Identification and Characterization of the Genes Encoding the Core Histones and Histone Variants of Neurospora crassa

RESULTS

Identification and sequence analysis of genes encoding the core histones and histone variants of N. crassa: To identify all of the hH3 and hH4 homologs in the N. crassa genome, we screened two genomic cosmid libraries with the previously identified hH3-hH4 gene pair (Woudtet al. 1983). Of the cosmids identified, only SV31:G9 from the V-Y cosmid library (Vollmer and Yanofsky 1986) was found to carry the hH3-hH4 gene pair. The other cosmids, G11:G5, G22:D6, and X18:H1 from the O-S cosmid library (Orbach 1994), were found to carry a second gene encoding H4, which we named hH4-2. The previously identified hH4 gene in the hH3-hH4 gene pair has been renamed hH4-1 (Perkinset al. 2001).

hH4-1 and hH4-2 encode identical proteins and are 96% identical (300/312) at the nucleotide level in their coding regions. Both genes contain two introns, at precisely conserved locations, but no similarity was found in the introns or in the 5′ or 3′ untranslated regions (UTRs). The intron lengths are also different. Introns 1 and 2 of hH4-1 are 69 and 68 bp, whereas the introns of hH4-2 are 316 and 65 bp, respectively (Figure 1).

To identify the genes encoding histones H2A and H2B, we first searched the two publicly available Neurospora EST databases (Nelsonet al. 1997; Roeet al. 2001b) by tBLASTn for corresponding cDNAs. We identified one set of ESTs for each and designated their corresponding genes hH2A and hH2B. Genomic copies of the genes were identified in O-S cosmid G25:G10, subcloned, and sequenced. We found that hH2A and hH2B lie 2.6 kb apart, are transcribed divergently, and are composed of three and four exons, respectively (Figure 1).

We failed to detect additional hH2A, hH2B, hH3, or hH4 genes in the N. crassa genome. Southern analysis of genomic DNA digested with a variety of restriction enzymes using hH2A, hH2B, hH3, hH4-1, and hH4-2 as probes detected only the bands expected for the known genes, as shown for hH2A (Figure 2). tBLASTn searches for genes encoding H2A, H2B, H3, and H4 in the EST databases (Nelsonet al. 1997; Roeet al. 2001b) detected numerous ESTs for hH2A, hH2B, hH3, hH4-1, and hH4-2, indicating that each of these genes is expressed (Figure 1). No other histone ESTs were found.

tBLASTn searches were also carried out using the databases of the two N. crassa genome sequencing projects, MNCDB (Schulteet al. 2001) and NSP (WICGR 2001). No new genes encoding core histones were found, but three histone variants were discovered. One was an H2A variant of the H2A F/Z family (Carret al. 1994), which we named hH2Az (Figure 3A). In the absence of ESTs for hH2Az, we used the high conservation within the hH2A F/Z family of variants to determine that the hH2Az coding region consists of three exons (Figure 1). We also found an H3 variant, which we named hH3v. It showed the greatest similarity to SpCENP-A (Figure 3B), a S. pombe homolog of CSE4, which is an essential gene of S. cerevisiae (Stoleret al. 1995). On the basis of similarity to other H3 variants, we suggest that the hH3v open reading frame is composed of two exons (Figure 1). The intron location in hH3v appears identical to the intron location in hH3. Without a cDNA sequence, we cannot be certain that hH3v does not have a second intron in the region of the expected N terminus, where there is low conservation among H3 variants (Figure 3B). We also identified a fragmented hH4 homolog, hH4v, which appears composed of six exons and five introns at positions different from those in hH4-1 and hH4-2 (Figure 1). The canonical methionine codon (ATG) of the potential hH4v translation product was replaced by a leucine codon (TTG) and its predicted sequence is only 62% identical to that of N. crassa H4 (Figure 3C). This is significantly lower than the >91% homology among H4 proteins across the fungal kingdom (Table 3), indicating that hH4v either produces a highly divergent H4 or is a pseudogene.

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Figure 1.

Structure of histone genes and number of associated ESTs identified in publicly available databases (Nelsonet al. 1997; Roeet al. 2001b). Solid boxes represent exons and open boxes represent introns. The gene structures were based on the similarity of the conceptually translated products with closely related proteins or were determined by comparing ESTs to the genomic sequences. Precise splice sites were assigned only if they matched the N. crassa splice site acceptor and donor consensus sequences (Radford and Parish 1997). Except for hH4v, numbering begins with the A in the initiating methionine codon and ends at the termination codon. For hH4v, numbering begins at a leucine codon that corresponds to the initiating methionine codon in the hH4 genes and ends at a glutamic acid codon that similarly corresponds to the hH4 termination codon (see Figure 3C). Due to the low similarity among H3 variants, the N terminus of hH3v is uncertain. Sizes of exons and introns (in base pairs) are shown above and below the diagrams, respectively. The exons are the same size in hH4-1 and hH4-2 because of conservation of intron positions, as indicated by the dashed lines.

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Figure 2.

Southern analysis for hH2A homologs. (A) Genomic DNA from strain N1089 was digested with the indicated restriction enzymes (P, PstI; N, NdeI; E, EcoRI; B, BamHI; A, ApaI) and probed for hH2A. The positions of size markers are shown on the right. No fragments <1 kb in size were detected. (B) Partial restriction map for the hH2A-hH2B genomic region. Fragment sizes resulting from digestion with each restriction enzyme are indicated. The probe used in A is represented by the open box below the map.

Genomic organization of the genes encoding the core histones and histone variants: The genomic location of the hH3-hH4-1 gene pair on the right arm of linkage group (LG) II was previously determined by restriction fragment length polymorphism (RFLP) mapping (Metzenberg and Grotelueschen 1987). The presence of this gene pair in the V-Y cosmid SV31:G9 (Vollmer and Yanofsky 1986), which carries the LG IIR gene aro-1 (Catchesideet al. 1985; Perkinset al. 2001), corroborates this assignment (Figure 4). The genomic locations of hH4-2 and the hH2A-hH2B gene pair were also determined by RFLP mapping using the standard sets of RFLP progeny (Metzenberget al. 1984). hH4-2 cosegregated with con-7 and trp-1 on LG IIIR and hH2A-hH2B cosegregated with ars-1 and the centromere of LG VII (Cen-VII; data not shown).

To map the histone and histone variant genes more precisely, we determined their location in the sequenced regions available from the NSP (Table 2) and MNCDB, searched for nearby genetic loci by BLASTn and BLASTx queries at NCBI and correlated matches with the Neurospora genetic map. The precise locations of all the histone genes were determined (Figure 4). The hH3-hH4-1 pair lies 15 kb centromere-proximal to aro-1 with hH4-1 lying centromere-distal to hH3. hH2Az also lies on LG II, 158 kb centromere-proximal to arg-5. Since no centromere-related sequences (Centola and Carbon 1994) were found between arg-5 and hH2Az, we conclude that it lies on the right arm of LG II and is transcribed toward the centromere. hH4-2 and hH3v were both found to lie on the right arm of LG III and are both transcribed toward the centromere. hH4-2 lies 38 kb centromere-distal to trp-1, whereas hH3v lies ≥121 kb centromere-proximal to ad-2. hH4v is located on the right arm of LG IV ≥416 kb centromere-distal to tol and is transcribed toward the centromere.

The hH2A-hH2B gene pair lies on supercontig 31 from the NSP (WICGR 2001), which also contains Cen-VII, in agreement with our RFLP mapping data. When we compared conceptual restriction digests of the contigs from supercontig 31 to published restriction maps of Cen-VII (Centola and Carbon 1994), we found that hH2A-hH2B lies to the right of the identified centromeric DNA with hH2A lying centromere-proximal to hH2B. Centola and Carbon (1994) originally determined the extent of the A + T-rich Cen-VII by constructing a restriction map of the centromere and flanking regions with the restriction enzyme PacI, which cleaves at an 8-bp recognition site composed solely of A:T pairs. The centromere was found to have a large concentration of PacI sites, while the relatively A + T-poor flanking regions were found to be devoid of PacI sites. To determine the distance between hH2A-hH2B and the centromere, we determined the concentration of PacI sites around hH2A-hH2B. We found that PacI sites were numerous both centromere-proximal and centromere-distal to it, suggesting that this gene pair may lie in centromeric heterochromatin (Figure 4).

Genetic identification of the histone gene complement: All of the core histones are essential in S. cerevisiae (Rykowskiet al. 1981; Schusteret al. 1986; Kayneet al. 1988; Mann and Grunstein 1992) and are presumably essential in all eukaryotes. Thus, as another test of whether we had identified the full complement of genes encoding each of the core histones of Neurospora, we attempted to inactivate each of the identified histone genes. Viability of strains bearing mutations in all known genes encoding a given histone would suggest that we had missed a functional histone gene, whereas lethality would suggest that we had not. Alternatively, lethality could result if redundant genes exist, but the expression level of each lies below a threshold for viability.

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Figure 3.

Alignments of the conceptually translated products of N. crassa histone variant genes with corresponding N. crassa histones, other fungal histone variants, and predicted mutant proteins. (A) Alignment of H2A, H2A^RIP, and H2Az from N. crassa with PHT1 from S. pombe and HTZ1 from S. cerevisiae. Numbering is relative to the H2Az sequence. (B) Alignment of H3v and H3 from N. crassa with fungal H3 variants SpCENP-A from S. pombe and CSE4 from S. cerevisiae. Numbering is relative to the H3v sequence. Due to the lack of homology in the extreme N terminus of H3v and the lack of ESTs, the N terminus shown is tentative. (C) Alignment of H4 with the conceptually translated products of hH4v (H4v), hH4-1^RIP1 (H4-1^RIP1), and hH4-1^RIP2 (H4-1^RIP2). Numbering is relative to H4. Turquoise residues are those that are identical between the core histone and the variant in N. crassa. Green residues are those that are identical between the N. crassa variant and at least one of the other fungal histone variants. Red residues are those that are identical between the core histone, the N. crassa histone variant, and at least one of the other fungal histone variants. Dark blue residues are those that are identical between any of the sequences, not including the N. crassa histone variant. Only the missense and nonsense mutations are shown in the mutant sequences. Asterisks indicate the position of stop codons.

In the case of H4, the test required us to inactivate both hH4-1 and hH4-2. For hH4-2, this was accomplished by replacing the hH4-2 gene with a functional inl gene (ΔhH4-2::inl⁺), which encodes an enzyme in the inositol biosynthesis pathway (Perkinset al. 2001). Two strains (N2014 and N2015) were transformed with pSH18, a plasmid carrying ΔhH4-2::inl⁺, and Inl⁺ strains were selected. Southern analysis of the transformants indicated that homologous replacement events occurred at a frequency higher than that normally reported for Neurospora (Fincham 1989; Aronsonet al. 1994): 24% (10/42) for N2014 and 14% (6/43) for N2015 (Figure 5). Two ΔhH4-2::inl⁺ transformants of N2014 (N2016 and N2017) and one of N2015 (N2018) were rendered homokaryotic by microconidiation (Pandit and Maheshwari 1993) and used in further tests. We compared the growth rates of N2018 and its host N2015 in race tubes and found no difference (data not shown). We also found that the transformants were fully fertile either as males or as females (data not shown). Apparently, either hH4-1 fully complements the loss of hH4-2 or hH4-2 is not a major contributor of H4.

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Figure 4.

Genomic locations of genes encoding core histones and histone variants. Solid circles represent centromeres. Distances between the genes in the two gene pairs were determined by sequencing the intervening regions. The distances to the other genetic loci were determined using available genome sequence data. Some distances are uncertain, due to gaps in the intervening sequenced regions. The hH2A-hH2B gene pair resides at the right edge of the A + T-rich DNA of the LG VII centromere.

To determine the importance of hH4-1 in the production of H4, we inactivated hH4-1 with RIP, a process in which duplicated sequences are peppered with G:C to A:T transition mutations during the premeiotic stage of crosses (Selker 1990). Many sequences altered by RIP are found methylated in vegetative cells (Selkeret al. 1993; Singeret al. 1995). We targeted hH4-1 to the his-3 locus, crossed the resulting hH4-1 duplication strains and analyzed the progeny for mutations and/or methylation at hH4-1. The nonduplication parent carried a nonfunctional arg-12 allele, which lies <1 map unit from aro-1 (Perkinset al. 2001). Because the hH3-hH4-1 gene pair lies between arg-12 and aro-1, crossovers between arg-12 and hH4-1 should be rare. Therefore, Arg⁺ progeny were selected, enriching for progeny whose native hH4-1 allele came from the duplication parent and therefore had the opportunity to undergo RIP. Two of 12 progeny screened for RFLPs and methylation, N2022 and N2023, exhibited methylation at hH4-1. N2023 also exhibited an altered digestion pattern with DpnII (data not shown). The hH4-1 gene at the native locus of each of the strains was sequenced and both alleles showed extensive mutations by RIP (Figure 3C). The hH4-1^RIP1 allele from N2022 exhibited 32 mutations in the 450 bp from the start codon through the stop codon, and allele hH4-1^RIP2 from N2023 showed 22 mutations in this span. A conceptual translation of hH4-1^RIP1 revealed 7 silent mutations and 13 missense mutations, whereas a conceptual translation of hH4-1^RIP2 revealed 6 silent mutations and 6 missense mutations. Both alleles include a nonsense mutation at Q94, which should cause a deletion of 10 amino acids from the C terminus. In S. cerevisiae, deletion of the 4 C-terminal amino acids of H4 is not lethal, whereas deletion of 6 C-terminal amino acids is (Kayneet al. 1988). Therefore, even disregarding the missense mutations, it seems unlikely that our H4 mutant alleles are functional. The growth rates of the hH4-1^RIP1 strains (N2022, N2023) and their parents were found to be equivalent, suggesting that hH4-2 supplied adequate H4. N2022 and N2023 were crossed as a male and female and with each other and in all cases were found to be fertile. Apparently, either hH4-1 or hH4-2 is sufficient for normal growth.

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Figure 5.

Homologous replacement of hH4-2 with inl. (A) Southern analysis of host (H) N2015 and its transformant (T) N2018 and of host N2014 and its transformants, N2017 and N2016. Genomic DNA was digested with BglII and probed with sequences flanking the replaced region. The native hH4-2 region yields a 21.4-kb BglII fragment, while a proper replacement event yields a 6.2-kb fragment. (B) Partial restriction map of the hH4-2 genomic region, the linearized pSH18 plasmid used in the transformations and the hH4-2 genomic region after a clean replacement event. In the genomic sequence the numbering is relative to the A in the initiation codon of hH4-2. In pSH18, solid lines represent sequences in common with the hH4-2 region, dashed lines represent vector DNA, and the boxed sequence represents the inl-bearing fragment from pOKE01. The precise location of inl in the fragment is unknown. Figure is not drawn to scale.

To confirm this conclusion and to verify that histone H4 is essential in Neurospora, we crossed strains carrying each hH4 mutant allele to determine whether or not hH4-1^RIP1;ΔhH4-2::inl⁺ progeny could be generated. Two crosses were carried out. In the first, N2024 × N2025, both parents carried defective inl alleles, so that the inl⁺ gene that replaced hH4-2 was the only functional inl gene in the cross. Furthermore, the ΔhH4-2::inl⁺ parent (N2025) carried arg-12, which is tightly linked to hH4-1. Ascospores from the cross were activated and plated directly on medium lacking inositol and arginine, thus selecting for ΔhH4-2::inl⁺ and the arg-12⁺ allele near hH4-1^RIP1. If strains lacking functional hH4-1 and hH4-2 genes could be viable, for example, due to a hypothetical third hH4 gene in the genome, the vast majority of progeny (>99%) should have been hH4-1^RIP1;ΔhH4-2:: inl⁺. Instead, analysis of 10 progeny by Southern hybridization revealed that all carried the wild-type hH4-1 allele, suggesting that the hH4-1^RIP1;ΔhH4-2::inl⁺ double mutant is not viable.

The second cross was equivalent to the first, except that a functional copy of hH4-2 was inserted at the his-3 locus of N2025 by transformation with pSH25, creating N2026. This artificially provided a third hH4 gene as a control. Progeny were selected as before, except that the functional hH4-2 at his-3 was selected by requiring histidine prototrophy. The majority of progeny (7/9) were hH4-1^RIP1;ΔhH4-2::inl⁺. Therefore, hH4-1^RIP1 and ΔhH4-2:: inl⁺ are null alleles and are synthetically lethal. On the basis of these results and our inability to detect other genes encoding H4, we conclude that hH4-1 and hH4-2 are the only functional hH4 genes in N. crassa.

We also attempted to recover an inactive hH3 allele by RIP. To provide a second copy of hH3 to activate RIP, we targeted hH3 to his-3 by transforming N1674 with pSH14, creating N2027, which was subsequently crossed with N2025. Progeny were selected using the same selection regime used in the hH4-1 RIP cross, described above, and 16 progeny were analyzed for hallmarks of RIP at hH3. No methylation or altered digestion patterns were observed (data not shown). Compared to the success rate of recovering null hH4-1^RIP alleles (2/12), the inability to recover hH3^RIP alleles (0/16) suggests that the loss of hH3 is lethal. On the basis of these results and our inability to identify other genes encoding H3, we conclude that hH3 is the only source of H3 in N. crassa.

To confirm genetically that hH2A and hH2B are unique in the genome, we employed the sheltered RIP strategy of Metzenberg and Grotelueschen (1992). This strategy relies on the inclusion of a mei-2 mutation in each parent to cause chromosome nondisjunction and thus production of aneuploid progeny. Disomic progeny break down rapidly to give heterokaryons with euploid nuclei that should be genetically identical, except for the chromosome that was subject to nondisjunction. One nuclear type should inherit this chromosome from one parent, while the other nuclear type should inherit this chromosome from the other parent. In this way a strain carrying an essential gene that has been disrupted by RIP can survive due to complementation by an extra wild-type allele.

In the sheltered RIP cross that we carried out, the two parents carried different mutations on the chromosome carrying hH2A-hH2B (LG VII). hH2A and hH2B were targeted to the his-3 locus of N1825 by transformation with pJS94 and pJS95 to create duplication strains N1822 and N1821, respectively. These transformants were crossed with N1679 and the resulting ascospores were germinated on medium lacking nicotinamide and arginine. This selected for the nic-3⁺ allele from N1821 and N1822 and the arg-10⁺ allele from N1679, resulting solely in the recovery of progeny disomic for LG VII. Eight progeny from the N1822 (hH2A-duplication) cross and 13 progeny from the N1821 (hH2B-duplication) cross were analyzed by Southern hybridization for hallmarks of RIP at hH2A and hH2B. One strain from the hH2A RIP cross, N2028, exhibited methylation at hH2A. Two of the progeny from the hH2B RIP cross exhibited methylation at hH2B and one of these (N2029) also exhibited an altered DpnII digestion pattern and was selected for further analysis (Figure 6).

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Figure 6.

Generation and propagation of hH2B^RIP1 allele. (A) Southern analysis of genomic DNA of N2035 (1), N2034 (2), N2029 (3), N1821 (4), and N1679 (5) digested with Sau3AI (S) or DpnII (D) and probed with hH2B. The higher molecular weight bands in the Sau3AI lanes are indicative of methylation. N2029 is the dikaryotic progeny of N1821 × N1679 and N2034 and N2035 are the dikaryotic progeny of N2029 × N1997. The letters to the left of the autoradiogram indicate the identity of the fragments as indicated in B. Positions of size markers are indicated on the right. No fragments >4 kb in size were detected. (B) DpnII/Sau3AI restriction map of hH2B. Vertical lines represent restriction sites; solid and open boxes represent the exons and introns of hH2B, respectively. The asterisk refers to the Sau3AI/DpnII site lost in hH2B^RIP1, resulting in the a+b fragment evident in A. The probe used in Southern blot in A is shown below the restriction map.

To test whether the hH2A^RIP and hH2B^RIP alleles were null and essential, we crossed N2028 and N2029 with N1997, a mei-2 strain with a genetically distinct LG VII (Figure 7). Ascospores from this second cross were germinated on medium containing hygromycin and arginine, which allows only two classes of progeny to survive: homokaryotic euploid progeny carrying the mutant histone allele and progeny disomic for LG VII, arising from a cross between N1997 and the nucleus bearing the mutant histone allele. Since the homokaryotic progeny would be Arg⁻ and the heterokaryotic progeny would be Arg⁺, we distinguished between these two expected classes by spot-testing on plates lacking arginine. In the cross involving the hH2B^RIP1 allele, all eight progeny were Arg⁺. The apparent requirement for a wild-type allele of hH2B in any strain carrying hH2B^RIP1 strongly argues that hH2B^RIP1 is null and that hH2B is essential, indicating that it is the only source of H2B in Neurospora.

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Figure 7.

Cross to determine whether the hH2A^RIP and hH2B^RIP (represented here only by hH2B^RIP) alleles are lethal. The markers shown are all carried on LG VII and the cross is homokaryotic for mei-2. The selection strategy allows only the growth of progeny carrying the LG VII with the mutant allele. Recovery of homokaryotic progeny indicates the mutant allele is not lethal.

In the case of the cross involving the hH2A^RIP allele, three of the seven progeny tested were Arg⁻, indicating either that the hH2A^RIP allele is not fully defective or that it is a null and there is a second hH2A gene in the genome that makes up for this deficiency. To distinguish between these two possibilities, we amplified the hH2A^RIP allele from the Arg⁺ progeny by PCR and sequenced it. Mutations from RIP were found, but it was not obvious that they would result in a null allele. In the 594-bp coding region and introns, only five mutations were found, three of which are silent mutations (Figure 3A). Since we were unable to generate a definite null allele of hH2A, we could not determine genetically the number of genes encoding H2A in the N. crassa. Nevertheless, since only hH2A was detected when genomic DNA blots or genomic cosmid libraries were screened with hH2A and since all of the H2A ESTs and genomic sequences that we found belong to hH2A, we conclude that hH2A is unique in the genome.

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Figure 8.

Most parsimonious tree of fungal histone H3 and H4 proteins. Branch lengths are proportional to amino acid substitutions. Percentages shown are from 1000 bootstrap replicates. Percentages <50 are not shown. Ascomycota, Zygomycota, and Basidomycota are sister phyla, whereas Euascomyctes are an order of Ascomycota. Basidiomycetes were used as the outgroup. The dotted portion of the Ascomycota line indicates that Zygomycota are not part of the Ascomycota.

Evolutionary relationship of the core histones of N. crassa to those of other fungi: Previous phylogenetic analyses of histones included few fungal representatives (Thatcher and Gorovsky 1994). To place the Neurospora histone genes in a fungal phylogeny, we searched the nonredundant databases at NCBI and several publicly available fungal EST databases to identify as many fungal core histone genes as possible. The amino acid sequences of the identified histone genes were then aligned and phylogenetic trees were constructed. All of the histones of Neurospora were found to group consistently with those of other Euascomycetes, as expected from previous phylogenetic studies based on morphological and molecular data (Spatafora 1995; Liuet al. 1999; Berbeeet al. 2000; Figure 8). As known for eukaryotes as a whole (Thatcher and Gorovsky 1994), H2A and H2B are the least highly conserved histones in fungi (Table 3) and most of their divergence is found in the N- and C-terminal tails. In contrast, H3 and H4 are more highly conserved (Table 3) and most of their divergence occurred in the structured globular domains.

Histone H4 of N. crassa is identical to one of the two H4 proteins of Botrytis fuckeliana and to the one H4 protein from F. sporotrichioides for which the entire amino acid sequence is available (Table 3). To estimate the time of divergence between hH4-1 and hH4-2 relative to their divergence from these counterparts, we constructed a phylogenetic tree of the coding sequences of the H4-coding genes from the euascomycetes. Although most of the tree is at low resolution, hH4-1 and hH4-2 cluster together with high confidence (data not shown). Thus, it appears that the two hH4 genes of Neurospora arose from a relatively recent gene duplication event. This is supported by an examination of intron locations between the two hH4 genes of Neurospora and the two H4-coding genes from E. nidulans, the only other euascomycete for which the genomic sequence of the H4 genes are known (Ehingeret al. 1990). Although the location of the second intron in hH4-1 and hH4-2 is not shared by the two H4 genes from E. nidulans, the first intron is located at the same position in all four genes.