National Institute for Basic Biology

Cell Biology

DIVISION OF CHROMATIN REGULATION

Professor

NAKAYAMA, Jun-ichi

Assistant Professor:

KATAOKA, Kensuke
KAWAGUCHI, Takayuki
Specially Appointed Assistant Professor:
HAYASHI, Aki
Technical Staff:
NISHIMOTO, Yuki
SOKENDAI Graduate Student: RAHAYU, Anisa
VALENTIROVIC, Olivera
NAKAMURA, Rinko
YOSHIDA, Takaki
OYA, Tomoyuki
Visiting Scientist:
HAMADA, Kyoko
Technical Assistant:
YOSHIMURA, Yuriko
ASAI, Yuriko
Admin Support Staff:
KIYOHARA, Megumi

The stable inheritance of gene expression or repressionstates is essential to cellular differentiation and development, and an epigenetic cellular-memory system derived
from higher-order chromatin structure plays a fundamentalrole in this process. The assembly of higher-order chromatin
structures has been linked to the covalent modifications ofhistone tails, chromatin-associated proteins, and non-coding
RNAs. However, the exact means by which this chromatinbased epigenetic information is established and faithfully
maintained across cell divisions and throughout development remains unclear. To try to gain a better understandingof the molecular mechanisms underlying chromatin-basedepigenetic phenomena, our lab uses model systems including
mammalian culture cells, fission yeast, Schizosaccharomyces
pombe (S. pombe), and ciliate Tetrahymena for studying the18

assembly of higher-order chromatin structures, known asheterochromatin. We are also attempting to determine thecellular functions of chromatin modifying factors so that we
can develop a better understanding of complicated epigenetic
phenomena in higher eukaryotic cells.

I. Establishment and maintenance of higherorder chromatin structures

1-1 Mechanisms regulating Clr4 histone methyltransferase activity

In eukaryotic cells, the assembly of heterochromatin plays
an important role in diverse chromosomal processes andepigenetic gene regulation. Heterochromatin is characterized by the methylation of histone H3 at lysine 9 (H3K9me).
H3K9me is catalyzed by SUV39H-family histone methyltransferases, and functions as a binding site for recruiting
heterochromatin protein 1 (HP1) family proteins. In fission
yeast, H3K9me is catalyzed by histone methyltransferase Clr4. Clr4 has two functional domains, the N-terminal
chromodomain (CD), which recognizes H3K9me, and theC-terminal SET domain responsible for Clr4’s enzymaticactivity (Figure 1A). Since Clr4’s uncontrolled activity leads
to inappropriate H3K9me and aberrant gene silencing, its
enzymatic activity needs to be strictly controlled. However,the detailed mechanisms regulating Clr4’s activity are poorlyunderstood.
A previous study showed that N-terminally deleted Clr4
mutant exhibits a stronger activity than full-length Clr4,
suggesting that the Clr4 N-terminal region negatively regulates the activity of C-terminal SET domain. To examine theregulation mechanisms of Clr4’s activity, we tested whether
Clr4 N-terminal region interacts with its C-terminal SET
domain. Pull-down assays using recombinant proteins revealthat the Clr4 N-terminal region physically interacts withthe SET domain and that this interaction requires both CDand its adjacent region. To determine the regions/residues
responsible for this interaction, we performed crosslinking

Figure 1. Characterization of intramolecular interactions of Clr4. (A) Domainorganization of Clr4. The N-terminal chromodomain (CD) recognizes
H3K9me, and the C-terminal SET domain is responsible for Clr4’s enzymatic activity. (B) Crosslinking mass spectrometry. After in vitro crosslinking
reaction, recombinant Clr4 proteins were separated by gel-filtration chromatography and analyzed by SDS-PAGE and silver staining. (C) Residues identified in the cross-linking mass spectrometry to be involved in the interaction
are mapped on 3D structure of Clr4 SET domain.

mass spectrometry (Figure 1B) and found that crosslinked
residues were concentrated in a region near the C-terminalend (Figure 1C). Interestingly, mutant Clr4 proteins containing amino-acid substitutions at this region exhibit higher
methyltransferase activity. These results suggest that Clr4’s
enzymatic activity is negatively regulated by intramolecularinteractions.

II. Roles of HP1 family proteins in assemblinghigher-order chromatin structure
2-1 Characterizing the role Chp2 in heterochromatin
assembly in fission yeast

Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that binds H3K9me, and plays
a crucial role in forming higher-order chromatin structure.
HP1 family proteins share a basic structure consisting ofan N-terminal CD and a C-terminal chromoshadow domain(CSD) linked by an unstructured henge region (Figure2A). The CD functions as a binding module that target
H3K9me, whereas CSD functions as a dimerization modulethat provides a recognition surface to bind and recruit otherproteins. In fission yeast, two HP1 family proteins, Swi6and Chp2, are involved in heterochromatic silencing. While
Swi6 is expressed abundantly and plays a dose-dependentrole, Chp2 is expressed at a lower level and displays a novel
ability to tightly bind chromatin-enriched fraction independently of H3K9me. To dissect how this Chp2-specificability contributes to its function, we performed Tandem
Affinity Purification (TAP) to identify Chp2-specific binding
partners and successfully identified novel candidates for
Chp2 binding partners. Furthermore, we examined Chp2specific DNA-binding activity by Electrophoretic Mobility
Shift Assay (EMSA) and found that both the hinge andCSD neighboring regions make distinct contribution to its
DNA binding. These results suggest that both the Chp2’sproperty to bind specific binding partners and its intrinsic
DNA binding ability contribute to its tight chromatin association and also to division of labor between Swi6 and Chp2.

2-2 M ultiple HP1-like protein-containing complex
regulates DNA elimination in Tetrahymena

Heterochromatin plays important roles in transposon (TE)silencing. A major type of heterochromatin contains chromatin, that is histone H3 methylated at lysine 9/27 (H3K9/27me),and its reader HP1 proteins, that recruit diverse proteins
onto the chromatin to silence the TEs. Although multipleHP1 proteins are co-expressed in many eukaryotic cells, theinterplay between these HP1 proteins has been elusive. Here,
we show that subset of the HP1 proteins form a complex
and play important roles to eliminate TE-related InternalEliminated Sequences (IESs) from the somatic genome
during macronuclear development in the ciliated protozoan
Tetrahymena. We tethered 7 HP1-like proteins individuallyto the artificially created locus by the LexA-LexO systemand found that only 4 of them including Pdd1 induced theelimination of the tethered site. This ectopic DNA elimination was exclusively achieved by their chromoshadow
domains (CSDs), indicating that the CSDs of the distinct typeof HP1-like proteins recruits all the proteins that are required
for DNA elimination. Immunoprecipitation of Pdd1 specifically enriched the other HP1-like proteins that were sufficientfor the ectopic DNA elimination. The chromodomains of asubset of HP1-like proteins showed strong affinity to both
H3K9me3 and H3K27me3. Overall, these results suggest thatmultiple HP1-like proteins cooperatively recognize the methylated histones and form core complex to recruit other effector
proteins for DNA elimination (Figure 3).

Figure 3. Model for how multiple HP1-like protein-containing complex
regulates DNA elimination in Tetrahymena.

2-3 The HP1-like protein Hpl8p is important for programmed DNA elimination in Tetrahymena

Figure 2. Chp2 possesses a strong DNA/RNA-binding activity. (A)Schematic diagram of Swi6 and Chp2. The chromodomain (CD) and thechromoshadow domains (CSD) are shown. (B) Representative results ofEMSAs performed with control and mutant Chp2.

Transposon mobility represents a threat for the genomeintegrity in all living things. To counter this, the ciliatedprotozoan Tetrahymena uses heterochromatin to selectively eliminate over 10,000 transposon loci from its somaticnucleus in the developmentally programmed process ofDNA elimination. To address how this process is coordinated by heterochromatin components, we focus on an
HP1-like protein named Hpl8p that localizes to the developing macronucleus during heterochromatin formation andDNA elimination (Figure 4). The chromodomain of Hpl8p
recognized both H3K9me3 and H3K27me3 in vitro. HPL8
disrupted mutants showed defect in vegetative growth and in
the progression of sexual reproduction. FISH analyses using
a probe against one of the transposons showed that more than19National Institute for Basic Biology

Cell Biology

90% of HPL8 disrupted cells did not complete its elimination, suggesting that Hpl8p plays important roles in DNA
elimination. Altogether, these results suggest that Hpl8p
binds the H3K9/27 methylation marks over transposon lociand facilitates the heterochromatin formation by interacting with heterochromatin components required for DNA
elimination. Furthermore, we propose that Hpl8p also plays
important roles in suppression of unwanted genes during thevegetative growth and sexual reproduction process possibly
by recognizing H3K27me3 in the transcriptionally active
macronucleus.

Figure 5. Analysis of chromatin dynamics in spore nuclei of fission yeast.
(A) A representative image of vegetative cells (veg.) and spores in asci. (B)Heatmap representation of proteins detected in spores and vegetative cells.

Publication List:

Figure 4. Cells expressing the Hpl8-mNeonGreen (green) and Pdd1pmCherry (red) at 8 and 13 hours post induction of mating are shown in(a) and (b), respectively. DNA was counterstained with DAPI. Arrows
and arrowheads indicate the parental and new MACs, respectively.

III. Analysis of chromatin dynamics in spore
nuclei of fission yeast

Gametogenesis is a crucial process for sexually reproducing organisms to produce haploid gametes from diploidcells. Generally, epigenetic memory is erased from zygote
nucleus, a phenomenon known as reprogramming, resulting in increased totipotency. On the other hand, it has been
demonstrated in some organisms that a part of epigenetic
memory can be inherited transgenerationally. The balance
between reprogramming and epigenetic inheritance plays a
key role in the developmental processes of offspring. It has
been also reported that some gametes, like plant sperm andmammalian spermatozoa, have highly compacted nuclei andhave altered histone modifications. However, the mechanisms underlying how epigenetic marks are regulated during
gametogenesis remain to be fully elucidated. To gain an
insight into epigenetic inheritance and chromosome structural change during gamete formation, we analyzed chromatindynamics in spore nucleus of fission yeast (Figure 5A). We
isolated fission yeast spores by density-gradient centrifugation and performed mass spectrometry analysis to determine
spore-specific nuclear proteins (Figure 5B). We further
examined the change in histone modifications of sporenuclei. Chromatin immunoprecipitation analysis revealed
that the levels of H3K9me at heterochromatic regions had
not been noticeably changed, but the levels of H3K4me associated transcriptionally active regions had clearly increasedin spore nuclei. We also found that H3K4me distribution atcoding regions in spore genome clearly different from thatof control vegetative cells. These results imply that dynamicchanges in active histone marks contribute to spore formation and epigenetic inheritance.

20〔Original paper〕

R., and Nakayama, J. (2022). Multiple interfaces to• Nakamura,recognize nucleosomal targets. J. Biochem. 171, 257–259. DOI:
10.1093/jb/mvab139