A reciprocal translocation involving Aspergillus nidulans snxAHrb1/Gbp2 and gyfA uncovers a new regulator of the G2-M transition and reveals a role in transcriptional repression for the setBSet2 histone H3-lysine-36 methyltransferase

Abstract

Aspergillus nidulans snxA, an ortholog of Saccharomyces cerevisiae Hrb1/Gbp2 messenger RNA shuttle proteins, is-in contrast to budding yeast-involved in cell cycle regulation, in which snxA1 and snxA2 mutations as well as a snxA deletion specifically suppress the heat sensitivity of mutations in regulators of the CDK1 mitotic induction pathway. snxA mutations are strongly cold sensitive, and at permissive temperature snxA mRNA and protein expression are strongly repressed. Initial attempts to identify the causative snxA mutations revealed no defects in the SNXA protein. Here, we show that snxA1/A2 mutations resulted from an identical chromosome I-II reciprocal translocation with breakpoints in the snxA first intron and the fourth exon of a GYF-domain gene, gyfA. Surprisingly, a gyfA deletion and a reconstructed gyfA translocation allele suppressed the heat sensitivity of CDK1 pathway mutants in a snxA+ background, demonstrating that 2 unrelated genes, snxA and gyfA, act through the CDK1-CyclinB axis to restrain the G2-M transition, and for the first time identifying a role in G2-M regulation for a GYF-domain protein. To better understand snxA1/A2-reduced expression, we generated suppressors of snxA cold sensitivity in 2 genes: (1) loss of the abundant nucleolar protein Nsr1/nucleolin bypassed the requirement for snxA and (2) loss of the Set2 histone H3 lysine36 (H3K36) methyltransferase or a nonmethylatable histone H3K36L mutant rescued hypomorphic snxA mutants by restoring full transcriptional proficiency, indicating that methylation of H3K36 acts normally to repress snxA transcription. These observations are in line with known Set2 functions in preventing excessive and cryptic transcription of active genes.

Keywords: H3K36me3; H3K4me3; cclABre2; gyfA; nsr1/nucleolin; nsrA; setB; snxAHrb1/Gbp2; GYF domain; translocation.

Fig. 1.

A reciprocal translocation interrupts AN6228-gyfA on chromosome IR and AN3739-snxA on chromosome IIL. Whole-genome sequences of the translocation regions in SWJ 5594 (snxA1) and SWJ 5586 (snxA2) were verified by Sanger sequencing of SWJ 5594, MDS250, SWJ 3404, and SWJ 5569 (snxA1), and SWJ 5585 and SWJ 5586 (snxA2), as described in the Materials and Methods. a) Translocation of the right arm of chromosome II onto the left arm of chromosome I. b) Translocation of the left arm of chromosome I onto the right arm of chromosome II. Key: Boxed areas indicate snxA or gyfA exons or neighboring genes. Nucleotide positions for chromosome I and II sequences, and for a 60-nt helitron fragment, are shown above and below the DNA sequences. In (a), 9-exon ATG-2 marks the beginning of a 42-nt sequence encoding the first 14 amino acids of the 9-exon snxA allele, located in the 547-nt second intron of the 11-exon snxA allele. This 42-nt codegenic sequence lies upstream of and includes the 3′ splice junction of intron 2 of the 11-exon snxA allele and continues in-frame with exon 3. In (b), backslashes in the center mark the position of the helitron fragment derived from one of 2 helitron sequences located on Chr II and Chr VIII. In the FGSC A4 reference, the helitron contains a G, shown above the DNA sequence, whereas in all 6 snxA1/A2 sequenced strains, a C occurred at this position. Candidate transcription startsites (TSSs) were mapped by Sibthorp et al. (2013).

Fig. 2.

Southern blot verification of reciprocal translocation. Genomic DNAs from 2 wild-type strains (PCS 439 and SO64), 2 snxA1 strains (SWJ 3404 and MDS 250), and 1 snxA2 strain (SWJ 5586) were digested with SacII and subjected to chemilluminescent Southern blotting using digoxigenin-labeled DNA probes, as depicted. a) Translocation of the left arm of chr I onto the right arm of chr II, T(IL; IIR), detected by 2 probes: AN6228 3′ probe and seq I probe. b) Translocation of the left arm of chr II onto the right arm of chr I, T(IIL; 1R), detected by 2 probes: AN6228 5′ probe and DF-AR probe. The seq I probe in (a) spans the tRNALeu gene. This 74-nt tRNA-Leu, tL(UAA)1, is the sole tRNA serving UUA codons (blastn e-value 4e⁻³² to itself). Next closest are 2 leucyl-tRNAs that serve CUA codons (blastn e-values 6e⁻⁰⁵). Therefore, no cross-hybridization on Southern blots would be expected.

Fig. 3.

Deletion of snxA (AN3739) partially rescues heat-sensitive G2–M defects of nimX^Cdk1 mutants. Growth phenotypes of single and double mutants on minimal medium (top panel) and rich medium (bottom panel) at the indicated temperatures. Strains were grown for 8 days at 21°C, and for 2.5 days at 32°C, 37°C, and 42°C. Strains: WT, SWJ 424; ΔsnxA, tSWJ 6620; snxA1, SWJ 5594; snxA2, SWJ 5585; nimX1, SWJ 1798; ΔsnxA nimX1, tSWJ 4578; snxA1 nimX1, SWJ 4202; snxA2 nimX1, SWJ 4203; nimX2, SWJ 1815; ΔsnxA nimX2, tSWJ 4460; snxA1 nimX2, SWJ 3710; snxA2 nimX2, SWJ 5619.

Fig. 4.

Mutations in AN6228-gyfA partially rescue heat-sensitive G2–M defects of nimX^Cdk1 mutants. Growth phenotypes of single and double mutants on minimal medium (top panel) and rich medium (bottom panel) at the indicated temperatures. ΔgyfA, complete deletion; gyfAΔaa1061-1506, reconstructed AN6228-gyfA truncated translocation allele. Strains were grown for 8 days at 21°C, and for 2.5 days at 32°C, 37°C, and 42°C. Strains: wild type, SWJ 424; ΔgyfA, tMLB 6498; gyfAΔaa1061-1506, tMLB 6744; nimX1, SWJ 1798; ΔgyfA nimX1, tSWJ 6590; gyfAΔaa1061-1506 nimX1, tKGW 6766; nimX2, SWJ 1815; ΔgyfA nimX2, tSWJ 6593; gyfAΔaa1061-1506 nimX2, tKGW 6767.

Fig. 5.

Mutations in gyfA partially rescue nimE6^cyclinB. The ability to rescue the temperature-sensitive lethal, G2–M-arresting nimE6^cyclinB mutation was assessed in 2 independent strains each for ΔsnxA, ΔgyfA, and the gyfAΔaa1061-1506 translocation allele. Strains were toothpicked onto minimal media and grown for 7 days at 21°C, and for 2.5 days at 32°C, 37°C, and 43°C. Strains: wild type, SWJ 4051; nimE6, SWJ 195; ΔsnxA, tSWJ 6944; ΔgyfA, tMLB 6501; gyfAΔaa1061-1506, tMLB 6744; ΔsnxA nimE6, tSWJ 4454 and tSWJ 4455; ΔgyfA nimE6, tMLB 6670 and tMLB 6671; gyfAΔaa1061-1506 nimE6, tMLB 6945 and tMLB 6946.

Fig. 6.

GYFA is a cytosolic protein. gyfA was C-terminally tagged with GFP and combined with histone H1::mCHERRY to create tMLB 6769 (see Materials and Methods). The strain was grown at 37°C for 6 h, and then live cell imaging was performed. GYFA::GFP localization was exclusively cytoplasmic in all germlings (n > 200). Yellow bar = 10 µM.

Fig. 7.

Allele-specific suppression of snxA1/A2 strains by setB point-mutations. setB point mutations and a setB deletion were tested for their ability to suppress the cold sensitivity of strains derived from snxA1 and snxA2. setB-sup26 (G255W) and setB-sup59 (G255V) were generated by 4-NQO mutagenesis in a snxA2 background, and then outcrossed twice to create double mutants with strains derived from snxA1 and snxA2. The setB-G255V and ΔsetB alleles were constructed by fusion PCR, followed by 1-step gene replacement at the setB locus. Fresh spores were inoculated by toothpick onto minimal agar media and grown for 6 days at 22°C, 2.5 days at 29°C, and 2 days at 37°C. Key to symbols: A1, snxA1; A2, snxA2; setB-G255V, genetically engineered knock-in mutant. Strains: wild type, SWJ 4052; snxA2, SWJ 5581; snxA1, SWJ 6343; snxA2 setB-sup26, SWJ 6092; snxA1, setB-sup26, SWJ 6202; snxA2 setB-sup59, SWJ 6082; snxA1 setB-sup59, SWJ 5998; snxA2 setB-G255V, tSWJ 6167; snxA1 setB-G255V, SWJ 6168; snxA2 ΔsetB, tSWJ 6164; snxA1 ΔsetB, tSWJ 6247; ΔsetB, tSWJ 6250.

Fig. 8.

A setB null mutation and a nonmethylatable histone H3K36L mutation rescue snxA transcriptional proficiency. Refer to Supplementary Fig. 9 and the methods for the transcript-specific qRT-PCR strategy. Relative snxA mRNA expression was measured in actively growing vegetative mycelia using primers to detect the 11- and 9-exon snxA transcripts in snxA+, snxA1, and snxA2 strains (1) lacking the setB histone H3K36 trimethyltransferase or (2) harboring a nonmethylatable histone H3K36L mutation or (3) both mutations. Relative expression was normalized to actA using the ΔΔCt method using 4 replicates in each of 2 independent RNA preps. Error bars, SEM. a) Relative normalized expression of 11-exon mRNA transcripts. b) Relative normalized expression of 9-exon mRNA transcripts and ability to suppress cold sensitivity in ΔsnxA, snxA1, and snxA2. Fresh conidiospores were toothpicked onto minimal media and grown at 37°C for 3 days, and at 21°C for 7 days. Strains: WT, SWJ 4052; snxA +  ΔsetB, tSWJ 6407; snxA+ H3K36L, tSWJ 6193; snxA +  ΔsetB H3K36L, tSWJ 6274; snxA1, tSWJ 5594; snxA1 ΔsetB, tSWJ 6247; snxA1 H3K36L, tSWJ 6214; snxA1 ΔsetB H3K36L, tSWJ 6353; snxA2, SWJ 5586; snxA2 ΔsetB, tSWJ 6164; snxA2 H3K36L, tSWJ 6171; snxA2 ΔsetB H3K36L, tSWJ 6267.

Fig. 9.

setB mutations do not fully rescue the snxA2 suppression of nimX2 temperature sensitivity. To determine if setB mutations can fully restore nimX2 temperature sensitivity in snxA2-derived translocation mutants, the growth of nimX2 and nimX2 snxA2 at restrictive temperatures of 37°C and 43°C was compared to triple mutant strains carrying nimX2 and snxA2 and one of 4 setB mutations. Fresh conidiospores were tooth picked onto minimal media and grown at 21°C for 8 days and at 37°C and 43°C for 3 days. Abbreviations: X2, nimX2; A2, snxA2. Strains: wild type, SWJ 424; snxA2, SWJ 5586; nimX2, SWJ 1607; nimX2 snxA2, SWJ 5619; nimX2 snxA2 ΔsetB, tMMC 6845; nimX2 snxA2 setB26-G255W, SWJ 6139; nimX2 snxA2 setB59-G255V, SWJ 6096; nimX2 snxA2 setB67, SWJ 6141.

Fig. 10.

SETB is the major A. nidulans histone H3K36 trimethyltransferase. Nuclear proteins were prepared from cultures grown at 37°C for 20 h. Five micrograms of each protein sample was separated in a 4–20% SDS-PAGE gel (Bio-Rad). Western blot analysis used primary antibodies against H3K36me3 (Abcam, 1:1,000), H3K36me2 (Abcam, 1:1,500), and pan-H3 (Millipore, 1:10,000). Secondary antibody was goat-α-rabbit-HRP (1:5,000). α-H3K36me3 (a) and (b): Samples from independently generated protein samples were detected with the H3K36me3 antibody. H3K36me2 and pan-H3 were from sample (a) Relative band intensities were measured using ImageJ and normalized against the lanes containing nonmethylatable H3K36L proteins. Strains: SWJ 424 (WT), tSWJ 5586 (snxA2; A2), tSWJ 6408 (ΔsetB), tSWJ 6164 (snxA2 ΔsetB; A2 ΔsetB), and tSWJ 6193 (nonmethylatable H3K36L mutant; K36L).

Fig. 11.

SET1/COMPASS function is essential in cells with reduced snxA expression. Genetic interactions between snxA1/A2 and the SET1/COMPASS complex were tested in double mutants carrying a deletion of cclA^Bre2, a key subunit of the SET1/COMPASS complex that mediates trimethylation of histone H3 lysine 4 (H3K4me3). Fresh conidiospores were seeded onto minimal agar media and grown at 35°C for 8 days. Strains: WT, SWJ 424; snxA1, tSWJ 5594; snxA2, SWJ 5586; ΔcclA^Bre2, tSWJ 5645; ΔsnxA, tSWJ 4395; snxA1 ΔcclA^Bre2, SWJ 5635; snxA2 ΔcclA^Bre2, tSWJ 5637.

Fig. 12.

The ΔnsrA suppressor mutation bypasses the requirement for snxA function at 21°C. a) Deletion of the nsrA^{Nsr1/nucleolin} gene suppresses the cold sensitivity of a ΔsnxA null mutation. Fresh conidiospores were point-inoculated on minimal media and grown for 3 days at 37°C and for 9 days at the restrictive temperature of 21°C. Strains: WT, SWJ 424; ΔnsrA, tAAM 6225; ΔsnxA, tSWJ 4412; snxA1, tSWJ 5594; snxA2, tLC 5602; ΔnsrA ΔsnxA, tSWJ 7104; ΔnsrA snxA1, tSWJ 7105; ΔnsrA snxA2, tAAM 6224. b) snxA expression in snxA2 nsrA39/49 double mutants was unchanged relative to a snxA2 control. Relative snxA mRNA expression was measured in actively growing vegetative mycelia using primers to detect the 9-exon snxA transcript in snxA+, snxA2, snxA2 nsrA39, and snxA2 nsrA49 strains. Relative expression was normalized to actA using the ΔΔCt method using 4 replicates. Error bars, SEM. Strains: WT, SWJ 4052; snxA2, SWJ 6034; snxA2 nsrA39 SWJ 6013; snxA2 nsrA49, SWJ 6014.