H1 linker histones silence repetitive elements by promoting both histone H3K9 methylation and chromatin compaction Sean E. Healtona,1,2, Hugo D. Pintoa,1, Laxmi N. Mishraa, Gregory A. Hamiltona,b, Justin C. Wheata, Kalina Swist-Rosowskac, Nicholas Shukeirc, Yali Doud, Ulrich Steidla, Thomas Jenuweinc, Matthew J. Gamblea,b, and Arthur I. Skoultchia,2 aDepartment of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461; bDepartment of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461; cMax Planck Institute of Immunobiology and Epigenetics, Stübeweg 51, Freiburg D-79108, Germany; and dDepartment of Pathology, University of Michigan, Ann Arbor, MI 48109 Edited by Robert G. Roeder, Rockefeller University, New York, NY, and approved May 1, 2020 (received for review December 15, 2019) Nearly 50% of mouse and human genomes are composed of repetitive mechanisms of this regulation have not been fully explored. To sequences. Transcription of these sequences is tightly controlled during further investigate the roles of H1 in epigenetic regulation, we have development to prevent genomic instability, inappropriate gene used CRISPR-Cas9–mediated genome editing to inactivate addi- activation and other maladaptive processes. Here, we demonstrate tional H1 genes in the H1 TKO ES cells and thereby deplete the H1 an integral role for H1 linker histones in silencing repetitive elements in content to even lower levels. mouse embryonic stem cells. Strong H1 depletion causes a profound Nearly 50% of the mouse and human genomes consist of re- de-repression of several classes of repetitive sequences, including major petitive sequences, including tandem repeats, such as satellite se- satellite, LINE-1, and ERV. Activation of repetitive sequence transcrip- quences, as well as interspersed elements, such as long interspersed tion is accompanied by decreased H3K9 trimethylation of repetitive nuclear elements (LINEs), short interspersed nuclear elements sequence chromatin. H1 linker histones interact directly with Suv39h1, (SINEs), and endogenous retroviruses (ERVs) (13). Transcription of Suv39h2, and SETDB1, the histone methyltransferases responsible for these sequences is tightly controlled during development (14). H3K9 trimethylation of chromatin within these regions, and stimulate Whereas transcription of certain repetitive sequences has been CELL BIOLOGY their activity toward chromatin in vitro. However, we also implicate reported to be required for normal cellular processes, such as het- chromatincompactionmediatedbyH1asanadditional,dominantre- erochromatin formation and embryonic development (15–17), ab- pressive mechanism for silencing of repetitive major satellite se- errant transcription of some repetitive sequences has been implicated quences. Our findings elucidate two distinct, H1-mediated pathways in disease states, including cancer (18, 19). for silencing heterochromatin. The establishment and maintenance of a repressive chromatin environment is an important mechanism for the silencing of epigenetics | linker histones | repetitive elements | chromatin these repetitive sequences. Silencing of these sequences in mouse ukaryotic genomes are packaged into chromatin, a complex Significance Epolymer of DNA, RNA, and protein that facilitates compaction of the long strands of linear DNA into the nucleus. The repeating unit of Eukaryotic genomes harbor a vast number of “selfish” DNA chromatin, the nucleosome, consists of a core histone octamer—two — elements, including transposable elements and repetitive se- each of the core histones H2A, H2B, H3, and H4 wrapped around quences. They constitute nearly 50% of the human genome 147 bp of DNA. A fifth histone, the linker histone H1, dynamically and need to be silenced to maintain the integrity of the ge- associates (1, 2) with the nucleosome core particle at the dyad axis nome. Aberrant expression of such sequences, possibly due to and facilitates the formation of more compact chromatin structures failure of silencing mechanisms, is associated with human dis- (3). In addition to controlling access to the underlying DNA via eases, including cancer. Silencing of these “selfish” DNAs in- steric hinderance, the histone proteins carry a multitude of post- volves methylation of specific lysine residues in the nucleosome translational modifications that influence many nuclear processes, core particles that help package these DNA elements into chro- including transcription, DNA repair, and replication (4). matin in the cell nucleus. Here we demonstrate that H1 linker Although linker histones are some of the most abundant nu- histones, the most abundant chromatin-binding proteins, are clear proteins, our understanding of their functions of remains very critical for silencing of these sequences, by promoting repressive incomplete. Mammals express multiple linker histone subtypes or lysine methylation and further compacting these elements into variants, including seven somatic subtypes and four germ cell- more condensed chromatin structures. specific subtypes (5). We previously generated mice inactivated for one or two H1 subtypes and discovered that cells can maintain Author contributions: S.E.H. and A.I.S. designed research; S.E.H., H.D.P., L.N.M., and J.C.W. their total H1 content through compensatory up-regulation of their performed research; H.D.P., G.A.H., K.S.-R., N.S., Y.D., U.S., T.J., and M.J.G. contributed remaining H1 genes (6). However, mice genetically ablated for new reagents/analytic tools; S.E.H., H.D.P., J.C.W., T.J., and A.I.S. analyzed data; and S.E.H. three highly expressed H1 subtypes exhibit embryonic lethality, and A.I.S. wrote the paper. demonstrating that linker histones are essential for mammalian The authors declare no competing interest. development (7). Embryonic stem (ES) cells derived from these This article is a PNAS Direct Submission. triple-knockout (TKO) embryos have a reduced (∼50%) total H1 Published under the PNAS license. content and display specific changes in gene expression due to See online for related content such as Commentaries. locus-specific alterations in DNA methylation and histone H3 lysine 1S.E.H. and H.D.P. contributed equally to this work. – 4methylation(8 10). These results, as well as data from numerous 2To whom correspondence may be addressed. Email: [email protected] or other studies (11, 12), have increasingly supported the view that in [email protected]. addition to their well-established structural roles in chromatin, H1 This article contains supporting information online at https://www.pnas.org/lookup/suppl/ linker histones are intimately involved in epigenetic regulation of doi:10.1073/pnas.1920725117/-/DCSupplemental. chromatin function. However, the extent, genomic contexts, and First published June 8, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1920725117 PNAS | June 23, 2020 | vol. 117 | no. 25 | 14251–14258 Downloaded by guest on September 29, 2021 ES cells involves trimethylation of histone 3 lysine 9 (H3K9me3) 15 AB**** catalyzed by three histone methyltransferases (HMTs): Suv39h1, 4 H1-enriched 10 Suv39h2, and SETDB1 (20–23). In mESCs null for both Suv39h1 Random **** 2 5 and Suv39h2, H3K9me3 is lost from pericentric heterochromatin, ns (IP/input) **** 2 Enrichment and major satellite and LINE elements are de-repressed (20, 24). 0 log 0 On the other hand, SETDB1 appears to be responsible for H3K9 (odds ratio) 2 -5 H1 enrichment -2 methylation and silencing of class I and II ERVs (22, 23). How- log Suv39h1 H3K9me3 SETDB1 G9a ever, H3K9 methylation alone is unlikely to be the sole determi- C -4 nant of the transcriptional status of the underlying repetitive H3K9me3 CH12.LX G9a H3K9me3 G1E sequence, because loss of H3K9 methylation does not always lead H2AX H3K9ac H3K9me3 megakaryocyte SETDB1 Suv39h2 Suv39h1 H3K56ac H3K27ac H3K4me3 H3K4me2 H3K4me1 H3K9me3 H2BK20ac H3K36me3 H3K79me2 to their de-repression (23). Furthermore, DNA methylation ex- H4K20me3 H3K27me3 H3K27me1 H2AK119ub macroH2A1 H3K9me3 erythroblast tensively decorates repetitive elements in both ES cells and so- 0 2 4 6 8 10 -log (adj. p-value) matic tissues and in some cases is lost concomitantly with H3K9 10 methylation (20, 24). However, genetic ablation of the three DNA DE300 methyltransferases does not lead to widespread de-repression of 1.5 200 repetitive element transcription (22); thus, the complex interplay Suv39-dependent 100 1.0 Suv39-independent between epigenetic marks and other nuclear factors in governing 10 Control (p-val.) 10 8 the transcription of these repetitive sequences has yet to be fully IP/input) 2 0.5 -log 6 elucidated. (log 4 GSAT_MM 0.0 2 Here we describe an integral role for H1 linker histones in si- Mean H1d Enrichment p<0.05 0.4 0.6 0.8 lencing repetitive element transcription in mouse ES cells. Using 102030 0102030 -2 -1 0 1 2 3 Distance from peak center (% total peak) Enrichment log (observed/expected) computational approaches, we uncover a set of H1 enriched do- 2 mains and show that they significantly overlap with constitutive Satellite LINE ERV F DNA SINE Other Simple repeats heterochromatin occupied by Suv39h1, Suv39h2, and SETDB1. 8 Next, using ES cells with only one functional H1 allele, we show that severe H1 depletion leads to a profound de-repression of 6 4 major satellite transcripts, to much higher levels than is seen in H1 % input Suv39h1/2 double-null cells, as well as de-repression of LINE-1 and IgG ERV transcripts. This de-repression is accompanied by a reduction 2 in H3K9me3