Spreading and epigenetic inheritance of heterochromatin require a critical density of histone H3 lysine 9 tri-methylation Amber R. Cutter DiPiazzaa, Nitika Tanejaa,1, Jothy Dhakshnamoorthya, David Wheelera, Sahana Hollaa, and Shiv I. S. Grewala,2 aLaboratory of Biochemistry and Molecular Biology, National Cancer Institute, NIH, Bethesda, MD 20892 Edited by Steven E. Jacobsen, University of California, Los Angeles, CA, and approved April 20, 2021 (received for review January 12, 2021) Heterochromatin assembly requires methylation of histone H3 domains coat pericentromeric, subtelomeric, and the silent mating- lysine 9 (H3K9me) and serves as a paradigm for understanding the type (mat) regions (9–12). Heterochromatin assembly is a multistep importance of histone modifications in epigenetic genome control. process that includes nucleation and spreading. The de novo nu- Heterochromatin is nucleated at specific genomic sites and spreads cleation of heterochromatin occurs at specific sites, such as repeat across extended chromosomal domains to promote gene silencing. elements within constitutive heterochromatin domains, from where Moreover, heterochromatic structures can be epigenetically inherited heterochromatin factors spread to surrounding sequences (13, 14). in a self-templating manner, which is critical for stable gene repres- RNAi machinery (13, 15), as well as factors involved in nuclear sion. The spreading and inheritance of heterochromatin are believed RNA processing and noncanonical RNA polymerase II termi- to be dependent on preexisting H3K9 tri-methylation (H3K9me3), nation (12, 16–19), nucleate heterochromatin by targeting the which is recognized by the histone methyltransferase Clr4/Suv39h multisubunit Clr4 methyltransferase complex (ClrC) (20) that is via its chromodomain, to promote further deposition of H3K9me. responsible for mono-, di-, and tri-methylation of histone H3K9 However, the process involving the coupling of the “read” and “write” (H3K9me1/2/3) (6, 21). Clr4 has a unique dual activity allowing it capabilities of histone methyltransferases is poorly understood. From to bind to methylated H3K9 via its chromodomain (“read”)and an unbiased genetic screen, we characterize a dominant-negative mu- catalyze H3K9 methylation (“write”), a feature that is essential for G13D tation in histone H3 (H3 ) that impairs the propagation of endog- heterochromatin to spread (22, 23). The H3K9me2/3 marks also GENETICS enous and ectopic heterochromatin domains in the fission yeast provide a binding site for other chromodomain proteins, including genome. H3G13D blocks methylation of H3K9 by the Clr4/Suv39h meth- the HP1 family members Chp2 and Swi6 (24), which in turn target yltransferase and acts in a dosage-dependent manner to interfere with effectors such as the Snf2-histone deacetylase (HDAC) repressor the spreading and maintenance of heterochromatin. Our analyses complex (SHREC) involved in transcriptional gene silencing (25, 26). show that the incorporation of unmethylatable histone H3G13D into The principle that preexisting heterochromatin can be epige- chromatin decreases H3K9me3 density and thereby compromises the netically inherited in a self-templating manner during both mitosis read-write capability of Clr4/Suv39h. Consistently, enhancing the af- and meiosis was uncovered through studying heterochromatin finity of Clr4/Suv39h for methylated H3K9 is sufficient to overcome G13D the defects in heterochromatin assembly caused by H3 .Ourwork Significance directly implicates methylated histones in the transmission of epige- netic memory and shows that a critical density threshold of H3K9me3 In multicellular organisms, a single genome gives rise to a multi- is required to promote epigenetic inheritance of heterochromatin tude of cell types by enforcing appropriate gene expression pat- through the read-write mechanism. terns. Epigenetic mechanisms involving modification of histones, includingtri-methylationofhistoneH3lysine9(H3K9me3),as- heterochromatin | epigenetic | histone methylation | gene silencing semble and propagate repressive heterochromatin to prevent untimely gene expression. Dysregulation of epigenetic gene- he assembly of distinct chromatin domains within eukaryotic silencing mechanisms is a hallmark of a variety of diseases in- Tgenomes facilitates diverse chromosomal processes, including cluding cancer. However, the requirements for epigenetic trans- the maintenance of genome stability, and the regulation of gene mission of heterochromatin are not well understood. This study expression, DNA replication and recombination. Histones and their reveals the mechanism by which methylated histones provide an posttranslational modifications are components of epigenetic mecha- epigenetic template for heterochromatin propagation. We dis- nisms that partition the genome into “open” euchromatin or “closed” cover that a critical threshold of H3K9me3 is required for ef- heterochromatin domains (1–3). Euchromatin domains, marked by fective chromatin-association of the histone methyltransferase, histone acetylation and histone H3 lysine 4 methylation (H3K4me), which binds to and catalyzes additional H3K9me to propagate are generally more accessible to transcription machinery, whereas heterochromatin and enforce stable gene silencing. repressive heterochromatin domains are hypoacetylated and contain the histone H3 lysine 9 methylation (H3K9me) mark (4, 5). Histone Author contributions: A.R.C.D. and S.I.S.G. designed research; A.R.C.D., N.T., and J.D. performed research; A.R.C.D. and J.D. contributed new reagents; A.R.C.D., N.T., D.W., H3 proteins bearing di- and tri-methylation marks (H3K9me2/3) S.H., and S.I.S.G. analyzed data; and A.R.C.D. and S.I.S.G. wrote the paper. are recognized by heterochromatin protein 1 (HP1) family proteins The authors declare no competing interest. (6–8), which together serve as a recruitment scaffold for multiple This article is a PNAS Direct Submission. effectors (2). Considering that histone modifications define gene This open access article is distributed under Creative Commons Attribution-NonCommercial- expression patterns specific to different cell types, determining their NoDerivatives License 4.0 (CC BY-NC-ND). role in the assembly and propagation of distinct chromatin domains 1Present address: Department of Molecular Genetics, Erasmus MC Cancer Institute, Erasmus is critical for understanding normal development and disease states. University Medical Center, 3000 CA Rotterdam, The Netherlands. The fission yeast Schizosaccharomyces pombe is an excellent 2To whom correspondence may be addressed. Email: [email protected]. model system for studying heterochromatin assembly. Small facul- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ tative heterochromatin islands target developmentally and environ- doi:10.1073/pnas.2100699118/-/DCSupplemental. mentally regulated genes, and large constitutive heterochromatin Published May 25, 2021. PNAS 2021 Vol. 118 No. 22 e2100699118 https://doi.org/10.1073/pnas.2100699118 | 1of10 Downloaded by guest on September 25, 2021 assembly at the silent mat region in S. pombe (13, 27, 28). Fur- and H3K9me2 occurred throughout the silent mat region in + ther insights into epigenetic transmission of chromatin structure EMS32 KΔ::ura4 cells (Fig. 1D). Additionally, EMS32 exhibited soon followed. The first major finding established the impor- reduced H3K9me at facultative heterochromatin islands (SI Ap- tance of Clr4 read-write activity in the epigenetic inheritance of pendix,Fig.S1) as well as changes in H3K9me levels at subtelomeric heterochromatin (22). This work showed that parental H3K9 meth- and pericentromeric regions (Fig. 1D). These analyses indicate that ylated nucleosomes and their associated factors are a “molecular EMS32 compromises heterochromatin assembly in cells defective in bookmark” and provide an epigenetic template for loading Clr4 (via de novo heterochromatin nucleation at the silent mat region, spe- the Clr4 chromodomain) to promote clonal propagation of hetero- cifically by affecting the propagation of heterochromatin domains. chromatin. Another key finding was that constitutive heterochroma- To map the EMS32 mutation, we backcrossed EMS32 to a non- tin domains show markedly lower turnover of histones compared to mutagenized parental strain and performed tetrad analyses in which + euchromatin domains and that the suppression of histone turn- silencing of the ura4 reporter was monitored to isolate wild-type over preserves epigenetic memory for the inheritance of hetero- (WT) and EMS32 segregants. Whole-genome sequencing and nu- chromatin (29). The HDAC Clr3 and the homolog of mammalian cleotide variant analysis of three WT and three EMS32 segregants SMARCAD1, Fft3, are recruited by HP1 proteins or other factors revealed a G-to-A nucleotide substitution unique to EMS32 mutants and stabilize nucleosomes carrying H3K9me, thus promoting stable that mapped to hht2.InS. pombe, the histone H3 protein is encoded + + + propagation of heterochromtin (29, 30). Moreover, the nuclear by three alleles, hht1 , hht2 ,andhht3 (40). Conventional DNA peripheral subdomain provides an ideal microenvironment for sequencing confirmed the G-to-A nucleotide conversion in hht2 loading factors that facilitate epigenetic inheritance (31, 32). De- (Fig. 2A), which changes glycine to aspartic acid at position 13 spite significant progress, evidence directly linking methylated his- (G13D) in the amino-terminal tail of histone