Article

Structure of the UHRF1 Tandem Tudor Domain
Bound to a Methylated Non-histone Protein, LIG1,
Reveals Rules for Binding and Regulation
Graphical Abstract

Authors
Satomi Kori, Laure Ferry,
Shohei Matano, ..., Yoichi Shinkai,
Pierre-Antoine Defossez, Kyohei Arita

Correspondence
pierre-antoine.defossez@
univ-paris-diderot.fr (P.-A.D.),aritak@yokohama-cu.ac.jp (K.A.)

In Brief
The interaction between UHRF1 and
LIG1K126me3 is essential for DNA
methylation maintenance. Kori et al.
determined the crystal structure of the
UHRF1 TTD bound to a LIG1K126me3
peptide, revealing the basis for the highTTD-binding affinity of LIG1K126me3,
regulation by phosphorylation, and that
LIG1K126me3 binding switches theoverall structure of UHRF1.

Highlightsd

The crystal structure of UHRF1 TTD domain bound to the
LIG1K126me3 was determinedd

Arg121 of LIG1 is a key residue for high-affinity binding to
the TTDd

Phosphorylation of LIG1T123 negatively regulates theinteraction with UHRF1d

LIG1K126me3 binding changes UHRF1 structure from closed
to openKori et al., 2019, Structure 27, 485–496
March 5, 2019 ª 2018 Elsevier Ltd.
https://doi.org/10.1016/j.str.2018.11.012

Structure

ArticleStructure of the UHRF1 Tandem Tudor Domain Boundto a Methylated Non-histone Protein, LIG1, Reveals
Rules for Binding and Regulation
Satomi Kori,1 Laure Ferry,2 Shohei Matano,1 Tomohiro Jimenji,1 Noriyuki Kodera,3,4,5 Takeshi Tsusaka,6
Rumie Matsumura,1 Takashi Oda,1 Mamoru Sato,1 Naoshi Dohmae,7 Toshio Ando,3,4 Yoichi Shinkai,6
Pierre-Antoine Defossez,2,8,* and Kyohei Arita1,5,8,9,*
1Graduate School of Medical Life Science, Yokohama City University, Yokohama 230-0045, Japan
2University of Paris Diderot, Sorbonne Paris Cité, Epigenetics and Cell Fate, UMR 7216 CNRS, 75013 Paris, France
3Bio-AFM Frontier Research Center, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
4WPI Nano Life Science Institute, Kakuma-machi, Kanazawa 920-1192, Japan
5JST, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
6Cellular Memory Laboratory, RIKEN Cluster for Pioneering Research, Wako, Saitama 351-0198, Japan
7Biomolecular Characterization Unit, Technology Platform Division, RIKEN Center for Sustainable Resource Science, Wako,
Saitama 351-0198, Japan
8Senior author
9Lead Contact*Correspondence: pierre-antoine.defossez@univ-paris-diderot.fr (P.-A.D.), aritak@yokohama-cu.ac.jp (K.A.)
https://doi.org/10.1016/j.str.2018.11.012

SUMMARY

The protein UHRF1 is crucial for DNA methylation
maintenance. The tandem Tudor domain (TTD) of
UHRF1 binds histone H3K9me2/3 with micromolar
affinity, as well as unmethylated linker regions within
UHRF1 itself, causing auto-inhibition. Recently, we
showed that a methylated histone-like region of
DNA ligase 1 (LIG1K126me2/me3) binds the UHRF1
TTD with nanomolar affinity, permitting UHRF1
recruitment to chromatin. Here we report the crystal structure of the UHRF1 TTD bound to a
LIG1K126me3 peptide. The data explain the basis
for the high TTD-binding affinity of LIG1K126me3
and reveal that the interaction may be regulated byphosphorylation. Binding of LIG1K126me3 switches
the overall structure of UHRF1 from a closed to a flexible conformation, suggesting that auto-inhibition isrelieved. Our results provide structural insight into
how UHRF1 performs its key function in epigenetic
maintenance.

INTRODUCTION
Histone modifications and DNA methylation are major epigenetic
marks that regulate diverse cellular events by modulating thestructure and function of chromatin (Allis and Jenuwein, 2016).
In mammals, DNA methylation occurs mostly at the fifth positionof cytosine in CpG dinucleotides and plays key roles in development, X chromosome inactivation, genome imprinting, and€beler, 2015). In proliferating cells, thecarcinogenesis (Schupattern of DNA methylation has to be re-established after eachcycle of DNA replication, and two proteins are known to be key

in this process, as their absence causes a similar phenotype ofprogressive DNA demethylation (Bostick et al., 2007; Sharif
et al., 2007; Smets et al., 2017; von Meyenn et al., 2016). The firstprotein involved is the maintenance DNA methyltransferase,
DNMT1, and the other is the protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1).
UHRF1 contains five annotated domains: ubiquitin-like (UBL),
tandem Tudor domain (TTD), plant homeodomain (PHD), SET
and RING associated (SRA), and RING, and their associatedlinkers (Figure 1A). The SRA is essential for function and specifically recognizes hemimethylated DNA, which is generated after
DNA replication (Arita et al., 2008; Avvakumov et al., 2008; Hashimoto et al., 2008; Liu et al., 2013). Subsequently the RING
domain, which has E3 ubiquitin ligase activity, ubiquitylates
K14, K18, and/or K23 on histone H3 (hereafter H3), which allows
DNMT1 recruitment and activation onto recently replicated sites(Ishiyama et al., 2017; Nishiyama et al., 2013; Qin et al., 2015).
The PHD and TTD work cooperatively to recognize the heterochromatin mark H3K9me2/3: the PHD recognizes the N-terminal
1
ARTK4 motif of the histone, while the TTD accommodates the
methylated H3K9me2/3 residue in an aromatic cage (Arita
et al., 2012; Cheng et al., 2013; Rajakumara et al., 2011; Rothbart
et al., 2013; Zhao et al., 2016). In addition, the TTD also interacts
with unmethylated lysine and arginine-rich linkers within UHRF1
itself: the ‘‘linker 2’’ between TTD and PHD finger (L2UHRF1) andthe ‘‘spacer,’’ which follows the SRA domain (spacerUHRF1) (Figure 1A) (Arita et al., 2012; Cheng et al., 2013; Fang et al., 2016;
Gao et al., 2018; Gelato et al., 2014). These intramolecular interactions lead to a "closed" overall structure of UHRF1 and theauto-inhibition of DNA binding and E3 activities (Fang et al.,2016; Gelato et al., 2014; Harrison et al., 2016).
We recently showed that the TTD of UHRF1 interacts with ahistone-like sequence within a replication protein, DNA ligase 1(LIG1) (Ferry et al., 2017). Molecularly, LIG1 contains an intrinsically disordered region at its N-terminus (residues 1–200), within
which residues 118–130 are similar to the H3 N-terminal tail
Structure 27, 485–496, March 5, 2019 ª 2018 Elsevier Ltd. 485

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486 Structure 27, 485–496, March 5, 2019

Table 1. Data Collection and Refinement Statistics
apo-TTD(PDB: 5YYA)

TTD:LIG1K126me3(PDB: 5YY9)P212121

P212121

Data Collection
Space group
Cell dimensions
38.8, 60.0, 68.9

27.1, 97.3, 132.5

Resolution (Å)

a, b, c (Å)

45.25–1.70(1.73–1.70)a

48.67–2.65(2.78–2.65)aRsym or Rmerge (%)

4.3 (22.6)a12.2 (70.2)a

I/s(I)
CC1/2
Completeness (%)

25.1 (5.3)

a8.5 (2.4)a99.9 (92.8)

a99.2 (89.1)a97.9 (80.2)

a97.0 (95.4)aaences, and how do they contribute to the higher affinity binding
to LIG1? What is the effect of LIG1 binding to the overall UHRF1
architecture and function?
To address these questions, we have solved the crystal structure of the UHRF1 TTD in complex with a methylated LIG1 peptide.
The structure, complemented by mutagenesis and functional assays reveals key residues for the interaction, sheds light on themechanism for high-affinity binding of LIG1 to TTD, and indicatesthat the binding event causes a large-scale molecular reorganization within UHRF1. Our data provide insight into UHRF1 binding toa methylated non-histone protein, and contribute to understanding its key role in epigenetic maintenance.
RESULTSa

Redundancy

6.5 (3.9)

Total reflections

117,437

3.8 (3.8)
40,019Unique reflections

17,94910,403

Resolution (Å)

34.35–1.70(1.81–1.70)a

40.23–2.65(2.79–2.65)a

No. of reflections

17,884 (1517)a

10,368 (989)a

Rwork/Rfree (%)

17.2/20.6

23.1/28.8

Protein

1,335

2,379

Ion

1

0

Water

244

24

Protein

21.19

44.41

Ion

27.75

–

Water

33.11

37.91

Bond lengths (Å)

0.004

0.005

Bond angles ( )

0.63

0.84

Refinement

No. atoms

B factors

RMSDa

Values in parentheses are for the highest-resolution shell.

(residues 1–12), and LIG1K126 is in a sequence environment
similar to H3K9. Furthermore, LIG1K126 is methylated by thelysine methyltransferases G9a (EHTM2) and GLP (EHTM1)
in vitro and in cells. We found that a LIG1K126me3 peptide
largely outcompetes H3K9me2/3 peptides for binding to the
UHRF1 TTD. We also reported that the aromatic cage of the
TTD was necessary for binding, yet a number of molecular questions remain open as to how UHRF1 interacts with this methylated non-histone protein. What are the similarities between
H3K9me2/3 and LIG1K126me3 binding? What are the differ-

The TTD in Complex with LIG1K126me3 Peptide Adopts
a Canonical Structure
We first produced and crystallized the wild-type (WT) human TTD(residues 123–285); a structure was obtained at 1.7 Å resolution(apo-TTD, Figure S1A; Table 1). The co-crystallization of thisWT construct with the LIG1K126me3 peptide was, however, unsuccessful. Based on the structure of the unliganded TTD (Figure S1A), we speculated that a flexible loop might be interferingwith co-crystallization, so we generated and tested mutant versions of the TTD lacking this loop. After optimization trials, forco-crystallization we used a variant TTD (vTTD from here on) in
which the loop is removed by deleting residues 167–175. The
vTTD has the same binding affinity for LIG1K126me3 as the WT
TTD, as measured by isothermal titration calorimetry (ITC) (Figure S1B). The LIG1 peptide contained residues 118–130 of thehuman protein, with the key lysine K126 trimethylated (K126me3).
We determined the crystal structure of vTTD with
LIG1K126me3 peptide at 2.65 Å resolution (Table 1); in the crystal, an asymmetric unit contained two vTTD:LIG1K126me3 complexes. The structures of the two vTTDs in the unit were identical(root-mean-square deviation [RMSD] of Ca atoms 1.4 Å over the
125 Ca atoms) (Figure S1A). The structure of LIG1K126me3 peptides in the asymmetric unit was also identical (RMSD of Ca
atoms 0.5 Å) (Figure S1C). The 12 successive residues from
Ile118 to Pro129 showed clear electron density in the jFoj 
jFcj omit map (Figures 1B and 1C), and are described hereafter.
The overall TTD structure was virtually identical with or without
the LIG1K126me3 peptide (RMSD of Ca atoms 0.8–1.6 Å) (Figure S1A), implying that binding of the LIG1K126me3 did notlead to the structural rearrangement of the TTD. As in previously
published structures (Fang et al., 2016; Nady et al., 2011), thefirst and second Tudor domains comprised a five-stranded bbarrel fold, and the two domains were separated by a groove

Figure 1. Structure of the UHRF1 TTD in Complex with a LIG1K126me3 Peptide(A) Schematic of the domain organization of UHRF1. The amino acid numbers in the human protein are indicated.
(B) Overall structure of TTD:LIG1K126me3 complex. The first and second Tudor domains are shown as yellow-green and pale-green surface models, respectively. LIG1 is depicted as a magenta stick model.
(C) The LIG1 peptide and jFoj  jFcj omit map contoured at 2 s are colored magenta and blue, respectively.
(D and E) Recognition of R125-K126me3 (D) and R121-A124 (E) of LIG1 by the TTD. Color schemes are the same as in (B). Water molecules are represented as
black balls.
(F) Schematic diagram of LIG1K126me3 recognition by the TTD. The LIG1 backbone and side chains are shown in black and TTD residues in green. Dotted lines
and black arcs with spokes indicate hydrogen bonds and hydrophobic interactions, respectively.
Supplemental data are provided in Figure S1.

Structure 27, 485–496, March 5, 2019 487

A

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C

(TTD groove from here on, Figures 1B and S1A). The
LIG1K126me3 peptide interacted in an extended conformation
with the TTD groove and, within the peptide, residues Arg121
to Lys126me3 contacted TTD residues (Figures 1B–1F).
Two Regions of the LIG1K126me3 Peptide Establish
Dense Contacts with the TTD
The structure showed that two clusters of dense contacts between TTD and LIG1 peptide participated in the stable complex
formation. First, the aromatic cage comprising Phe152, Tyr188,
and Tyr191 of UHRF1 interacted with the tri-methyl moiety of
K126me3 in LIG1 (Figures 1D and 1F). Second, the side chainof LIG1Arg121 was inserted into a depression of the TTD groove,
designated as an ‘‘Arg-binding cavity’’; there, the guanidinogroup and aliphatic portion of Arg121 were recognized by multiple hydrogen bonds and hydrophobic interactions with side
chains of UHRF1-Asp142 and Met224, Trp238, and Phe278,
respectively (Figures 1E and 1F). Of these positions, Asp142,
Met224, and Trp238 are almost invariant within UHRF1 orthologs
found in animal species, whereas Phe278 is less strictly
488 Structure 27, 485–496, March 5, 2019

Figure 2. Structural Comparison of TTD
Binding Partners(A) Sequence alignment of LIG1, H3, spacerUHRF1,
and L2UHRF1. The residues underlined have a side
chain that interacts with the TTD.
(B and C) Structure around the TTD groove incomplex with LIG1K126me3 (B) and H3K9me3 (C)(PDB: 2L3R). Each peptide is shown as a stickmodel and the color schemes are same in (A).
Red and blue on the surface of TTD indicatehydrophilic and hydrophobic interaction residues,
respectively. Right panel is the structure aroundArg-binding cavity in TTD complexed with
LIG1K126me3 (B) and H3K9me3 (C). Color schemeof LIG1 and H3 are same as in (A) and TTD residues
are shown as gray stick model.
Supplemental data are provided in Figure S2.

conserved (Figure S2A). In addition tothese dense contacts, the side chains of
Arg122 and Arg125 of LIG1 were recognized by the side chain of Glu276 and themain chain of Asp190 of TTD, respectively(Figures 1D–1F). Glu193 of TTD also
supported the binding to LIG1Arg125
by long-range electrostatic interaction(3.7 Å) (Figure 1D). The side chain of
LIG1Thr123 formed additional hydrogenbonds with the side chain of UHRF1Trp238 (Figures 1E and 1F). Finally, themain chains of Arg121, Arg122, Ala124,
and K126me3 in LIG1 were also involvedin the interaction with TTD (Figures 1D–1F).
LIG1R121 Plays a Key Role for theHigh-Affinity Interaction between
TTD and LIG1K126me3
Our published work (Ferry et al., 2017), as
well as data presented here, suggest that
the TTD of UHRF1 has much higher affinity for methylated LIG1
than for its other reported interactors: H3K9me3, spacerUHRF1,
and L2UHRF1 (Arita et al., 2012; Fang et al., 2016; Gelato et al.,2014; Nady et al., 2011). To understand the basis for this preference, we compared the peptide sequences and structural data
for these interactions (Figures 2A–2C). Of all the interacting peptides, H3 has the clearest sequence similarity to LIG1 (KQTARK
versus RRTARK, see Figure 2A); it also has a similar binding
mechanism, interacting both with the aromatic cage and Argbinding cavity of the TTD (Figures 2B and 2C). However, thebinding affinity of LIG1K126me3 for the TTD, determined by
ITC, was approximately 180 times higher than that of
H3K9me3 (KD = 1,620 nM for H3K9me3 versus KD = 9.1 nM for
LIG1K126me3, Table 2, and ITC thermograms in Figures S1B
and S2B). The major sequence differences between peptides
are Lys4 and Gln5 of H3, which are in the position of Arg121
and Arg122 of LIG1, respectively, so we mutagenized these
positions within an H3K9me3 peptide and determined the consequences for TTD binding (Table 2; Figure S2B). Replacing
H3K4 by Arg dramatically increased the binding affinity for TTD

Table 2. Summary of ITC Measurements
TTD

LIG1 or H3

KD (nM)

WT

LIG1K126me3

9.1 ± 3.8

WT

LIG1K126me0

250 ± 35

D142A

LIG1T123ph/K126me3>104,500

LIG1R121A/K126me3>55,000

LIG1R125A/K126me3

21.4 ± 1.1

LIG1K126me3

ND

Y188A/Y191A

5,537 ± 490

E193A

23.0 ± 8.2

E276A

49.7 ± 11

W238A
WT

1,056 ± 35
H3K9me3

1,620 ± 111

H3K4R/K9me3

22.2 ± 4.4

H3Q5R/K9me3

1,440 ± 17

H3K4R/Q5R/K9me3

13.8 ± 0.8

ND, not determined.

(KD = 22.2 nM, similar to that of LIG1K126me3, KD = 9.1 nM); in
contrast, replacing H3Q5 by Arg had no effect on the binding
affinity (KD = 1,440 nM). The double-substituted peptide
H3K4R/Q5R behaved like the single K4R-substituted peptide(KD = 13.8 nM).
These experiments show that the presence of an arginine residue at position 121 of LIG1 (in contrast to the lysine residue
found at the equivalent H3 position 4) is a key contributor tothe high-affinity binding. This can be explained by comparing
our structure to the previously published TTD:H3K9me3 structure (Figures 2B and 2C). Indeed, the side-chain conformationsof Arg121 in LIG1K126me3 and Lys4 in H3K9me3 markedly
differ: Arg121 establishes hydrogen bonds with TTD-Asp142
via its guanidine group, and CH-p interactions with TTDTrp238 and TTD-Phe278 via its alkyl group, all of which likely
contribute to the high-affinity interaction (Figure 2B). The difference in side-chain length between lysine and arginine also
affects the main-chain conformation, allowing a higher numberof interactions for LIG1, as the side chains of residues Arg122
and Arg125 form with the TTD contacts not formed by H3 (Figures 2A and 2B).
Mutational Analysis Validates the Structural Data andUncovers a Phospho-Switch Regulation
To validate our structural data and quantify the contribution ofindividual residues to the interaction, we performed ITC experiments using WT or mutated versions of the TTD and LIG1
peptide (Table 2; Figure S3A). The values obtained with WT partners were consistent with our previous report (Ferry et al., 2017):
the TTD bound LIG1K126me3 with a KD = 9.1 nM, and
LIG1K126me0 with a KD = 250 nM (Table 2; Figures S1B
and S3A).
We tested several mutations of the TTD: the mutation with themost deleterious effect was D142A, which obliterated any
detectable binding. The second most deleterious change wasinactivation of the aromatic cage, using the double mutation
Y188A/Y191A; this resulted in detectable binding but vastly
reduced affinity for LIG1K126me3 (KD = 5,537 nM). Mutation

W238A also had a large effect, reducing the affinity by 100-fold(KD = 1,056 nM). Finally, mutations E193A and E276A had
smaller but measurable effects on the UHRF1/LIG1K126me3
interaction. These results are consistent with our structural data.
Next, we introduced mutations in the LIG1K126me3 peptide.
The R121A change had a severe effect, reducing binding by at least
6,000-fold (KD > 55,000 nM); in contrast, the R125A mutation only
led to a slight binding reduction (KD = 21.4 nM). Finally, it has been
observed that phosphorylation of Thr6 of H3, Ser298 of L2UHRF1,
and Ser651 of spacerUHRF1 inhibits the interaction with the TTD(Arita et al., 2012; Fang et al., 2016; Gelato et al., 2014; Rothbart
et al., 2013), so we tested whether a similar effect might also occur
when LIG1 is phosphorylated on the equivalent residue, Thr123.
We synthetized a peptide in which LIG1K126 was trimethylated,
LIG1T123 phosphorylated; this peptide interacted extremely
poorly with the TTD (KD > 104,500 nM), establishing that phosphorylation at Thr123 is indeed inhibitory to the interaction.
We next sought to identify a kinase that could phosphorylate
LIG1T123. The equivalent position in H3, H3T6, is phosphorylated by protein kinase Cb (PKCb) (Metzger et al., 2010), so wetested whether PKCb also phosphorylates LIG1T123. In vitro
phosphorylation assay combined with mass spectrometry
demonstrated that LIG1T123 was indeed efficiently phosphorylated by the kinase, independent of the Lys126 methylation
status (Figure 3A), suggesting that PKCb is a candidate for
LIG1T123 phosphorylation in the cell.
Validation of the Structural Data by a Cellular Assay
Reveals the Importance of LIG1R125 for Methylation of
LIG1K126
We also examined the interaction by an independent technique,the fluorescent three-hybrid assay (F3H) (Herce et al., 2013). In
this approach, proteins bearing fluorescent tags are co-expressed in an engineered mammalian cell line, which is designed
so that the GFP-tagged protein will be recruited to a nuclear
spot. The percentage of cells in which the RFP-tagged protein
forms a spot co-localizing with the GFP spot is then recorded,
and provides a direct estimate of the interaction propensity incells (schematic in Figure 3B). We carried out this assay withfull-length UHRF1 fused to GFP, and full-length LIG1 fused todsRed, using WT proteins or introducing the mutations studied
by ITC. The F3H results agreed very well with ITC: mutation
D142A in UHRF1 had a severe effect, W238A was less marked,
and E193A and/or E276A had smaller effects (Figures 3C and
S3B). Within LIG1, the R121A mutation totally abrogated interaction, and so did the phosphomimetic T123D mutation. A striking
difference with the ITC results is that the LIG1R125A mutation,
which had little effect in ITC, led to total loss of interaction in
the F3H assay (Figures 3C and S3B). G9a and GLP favor an
RK motif in their targets to efficiently catalyze lysine methylation(Rathert et al., 2008), so we postulated that the LIG1R125A
mutation could affect the interaction with the TTD indirectly, bydecreasing LIG1K126 methylation.
We tested this hypothesis in vitro by incubating LIG1 peptides
with recombinant G9a, and then detecting lysine methylation by
MALDI-TOF (Figure 3D). As described previously (Ferry et al.,2017), G9a efficiently trimethylated the LIG1 peptide. The
LIG1R121A mutation had very little effect on methylation by
G9a. In contrast, the LIG1R125A mutation had a major effect,
Structure 27, 485–496, March 5, 2019 489

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D

(legend on next page)490 Structure 27, 485–496, March 5, 2019

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B

Figure 4. Stepwise Binding of LIG1K126me3
to the TTD-PHD Module(A) Superimposition of enthalpy change plots for (A)binding of LIG1K126me3 peptide to TTD-PHD(abbreviated TP) with the indicated mutations and
LIG1K126me0 to WT TP. The binding affinities andsamples in the syringe (Sy) and cell are indicated.
(B) Binding of H3K9me3 peptide to TP with theindicated mutations.
(C) Binding of H3K9me0 or H3K9me3 peptides to
TTD-PHD in the presence of LIG1K126me3.
(D) Binding of LIG1K126me3 to TTD-PHD in thepresence of H3K9me0 or H3K9me3.
Supplemental data are provided in Figure S4.

C

D

totally preventing trimethylation by G9a (Figure 3D). Similar results
were obtained with GLP (not shown). These in vitro data strongly
suggest that reduced methylation of LIG1K126 is the reason why
the LIG1R125A mutant fails to interact with the UHRF1 TTD in acellular assay. Altogether, the data from ITC and F3H are fully
consistent with the structure we obtained: they show that the ionicinteraction between Asp142 of TTD and Arg121 of LIG1 plays aforemost role in the binding, followed by the hydrophobic interaction between LIG1K126me3 and the TTD hydrophobic cage.
Lastly, we have shown that the interaction can be negatively regulated by phosphorylation of LIG1T123.
LIG1K126me3 Binds to TTD-PHD in a Way Distinct
from H3
The adjacent TTD and PHD domains of UHRF1 form a functional
unit for binding H3K9me3, in which L2UHRF1 interacts with
the TTD groove, the N-terminus of H3 binds the PHD, andthe H3K9me3 residue inserts into the aromatic cage of the TTD(Arita et al., 2012; Fang et al., 2016; Gelato et al., 2014). We
next asked whether LIG1 binding obeyed similar rules, by performing binding assays with WT or variant TTD-PHD (TP) units.
The LIG1K126me3 peptide bound to the WT TP with a KD =124 nM, whereas the LIG1K126me0 peptide did not show any
detectable binding (Figures 4A, S4A, and S4B). As we previously
found that the LIG1K126me0 peptide binds the isolated TTD witha KD = 250 nM (our previous work [Ferry
et al., 2017] and Table 2), we conclude
that its binding to TP is prevented, presumably by L2UHRF1 occupying the TTD
groove.
To discern the relative contributions of
TTD and PHD to the binding, we used two
mutant forms of the TP: the Y188A/Y191A
mutant that has an inactivated TTD aromatic
cage (hereafter TPmTTD), and the D334A/
D337A mutant that loses the PHD function(TPmPHD). When incubated with LIG1K126me3, the TPmTTD showedno detectable binding, while TPmPHD behaved like the WT form (Figures 4A and S4A). Conversely, when incubated with H3K9me3,
TPmTTD bound the peptide, but TPmPHD did not (Figures 4B and
S4C), consistent with the published finding that H3K9me3 must
engage the PHD to bind TP (Cheng et al., 2013). These results imply
that binding of LIG1 to the TP is strictly dependent on K126 methylation and is limited to the TTD moiety. In support of this hypothesis,
we found that LIG1K126me3 could not bind the isolated PHD (Figure S4D). This might be explained by the fact that LIG1 has no basic
residue equivalent to H3R2, a critical contributor to PHD binding by
H3 (Figure 2A).
Next, we examined whether H3K9me3 and LIG1K126me3
could simultaneously bind to TP; for this, we pre-complexed
the TP with peptides of interest, and measured binding to theother peptides (Figures 4C, 4D, S4E, and S4F). H3K9me0 andH3K9me3 peptides could bind to the TP pre-complexed with
LIG1K126me3 with a KD of 1,880 and 2,141 nM, respectively.
These affinities are comparable with that of the isolated PHD
for the N-terminus of H3 (Arita et al., 2012), suggesting that
LIG1K126me3 bound to the TTD moiety does not prevent N-terminus of H3 binding the PHD moiety (Figures 4C and S4E). Precomplexing the TP with H3K9me0 did not decrease its affinity for
LIG1K126me3 with a KD of 159 nM, while pre-complexing with
H3K9me3 totally abolished binding (Figures 4D and S4F).

Figure 3. Mutation Analysis Confirms the Structural Data and Uncovers a Negative Regulation by Phosphorylation(A) In vitro phosphorylation assay of LIG1118-130 peptides by PKCb. The mass shift of +80 Da detected by MALDI-TOF mass analysis, indicates phosphorylation.
(B) Principle of the F3H assay.
(C) Results of the F3H assay: mutations in the UHRF1 TTD affect interaction with LIG1 and mutations in LIG1 affect interaction with the UHRF1 TTD. The scale bar
represents 10 mm.
(D) In vitro methylation assay of LIG1118-130 peptides by G9a. Each methylation event adds 14 Da to the peptides. The R125A mutation inhibits methylation by G9a.
Supplemental data are provided in Figure S3.

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B

C

A possible interpretation of the results is that the binding of
H3K9me3 stabilizes the TTD-PHD in a "locked" conformationin which L2UHRF1 occupies the TTD groove, preventing accessof LIG1K126me3. Alternatively, the data could indicate that afree TTD aromatic cage is crucial for the binding of LIG1 to
UHRF1. This would suggest a two-step model of LIG1K126me3
binding to the TP, the first step being access of LIG1K126me3
to the aromatic cage of the TTD, and the second being
LIG1R121 inserting into the Arg-binding cavity of the TTD
groove.
Binding to LIG1 Changes the Arrangement of the TTDPHD Module and the Overall UHRF1 Structure
Next, we calculated the different thermodynamic parameters of
LIG1K126me3 and H3K9me3 binding to the TP from ITC experiments, as these can give insight into the nature of the binding
mode (Du et al., 2016). We found that these parameters weremarkedly different: the binding of H3K9me3 was enthalpy driven,
492 Structure 27, 485–496, March 5, 2019

Figure 5. LIG1 Binding Decreases
Compactness of TTD-PHD

the(A) Thermodynamic data of binding of LIG1K126me3
and H3K9me3 peptide to the TTD-PHD determinedby ITC. Data are represented as mean ± SD.
(B) SAXS experiments on the TTD-PHD module(TP). P(r) functions are shown for apo-TP (blue) its
complex with H3K9me3 (green) and LIG1K126me3
peptides (red) and TP harboring D142A mutant(orange). Rg and Dmax values are also shown.
(C) Low-resolution ab initio models derived fromSAXS data.
Supplemental data are provided in Figures S5–S8.

with a DH of large negative value(9.9 kcal/mol, Figure 5A), while the binding of LIG1K126me3 was entropy-driven,
with a positive TDS value (6.1 kcal/mol,Figure 5A). The enthalpy-driven bindingof H3K9me3 is consistent with its known
mode of interaction, in which the binding
occurs without conformational change ofTP (Arita et al., 2012). The entropy-driven
binding of LIG1K126me3, in contrast, issuggestive of a rearrangement of the TP,
and a likely candidate for this is adisplacement of L2UHRF1 from the TTD
groove.
To verify that a structural change in
the TP does take place upon binding of
LIG1K126me3, we used size-exclusion
chromatography in line with small-angle
X-ray scattering (SEC-SAXS) (Figures 5B,
S5, and S6; Table S1). Purified TP, in its unliganded apo form, or in complex with
H3K9me3 or LIG1K126me3 peptide, wasloaded on the SEC and the eluted fractions
were exposed by X-ray beam. We also
included a mutant TP with the D142A,
which prevents interaction between the
TTD groove and the L2UHRF1, thereby
opening the structure (Fang et al., 2016; Gelato et al., 2014; Harrison et al., 2016).
The molecular mass of the samples estimated by comparing
I(0)/c (where c is the protein concentration) of the standard sample(bovine carbonic anhydrase) established that there was no aggregation of the measured samples (Figure S5). The radius of gyration(Rg), the distance distribution function P(r), and the maximum
dimension of particles (Dmax) for each scattering image at thepeak of absorption at 280 nm (A280), and the I(0) of the each sample
were calculated (Figures 5B and S6). The numbers obtained forunliganded TP were similar to those previously reported (Arita
et al., 2012; Houliston et al., 2017), and they were not affectedby the addition of H3K9me3 peptide, consistent with an absenceof overall structural rearrangement (Figures 5B, S5A, S5B, S6A,
and S6B). In contrast, the addition of LIG1K126me3 caused amarked increase in the Rg and Dmax values, implying a rearrangement of the TTD-PHD module to a more extended form. The
structural change was very similar to that seen upon the D142A

Figure 6. Schematic Model for the Different
Binding Characteristics of LIG1 and H3 toUHRF1 TTD-PHD
Summary and interpretation of the data. The yellow
sphere and cyan box represent methylated lysine
and H3 N-terminal 1ARTK4 residues, respectively.

mutation (Figures 5B, S5C, S5D, S6C, and S6D). An ab initio shapereconstruction from scattering data yielded highly similar shapes
for unliganded TP and TP bound to H3K9me3 (Figure 5C). In
contrast, the shape of TP bound to LIG1K126me3 was more
extended, and similar to that of TP D142A mutant (Figure 5C).
This is compatible with the possibility that LIG1K126me3 binding
displaced L2UHRF1 from the groove (Figure 6).
We also examined the overall structure of full-length UHRF1.
As full-length UHRF1 tends to aggregate (our unpublisheddata), it was not amenable to SEC-SAXS, but we could employ
an independent approach, high-speed atomic force microscopy (HS-AFM). We found that unliganded full-length UHRF1
appeared as a compact molecule, whereas it became more
extended and its conformational flexibility increased upon binding of LIG1K126me3 (Figures S7 and S8; Videos S1, S2,
and S3).
These data show that LIG1 binding alters the overall structure and dynamics of the TTD-PHD module but also of full-length
UHRF1.

DISCUSSION
We report the structure of the UHRF1 TTD bound to a methylated non-histone protein, LIG1. The UHRF1/LIG1 interaction is
physiologically important, as it permits the recruitment of
UHRF1 to replicating DNA, and the maintenance of DNA
methylation, an essential epigenetic mark in mammals (Ferry
et al., 2017). Our structural data are validated by independent
biochemical and cellular approaches, and they shed light onthree important questions: what distinguishes LIG1 interaction
from interaction with other UHRF1 TTD binders? What mechanisms may modulate the LIG1/UHRF1 interaction? Finally,
how does this interaction affect the overall structureof UHRF1?

Structure of the UHRF1 TTD
Complexed with LIG1 Reveals
Commonalities and Differenceswith Other Interactors
The TTD of UHRF1 binds histone andnon-histone proteins. This property
has already been described for a few
other cases, and one well-characterized example is 53BP1: its TTD canbind H4K20me2 (Botuyan et al., 2006),
but also RbK810me2 (Carr et al.,2014) and p53K370me2 (Tong et al.,2015). We present an example of a
TTD binding a non-histone proteinwith an affinity far greater than its affinity for histones, and moreover we can explain this binding
behavior structurally.
A first conclusion from our data is that the TTD engages LIG1 in
a manner similar to its other targets: the LIG1 peptide
was extended in the TTD groove as previously reported for H3,
the L2UHRF1, and spacerUHRF1. Another commonality between
the various interactions is that they all involve a basic residue(Arg or Lys, R121 in the case of LIG1) in the binder, which penetrates deep into the Arg-binding cavity of the groove and forms
an electrostatic interaction with Asp142 of UHRF1, a residue
highly conserved through evolution. It is noteworthy thatArg121 is in position equivalent to H3K4, and that lack of methylation at H3K4me0 is a prerequisite for DNA methylation to occur.
Finally, another similarity with previous structures is that the
methylated lysine (LIG1K126me3) is accommodated by the aromatic cage formed of UHRF1 Phe152, Tyr188, and Tyr191, as is
the case for H3K9me3 (Arita et al., 2012; Cheng et al., 2013;
Nady et al., 2011).
These similarities imply that the binding of the TTD to its
various interactors must be mutually exclusive—something
we have verified for H3K9me3 (Figure 4)—raising the questionof how LIG1 can outcompete the other binders. Indeed,
L2UHRF1 and spacerUHRF1 form with the TTD intramolecularinteractions, which should have a much higher probability ofcontact, especially at lower UHRF1 concentrations. As forH3K9me3 molecules, they form intermolecular interactions
with the TTD, but they outnumber LIG1 by a factor of 100
(Ferry et al., 2017). To be able to engage the TTD, it is expectedthat LIG1 should have an affinity significantly higher than theother binders, and this is in fact what we observe (Ferry et al.,2017). In the crystal structure, Arg122 and Arg125 of LIG1
formed interactions not seen for H3K9me3, L2UHRF1, or spacerUHRF1. Based on these observations, it seems likely that thehigh avidity of LIG1 for the TTD results from a combination ofseveral hydrophilic and hydrophobic interactions that are notformed by the other binders.
Structure 27, 485–496, March 5, 2019 493

Possible Modes of Regulation
The interaction between LIG1 and UHRF1 shows high affinity
in vitro, and is easily detectable in cells. Nevertheless, it seems
likely that the complex should undergo dissociation at somepoints in time and space, so that LIG1 can fulfill its catalytic activity—the ligation of Okazaki fragments—while UHRF1 remains
on replicated DNA to ensure H3 ubiquitylation, DNMT1 recruitment, and allosteric DNMT1 activation (Bashtrykov et al., 2014;
Berkyurek et al., 2014; Ishiyama et al., 2017; Nishiyama et al.,2013; Qin et al., 2015). How may the interaction between LIG1and UHRF1 be dissociated?
A first and obvious way to dissociate the complex is to demethylate LIG1K126. Of note, 80% of LIG1 molecules carry
Lys126 methylation in cells (Ferry et al., 2017), suggesting that
if Lys126 demethylation occurs it is a transient event. Future
work may reveal if such an event occurs, and which enzyme isinvolved.
Our data reveal at least three additional possibilities to negatively regulate the UHRF1/LIG1 interaction. The first possibility
is methylation of LIG1R121, which is predicted to disrupt the
key electrostatic interactions with UHRF1D142. The second
possibility is methylation of LIG1R125, which is predicted to prevent G9a from methylating LIG1K126 (Rathert et al., 2008), andshould therefore phenocopy our LIG1R125A mutation. In this regard, FEN1, the flap endonuclease acting just before LIG1 during
replication, is regulated by arginine methylation (Guo et al.,2010), and it could be of interest in the future to ask whether
LIG1 is also targeted on Arg121 and Arg125 by arginine methyltransferases. Finally, a third event that may disrupt UHRF1/LIG1
complexes is phosphorylation of LIG1T123; we experimentally
showed that it decreases affinity for the TTD 10,000-fold. Interestingly, this mechanism could be conserved between interactions, as phosphorylation of the equivalent residues (Thr6 in
H3, Ser298 in L2UHRF1, and Ser651 in spacerUHRF1) has a similareffect (Arita et al., 2012; Fang et al., 2016; Rothbart et al., 2012).
We find that PKCb, which is known to phosphorylate H3T6
(Metzger et al., 2010), efficiently phosphorylates LIG1T123, irrespective of the methylation status of LIG1K126, thus this kinase
is a candidate that might regulate the dissociation of the UHRF1/
LIG1 complex in cells. Interestingly, it has previously been
observed that activation of PKC decreases DNA methylation(Lavoie et al., 2011).

module, in which the PHD is precisely positioned relative to the
TTD by L2UHRF1, which binds the TTD groove. The addition of
H3K9me3 does not disrupt this architecture: instead H3K9me3
adopts a constrained conformation so that its N-terminus binds
the PHD, and its C-terminus binds the TTD (Arita et al., 2012;
Rothbart et al., 2013). In clear contrast, our SEC-SAXS experiments show that LIG1 does modify the TTD-PHD module, toward a more open conformation; presumably this happens bydisplacing L2UHRF1 from the groove. Our ITC experiments also
show that the PHD does not detectably bind the LIG1 peptide,and this is consistent with expectations as LIG1 does not havethe free N-terminal 1ARTK4 motif that is critical for interaction
with the PHD (Arita et al., 2012; Hu et al., 2011; Lallous et al.,2011; Rajakumara et al., 2011). Our data show that, when
UHRF1 is bound to LIG1, the PHD domain is free to bind
H3K9me0 and H3K9me3, with similar affinities. Interestingly,
we also find that, once the TTD-PHD is bound to H3K9me3,
LIG1K126me3 can no longer bind. This mechanism could
possibly ensure a unidirectional handing-over of UHRF1 from
LIG1 to H3K9me3 behind the replication fork. These complex
dynamics may be necessary to order, in space and time, thedifferent functions that UHRF1 has to fulfill: recruitment torecently replicated DNA, methylation of histones, and activationof DNMT1.
Taken together, our results contribute to a better understanding of the critical epigenetic regulator UHRF1, and will guide
future experiments to further study its functions. More generally,
they provide an example of the flexibility deployed by structural
domains in the recognition of methylated peptides, of the complex intermolecular and intramolecular events modulating protein structure, and of how these properties are used biologically.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILSd METHOD DETAILSB Peptide Preparation
B Crystallography

LIG1 Modifies the Overall Structure of UHRF1:
Functional Consequences
Several reports have unambiguously established that full-length
UHRF1 undergoes intramolecular interactions, causing it to present a compact physical aspect, and leading to the inhibition ofits molecular activities (Fang et al., 2016; Gelato et al., 2014; Harrison et al., 2016; Vaughan et al., 2018). Using SEC-SAXS and
HS-AFM, we confirm that UHRF1 is compact, and further
observe that its conformation becomes more extended andmore dynamic once it interacts with LIG1. Therefore, LIG1 canbe added to phosphatidylinositol 5-phosphate, hemimethylatedDNA, and H3K9me3 (Fang et al., 2016; Gelato et al., 2014; Harrison
et al., 2016) as a binder that affects the overall UHRF1 structure.
In spite of this similarity, LIG1 also presents a key differencewith H3K9me3 with respect to its effect on the TTD and PHD
domains. Indeed these two domains can form a functional
494 Structure 27, 485–496, March 5, 2019of TTD and Its Complex with
LIG1K126me3 Peptide
B ITC Measurements
B Fluorescent Three-Hybrid Assay (F3H)
B In Vitro Methylation Assay
B In Vitro Phosphorylation Assay
B SEC-SAXS
B HS-AFM Observations
B Analysis of AFM Images
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND SOFTWARE AVAILABILITY
SUPPLEMENTAL INFORMATION
Supplemental Information includes eight figures, one table, and three videos
and can be found with this article online at https://doi.org/10.1016/j.str.2018.
11.012.

ACKNOWLEDGMENTS
We would like to thank the beamline staff at the Photon Factory for X-ray datacollection. We also thank Dr. T. Uchihashi for 2D-correlation coefficients analysis of HS-AFM. PAD thanks Allison Bardin for useful comments on the manuscript. We express our thanks to Dr. David Spector for sharing the BHK-LacOp
cells. This study was supported by a PRESTO (14530337) from JST and MEXT,
Grant-in-Aid for Scientific Research (B), 18H02392, (to K.A.). P.A.D. was supported by Association pour la Recherche contre le Cancer (ARC2014), by
Agence Nationale de la Recherche (ANR-15-CE12-0012-01 and ANR-11LABX-0071 under ANR-11-IDEX-0005-01), and by Institut National Du Cancer(INCa PLBio 2015-1-PLBio-01-DR A-1).
AUTHOR CONTRIBUTIONS
K.A. and P.-A.D. conceived the study and experimental design, analyzed experiments and co-wrote the manuscript. S.K., S.M., R.M., and K.A. analyzed
the structural basis of the TTD:LIG1K126me3 complex. S.K., T.J., T.O.,
M.S., and K.A. performed SAXS measurements and analyzed the data. S.K.,
S.M., R.M., and K.A. analyzed interaction between TTD and LIG1 by ITC.
L.F. and P.A.D. performed F3H assay. S.K., N.K., T.A., and K.A. performed
HS-AFM experiments and analyzed the data. T.T., N.D., and Y.S. performedin vitro methylation assay.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: September 13, 2018
Revised: October 19, 2018
Accepted: November 28, 2018
Published: January 10, 2019
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(2013). Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA
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7, 12464.

STAR+METHODSKEY RESOURCES TABLEREAGENT or RESOURCESOURCEIDENTIFIER

Novagen

Cat#71400-3CN

Recombinant UHRF1 TTD (residues 123-285)

Arita et al. (2012)

N/A

Recombinant UHRF1 vTTD (residues 123-285 D167-175)

This study

N/A

Recombinant UHRF1 TTD D142A (residues 123-285)

Ferry et al. (2017)

N/A

Recombinant UHRF1 TTD Y188A/Y191A(residues 123-285)

Ferry et al. (2017)

N/A

Recombinant UHRF1 TTD E193A (residues 123-285)

This study

N/A

Recombinant UHRF1 TTD E276A (residues 123-285)

This study

N/A

Recombinant UHRF1 TTD W238A (residues 123-285)

This study

N/A

Recombinant UHRF1 PHD (residues 299-366)

Arita et al. (2012)

N/A

Recombinant UHRF1 TTD-PHD (residues 123-366)

Arita et al. (2012)

N/A

Recombinant UHRF1 TTD-PHD D142A(residues 123-366)

This study

N/A

Recombinant UHRF1 TTD-PHD Y188A/Y191A(residues 123-366)

This study

N/A

Recombinant UHRF1 TTD-PHD D334A/D337A(residues 123-366)

This study

N/A

Recombinant full length His-tagged UHRF1

Nishiyama et al. (2013)

N/A

Recombinant full length His-tagged UHRF1 D142A

This study

N/A

Chemical synthesized LIG1K126me3 (residues 118-130)

Ferry et al. (2017)

N/A

Chemical synthesized LIG1K126me0 (residues 118-130)

Ferry et al. (2017)

N/A

Chemical synthesized LIG1R121A (residues 118-130)

This study

N/A

Chemical synthesized LIG1T123ph (residues 118-130)

This study

N/A

Bacterial and Virus Strains
E.Coli RosettaTM 2 (DE3)
Chemicals, Peptides, and Recombinant Proteins

Chemical synthesized LIG1R125A (residues 118-130)

This study

N/A

Chemical synthesized LIG1R121A/K126me3(residues 118-130)

This study

N/A

Chemical synthesized LIG1T123ph/K126me3(residues 118-130)

This study

N/A

Chemical synthesized LIG1R125A/K126me3(residues 118-130)

This study

N/A

Chemical synthesized H3K9me3 (residues 1-12)

Arita et al. (2012)

N/A

Chemical synthesized H3K4R/K9me3 (residues 1-12)

This study

N/A

Chemical synthesized H3Q5R/K9me3 (residues 1-12)

This study

N/A

Chemical synthesized H3K4R/Q5R/K9me3(residues 1-12)

This study

N/A

Recombinant GST-SENP protease

Arita et al. (2008)

N/A

PreScission protease

GE Healthcare

Cat#27084301

Recombinant G9a

Tachibana et al. (2001)

N/A

Recombinant GLP

Tachibana et al. (2001)

N/A

Recombinant human PKCb2 protein

abcam

Cat#ab60841

KOD-Plus Mutagenesis kit

ToyoboCat#SMK-101

Gibson Assembly Cloning kit

NEB

Cat#E5510

Critical Commercial Assays(Continued on next page)

Structure 27, 485–496.e1–e7, March 5, 2019 e1

Continued
REAGENT or RESOURCESOURCEIDENTIFIER

Crystal structure of apo-TTD

This study

PDB: 5YYA

Crystal structure of TTD in complex with LIG1K126me3

This study

PDB: 5YY9NMR structure of TTD in complex with H3K9me3

Nady et al. (2011)

PDB: 2L3R

Crystal structure of TTD in complex with H3K9me3

Nady et al. (2011)

PDB: 3DB3

Crystal structure of TTD-PHD in complex with H3K9me3

Arita et al. (2012)

PDB: 3ASK

Herce et al. (2013)

N/A

Primer: UHRF1 D167-175 Forward: 5’-GCCCCCTCCCGGCCG
GCGCTGGAGGAGGACGTCATT

Hokkaido System
Science Co.,Ltd.

N/A

Primer: UHRF1 D167-175 Reverse:5’-CTCCAGCGCCGGCCG
GGAGGGGGCCTTCCGCGTCAC

Hokkaido System
Science Co.,Ltd.

N/A

Primer: UHRF1 E193A Forward: 5’-CTACCCGGCAAACGGCGT
GGTCCAGATGAA

Hokkaido System
Science Co.,Ltd.

N/A

Primer: UHRF1 E193A Reverse: 5’-CGCCGTTTGCCGGGTAGTCGTCGTATTTCA

Hokkaido System
Science Co.,Ltd.

N/A

Primer: UHRF1 E276A Forward: 5’-CGTGGACGCAGTCTTCAAGATTGAGCGGCC

Hokkaido System
Science Co.,Ltd.

N/A

Primer: UHRF1 E276A Reverse: 5’-TGAAGACTGCGTCCACGAAGATGATCCGAC

Hokkaido System
Science Co.,Ltd.

N/A

Primer: UHRF1 W238A Forward: 5’-GGGCTTCGCGTACGACG
CGGAGATCTCCAG

Hokkaido System
Science Co.,Ltd.

N/A

Primer: UHRF1 W238A Reverse: 5’-CGTCGTACGCGAAGCCC
CGCTCCTTGGGGT

Hokkaido System
Science Co.,Ltd.

N/A

Primer: UHRF1 D334A/D337A Forward: 5’-TGTGCGCTGAGTGCGCCATGGCCTTCCACA

Hokkaido System
Science Co.,Ltd.

N/A

Primer: UHRF1 D334A/D337A Reverse: 5’-CCATGGCGCACTCAGCGCACATGAGCTGCT

Hokkaido System
Science Co.,Ltd.

N/A

pGEX-ST1-UHRF1 TTD WT (residues 123-285)

Arita et al., 2012

N/A

pGEX-ST1-UHRF1 vTTD (residues 123-285 D167-175)

This study

N/A

pGEX-ST1-UHRF1 TTD D142A (residues 123-285)

Ferry et al., 2017

N/A

pGEX-ST1-UHRF1 TTD Y188A/Y191A (residues 123-285)

Ferry et al., 2017

N/A

pGEX-ST1-UHRF1 TTD E193A (residues 123-285)

This study

N/A

pGEX-ST1-UHRF1 TTD E276A (residues 123-285)

This study

N/A

Deposited Data

Experimental Models: Cell Lines
BHK-LacOp cells (Baby Hamster Kidney, female)
Oligonucleotides

Recombinant DNA

pGEX-ST1-UHRF1 TTD W238A (residues 123-285)

This study

N/A

pGEX-ST1-UHRF1 PHD WT (residues 299-366)

Arita et al., 2012

N/A

pGEX-ST1-UHRF1 TTD-PHD WT (residues 123-366)

Arita et al., 2012

N/A

pGEX-ST1-UHRF1 TTD-PHD D142A (residues 123-366)

This study

N/A

pGEX-ST1-UHRF1 TTD-PHD Y188A/Y191A(residues 123-366)

This study

N/A

pGEX-ST1-UHRF1 TTD-PHD D334A/D337A(residues 123-366)

This study

N/A

pGEX-6P-1-full length His-tagged UHRF1 WT

Nishiyama et al. (2013)

N/A

pGEX-6P-1-full length His-tagged UHRF1 D142A

This study

N/A

pEGFP-C2- full-length UHRF1 WTFerry et al. (2017)

PAD1543

pEGFP-C2- full-length UHRF1 D142A

This study

N/A

pEGFP-C2- full-length UHRF1 E193A

This study

N/A

pEGFP-C2- full-length UHRF1 E276A

This study

N/A

pEGFP-C2- full-length UHRF1 W238A

This study

N/A(Continued on next page)e2 Structure 27, 485–496.e1–e7, March 5, 2019

Continued
REAGENT or RESOURCESOURCEIDENTIFIER

pmRFP-C2 Full-length human LIG1 WTFerry et al. (2017)

PAD1766

pmRFP-C2 Full-length human LIG1 R121A

This study

N/A

pmRFP-C2 Full-length human LIG1 T123DThis study

N/A

pmRFP-C2 Full-length human LIG1 R125A

This study

N/A

Coot

Emsley et al. (2010)

http://www2.mrc-lmb.cam.ac.uk/
personal/pemsley/coot

PHENIX

Afonine et al. (2012)https://www.phenix-online.org/
documentation/index.html

PHASER

McCoy et al. (2007)

http://www-structmed.cimr.cam.ac.uk/
phaser/XDS

Kabsch (2010)

http://xds.mpimf-heidelberg.mpg.de/

AIMLESS

Evans and Murshudov(2013)

http://www.ccp4.ac.uk/html/aimless.html

Pymol

http://www.pymol.org

http://www.pymol.org

SAngler

Shimizu et al. (2016)

http://pfwww.kek.jp/saxs/SAngler.html

PRIMUS

Konarev et al., 2003

https://www.embl-hamburg.de/biosaxs/
primus.html

DAMMIF

Franke and Svergun(2009)

https://www.embl-hamburg.de/biosaxs/
dammif.html

DAMAVER & DAMMIN

Volkov and Svergun, 2003

https://www.embl-hamburg.de/biosaxs/
damaver.html

Software and Algorithms

Origin 5.0, 7.0 and Origin Pro 9.1

https://www.originlab.com/

Other
Glutathione Sepharose 4B

GE Healthcare

HiTrap Heparin HP column

GE Healthcare

Cat#17075605
Cat#17040701

HiTrap Q HP column

GE Healthcare

Cat#17115401

HiLoad 26/600 Superdex 75 column

GE Healthcare

Cat#28989334

HiLoad 26/600 Superdex 200 column

GE Healthcare

Cat#28989336

Superdex 200 Increase 10/300 GL

GE Healthcare

Cat#28990944

Superdex 200 Increase 5/150 GL

GE Healthcare

Cat#28990945

RIGAKU R-AXIS IV++ with MicroMax 007

RIGAKU

MicroCal VP-ITC Isothermal Titration Calorimeter

Malvern

Cat#MicroCal VP-ITC

Q Exactive mass spectrometer

Thermo Fisher Scientific

MALDI-TOF/MS

Brucker: autoflex-YS

Atomic force microscope

Ando et al., 2001, 2008

N/A

Cantilevers for AFM

Olympus

BL-AC10DS-A2

CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Kyohei
Arita (aritak@yokohama-cu.ac.jp)
EXPERIMENTAL MODEL AND SUBJECT DETAILSAll recombinant proteins (UHRF1 full-length, TTD, PHD and TTD-PHD) were expressed in E. coli RosettaTM 2 (DE3) (Novagen). Cells
were grown in Luria-Bertani (LB) medium at 37 C shaking at 150 rpm in an INNOVA 42 shaker (New Brunswick Science) and inducedwith 0.2 mM isopropyl-b-D-thiogalactopyranoside (IPTG) when they reached an optical density of 0.60.7 at 660 nm. The cells wereincubated at 15 C overnight or 30 C for 6 hours.
For fluorescent-3-hybrid experiments, we used BHK cells (Baby Hamster Kidney cells, female, ATCC CCL-10), modified to containmultiple copies of the Lac Operator (Herce et al., 2013). The cells were grown in DMEM/10% Fetal Calf Serum and reverse-transfected in 24-well plates using Lipofectamine 2000 (ThermoFisher, Cat# 11668027).
Structure 27, 485–496.e1–e7, March 5, 2019 e3

METHOD DETAILS
Peptide Preparation
The human LIG1 (UniProt ID: P18858) peptides, residues 118-130 (NH2-118IPKRRTARKQLPK130-COOH) harboring K126me3,
R121A-K126me3, R125A-K126me3 or T123ph-K126me3, and histone H3K9me3 (NH2-1ARTKQTAR-K(me3)-STG12-COOH), K4RK9me3, Q5R-K9me3 and K4R-Q5R-K9me3 were purchased from Toray Research Center (Tokyo, Japan).
Crystallography of TTD and Its Complex with LIG1K126me3 Peptide
The TTD (residues 123-285) of human UHRF1 (UniProt ID: Q96T88) was expressed as a fusion protein with glutathione S-transferase(GST) and small ubiquitin like modifier-1 (SUMO) at its N-terminus (Arita et al., 2012). The protein was expressed in E. coli strain RosettaTM 2 (DE3) (Novagen). Cells were grown at 37 C in Luria-Bertani (LB) medium and induced with 0.2 mM isopropyl-b-D-thiogalactopyranoside (IPTG) when they reached an optical density (O.D.) of 0.6 at 660 nm, and incubated at 15 C for 15 hours. The cells
were re-suspended with a lysis buffer (40 mM Tris-HCl (pH8.0), 300 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine Hydrochloride(TCEP), 1 mM EDTA, 10% glycerol and protease inhibitor cocktail (Nacalai tesque, Cat# 03969-21)) and sonicated with the cycle ofpulse on for 5 seconds and pulse off for 1 minute (total pulse on time; 5 minutes). Insoluble debris was removed by centrifugation at
19,000 rpm for 40 minutes at 4 C using Avanti J-E with a rotor JA-20 (BECKMAN COULTER). The GST-SUMO-tagged TTD was
loaded to Glutathione Sepharose 4B (GS4B; GE Healthcare, Cat# 17075605), and the unbound proteins were washed by the lysis
buffer. The GST-SUMO-tagged TTD was eluted by an elution buffer; 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM dithiothreitol(DTT), 10 % glycerol, 20 mM reduced glutathione (GSH). The GST-SUMO tag was cleaved by the SUMO specific protease, SENP
(purified in house), at room temperature for 56 hours. The TTD was further purified by anion-exchange chromatography, HiTrap
Q (GE Healthcare, Cat# 17115401) using gradient elution from 100 – 500 mM NaCl in 20 mM Tris-HCl (pH 8.0) buffer containing
0.5 mM DTT and 10% glycerol. Finally, the protein was purified with Hiload 26/600 Superdex 75 size exclusion chromatography(GE Healthcare, Cat# 28989334) equilibrated with 20 mM Tris-HCl (pH 8.0), 150 mM NaCl and 0.2 mM DTT. Cocrystallization ofthis wild-type TTD with the LIG1K126me3 peptide was unsuccessful, so we used instead a modified version of the TTD, designatedvTTD, deleting residues 167-175. The protocols of cell culture and purification of vTTD are the same as for wild-type TTD.
Crystal of apo-TTD were obtained by using a reservoir solution containing 0.1 M Bis-Tris (pH 6.5), 200 mM ammonium acetate and
25% (w/v) polyethylene glycol (PEG) 3,350 at 4 C. The crystals were cryoprotected by 20% (v/v) ethylene glycol. Diffraction data werecollected at a wavelength of 1.5418 Å on a RIGAKU R-AXIS IV++ equipped with MicroMax 007 (RIGAKU). Data were processed with
program XDS package (Kabsch, 2010) and AIMLESS (Evans and Murshudov, 2013) at 1.7 Å resolution. Data were processed in spacegroup P212121 and Matthews coefficient suggested one molecule of apo-TTD in the asymmetric unit. The structure of apo-TTD wassolved by molecular replacement method using the coordinates of the human UHRF1 TTD (PDB; 3DB3 (Nady et al., 2011)) as asearch model. Molecular replacement and model refinement were performed using PHASER (McCoy et al., 2007) and PHENIX (Afonine et al., 2012), respectively. After rigid body refinement and simulated annealing by PHENIX, 2jFoj - jFcj difference Fourier map
corresponding to the flexible loop, residues 160-179, was unambiguously observed, which has not seen in search model. Manualmodel building was performed with Coot (Emsley et al., 2010). After several cycles of refinement with PHENIX, the model converged
well at 1.7 Å resolution with a crystallographic R-factor of 17.2 % and free R-factor of 20.6%.
The vTTD:LIG1K126me3 complex was prepared by adding a 1.5-molar excess of the LIG1K126me3 peptide to the protein before
concentration using an Amicon concentrator with a 10,000 Da cutoff (Millipore). The crystal was obtained using a 30 mg/ml concentration of the complex at 20 C and the hanging drop vapor diffusion method with a reservoir solution containing 100 mM Tris-HCl(pH 7.0), 200 mM tri-potassium phosphate and 20% (w/v) PEG3,350. The crystal was directly frozen in liquid nitrogen using a cryoprotectant containing 20% (v/v) ethylene glycol. The X-ray diffraction data were collected at a wavelength of 0.98000 Å on a Pilatus3
6M detector in beam line BL-17A at Photon Factory (Tsukuba, Japan) and scaled at 2.65 Å resolution with the program XDS package
and Aimless. Data were processed in space group P212121 and Matthews coefficient suggested two molecules ofvTTD:LIG1K126me3 complex in the asymmetric unit. Molecular replacement was performed by PHASER using apo-TTD structure
determined in this study as a search model. Manual model building was performed with Coot. After rigid body refinement and simulated annealing by PHENIX, 2jFoj - jFcj difference Fourier map corresponding to LIG1K126me3 were unambiguously observed. Afterseveral cycle of model refinement by PHENIX, the final model converged at 2.65 Å resolution with a crystallographic R-factor of
23.1% and a free R-factor of 28.8%. The crystallographic data and refinement statistics are given in Table 1. The figures were generated using Pymol (http://www.pymol.org).
ITC Measurements
Human UHRF1 PHD (residues 299-366) and TTD-PHD (residues 123-366) were expressed as GST-SUMO fusion protein and purified
in the same experimental conditions. The proteins were expressed in E. coli strain RosettaTM 2 (DE3). The protein expression was
induced with 0.2 mM IPTG at O.D660 = 0.60.7. In the case of the PHD, the cells were incubated at 30 C for 6 hours; for the TTDPHD, the cells were incubated at 15 C for 15 hours. The cells were re-suspended with a lysis buffer (40 mM Tris-HCl (pH8.0),
300 mM NaCl, 0.5 mM TCEP, 30 mM zinc acetate, 10% glycerol and protease inhibitor cocktail) and sonicated with the same cycle

e4 Structure 27, 485–496.e1–e7, March 5, 2019

as TTD. Insoluble debris was removed by centrifugation at 19,000 rpm for 40 minutes at 4 C. The GST-SUMO-tagged protein was
loaded to GS4B and eluted by the elution buffer: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM DTT, 10 % glycerol, 20 mM GSH. The
GST-SUMO tag was cleaved by the SENP at room temperature for 56 hours. The TTD-PHD or PHD was further purified by anionexchange chromatography, HiTrap Q using gradient elution from 100 -500 mM NaCl in 20 mM Tris-HCl (pH 8.0) buffer containing
0.5 mM DTT and 10% glycerol. Finally, the protein was purified with Hiload 26/600 Superdex 75 size exclusion chromatography equilibrated with 10 mM Tris-HCl (pH 8.0), 150 mM NaCl and 0.2 mM TCEP. For binding experiments, we introduced the following mutations in the context of WT TTD (residues 123-285): D142A, Y188A/Y191A, E193A, E276A, W238A. We also introduced the following
mutations within the TTD-PHD: D142A, Y188A/Y191A, D334A/D337A. All the constructs were obtained by the Quickchange mutagenesis method (Agilent Technologies).
Purified UHRF1 WT TTD, mutants of the TTD (D142A, Y188A/Y191A, E193A, E276A, W238A), vTTD, PHD, TTD-PHD, and mutantsof the TTD-PHD (D142A, Y188A/Y191A, D334A/D337A) were buffer-exchanged using Superdex 200 Increase 10/300 GL (GE Healthcare, Cat# 28990944) equilibrated with the ITC buffer (10 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 0.25 mM TCEP). Lyophilized
LIG1 peptides (residues 118-130) and H3K9me3 peptide (residues 1-12) were dissolved in the ITC buffer. A MicroCal LLC calorimeter, VP-ITC (MicroCal), was used for the ITC measurements. The 1020 mM of protein solution in the calorimetric cell was titrated
with the 125250 mM of peptide solution at 293 K. The data were analyzed with the software ORIGIN (MicroCal) using a one-site
model. The first data point was excluded from the analysis. For each interaction, we performed at least three independent
experiments.
Fluorescent Three-Hybrid Assay (F3H)
The starting plasmids for F3H were: full-length human UHRF1 cloned in pEGFP-C2 (plasmid PAD1543) (Ferry et al., 2017) and fulllength human LIG1, cloned in pmRFP-C2 (plasmid PAD1766) (Ferry et al., 2017). The various mutations (UHRF1 D142A, E193A,
E276A, W238A; LIG1 R121A, T123D, R125A) were introduced by Gibson Assembly Cloning. The F3H assay were performed as previously described (Herce et al., 2013): we used BHK cells containing multiple copies of the Lac Operator (a kind gift of David Spectorand Heinrich Leonhardt). The cells were grown in DMEM/10% Fetal Calf Serum and reverse-transfected in 24-well plates using Lipofectamine 2000. Each well received equal amounts of three plasmids encoding: the LacR-GFP binder fusion protein (plasmid kindly
given by Heinrich Leonhardt), the GFP-UHRF1 fusion (or its mutant derivatives), and the LIG1-mRFPC2 fusion (or its mutant derivatives). Eighteen hours after transfection, the coverslips were washed with PBS, fixed with 4% paraformaldehyde (10 minutes, room
temperature), permeabilized with 0.5% Triton X-100 in PBS (4 minutes at 4 C), and stained with DAPI. They were mounted andobserved at 40X magnification on a fluorescence microscope. Cells expressing both GFP and RFP were scored for colocalization.
Each experiment was carried out at least twice independently, with 100 cells scored in blind in each repetition.
In Vitro Methylation Assay
Methylation assays were performed in 50 mM Tris-HCl (pH 8.0), 10 mM LIG1 peptide (residues 118-130; WT, R121A, T123ph, R125A),
0.1 mg/ mL enzyme, 200 mM S-adenosyl-L-methionine at 30 C. The reactions were stopped by the addition of trifluoroacetic acid to
0.5%. Samples were then analyzed by mass spectrometry. Recombinant G9a and GLP were prepared as described previously(Tachibana et al., 2001). The following peptides were used: hLIG1(118-130) WT, IPKRRTARKQLPK; hLIG1(118-130) T123ph,
IPKRRT(ph)ARKQLPK; hLIG1(118-130) R125A, IPKRRTAAKQLPK; hLIG1(118-130) R121A, IPKARTARKQLPK.
The samples of methylation assays were diluted to 1/100 with 0.1% formic acid and applied to nano-liquid chromatography-tandem mass spectrometry using a Q Exactive mass spectrometer (Thermo Fisher Scientific). The peptide mixtures (2 mL) were separated by nano ESI spray column (75 mm [ID] 3 100 mm [L], NTCC analytical column C18, 3 mm, Nikkyo Technos) with a linear gradientof 0 - 35% buffer B (acetonitrile with 0.1% (v/v) formic acid) in buffer A (MilliQ water with 0.1% (v/v) formic acid) at a flow rate of
300 nL/min over 10 minutes (EAST-nLC 1000; Thermo Fisher Scientific). The mass spectrometer was operated in the positive-ion
mode for MS and MS/MS, and the MS/MS spectra were acquired using an inclusion list containing methylation specific peptideions (WT: triply charged none-, mono-, di- and tri-methyl IPKRRTARKQLPK ion m/z= 531.346, 536.018, 540.690 and 545.362,
respectively. T123ph: triply charged none, mono-, di- and tri-methyl IPKRRT(ph)ARKQLPK ion m/z= 558.001, 562.673,
567.345 and 572.017, respectively. R125A and R121A: triply charged none-, mono-, di- and tri-methyl IPKRRTAAKQLPK and
IPKARTARKQLPK ion m/z= 502.991, 507.663, 512.335 and 517.007, respectively.) The MS chromatograms of these peptide ionswere drawn using Qual Browser, Thermo Xcalibur 3.1.66.10 and each peptide amount was estimated by the peak area.
In Vitro Phosphorylation Assay
Phosphorylation assays were performed in 20 mM HEPES-NaOH (pH 7.5), 1 mM DTT, 1 mM b-Glycerophosphate, 0.05 mM Na3VO4,
1 mM ATP, 10 mM MgCl2, 10 mM LIG1 peptide (residues 118-130; WT and K126me3), 0.02 mg/ mL human PKCb (Abcam, Cat#ab60841) at 30 C for 15 hours. Samples were then analyzed by MALDI-TOF/MS (Brucker: autoflex-YS).
SEC-SAXS
Protein preparation of the WT and D142A mutant of TTD-PHD, residues 123-366, were described as before. SAXS data werecollected on Photon Factory BL10C (Tsukuba, Japan) using UPLC ACQUITY (Waters) integrated SAXS set-up. 50 ml of 6 mg/ml
sample was loaded onto a 15/150GL INCREASE Superdex 200 (GE Healthcare, Cat# 28990944, column volume; 3 ml) pre-equilibrated by 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM DTT, 10 mM zinc acetate and 5% glycerol at a flow rate of 0.25 ml/min
Structure 27, 485–496.e1–e7, March 5, 2019 e5

at 4 C. The flow rate was reduced at 0.025 ml/min at elution volume of 1.85-2.30 ml. X-ray scattering was collected every 20 seconds
on a PILATUS3 2M detector over an angular range qmin = 0.00690 Å1 to qmax = 0.27815 Å1. UV spectra at a range 200 nm to 450 nm
was recoded every 10 seconds. Circular averaging and subtraction including radius of gyration Rg and I(0) calculations were carriedout using program SAnglar (Shimizu et al., 2016) to obtain one-dimensional scattering data I(q) as a function of q (q = 4psinq/l, where
2q is the scattering angle and l is the X-ray wavelength 1.5 Å). To obtain scattering intensity on an absolute scale, measured scattering intensities were calibrated based on a scattering intensity of water (Orthaber et al., 2000). Estimation of the molecular weight ofsamples was calculated from I(q) data of bovine carbonic anhydrolase (Sigma) at the most highest value of I(0). The radius of gyration
Rg and forward scattering intensity I(0) were estimated from the Guinier plot of I(q) in the smaller angle region of qRg < 1.3. The distance distribution function P(r) of the sample at the highest peak of A280 and I(0) was calculated in the program GNOM, where theexperimental I(q) data were used in a q-range from 0.00885 to 0.17670 Å-1. The maximum particle dimension Dmax was estimatedfrom the P(r) function as the distance r for which P(r) = 0. ab initio three-dimensional shape reconstruction was performed using
the DAMMIF (Franke and Svergun, 2009). In total, 10 models were averaged and the resulting structures were compared with the
crystal structure of TTD-PHD in complex with H3K9me3 (PDB: 3ASK) in PyMOL. The SAXS data statistics are given in Table S1.
HS-AFM Observations
N-terminal 63histidine (His6) tag full-length human UHRF1 was sub-cloned into pGEX6P-1 (GE Healthcare, Cat# 28954648). The
UHRF1 wild type and D142A mutant were expressed in E. coli Rosettta2 (DE3) and induced with 0.2 mM IPTG at O.D660 = 0.7.
The cells were further incubated at 15 C for 15 hours. The cells were re-suspended with a lysis buffer (40 mM Tris-HCl (pH8.0),
300 mM NaCl, 0.5 mM TCEP, 30 mM zinc acetate, 10% glycerol and protease inhibitor cocktail) and sonicated with the same cycle
as TTD. Insoluble debris was removed by centrifugation at 19,000 rpm for 40 minutes at 4 C. The soluble proteins were loaded to
GS4B and eluted by the elution buffer: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM DTT, 10 % glycerol, 20 mM GSH. The GST
tag was cleaved by the PreScission protease (GE Healthcare, Cat# 27084301) at 4 C for overnight. The UHRF1 was further purified
by HiTrap Heparin (GE Healthcare, Cat# 17040701) using gradient elution from 150 - 800 mM NaCl in 20 mM Tris-HCl (pH 8.0) buffer
containing 0.5 mM DTT and 10% glycerol. Finally, the protein was purified with Hiload 26/600 Superdex 200 size exclusion chromatography equilibrated with 10 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10% glycerol 0.2 mM TCEP and 10 mM zinc acetate.
HS-AFM imaging was performed in solution at room temperature using a laboratory-built HS-AFM setup (Ando et al., 2001, 2008)
as described previously (Uchihashi et al., 2012). In brief, a glass sample stage (diameter, 2 mm; height, 2 mm) with a thin mica disc(1 mm in diameter and 0.05 mm thick) glued to the top by epoxy was attached onto the top of a Z-scanner by a drop of nail polish. A
freshly cleaved mica surface was prepared by removing the top layers of mica using Scotch tape and coated by 2 ml of 5 mM NiCl2.
Then, a drop (2 ml) of diluted protein sample (ca. 3 nM) in dilution buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl and 10% glycerol) was
placed on the mica surface. After incubation for 3 minutes at room temperature, the mica surface was rinsed with 20 ml of the observation buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl) to remove floating samples. The sample stage was then immersed in a liquidcell containing 60 ml of the observation buffer with and without 150 nM LIG1K126me3 peptide. AFM Imaging was carried out in thetapping mode, using small cantilevers (Olympus, Cat# BL-AC10DS-A2); resonant frequency, 0.5 MHz in water; quality factor, 2 inwater; spring constant, 0.1 N/m. The cantilever’s free oscillation amplitude A0 and set-point amplitude As were set at 12 nm
and 0.9 3 A0, respectively. The imaging rate, scan size and the pixel size for each AFM image are 150 ms/frame, 60 3 60 nm2
and 80 3 80 pixels, respectively.
Analysis of AFM Images
For analysis, AFM images were pretreated with a low-pass filter to remove spike noise and with a flatten filter to make the overall
xy-plane flat, using a laboratory built software as described before (Ngo et al., 2015; Uchihashi et al., 2012). This software is available
at https://elifesciences.org/content/4/e04806/article-data-fig-data-supplementary-material. The heights of molecules were
measured semi-automatically using the following steps. First, the most probable highest point near the highest point of the molecule
was selected manually. Second, the actual highest point was automatically determined by searching a 10 3 10 pixel area (typically
7.5 3 7.5 nm2) around the selected point.
2D correlation coefficients were calculated between the HS-AFM images of the first frame and each of the frames within the Regionof Interest (ROI) (i.e., the first frame is the reference) (Uchihashi et al., 2011). The sizes of the ROIs were about 25325 nm2. The 2D
correlation coefficient was calculated frame-by-frame for each ROI. The 2D correlation coefficient r is defined as,


P P 
n  H Rm
n R
m
n Hm
r = rﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
P P 
2  P P 
2
n H
n R
m
n Hm
m
n Rm
in which Hmn and Rmn are the heights at the pixel point (m, n) in the ROI to be analyzed and the reference ROI of the reference frame,
respectively. H and R are the mean values of the height matrices H and R, respectively.

e6 Structure 27, 485–496.e1–e7, March 5, 2019

QUANTIFICATION AND STATISTICAL ANALYSIS
Statistics of the X-ray crystallographic and small angle-X-ray scattering data processing, refinement and structure validation aresummarized in Table 1 and S1. ITC measurements were performed at least three times and the quantified data represent mean ± SD.
DATA AND SOFTWARE AVAILABILITY
The PDB accession number for apo-TTD is 5YYA and its complex with LIG1K126me3 peptide is 5YY9.

Structure 27, 485–496.e1–e7, March 5, 2019 e7