LSD1 prevents aberrant heterochromatin formation in Neurospora crassa

Abstract

Heterochromatin is a specialized form of chromatin that restricts access to DNA and inhibits genetic processes, including transcription and recombination. In Neurospora crassa, constitutive heterochromatin is characterized by trimethylation of lysine 9 on histone H3, hypoacetylation of histones, and DNA methylation. We explored whether the conserved histone demethylase, lysine-specific demethylase 1 (LSD1), regulates heterochromatin in Neurospora, and if so, how. Though LSD1 is implicated in heterochromatin regulation, its function is inconsistent across different systems; orthologs of LSD1 have been shown to either promote or antagonize heterochromatin expansion by removing H3K4me or H3K9me respectively. We identify three members of the Neurospora LSD complex (LSDC): LSD1, PHF1, and BDP-1. Strains deficient for any of these proteins exhibit variable spreading of heterochromatin and establishment of new heterochromatin domains throughout the genome. Although establishment of H3K9me3 is typically independent of DNA methylation in Neurospora, instances of DNA methylation-dependent H3K9me3 have been found outside regions of canonical heterochromatin. Consistent with this, the hyper-H3K9me3 phenotype of Δlsd1 strains is dependent on the presence of DNA methylation, as well as HCHC-mediated histone deacetylation, suggesting that spreading is dependent on some feedback mechanism. Altogether, our results suggest LSD1 works in opposition to HCHC to maintain proper heterochromatin boundaries.

Figure 1.

Identification of LSDC in Neurospora crassa. (A) WGBS tracks displaying DNA methylation in WT (blue) and Δlsd1 (red) strains over LG VIIL (additional tracks for other chromosomes can be found in Supplemental Figure S2). NCU02455 is indicated with an asterisk. (B) Schematic representation of the identified members of LSDC with their predicted domains and length (amino acids) indicated. (C) LSDC knockouts exhibit hypermethylation. Genomic DNA from WT, a strain lacking DNA methylation (Δdim-2), and LSDC knockouts (Δlsd1, Δphf1 and Δbdp-1) were digested with the 5mC-sensitive restriction enzyme AvaII and used for Southern hybridizations with the indicated probes for a euchromatin control (his-3), an unaffected heterochromatin control (8:A6), and two Δlsd1-sensitive regions (Tel VIIL and NCU02455). Strains (left to right): N3753, N4711, N5555, N6411, N6221, N6414, N6220 and N6416.

Figure 2.

Variable hypermethylation and growth rates in strains with knockouts of LSDC members. (A) DNA from asexually-propagated Δlsd1 strains exhibit variable hypermethylation. Genomic DNA from the FGSC knockout of lsd1 and nine asexually-propagated strains was tested for DNA methylation by Southern hybridization, as in Figure 1C. The pedigree for these strains, relative to the initial strain (marked with an asterisk), is displayed on the right, and the pedigree provides a key to the numbers above the lanes in the Southern hybridization and to the strains in panel C. WT strain: N3753; Δdim-2 strain: N4711. (B) Linear growth rates of strains with LSDC member knockouts at 32°C. Strains (left to right): N3753, N5555, N7979, N6221, N6222, N6219 and N6220. (C) Linear growth rates of asexually-propagated Δlsd1 strains, relative to the initial ‘parental’ strain (N5555; indicated by an asterisk) at 32°C. The quadruple asterisks indicate strains that grew significantly slower than the other strains (P ≤ 0.0001).

Figure 3.

LSD1 prevents DNA methylation-dependent hyper H3K9me3. (A) H3K9me3 ChIP-seq and WGBS tracks showing H3K9me3 enrichment and DNA methylation respectively at two unaffected heterochromatin regions (Cen IIIL and 8:A6) and two Δlsd1-sensitive regions (NCU02455 and Tel VIIL) in the indicated strains. dim-2* denotes the C926A catalytic null mutation in dim-2 (42). NCU02455 is indicated with an asterisk. (B) Linear growth rates in WT, Δlsd1, and Δlsd1 strains bearing a deletion (Δ) or catalytic null (*) mutation in dim-2. Strains (left to right): N3753, N5555, N7979, N6337, N8081, N8082, N6679, N8083 and N8084. All Δlsd1; Δdim-2 and Δlsd1; dim-2* strains are siblings from separate crosses. (****) P ≤ 0.0001; (**) P ≤ 0.01. (C) Loss of LSD1 catalytic activity causes hyper DNA methylation. (Top panel) Sequence alignments of human, S. pombe, and Neurospora LSD1 homologs centered on a lysine essential for catalytic activity (highlighted). The NK982,983AA mutation introduced to create the presumptive catalytic null lsd1 Neurospora strains is displayed above. (Bottom panel) DNA methylation-sensitive Southern hybridization analysis (as in Figure 1C) on 3xFLAG-tagged and catalytic null (indicated by *) 3xFLAG-tagged LSD1 strains. Strains (left to right): N3753, N4711, N5555, N6300 and N7899.

Figure 4.

HCHC catalytic activity is necessary for Δlsd1-induced hyper DNA methylation. (A) WGBS tracks displaying DNA methylation in wild-type (WT), Δhda-1, and Δlsd1 strains at selected Δlsd1-sensitive regions. (B) DNA methylation-sensitive Southern hybridizations probing the regions illustrated in panel A showing the loss of Δlsd1-induced hypermethylation in a hda-1 catalytic-null background in sibling strains. The Southern probes used are indicated by a red bar in panel A. Strains (left to right): N3753, N4711, N3998, N6412, N8089 and N8090. (C) ChIP-seq tracks showing H3K9me3 enrichment in WT, Δlsd1 and Δlsd1; hda-1^D263N strains at selected Δlsd1-sensitive regions.

Figure 5.

HCHC catalytic activity and DNA methylation become necessary for heterochromatin formation with increasingly GC-rich DNA. (A) Metaplot displaying the averaged profile of H3K9me3 enrichment in WT and Δhda-1 strains as determined by ChIP-seq and averaged %GC-content over all constitutive heterochromatin domains in wild type. (B) Model showing interactions between factors involved in establishing heterochromatin with decreasing DNA AT-content. (Left) In the absence of HCHC catalytic activity, the significant AT-richness of the interior of heterochromatin domains is sufficient to recruit the histone lysine methyltransferase complex, DCDC, to establish H3K9me3 and subsequent methylation of underlying DNA. However, the abruptly decreasing AT content at heterochromatin borders is insufficient for DCDC-induced heterochromatin, and without HCHC, heterochromatin is unable to properly spread over the canonical domain. (Middle) In a wild-type scenario, HCHC is able to localize to heterochromatin boundaries through binding of H3K9me3 catalyzed by DCDC, as well as through AT-hook domains in the CHAP subunit. Histone deacetylation activity by HCHC is able to recruit DCDC to further mark H3K9me3 on neighboring chromatin and establish a propagating feedback loop capable of spreading heterochromatin across the entirety of the canonical domain. Here, factors such as LSD1 that limit heterochromatin spreading act to keep the expansion in check within proper limits of what should be heterochromatin. (Right) In the absence of such limiting factors where heterochromatin spreads over its boundary into euchromatin, or in DLDM where convergent transcription induces H3K9me3 and DNA methylation, heterochromatin is established over DNA with AT-content well below the level for RIP-induced DNA methylation. Typically, DNA methylation is dependent on the H3K9me3 mark and loss of DNA methylation has no impact on H3K9me3 (21,37,38). In instances of heterochromatin over low (∼50%) AT-richness, DNA methylation now becomes essential for H3K9me3 and further spreading.