb heterochromatin.

To investigate the binding of CKII to heterochromatin, we ectopically expressed GFP-tagged CKA and RFP-tagged HP1 in the WT strain. Consistent with previous reports (58), we found that CKA-GFP is widely distributed within the cell. The CKA-GFP could localize to heterochromatin, as shown for example by its colocalization with foci of HP1-RFP, which binds to H3K9me3 in vivo (16) (Supplementary Figure S5A). Furthermore, we generated antiserum specifically against the N. crassa CKA protein (Supplementary Figure S5B) and carried out a ChIP assay. Consistent with the fluorescence microscopy results, the CKA protein was highly enriched in the 5-kb heterochromatin (primer pairs 2–4) and its boundaries (primer pairs 1 and 5) in the WT and ckb1KOckb2KO strains, whereas the enrichment was significantly reduced in the ckaD149A and H3T11A strains and slightly reduced in the ckaK61A strain (Figure 3I), suggesting that the association of CKA protein with the heterochromatin is regulated by CKA catalytic activity and its substrate availability. Taken together, these results suggest that CKII can bind to the 5H-cat-3 domain and affect heterochromatin formation by regulating H3T11 phosphorylation.

In budding yeast, Tda1, Sch9 and Pyk1 participate in H3T11 phosphorylation (28,30,31,59). We further investigate the role of these kinases in H3T11 phosphorylation in N. crassa by the H2O2 sensitivity assay. The homologs of Pyk1 and Sch9 in N. crassa are encoded by ace-8 (NCU06075) and stk-10 (NCU03200), respectively. Specifically, ace-8 is an essential gene, and three purified heterozygotes were tested for H2O2 sensitivity. The Tda1 protein shares partial homology with several proteins encoded by genes camk-2 (NCU02283), camk-1 (NCU09123), stk-53 (NCU09064), stk-13 (NCU00108) and prk-9 (NCU04096) in N. crassa. As shown in Supplementary Figure S6A–C, we did not identify mutants with notable H2O2 resistance and elevated CAT-3 activity.

CKII-dependent H3T11 phosphorylation is also required for the formation of the 50-kb heterochromatin region adjacent to the met-8 gene

To determine whether CKII-mediated H3pT11 is involved in general heterochromatin formation in the N. crassa genome, we examined its involvement at another heterochromatin locus. We previously found that the formation of a 50-kb heterochromatin region is required for high expression of the adjacent methionine synthase gene (met-8) (41) (Figure 4A). We first examined whether the CKA protein can also bind to the 50-kb heterochromatin region in the WT, ckb1KOckb2KO, ckaD149A, H3T11A and ckaKO strains. ChIP results showed that the CKA protein was highly enriched in the 50-kb heterochromatin (primer pairs 2–6) in the WT and ckb1KOckb2KO strains and the enrichment was significantly reduced in the ckaKO, ckaD149A and H3T11A mutants (Figure 4B). As predicted, compared with the WT strain, loss of CKA also resulted in significant decreases in the depositions of H3pT11 (Figure 4C), H3K9me3 (Figure 4D) and HP1 (Figure 4E) within the 50-kb heterochromatin region, as well as a remarkable reduction of the met-8 gene expression (Figure 4H and I). Moreover, the mutations of H3T11A and H3T11E also resulted in significant reductions of H3K9me3 (Figure 4F) and HP1 (Figure 4G) depositions within the 50-kb heterochromatin region, as well as downregulation of the met-8 gene expression (Figure 4H and I). Together, these results suggest that the CKA-mediated H3pT11 is a general mechanism in heterochromatin regulation in the N. crassa genome.

Figure 4. CKII-dependent H3T11 phosphorylation is also required for the formation of the 50-kb heterochromatin region. (A) Schematic diagram displaying a 50-kb heterochromatin domain upstream of the met-8 gene on linkage group III of the N. crassa genome. Primer pairs from 1 to 7 under the schematic diagram indicate the regions tested by ChIP-qPCR. (B) ChIP assay with the CKA antibody displaying the binding profiles of CKA protein at the 50-kb heterochromatin domain in the WT, ckb1KOckb2KO, ckaD149A and H3T11A mutant strains; the ckaKO mutant was used as the negative control. Error bars indicate SD (n = 3). ***P < 0.001. Unpaired Student’s t-test was used. (C) ChIP assay with H3pT11 antibody showing the enrichment of H3pT11 at the 50-kb heterochromatin domain in the WT and ckaKO strains. The H3T11A strain was used as the negative control. Error bars indicate SD (n = 3). ***P < 0.001. Unpaired Student’s t-test was used. ChIP assays revealing the enrichment profiles of H3K9me3 (D) and HP1 (E) at the 50-kb heterochromatin in WT and ckaKO strains. The H3K9L and hpoKO strains were used as the negative controls in panels (D) and (E), respectively. Error bars indicate SD (n = 3). ***P < 0.001. Unpaired Student’s t-test was used. ChIP assays revealing the enrichment profiles of H3K9me3 (F) and HP1 (G) at the 50-kb heterochromatin in WT, H3T11A and H3T11E strains. The H3K9L and hpoKO strains were used as the negative controls in panels (F) and (G), respectively. Error bars indicate SD (n = 3). ***P < 0.001. Unpaired Student’s t-test was used. (H) RT-qPCR assay analyzing the levels of met-8 mRNA in WT, ckaKO, ckaK61A, ckaD149A, ckb1KOckb2KO, H3T11A, H3T11E and dim-5KO strains. Error bars indicate SD (n = 3). ***P < 0.001. Unpaired Student’s t-test was used. (I) Western blot analysis of MET-8 protein in WT, ckaKO, ckaK61A, ckaD149A, ckb1KOckb2KO, H3T11A, H3T11E and dim-5KO strains. The membrane stained by Coomassie blue served as the loading control.

CKII-mediated H3T11 phosphorylation is required for global H3K9me3

To investigate whether CKII-mediated H3T11 phosphorylation regulates global heterochromatin formation, we performed western blot analyses on nuclear extracts of WT and mutant strains using antibodies that recognize specific histone H3 modifications. As shown in Figure 5A, compared with the WT strain, the levels of H3pT11 and H3K9me3 were significantly reduced in the ckaKO and ckaD149A strains, and ectopic expression of Myc-tagged CKA complemented these deficiencies. On the other hand, the H3K9R and H3S10A mutations abolished the modifications of H3K9me3 and H3pS10 but not H3pT11, while the H3T11A mutation abolished H3K9me3, H3pS10 and H3pT11 levels (Figure 5A). These results indicate that CKII-mediated H3T11 phosphorylation promotes global H3K9me3. In addition, compared with the WT strain, the CKA protein level was decreased in the ckb1KOckb2KO strain (Figure 5B), whereas the H3pT11 level did not change significantly (Figure 5A), suggesting that the regulatory subunits affect the protein level of CKA. To rule out the possibility that CKA indirectly regulates H3K9me3 through the histone methyltransferase complex DCDC (DIM-5/-7/-9, CUL4/DDB1 complex) (40,42,60,61), we examined the expression levels of DCDC subunits in the CKA mutant strains. As shown in Figure 5C and D, the expression level of DIM-5, but not other subunits, was significantly increased in CKA mutants compared with the WT strain. Further detection found that the dim-5 mRNA was also increased in H3T11A, H3T11E, ddb1KO and cul4KO strains (Supplementary Figure S7A), indicating that heterochromatin can affect the expression of the dim-5 gene. Collectively, these results suggest that CKA-mediated H3pT11 is required for global H3K9me3.

Figure 5. CKII-dependent H3T11 phosphorylation is required for global H3K9me3. (A) Nucleoproteins from WT, ckaKO, ckaK61A, ckaD149A, ckb1KOckb2KO and ckaKO, CKA strains were analyzed for H3K9me3, H3pS10 and H3pT11 by immunoblotting analyses; the H3K9R, H3S10A and H3T11A mutants served as the negative controls, respectively. Western blot for histone H3 acted as the loading control. (B) Western blot analysis of the CKA protein in WT, ckaK61A, ckaD149A, ckb1KOckb2KO, ckaKO, CKA, H3K9R, H3S10A and H3T11A strains; the ckaKO strain served as the negative control. The membrane stained by Coomassie blue served as the loading control. (C) RT-qPCR assay analyzing the mRNA levels of DCDC subunits in WT, ckaKO, ckaK61A and ckaD149A strains. Error bars indicate SD (n = 3). *P < 0.05 and ***P < 0.001. Unpaired Student’s t-test was used. (D) Western blot analysis of the DIM-5 protein in WT, ckaKO, ckaK61A and ckaD149A strains; the dim-5KO strain served as the negative control. The membrane stained by Coomassie blue served as the loading control. (E) Nucleoproteins from WT, dim-5KO, cul4KO, ddb1KO and dcaf26KO strains were analyzed for H3K9me3, H3pS10 and H3pT11 by immunoblotting analyses. Western blot for histone H3 acted as the loading control. (F) Western blot analysis of the CKA protein in WT, dim-5KO, cul4KO, ddb1KO and dcaf26KO strains. The membrane stained by Coomassie blue served as the loading control.

To further dissect the relationship between H3K9me3 and H3pT11, we detected the global histone modifications from nuclear extracts of the WT, dim-5KO, cul4KO, ddb1KO and dcaf26KO strains. Western blot analyses revealed that the levels of CKA protein in DCDC mutants are comparable to those of the WT strain, while the phosphorylation of H3T11 has only slightly decreased (Figure 5E and F). The ChIP assay showed a reduction in H3T11 phosphorylation within the 5H-cat-3 domain and the 50-kb heterochromatin upstream of the met-8 gene in DCDC mutants (Supplementary Figure S7B and C). Despite the decrease in H3T11 phosphorylation levels in the DCDC mutants, the reduction is not as pronounced as that of H3K9me3 in the ckaKO strain, as shown by ChIP and western blot analyses. These results indicate that H3T11 phosphorylation precedes the formation of H3K9me3, and H3K9me3 may also feed back to regulate H3T11 phosphorylation. Different from the H3K9R strain, the global levels of H3pS10 were significantly increased in the DCDC mutants compared to those in the WT strain (Figure 5E), confirming the antagonistic relationship between H3K9me3 and H3pS10 (62). The difference between the H3K9R and DCDC mutants is due to the H3K9R strain, which not only mimics the absence of H3K9me3 but also indicates a mutation at the K9 residue. Such a mutation may affect the binding and activity of kinases targeting the adjacent H3S10. Taken together, these results demonstrate that CKII-mediated H3pT11 is required for global H3K9me3.

CKII-mediated H3pT11 is required for DNA methylation in most RIP’d relics

The globally decreased H3K9me3 in the CKA mutants prompted us to perform whole genome bisulfite sequencing (WGBS) to examine the genome-wide DNA methylation. Consistent with the absence of H3K9me3, DNA methylation was also completely lost across seven linkage groups in the H3T11A strain (Figure 6A and B, and Supplementary Figure S8), indicating that the H3T11 residue is necessary for global H3K9me3 and DNA methylation. Importantly, the levels of DNA methylation at RIP’d relics across seven linkage groups were dramatically reduced in the ckaKO and ckaD149A mutants compared to the WT and ckb1KOckb2KO strains, and ectopic expression of the CKA protein restored global DNA methylation to the WT levels (Figure 6A and B, and Supplementary Figure S8). These results indicate that CKII-dependent H3pT11 is required for genome-wide RIP-induced DNA methylation.

Figure 6. CKII-dependent H3pT11 is required for DNA methylation in most RIP’d relics. WGBS assays showing the distribution of DNA methylation in WT, ckaKO, ckaD149A, ckaKO, CKA, ckb1KOckb2KO and H3T11A strains over the linkage groups I (A) and II (B), respectively (additional tracks for other chromosomes can be found in Supplementary Figure S8A). The horizontal lines in the RIP index panel indicate the threshold (<0.7) to trigger DNA methylation by RIP. The red regions of the broken line represent the AT-rich regions. (C) Metagene analyses displaying the average profiles of 5mC enrichment in WT, ckaKO and ckb1KOckb2KO strains as determined by WGBS analyses and averaged %GC content over all constitutive heterochromatin domains in WT. Pie charts showing the proportions of increased, unchanged and decreased DNA methylation sites in AT-rich regions over 1 kb of the genome in ckaKO (D) or ckb1KOckb2KO (E) strains compared with the WT strain. The threshold for change is defined as 1.5 times; otherwise, it is defined as unchanged.

Since DNA methylation mainly occurred at AT-rich regions that are relics of the RIP, we used the RIP index as an indicator to define the methylated regions (63). To minimize the noise from the RIP index, we focused on 834 RIP’d regions whose lengths are longer than 1 kb in the N. crassa genome. As shown in Figure 6C, the DNA methylation levels were generally decreased within the RIP’d domain in the ckaKO strain but not in the ckb1KOckb2KO strain. Moreover, compared with the 834 RIP’d regions in the WT strain, 694 (83.21%) regions in the ckaKO strain (Figure 6D) and 38 (4.56%) regions in the ckb1KOckb2KO strain (Figure 6E) exhibited decreased DNA methylation levels. Among the 38 such regions in the ckb1KOckb2KO strain, 35 overlapped with those in the ckaKO strain (Supplementary Figure S9A). Moreover, DNA methylation levels at 129 (15.47%) RIP’d regions in the ckaKO strain (Figure 6D) and 771 (92.45%) RIP’d regions in the ckb1KOckb2KO strain (Figure 6E) (116 overlapped regions) (Supplementary Figure S9B) were similar to those in the WT strain. Additionally, previous studies have also detected DNA methylation at some non-RIP’d regions (64,65). Then, we focused on 535 methylated non-RIP’d regions whose lengths are longer than 1 kb in N. crassa genome. Compared with the 535 methylated non-RIP’d regions in the WT strain, 469 (87.66%) regions in the ckaKO strain (Supplementary Figure S9C) and 512 (95.70%) regions in the ckb1KOckb2KO strain (Supplementary Figure S9D) showed similar DNA methylation levels. Together, these results demonstrate that CKII-dependent H3pT11 is required for DNA methylation in most RIP’d relics.

Discussion

Constitutive heterochromatin is an important component of the eukaryotic nucleus. Here, we have revealed that the disruption of the 5H-cat-3 domain in the ckaKO strain results in robust cat-3 gene activation, which exhibits an H2O2-insensitive phenotype. The kinase CKII regulates H3T11 phosphorylation within the 5H-cat-3 domain, which is required for the proper formation of the 5-kb heterochromatin. Furthermore, we demonstrate that CKII-dependent H3T11 phosphorylation is required for global H3K9me3 and DNA methylation. Thus, our study revealed that CKII is an important regulator of heterochromatin assembly by regulating H3T11 phosphorylation at RIP’d relics in Neurospora.

The requirement of H3K9me for constitutive heterochromatin formation is well established in animals, plants and fungi (66). In N. crassa, the DIM-5 complex mediates H3K9me3 modifications to create binding sites for HP1 at RIP’d DNA sequences, which are required for the assembly and spreading of heterochromatic structures (15,16,18). However, the intricate mechanism by which DIM-5 is recruited to RIP’d relics remains unknown. Here, we found that H3T11 phosphorylation is colocalized with H3K9me3 and HP1 within the 5H-cat-3 domain and is crucial for the precise assembly of the heterochromatin. Similar results were also obtained in the 50-kb heterochromatin upstream of the met-8 gene. Most importantly, the global levels of H3K9me3 and DNA methylation were dramatically reduced in the ckaKO and H3T11A strains compared to those in the WT strain, suggesting that H3T11 phosphorylation is required for genome-wide H3K9me3 and DNA methylation. Moreover, the overall levels of H3T11 phosphorylation were not significantly affected by the loss of H3K9me3 in DCDC mutants, suggesting that H3T11 phosphorylation is an upstream event of H3K9me3. Additionally, WGBS analyses showed that CKII-dependent H3pT11 specifically affects RIP-induced DNA methylation. Overall, these results suggest that H3T11 phosphorylation is involved in the initiation of heterochromatin by promoting H3K9me3 levels at RIP’d DNA sequences in N. crassa.

In budding yeast, H3T11 phosphorylation maintains telomeric silencing by promoting SIR complex occupancy (28). In the mammalian zygote, H3T11 phosphorylation cooperates with H3S10 phosphorylation to stabilize histone and DNA methylation during the first cell cycle (67). It will be interesting to investigate the underlying mechanism by which H3pT11 promotes the establishment of H3K9me3 in Neurospora. A previous study showed that the exchange of H3 tail residue T11 by E or A strongly reduced H3K9me by DIM-5 in vitro, suggesting that the phosphorylation of T11 may regulate the activity of DIM-5 (27). Consistently, our results revealed that the H3T11A mutation abolished global H3K9me3 and H3pS10 modifications in vivo, which indicate that the exchange of T11 by A of the H3 tail makes it unsuitable as a substrate for DIM-5 to catalyze H3K9me3 or kinases to catalyze H3S10 phosphorylation. In vitro experiments revealed that DIM-5 recognizes R8–G12 residues of the histone H3 tail, with T11 and G12 residues being the most significant specificity determinants (27). In addition, the main chain carbonyl of H3T11 hydrogen binds the side chain of DIM-5 residue Q285 (26), and the phosphorylation modification at the T11 site most probably affects this interaction. That is most likely why, in ckaKO strain with deficient H3T11 phosphorylation, DIM-5 cannot effectively methylate H3K9 residue despite increased expression. Therefore, T11 phosphorylation of histone H3 may directly affect the binding and activity of DIM-5 to regulate the catalysis of H3K9me3.

In budding yeast, CKII is involved in the regulation of H3T11 phosphorylation by affecting the nuclear localization of the kinase Tda1 (30). In this study, CKA protein and H3T11 phosphorylation were found to be colocalized within the 5H-cat-3 domain and the 50-kb heterochromatin upstream of the met-8 gene. Other unknown factors may be involved in the recruitment of CKII, enabling CKII-mediated H3T11 phosphorylation to specifically take place in AT-rich regions. Given that A:T-rich DNA sequences typically trigger DNA methylation in Neurospora (13), proteins with AT-hook motifs might play a role in the recruitment of CKII protein to RIP’d relics. Interestingly, the binding of CKA protein to AT-rich region is modulated by its catalytic activity and substrate availability. Given that other factors may play a role in recruiting CKII to the RIP’d relics, the lack of CKII enzymatic activity might impair the recruitment process mediated by these factors. Additionally, the mutation at H3T11A may affect CKII’s ability to recognize and bind its phosphorylation substrates, thereby leading to a significant reduction in its recruitment levels. ChIP assays indicate that CKA deficiency resulted in a marked reduction in H3pT11 levels within the detected heterochromatic regions. Furthermore, western blot analyses revealed that the overall levels of H3pT11 were significantly reduced but not completely lost in the ckaKO and ckaD149A strains, indicating that there are other kinases involved in H3T11 phosphorylation in N. crassa. We further explored the role of other kinases in H3T11 phosphorylation by the H2O2 sensitivity assay but did not identify mutants exhibiting significant H2O2 resistance. Although we cannot exclude the possibility that CKII indirectly regulates H3T11 phosphorylation, there is no doubt that CKII plays a key role in the regulation of H3T11 phosphorylation, which in turn triggers H3K9me and heterochromatin assembly at RIP’d relics.

Supplementary Material

gkae664_Supplemental_File

Acknowledgments

We are very grateful to Miss Moater Altaf for the crucial revision of this manuscript.

Data availability

Complete WGBS files have been deposited in NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE252700.

Supplementary data

Supplementary Data are available at NAR Online.

Funding

National Natural Science Foundation of China [32370638 to Q.H., 32171262 and 31871254 to Y.D.]; National Key Research and Development Program of China [2018YFA0900500 to Q.H.]; National Institutes of Health [R35GM118118 to Y.L.]; Welch Foundation [I-1560 to Y.L.].Funding for openaccess charge:National Natural Science Foundation of China [32370638 to Q.H., 32171262 and 31871254 to Y.D.]; National Key Research and Development Program of China [2018YFA0900500 to Q.H.]; National Institutes of Health [R35GM118118 to Y.L.]; Welch Foundation [I-1560 to Y.L.].

Conflict of interest statement. None declared.

Notes

Present address: Chengcheng Zhang, Department of Physiology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
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