Linker histones consolidate heterogenous nucleosome fiber contacts by linking together multiple nucleosomes

Domain: chemistry
Published: 2026-03-05
Editorial outcome: major_revision

Abstract
The consensus mode for linker histone (H1) association coincides with ‘on-dyad’ binding to
an individual nucleosome, making it challenging to rationalize the chromatin dynamics and
compacting activities of H1 in the context of a highly heterogenous structural scaffold. Here,
we investigate the activity of the somatic H1 variants by conducting crystallographic analysis
of nucleosomal assemblies and characterization of nucleosome array condensates, which
recapitulate long-range nucleosome fiber interactions in chromatin. H1 is observed to
associate variant-dependently with nucleosomes through a diversity of binding modes that
include linking multiple nucleosomes/fibers together. Binding versatility is facilitated by the
proclivity of the H1 globular domain to recognize DNA structural motifs, which are similar
between an individual nucleosome and specific niches within clusters of nucleosomes. We
propose that linker histones support a structurally and functionally complex repertoire for
chromatin regulation by assuming a variety of context-and variant-dependent DNA binding
modes.

Introduction
The eukaryotic genome consists of chromatin fibers, which can comprise more than one
million nucleosomes in tandem for a given chromosome1,2. Condensing nucleosomes into compact
states provides a means for gene silencing in heterochromatin, maintaining genomic integrity and
generating mitotic chromosomes for cell division. Nucleosome compaction is facilitated by the
action of a variety of chromatin architectural factors, which include linker histone (LH), cohesin,
condensin, and HP1, amongst other factors, that appear to act at different levels of the chromatin
structural hierarchy. While it has come to light in recent years that compact chromatin structure in
vivo generally consists of irregular interdigitated nucleosome fibers with zigzag features, as
opposed to highly regular ‘30 nm fiber’ structures, the mechanisms by which architectural factors
operate to shape local chromatin 3D organization are not well understood3.
As the most abundant chromatin-binding protein, LHs are essential factors in higher
eukaryotes, with eleven H1 variants present in mammals, of which seven (H1.1–H1.5, H1.0 and
H1x) are somatic and four (H1oo, H1t, H1t2 and HILS1) are germ cell-specific4. LH variants
display specialized genomic functions and gene-specific regulatory activities as well as cell-
dependent expression and differential chromatin localization5. Notably, H1.0 has been shown to
play a key role in controlling proliferative potential and differentiation status of tumor cells6, while
cancer-associated mutations in the H1.2–H1.5 variants can act as oncogenic drivers, rendering the
LHs bone fide tumor suppressors7.
LH structure is generally conserved across protein variants in metazoans, with a short N-
terminal domain (~30 residues; LH ) and a long C-terminal domain (~110 residues; LH ) that
NT CT
are unstructured in solution, linked by a globular domain (LH ) of about 80 amino acids4,8,9. The
GD
LH consists of a so-called winged helix motif, having three off-parallel packed -helices.
GD
Although the LH is essential and alone sufficient for nucleosome association, the LH is
GD CT
required for high affinity chromatin binding and condensing activity10,11,12,13. There have been
several models proposed for how the LHs bind to chromatin, which differ largely in whether the
LH assumes a roughly central position on the nucleosome dyad, interacting with both linker
GD
DNA arms (on-dyad), or is situated off to one side, interacting preferentially with one of the linker
DNA arms (off-dyad)9.
In conjunction with advances in cryogenic electron microscopy (cryo-EM), there have been
many structures solved of nucleosomes assembled with different LH variants and truncatants
(reviewed in ref. 14), all of which have on-dyad association of the LH , with two exceptions.
GD
Cryo-EM structures of glutaraldehyde-fixed 12-nucleosome arrays assembled with either human
H1.4 or H5 (a.k.a. avian H1.0) display a 2-start, zig-zig 30 nm fiber configuration, in which the
LH associates in an off-dyad fashion15,16. Although footprinting analyses of H1.5 or H1.0
GD
binding to multi-nucleosome (3- or 6-nucleosome) arrays have instead shown on-dyad
association17,18, this off-dyad H1 binding is analogous to the binding mode elucidated in an earlier
solution NMR study yielding a Drosophila H1 (dH1 )–nucleosome structure19. However, with
GD GD
exception of the 30 nm fiber, cryo-EM structures have been based on individual nucleosome-LH
assemblies (chromatosomes) in isolation, and the few crystal structures reported coincide with
discontinuity/interruption of the double helix between chromatosomes in the lattice as well as
limited resolution.
It appears to have been largely assumed that the physiological substrate for a LH is a single
nucleosome, but this may not be accurate considering the salient chromatin compacting activities
of LHs and the fact that diverse domains of the core histones participate avidly in the condensation
of chromatin fibers by mediating internucleosomal interactions20. Therefore, from the standpoint
of chromatin association, it remains to be established what LH binding modes are prevalent in
vivo, in the context of crowded, condensed, or compact poly-nucleosomal states.
In order to shed light on the nature of LH association with nucleosome fibers as well as the
structure of compacted chromatin, we developed a strategy for designing nucleosomes to self-
assemble into continuous fibers21. Application of this approach so far entailed crystallizing
nucleosomes or dinucleosomes having 4-nucleotide overhangs, which foster Watson-Crick base
pairing to yield a continuous double helix that comprises linker DNA connecting nucleosomes in
the lattice. Two of the dinucleosome constructs yielded near-atomic resolution structures in which
zigzag configured nucleosome fibers span the length of the crystals and are interdigitated with one
another in the lattice22 (HD animation, ref. 23). When co-crystallized with LH, in addition to on-
dyad binding of H1.0, another copy of H1.0 was found associated in a newly observed ‘linking’
mode22 (formerly referred to as the ‘non-dyad’ mode; HD animation, ref. 24). This linking mode
entails the LH associating with three separate nucleosome fibers, thereby stabilizing compaction
GD
and interdigitation. This led us to propose that LHs may foster chromatin compaction by
associating with a variety of structural niches inherent to condensed nucleosome fibers.
In this study, we analyzed LH association from the crystal structures of a variety of
nucleosome-LH assemblies that stem both from our recent21,22 as well as unpublished works. In
conjunction with in vitro nucleosome and nucleosome array condensate experiments, the results
suggest that condensed chromatin harbors a variety of inter-nucleosomal motifs to which LHs can
associate in a variant-dependent fashion. The existence of alternative LH binding modes, which
include multi-nucleosome association, is consistent with the observed promiscuity and plasticity
of LHs as well as their ability to bind nucleosomes with greater than unity stoichiometry and
promote interfiber crosslinking in condensates.

Results
Nucleosome-Linker Histone Assembly Structures
We screened 24 different cohesive-ended mononucleosome and dinucleosome constructs,
of which 10 yielded well diffracting crystals (Table 1 in ref. 21)21,22. Various constructs were also
co-crystallized with different LH variants, yielding instances of LH-associated electron density for
H1.0, H1x, H5, and H1.3 that was sufficiently clear to permit explicit modeling of the LH
GD
(Table 1; Supplementary Tables 1 and 2; Supplementary Figs. 1–4). Co-crystallization trials with H1.1,
H1.2, H1.4, or H1.5 also yielded many near-atomic resolution data sets, but with insufficiently
clear electron density associated with the LH proteins to permit model building. Generally, across
all assemblies for which we were able to obtain data of sufficient resolution to allow a nucleosome-
based molecular replacement phasing solution, LH could not be explicitly modeled (given the
presence in the crystal of at least 1 bound LH molecule/nucleosome; see Methods). Nonetheless,
in many data sets, residual features in the electron density maps suggest DNA-bound LHs that are
associated with static and/or dynamic disorder.
With one particular type of construct, which consists of a 165 bp double helix having 4-
nucleotide overhangs at both termini, we were able to solve LH-nucleosome assembly structures
from crystals grown in the presence of H1x, H1.0, H5, or H1.3. The same overall lattice
arrangement was obtained using three different 169 ‘bp’ constructs (169a, 169ak, 169an) that differ
by virtue of the DNA sequence associated with the single-stranded termini. The 169 nucleosomes
dimerize to form pairs, in which the self-complementary, 4-nucleotide overhangs from each of the
linker DNA termini are associated with Watson-Crick base pairing (Figs. 1a and 1b). This results
in a pseudo-continuous double helix of 338 bp encompassing the nucleosome dimer, which also
usually corresponds to the asymmetric unit of the crystal. As such, the two 145 bp-core
nucleosomes are connected on either end by a 24 bp-linker DNA arm.
The 169 nucleosome dimers are arranged in layers in the crystal, with nearly fully
overlapping face-to-face stacking of the nucleosomes perpendicular to the layer planes (Fig. 1c).
The face-to-face stacking from one layer to the next involve roughly opposite nucleosome
orientations, such that the two nucleosomes within a dimer each interact with two different dimers
from adjacent layers. This coincides with a zig-zag disposition of dimers along the stacking plane,
and in the orthogonal directions, the dimers are again offset, such that the two nucleosomes of a
given dimer are alternatively positioned in the recesses within and between dimers (Fig. 1d).
Depending on the LH variant and crystallization outcome, the LHs can be situated either between
the linker DNA sections of a given nucleosome (Fig. 1a, LH1, magenta; on-dyad mode) or within
the roughly cube-shaped cavity formed by DNA boundaries from eight different nucleosomes over
three consecutive layers (Figs. 1b–d; linking modes).
A total of 11 crystallographically distinct LH structures for H1x, H1.0, H1.3, and H5
GD
from 7 different nucleosome-LH assembly crystals were solved. The nucleosome-LH assembly
crystals coincide with nearly identical nucleosome packing (lattice) configurations, while the
LH structures all adopt the winged helix fold and are overall similar to each other
GD
(Supplementary Fig. 5). The three -helices (1–3) forming the N-terminal core module are
connected by loop regions (l1, l2), and the C-terminal element consists of two strands (s1, s2),
which fold back over the core module and interact with the C-terminal portion of l1. A third loop
(l3) connects s1 and s2. In spite of conservation of the winged helix motif, the different LH
GD
structures display significant conformational variability in the loop regions, both between and
within variants— in particular for l3. This results from an overall degree of plasticity that allows
accommodation of differences in binding context (see below).
On-Dyad Consensus Mode Across Nucleosomal Constructs
In our earlier structural solutions of a diverse set of nucleosomal constructs, in cases where
LH could be explicitly modelled, we consistently observed binding in the on-dyad mode (Fig. 2).
This includes three different di-nucleosomal constructs with distinct linker DNA lengths that
crystallize as closed/circular di-nucleosomes or tetra-nucleosomes, or as continuous nucleosome
fibers. The LH is known to have three major DNA binding surfaces/motifs that interface with
GD
the nucleosome dyad region and the two linker DNA arms. This entails the 2/3/l3, 1/l2/3,
and l1/s1/s2 motifs, which interact respectively with the central (widened) minor groove at the
nucleosome dyad, a minor-and-major groove section of linker DNA arm 1, and phosphodiester
elements (generally over a minor groove region) of linker DNA arm 2 (Figs. 2 and 3).
In our structures of 343c, 349c, and 353e assembled with H1.0, we observed 5 instances of
on-dyad binding (of a total of 8 potential sites), which nonetheless coincide with substantial
conformational differences in the linker DNA arms (Fig. 2). However, the extent of conformational
variability of linker DNA arm 2 is substantially greater than that of either linker DNA arm 1 or the
bound dyad region, which essentially has a fixed conformation. This is consistent with the
relatively sparse number of DNA contacts associated with the l1/s1/s2 interface relative to either
the 2/3/l3 or 1/l2/3 interfaces (Fig. 3; Supplementary Fig. 6; Supplementary Data 1).
For the 169 construct assemblies, we also observe at least one example of on-dyad binding.
We had originally tested this class of construct, having short linker DNA arms, to ensure that high
affinity LH binding to a single nucleosome can be achieved. H1x, H1.0, H1.3, and H5 all display
very similar kinetics and high affinities towards the 169a nucleosome in solution. The dissociation
constants (K ) span a narrow range of between 15 and 45 pM (Supplementary Table 3;
D
Supplementary Fig. 7). In the H5-169a crystal structure, on-dyad binding is observed at one of the
two paired nucleosomes, although the H5 assumes the orientation whereby the 1/l2/3 motif
GD
also interacts with elements of the major groove that is formed by the annealed overhangs from
the paired nucleosome (Figs. 4a and 4b).
Linking and On-Dyad Modes Adopted in Parallel
In the H5-169a structure, the nucleosome paired with that having the on-dyad-bound LH
does not serve as host to another instance of on-dyad binding. Instead, a second H5 molecule is
associated via a linking mode, with DNA elements from four different nucleosomes, comprising
the SHL 4/4’ DNA supergroove of one nucleosome (N4), the SHL 5.5 major groove of a second
nucleosome (N3), and the paired linker DNA section of the neighboring nucleosome dimer (N1/N2;
Figs. 4a and 4c). The major groove interface entails extensive DNA base contacts with C-terminal
portions of the H5 1 and 2 that would suggest the potential for preferential recognition (direct
GD
readout) of the DNA sequence (Fig. 4d).
The linking mode interactions displayed by the H5 are analogous to those of H1.0, which
GD
was seen linking together three separate fibers/nucleosomes by interacting with five different DNA
elements, in our earlier dinucleosome (349c) crystal structures22-24 (Figs. 3, 4e and 4f). The H1.0
GD
forms interactions with nucleosome core DNA elements that are configured in a fashion similar to
that which supports on-dyad binding within a single nucleosome. This is apparent from structural
comparison as well as by the observation that the residues involved in H-bonding with the DNA
are nearly identical between the on-dyad and linking modes (H1.0D and H1.0L2, respectively, Fig.
3 and Supplementary Data 1). In fact, the H1.0 -DNA contact area for the linking mode (1,534
GD
Å2; calculated as the change in solvent-accessible surface area; Supplementary Table 4) is very
similar compared to that for the on-dyad mode, being either slightly higher (1,569 Å2) or lower
(1,425 Å2) depending on whether one includes the contribution from a close contact ‘above’ with
the linker DNA that is shared between the N+1 and N+2 nucleosomes (Fig. 4e).
In the H5-169a structure, the GD-DNA contact areas are also very similar between the on-
dyad (2,199 Å2) and linking (2,188 Å2) modes (Supplementary Table 4). Moreover, the H1.0-349c
and H5-169a linking modes share application of the 2/3/l3 and l1/s1/s2 motifs in a manner
similar to the corresponding on-dyad modes as well as engage additional GD residues to achieve
contacts with DNA elements outside of the on-dyad binding-triad (Figs. 3 and 4 and
Supplementary Data 1). In fact, in either case, the 2/3/l3 motif is associated with a widened
minor groove nucleosome core element, which— as a consequence of histone-mediated bending
into the (inward-facing) major groove— is nearly identical in structure to that at the dyad
(nucleosome center; Figs. 4g and 4h). On the other hand, while the 1/l2/3 motif is seen to
recognize a similar minor-and-major groove element for the H1.0-349c on-dyad and linking modes
and the H5-169a on-dyad mode, in the case of the H5-169a linking mode, it is only partially
utilized and in a split fashion. More specifically, the central region of the H5 1/l2/3 surface
remains unengaged, while the peripheral portions associate with two separate DNA elements (Fig.
4h). In this way, a novel interface is created, whereby the 1/l1/2 surface binds to an extended
major groove section with shape/electrostatic base-complementarity (Fig. 5a). This fourth
interaction surface of the GD allows for a tetrahedral-like DNA binding sphere, as it is also
observed for the H1.0-349c on-dyad mode, where it is utilized (albeit only sparingly) to contact a
neighboring section of linker DNA (Fig. 4e). Overall, the H1.0 and H5 GDs preside over DNA
binding elements beyond the canonical dyad and linker DNA arm 1 and 2 motifs that include both
electropositive and electronegative patches (Figs. 3, 5a and 5b). In spite of sharing >80% sequence
identity, the H1.0 and H5 display distinctions in charge distribution, amongst other features,
GD GD
which likely contribute to differences in DNA binding preferences (see also below).
Variant-Dependency in Linking Mode Binding
The ability of H5 to associate with the 169a nucleosome dimer via the on-dyad mode
indicates that this LH binding mode is permissible within 169 crystals, in spite of structural
constraints that may be imposed by the nature of nucleosome pairing. Nonetheless, the absence of
on-dyad H5 binding in the second, opposing, nucleosome of the dimer may be a consequence of
either a special favorability associated with the observed H5 occupancy at the linking-mode
binding site or an on-dyad-disfavoring/incompatible nucleosome conformation (although a
significant degree of plasticity is inherent in the nature of on-dyad LH -nucleosome interactions;
GD
Fig. 2). In any case, for the remainder of the LH-169 assembly crystals analyzed, no further
instances of on-dyad binding were observed (Figs. 5c–h).
For the H1x, H1.0, and H1.3 variants, we analyzed many data sets across a variety of
nucleosomal constructs. Crystals of H1x-169a consistently displayed a type of linking mode
binding that is distinct from that described above for H1.0 and H5. Although interactions are
formed between the H1x and three nucleosomes (four DNA sections), there is only a single
GD
extensive interface, which involves the 1/l2/3 motif (Figs. 5c and 5d). This interface accounts
for the majority of the contact area (819 Å2 of the 1,209 Å2 total; Supplementary Table 4), while
the three other contact points encompass sparse interactions. The main interface involves
positioning of 3 within the minor groove around SHL 6, accompanied by concerted binding of
the N-terminal region of 1 to the DNA backbone (Fig. 6). This interface in its entirety spans over
a turn of the double helix as N-terminal (and in one H1x copy also C-terminal) residues emerging
from the GD track further along the DNA to provide supporting interactions (Supplementary Data
1; Fig. 6a). The result is a pronounced conformational change entailing a widened minor groove,
which permits extensive hydrogen bonding and hydrophobic contacts with the sugar-phosphate
backbone as well as a hydrogen bond between the N3 ring nitrogen atom of a guanine base and
the penetrating side chain of Y91. Nonetheless, this SHL 6 interaction alone is not sufficient to
allow specific association with the corresponding NCP, and therefore the presence of linker DNA
is essential for specific H1x binding to individual nucleosomes in solution (Supplementary Fig. 8).
Hydroxyl radical footprinting analysis of the 169a-LH assembly indicates that H1x interacts with
both dyad and linker DNA regions but not SHL ±6 (Supplementary Fig. 9), which is consistent
with H1x binding to mononucleosomes in the on-dyad mode, as observed by cryo-EM25.
The nucleosome context-dependent binding of H1x— evident from the on-dyad mode of
association to individual (169a) nucleosomes in solution versus the type of linking mode adopted
in the dense multi-nucleosomal environment in the crystals— indicates a context sensitivity that
is also seen in a 2.5 Å resolution structure of H1x assembled with a 338 bp-dinucleosome (338b)21.
This construct was engineered to mimic the nucleosome-LH dimer configuration of the 169
crystals (Figs. 1 and 5c), and the ensuing dinucleosome crystal packing creates a similar
environment (Supplementary Fig. 10). However, only one H1x molecule per dinucleosome can be
resolved, although it binds in the identical linking mode as seen in the H1x-169a structure. The
absence of a second H1x copy binding in a side-by-side fashion coincides with a shift in the
disposition of the corresponding interacting nucleosomes, which would preclude association in the
same fashion (Supplementary Fig. 10).
In screening through many H1.0-169 data sets, we discovered three different crystal types
where H1.0 assumes distinct binding modes. One of these coincides with the same linking mode
seen in the H1x-169a crystals. The GD in the H1.0-169a structure interacts with the four
surrounding DNA elements in nearly identical fashion as H1x (Fig. 5e), although this is achieved
with many differences in amino acid sequence between the two variants for the interfacial residues
(Fig. 3; Supplementary Data 1). Like H1x, most of the H1.0 -DNA interfacial area (783 Å2 of
GD
the 1,336 Å2 total; Supplementary Table 4) stems from the 1/l2/3 motif interaction with the
SHL 6 widened minor groove.
Given the common character of the single-major-interface LH binding mode in these
H1x- and H1.0-169a structures (where the 1/l2/3 motif is the apparent driver), we assign this
as a linking mode 1 (L1) category, with which to distinguish between the other linking mode
configurations (Fig. 7). This can be contrasted with the H1.0 and H5 linking modes outlined in the
previous sections, whereby the overall commonality is the presence of two major interfaces that
involve both the 1/l2/3 and the 2/3/l3 motifs, or in the case of the H5-169a structure,
2/3/l3 together with an alternative LH motif. As such, we assign this as the L2 category,
GD
which is more closely analogous to the on-dyad binding mode that is characterized by two major
DNA interfaces based on the 1/l2/3 and the 2/3/l3 motifs.
Regarding the other two H1.0 binding modes observed in 169 crystals, one corresponds to
a crystal growth polymorph of the H1.0-169a assembly that coincides with a type of binding
referred to as L1b, since it appears as a variant of the L1 mode (Figs. 5f and 7a; Supplementary
Fig. 11). In either case, an 1/l2/3-DNA minor groove interaction is the major interface that is
supported with otherwise sparse contacts to several surrounding DNA elements. However, for the
L1b mode, the engagement of the 1/l2/3 motif is with an SHL 4 (as opposed to SHL 6 in the
L1 mode) minor groove and coincides with a less invasive interaction that results in a significantly
reduced total interfacial area (849 Å2 [L1b] versus 1,336 Å2 [L1]; Supplementary Table 4). A
distinct linking mode example, designated as L2b, was also obtained for crystals of H1.0
assembled with the 169an construct (Figs. 5g and 7b). It is assigned to the L2 class, as it has the
analogous profile as seen for the L2 mode of H1.0-349c crystals, by virtue of having two major
interfaces based on the 1/l2/3 and the 2/3/l3 motifs together with additional sparse contacts
to surrounding DNA elements. Indeed, the total interfacial areas for the two systems (1,366 Å2
[169an] versus 1,534 Å2 [349c]; Supplementary Table 4) are both similar and larger than those
seen for the L1/L1b examples (Fig. 7c).
In contrast to the binding displayed by H1x and H1.0, the 169a structure obtained for H1.3
shows an off-dyad type mode, in which the H1.3 is situated over SHL 0.5 (directly adjacent to
GD
the dyad, i.e., SHL 0), where it makes contacts with either end of the major groove (Fig. 5h). In
spite of this off-the-dyad positioning, it is still able to form interactions with both linker DNA
sections, but with contacts to one arm (comprising 982 Å2 of the 1,562 Å2 total interfacial area;
Supplementary Table 4) being significantly more extensive than the other. The extensively
contacted linker DNA arm involves 1/l2/3 elements in a manner similar to that seen for on-
dyad binding, such as in the H5-169a structure. However, for H1.3, the 1 N-terminus is inserted
into the major groove, where hydrophobic contacts to 3 thymine bases of the poly-A/T track are
formed with a leucine and valine side chain. The nature of these contacts suggests that the DNA
sequence could play a direct role in LH binding, and the corresponding V SEL sequence motif
41 44
is in fact unique to the H1.1–H1.5 variant subfamily (Fig. 3).
Nucleosome Core Double Helix Remodeling
In the L1 mode H1x- and H1.0-169a structures, both of the shared linker DNA sections are
relatively straight, until around SHL 6, at which point there is a sharp kink of about 40° in each
nucleosome (Fig. 6d). This differs from the respective symmetry-related halves (SHL 6ʹ), where
there is only about a 20° bend from histone octamer association (Fig. 6c). The distinct 40° kinks,
which are directed into the major groove, are a consequence of the invasive 1/l2/3-nucleosome
(N1) interactions (Figs. 5d, 5e and 6). The insertion of the LH 3 helix into the minor groove and
the associated supporting motifs result in remodeling the double helix around SHL 6 to have a
pronounced bulge with substantial A-form-like character (Fig. 6a). In particular, a triad of tyrosine
residues stack against each other in H1x— which are correspondingly a tyrosine, glutamine, and
a leucine residue in H1.0 (Fig. 3)— and in either case these three residues stack against the exposed
face of the splayed-open minor groove, with two adjacent nucleotides adopting C3’-endo (A-form)
sugar pucker. The DNA structural remodeling renders the double helix with closer contacts to
histone octamer elements around the nucleosome core terminus (SHL 6.5; Fig. 6b).
Linker Histones Link Together Multiple Nucleosome Fibers
Given our earlier observation that H1.0 binds in a linking (L2) mode by interacting with
multiple nucleosome fibers in dinucleosome crystals22-24, in conjunction with the above results, we
conducted LH binding experiments with condensates generated in vitro. The condensates, which
are formed under conditions that promote self-association of nucleosomal arrays, have been shown
to provide an excellent model system for studying the condensed chromatin environment present
in interphase chromosomes26,27. When varying amounts of LH are added to preformed
condensates, binding to nucleosomes does not cease at unitary (1:1 LH:nucleosome)
stoichiometry but instead LH continues to associate with the condensates up to and potentially
beyond 6:1 added H1 ratios (Fig. 8a). The binding profiles and points of plateau differ between H1.0
and H1x, which moreover display distinct saturation behavior when the amount of bound LH in the
arrays is quantified (Supplementary Figs. 12 and 13). For H1.0, association levels approach nearly 3 LH
molecules bound per nucleosome and do not appear to have achieved saturation by 6:1 added LH.
In contrast, for H1x, the condensates harbor binding sites for up to about 4 molecules per
nucleosome, which appears to also correspond to a saturation point. Analogous behavior, with
variant-specific differences in loading and saturation profiles, are seen for the DNA replication-
dependent members (H1.1–5 variants) of the somatic LH family (Supplementary Fig. 14).
This indicates that there are multiple LH binding sites within condensed nucleosome fiber assemblies,
and at least some of these binding motifs are occupied in a variant-dependent fashion.
We also conducted microscopic imaging of condensates with arrays that had been
fluorescently labelled (Figs. 8b and 8c; Supplementary Figs. 15 and 16). In the absence of LH or
in the presence of LH at low stoichiometry, different condensates have a nearly identical
appearance, entailing a typically spherical geometry with a diameter of 260±100 nm. However, at
higher LH stoichiometry, the spherical condensates are seen to aggregate into much larger
assemblies resembling beads on a string in varying states of compaction. The effect is apparent
already at around 1:1 LH:nucleosome stoichiometry for some of the H1.1–5 variants and for H1.0
(Fig. 8c; Supplementary Figs. 15 and 16), which displays overall greater condensate-linking
activity relative to H1x, but in any case, the extent of aggregation increases with the amount of
added LH. These observations indicate that H1 is able link multiple nucleosome fibers together by
mediating complex internucleosomal contacts, consistent with our body of crystal structures.

Methods
Crystallization and Data Collection
H. sapiens core histones and LHs as well as DNA fragments and chicken H5 were
overexpressed individually in E. coli and processed/purified, followed by core histone octamer
refolding and nucleosome reconstitution21,22. The DNA fragments are described in detail in ref. 21.
Briefly, the 169 fragments are cohesive-ended and the 338b (b=second-generation) fragment is
blunt-ended. The 169a (a=first-generation) and 169ak (k=KpnI) fragments have cohesive ends
generated by PstI and KpnI restriction digestion, respectively. The cohesive ends of the 169an
(n=non-equivalent termini) fragment are generated by KpnI and SacI double restriction digestion.
169 and 338b nucleosome-LH assemblies were prepared for crystallization by mixing LH and
nucleosome with a slight excess of LH (1.2–1.4 molar stoichiometry) in a buffer consisting of 76–
90 mM CaCl , 50 mM KCl and 20 mM Na-acetate [pH 4.5] to give a final nucleosome-LH
2
concentration of 4 mg ml−1. Crystals of nucleosome-LH assembly were grown by the hanging-
droplet vapor diffusion method through salting-in via equilibration against a reservoir solution
containing 38–45 mM CaCl , 25 mM KCl and 10 mM Na-acetate [pH 4.5], with incubation at
2
18°C.
Nucleosome-LH assembly crystals were harvested and stabilized in a buffer consisting of
20 mM CaCl , 12.5 mM KCl, 10 mM Na-acetate [pH 4.5], 25% 2-methyl-2,4-pentanediol (MPD)
2
and 2% trehalose. Prior to data collection, the MPD concentration was increased gradually to 65%,
yielding a pronounced gain in diffraction quality. Single crystal X-ray diffraction data were
recorded, subsequent to mounting stabilized crystals directly into the cryocooling N gas stream,
2
at beam line X06DA of the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland)
using a Pilatus 2M-F detector and an X-ray wavelength of 1.0 Å.
Structure Solution and Refinement
Diffraction data were indexed, integrated, merged, scaled and evaluated with a
combination of iMosflm45,46, XDS47, SCALA48, and AIMLESS49 from the CCP4 package50,51 and
the in-house data processing pipeline, go.com, developed by the Swiss Light Source
macromolecular crystallography beamlines (Paul Scherrer Institute, Villigen, Switzerland). Initial
phases for solving the structure of the H1x-169a assembly were obtained by molecular replacement
using the program PHASER52, with the 2.2 Å resolution crystal structure of NCP composed of the
601L DNA fragment (pdb code 3UT9)53 serving as the search model. Additional search model
elements for molecular replacement included the H 1x NMR structure (pdb code 2LSO), lacking
the s1-l3-s2 motif. For subsequent initial structural solution of the other nucleosome-LH
assemblies, search models for molecular replacement included the nucleosome from the H1x-169a
structure as well as the LH from the H1x-169a structure or the H5 from the assembly with a
GD GD
167 bp nucleosome crystal structure (pdb code 4QLC)28.
Atomic refinement and model building were carried out with REFMAC54 and COOT55,
respectively, from the CCP4 suite50,51. Data collection and structure refinement statistics are given
in Supplementary Tables 1 and 2.
Structural Analysis
Solvent accessible surface area calculations were conducted using GETAREA56. Double
helix conformational parameters and reconstructions were obtained with 3DNA57,58. Graphic
figures were prepared with PyMOL (DeLano Scientific LLC, San Carlos, CA, USA), CCPmg59
and DSSR60.
Hydroxyl Radical Footprinting Analysis
DNA or nucleosome was 5’ end-labeled with 32P by mixing 12 µg of nucleosome or 6 µg
of DNA, 10 units of T4 polynucleotide kinase (PNK) and 10 µCi of γ32P-ATP (PerkinElmer,
Waltham, MA, USA) in a 20 µl volume with T4 PNK buffer (70 mM Tris-HCl [pH 7.5], 1 mM
MgCl , 5 mM DTT). After 1 h incubation at 37°C, the reaction was arrested by passing the sample
2
through a DyeEx 2.0 spin column (Qiagen, Hilden, Germany) to remove unbound γ32P-ATP. 32P-
labeled nucleosome or DNA was buffer-exchanged to 20 mM K-Cacodylate [pH 6.0] using
Amicon Ultra-0.5 ml centrifugal filters (Merck Millipore, Carrigtwohill, Co. Cork, Ireland).
nucleosome-LH assembly was generated by mixing H1x with nucleosome in 1.0 or 1.2 molar
stoichiometry, followed by incubation on ice for 15 m.
Hydroxyl radical footprinting was carried out based on an established protocol61. The
reaction was achieved by mixing 1 µl of 100 mM sodium ascorbate, 1 µl 0.6% H O and 1 µl Fe2+-
2 2
EDTA (4 mM Fe2+ / 8 mM EDTA) on the side of an Eppendorf tube, which was then immediately
added to a 10 µl solution containing either 1 µg DNA, 2.2 µg nucleosome or 2.4 µg nucleosome-
LH assembly (in each case an equivalent quantity of DNA). After incubation for 10 m at room
temperature, the reaction was arrested by adding 1 µl of 100 mM thiourea. DNA was purified by
phenol:chloroform:isoamyl alcohol (25:24:1) extraction and ethanol precipitation. After a 70%
ethanol wash, the pellet was dissolved in formamide loading buffer (96% formamide, 20 mM
EDTA, 0.05% (w/v) bromophenol blue). To serve as markers, a Maxam-Gilbert purine sequencing
standard was prepared62. Samples were heated at 95°C for 3 m before being loaded onto a
denaturing 10% polyacrylamide gel, run in 0.5x TBE. Gels were dried, exposed to Imaging Screen-
K (Bio-Rad Laboratories, Hercules, CA, USA) and scanned with a Typhoon Trio variable mode
imager (GE Healthcare, Chicago, IL, USA).
Kinetics and Affinity Measurements
Nucleosome-LH binding kinetics and affinity measurements were carried out using the
Octet RED96e Bio-layer interferometry system (Pall Fortebio, Fremont, CA, USA). Purified 169a
DNA fragment was biotinylated using enzymatic labeling, whereby biotin-14-dCTP (Thermo
Scientific, Waltham, MA, USA) was coupled to the DNA 3’ end by terminal deoxyribonucleotide
transferase (Promega, Madison, WI, USA). Biotinylated 169a DNA was subsequently used to
reconstitute nucleosome. Biotinylated nucleosome was captured on streptavidin biosensors (Pall
Fortebio, Fremont, CA, USA), pre-equilibrate with PBST buffer (8 mM Na HPO , 150 mM NaCl,
2 4
2 mM KH PO , 3 mM KCl, 0.05% Tween-20 [pH 7.4]), to the extent that all sensors reached a
2 4
0.15 nm binding threshold. Captured nucleosome was stable on the surface without any drift in the
baseline after loading. All binding experiments were performed at 30° C in duplicate. LHs were
determined to have no non-specific interaction with the streptavidin biosensors in PBST buffer.
Single cycle kinetics (or kinetic titration) was performed with increasing concentrations of LH
(0.625, 1.25, 2.5, 5, and 10 nM). Association and dissociation phases were both measured for 300
s at each concentration. One of the biosensors was dipped in PBST buffer during both association
and dissociation phases to serve as the reference channel. The data were analyzed using
BIAevaluation v4.1 software (GE healthcare, Chicago, IL, USA). The kinetic titration series
obtained from the Octet was referenced and fit with a 1:1 kinetic titration model.
Linker Histone-Condensate Binding Assays
Purification of 601-207x12 Template DNA. pUC19 plasmid containing the 207x12 DNA template
insert, composed of 12 repeats of the 601 Widom high affinity nucleosome binding sequence63,
was purified from E. coli through alkaline lysis. Plasmid was digested using Xba1 and HindIII to
cut out the template, and HaeII and Dra1 was used to further digest the remaining plasmid
fragments (New England Biolabs, Ipswich, MA, USA). Digested plasmid fragments were
separated through size exclusion chromatography on a Sephacryl S-1000 gravity fed column.
Fractions containing the 601x12 2500 bp fragment were combined and alcohol precipitated. The
template DNA was then resuspended in 10 mM Tris-HCl (pH 7.8) and 0.1 mM EDTA buffer to
yield a DNA concentration of 1 mg/mL.
Assembly and Purification of Histone Octamer. Recombinant Xenopus core histones were
purchased through the Colorado State University (CSU) Protein Expression and Purification
facility. Lyophilized histones H2A, H2B, H3, and H4 were resuspended in 6 M guanidinium HCl,
20 mM Tris-HCl (pH 7.5), and 5 mM DTT for 2 hours at 25°C. Histones were mixed at equimolar
ratios to a final concentration of 1 mg/mL. The mixture was dialyzed against 2 M NaCl, Tris-HCl
(pH 7.5), 1 mM EDTA, and 5 mM β-mercaptoethanol, with fresh buffer being changed three times
every 4 hours64. Folded octamer was concentrated using Amicon Ultra-15 centrifugal filters
(Merck Millipore, Carrigtwohill, Co. Cork, Ireland), and purified using size exclusion
chromatography with a HiLoad 16/6- Superdex 200 column on the AKTA start liquid
chromatography system (GE Healthcare, Chicago, IL, USA). Fractions collected from the peak
were assessed using SDS-PAGE, then combined and concentrated to 5 mg/mL with an Amicon
Ultra 15 centrifuge filter.
Alexa 488 H4E63C Labeling and Labeled Histone Octamer Assembly. Recombinant H4E63C was
purchased from the CSU Protein Expression and Purification facility. The addition of the Alexa
488 fluorophore was achieved by adding an equimolar concentration of Alexa 488 C5 maleimide
(Thermo Scientific, Waltman, MA, USA) to histone H4E63C in 6 M guanidinium-HCl, 20 mM
Tris-HCl (pH 7.5), and 0.7 mM tris(2-carboxyethyl)phosphine (TCEP). The fluorophore and
histone solution was covered and placed at 4°C overnight in a rotator. Fluorescently labeled H4
was then mixed with H2A, H2B, and H3C110A (to prevent additional fluorophore labeling) in
equimolar ratios and subjected to the same dialysis and size exclusion chromatography purification
protocol described above for unlabeled octamer assembly, with the exception that the solution was
kept in the dark at all times.
Assembly of Unlabeled Nucleosomal Arrays and Alexa 488-Labeled Nucleosomal Arrays.
Nucleosomal arrays were reconstituted using equimolar combinations of the purified 601x12 DNA
template with reconstituted histone octamer at a final concentration of 0.3 mg/mL. Gradual
nucleosome formation was achieved with a decreasing salt dialysis protocol at 4°C, starting at 2
M NaCl, followed by 1M NaCl, then 0.75 M NaCl, and ending at 2.5 mM NaCl in 10 mM Tris-
HCl (pH 7.8), 0.25 mM EDTA65. The resulting arrays were sedimented using a Beckman XL-I
analytical ultracentrifuge (Beckman Coulter, Brea, CA, USA) and analyzed using UltraScanIII
software. Only arrays that sediment between 27S and 30S, which contain between 11 and 12
octamer/template66, were used for experiments.
Chromatin Condensate Formation. Condensates were formed by adding nucleosome arrays to a
10 mM Tris-HCl (pH 7.8) and 1 mM EDTA solution containing >4 mM MgCl , to yield a final
2
array concentration of 10 nM. This was followed by incubation for 20 minutes at 25°C.
Differential Centrifugation Pelleting Assay. Following condensate formation, nucleosome arrays
were incubated with linker histone for 30 minutes at 25° C (Supplementary Fig. 12). A given
linker histone variant was mixed at a stoichiometry range encompassing 0.25, 0.5, 0.75, 1, 1.5, 2,
2.5, 3, 3.5, 4, 4.25, 4.5, 5, 5.25, 5.5, and 6 H1:nucleosome to investigate the occurrence of multiple
binding modes. After incubation, condensate and linker histone mixtures were centrifuged at
20,000 g for 5 minutes, at which point condensates and condensate-bound proteins pellet while
unassociated arrays and free proteins remain in the supernatant. Supernatant was removed and the
pellet was washed with a 5 mM MgCl solution twice before pellet and supernatant were analyzed
2
via SDS-PAGE (Supplementary Fig. 13).
Quantitation of Condensate-Bound H1. To quantify the amount of linker histone bound to
condensates, standards of known H1 quantity (µg) along the condensate pellets of various
H1:nucleosome stoichiometry were analyzed by SDS-PAGE (Invitrogen Novex Wedgewell 4-20%
Tris-Glycine Gels, stained with Coomassie blue; Supplementary Fig. 13). Gel images were
captured using ImageQuant LAS 500 (GE Healthcare, Chicago, IL, USA). Images were analyzed
via gel band densitometry using the NIH public domain Java program, ImageJ, employing the
rolling ball background subtraction. Standard curves were generated for each gel based on the H1
standards and subsequently applied to determine the amount of protein in µg. At least three, and
up to six, biologically independent experiments were carried out for each H1:nucleosome
stoichiometry tested. In order to allow for complete and redundant coverage of all the samples,
many gels were run, from which the standard slopes were calculated and applied to the condensate
samples. Given the number of data points required to explore a finely sampled saturation curve, it
was not possible to obtain a full range of samples and standards within a single gel. Therefore,
many gels were used to quantify condensate samples by fitting to the most proximal standard
slopes.
Fluorescence Microscopy. Alexafluor-488-H4-labeled condensate samples were incubated
(covered) with varying stoichiometric ratios of unlabeled H1 variant (r=0, r=0.5, r=1.0, r=2.0, and
r=4.0) for 10 minutes at 25°C. Samples were then placed on a MatTek glass bottom 35 mm petri
dish with a 14 mm microwell 1.5 cover glass (MatTek, Ashland, MA, USA), covered and given
an additional 20 minute incubation and settling time. Condensates were imaged using the Zeiss
LSM 900 Confocal Microscope with Airyscan 2, with a 63x/1.40 objective/numerical aperture
(Zeiss, White Plains, NY, USA). Zen 3.1 software was used for both capture and image analysis.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Adhireksan et al. is an extension of their earlier work published in Nat. Commun. 2020 & Nucleic Acids Res. 2021. The current study aimed to study the association of nucleosome and linker histone subtypes (Human H1x, H1.0, H1.3 and Chicken H5) in oligo-nucleosome chromatin fibers at an atomic level using X-ray crystallography. The authors found that LHs bind in ‘non-canonical’ off-dyad modes, associating with the inter-nucleosomal linker DNA and other positions on the nucleosome. They find that in many cases LHs engage in multiple bridging interactions between DNA segments nucleosomes, with some variation between the LH subtypes studied. The study reports the first structural visualization of alternative H1 binding modes (beyond the canonical on-dyad mode) in a chromatin complex, extending our knowledge of how LH subtypes interact in non-canonical ways in chromatin, especially in situations where there is stoichiometric excess of LH over nucleosomes. However, enthusiasm is somewhat damped by the low resolution for two of the four structures, some issues with the way the data is analyzed and presented, some lack of connection to prior work, and discussion of limits of the current study.\n\nComments:\nIn the abstract and other places the authors state that the commonly accepted mode of LH binding is based on studies of individual nucleosomes. However, the paper ignores studies of H1-di- and tri-nucleosome complexes (Syed et al., 2010 PNAS) and the crystal structure of a 6-nucleosome array bound to histone H1 (Garcia-Saez, 2018 Mol. Cell), both showing on-dyad binding to oligonucleosomes is identical to full-length H1 binding to mononucleosomes.\n\nRelatedly, the ‘closed-loop’ dinucleosomes structures appear to force a full cross-over of linker DNA, perhaps interfering with some LH binding to the canonical on-dyad site. Is this the case, i.e. are there steric hinderances or distortions that would inhibit LHG domain fitting in at the canonical on-dyad position? This should be discussed/presented in the MS.\n\nResults: The authors show an ‘on-dyad’ mode of binding for H5, but there is no comparison to previous reports of on-dyad binding modes for this or other LHs (Zhou et al., 2015 Mol. Cell; Bednar et al., 2017 Mol. Cell; Zhou et al., 2021 Mol. Cell).\n\nPage 7, Authors statement that “compaction of Ns within the crystal is fostered by DNA binding of LH, N- and C-terminal tails of the core histones and Ca2+ ions, which function in large part to provide electrostatic counterparts for the double helix”. The authors should show or document a comparison of structures in absence and presence of LH to support this conclusion.\n\nPage 7, Author’s mentioned that based on crystallization outcome LHs present between the linker DNA or within the roughly cube shaped cavity. Please label this cavity in Fig. 1. In addition, it would be helpful to the reader to see an animation/video of the different LHs positioning between the linker DNAs and in the cavity to support Fig. 2 (besides Ref. 20&21), and furthermore tabulate the distances/interactions between LHs and nucleosomes/DNA for Fig. 3, 4, and 5b to provide better understanding of the interacting residues at an atomic level beside their surface accessibility as mentioned in Table S2.\n\nFigure S3, Some of the electron density for LHNT and LHCT has been found and the region has been modelled and marked to interact with the distal nucleosome. It would be interesting to show a pictorial view of the interaction and tabulate the distances of the interacting residues as well. Similarly, indicate the LHNT interactions for Figure S11.\n\nFigure 8 should be extended by showing individual orientation with interacting residues followed by a combined model, it’s always nice to show the interacting partners/resides in tabulated form.\n\nThe authors report extremely tight binding for H1-N interactions. Given that the authors detail multiple alternative binding modes for these LHs, how well does the 1:1 kinetic binding model fit the data? If >1 H1 binding sites exist per nucleosome, how does this affect the calculation of overall affinity?\n\nDiscussion: Is there potential for numerous additional alternative H1-binding modes in chromatin beyond what is reported here?\n\nMinor comments:\nResults, p. 5 bottom: The authors state: “Various constructs were also co-crystallized with different LH variants, which included human H1x, H1.0, H1.1–H1.5 and chicken H5 (a.k.a. avian H1.0)”. I found this presentation to be confusing when no results are presented for most of these proteins. Simply say that ‘similar attempts were made with LHs X, Y, and Z, but insufficient density was detected to allow analysis for these proteins’?\n\nFig. 1A. What does the cloud around the LH cartoon structures indicate? This should be noted in the figure legend.\n\nFig. 2. The current representation of Fig. 2a-b is somewhat crowded and its hard to the layering. Again, a video or animation would help the reader here.\n\nSimilarly, the authors state: “With one particular type of construct, which consists of a 165 bp double helix having 4-nucleotide overhangs at either terminus, we were able to solve many structures with the four different LH variants.” Again, confusing. What is meant by “many structures”? Is the linker DNA evenly distributed about the nucleosome core binding site? Shouldn’t “either terminus” be “both termini”?\n\nResults, p. 6, top: Related, the authors state “However, the best diffracting crystals were typically the ones co-crystallized with H1.0 or H1x, and these two variants together with H5, and to a lesser extent H1.3, were the only LH types for which the associated electron density was sufficiently clear to permit explicit modeling of at least the LHGD (Table S1 & Figs. S1–S8). This is a long and confusing sentence.\n\nRelated, p. 7, middle para: “A total of 11 LHGD of H1x, H1.0, H1.3 and H5 across 7 different N-LH structures, encompassing nearly identical N packing (lattice) configurations, all adopt the winged helix fold and are overall similar to each other (Fig. 2c). Again, long, cumbersome, confusing to the reader.\n\nWhat are the DNA sequences for the templates, and specifically what do a, ak, and an mean in terms of sequence?\n\nFig. S1-S8: Choose contrast color and bolding for numeric labelling of residues for better visualization.\n\nPage 7, in my opinion the statement “Electron density maps stemming from H1x-169a assembly crystals are especially well defined” should be specified: ... .well defined H1x globular domain.\n\nFigure S9, please mark the interface with a dotted line and labelled the respective nucleosomes as shown in S9a.\n\nPlease expand what the author means in the header of Table S1. I understand it makes more sense to show the data set of H1x-169aP, H1.0-169aN1, H1.0-169anR, H1.3-169aF, H5-169aD/N2 but what about the data set with similar nucleosome conditions (mono or di)?", "reviewer_2": "The manuscript by Adhireksen et al. builds on previous work by the authors reported in ref. 18, in which they engineered a number of nucleosomal constructs with cohesive-ended DNA that allowed them to crystallize nucleosomes in a circular or continuous fibre configuration. The current manuscript reports five additional such structures at resolutions ranging from 2.7 to 3.5 Å. These describe nucleosomes in complex with four different linker histone (LHs) - the human histones H1.0 (two structures), H1x and H1.3 as well as chicken histone H5. The new structures all comprise a circular configuration of a nucleosome dimer, in which the entry and exit linker arms of one nucleosome connect to the exit and entry arms of the other, respectively. These structures show similar crystal packing and contain the same palindromic DNA sequence (169a), except for an H1.0 structure that contains a non-palindromic sequence (169an). The crystal structures capture the LHs in three previously observed configurations (on-dyad, off-dyad and peripheral) as well as three new configurations that the authors term reverse peripheral, non-dyad(1) and non-dyad(2).\n\nThe authors also report complementary data that include: bio-layer interferometry data showing that the four LHs bind the 169a mononucleosome with similar affinity; a hydroxyl radical footprint showing that H1x binds to the dyad and linker DNA of the 169a nucleosome; fluorescence microscopy data showing that the addition of H1x or H1.0 alters the appearance of nucleosomal condensates; and binding data indicating that H1x and H1.0 can associate with condensates in a molar ratio exceeding 1 LH per nucleosome.\n\nOverall, the reported structures represent an impressive technical achievement, with the new observed LH configurations providing novelty. However, to what extent the nucleosomal packing in these engineered crystal forms is relevant to the cellular chromatin context is unclear, as is the physiological relevance of the novel observed LH configurations. Also, the authors observe certain LH configurations associated with specific H1 variants. However, whether this H1 variant dependence sheds light on the functional diversity of H1 variants in the cell or is merely a consequence of the specific crystal packing constraints associated with the engineered nucleosome constructs is an open question. The link between the different reported LH configurations and the condensate binding data reported in Figure 7 is also somewhat tenuous.\n\nMost remarkably, the authors make the claim that they could trace the entire N- and C-terminal domains of H1.x. If true, this would be a highly significant and original result. Unfortunately, 2Fo-Fc and omit maps calculated from the data deposited by the authors (PDB 7xx7) reveal that the claim is unfounded. The density for residues 1-35 and 120-213 is uninterpretably poor. The atomic model for these regions contains long stretches of residues devoid of any secondary structure or of any contacts with neighbouring molecules, rendering a unique stable conformation highly improbable. The geometry for these regions is also very poor. In short, the atomic model proposed for the H1.x N- and C-terminal domains appears inconsistent with the diffraction data and with stereochemical considerations.\n\nSpecific points.\n1. On p.7 the authors state: \"A total of 11 LHGD copies of H1x, H1.0, H1.3 and H5 across 7 different N-LH structures, encompassing nearly identical N packing (lattice) configurations, all adopt the winged helix fold and are overall similar to each other (Fig. 2c).\" To which 11 LHGD copies and 7 N-LH structures are the authors referring? Table S1 lists only 5 PDB structures and inspection of these reveals a total of only 8 LH polypeptide chains.\n\n2. Related to the previous point, Fig. 2c shows H1.0 as adopting three configurations: non-dyad, peripheral and reverse-peripheral. However, the two corresponding PDB structures (7xx6 and 7xvl) only show the non-dyad configuration (occuring twice) and the reverse peripheral configuration. Which structure shows H1.0 as adopting a peripheral configuration? (Is it 6lab from ref.18 ? If so, this should be indicated.)\n\n3. On p.10, the authors state \"Nonetheless, the α1/α3 motif is in fact employed in a distinct fashion, whereby the association is with an SHL 4 minor groove and coincides with remodeling of the H1.0GD conformation to accommodate the distinct binding context of this mode (Figs. 3, 4 & 5a). Specifically, the register of α3 is rotated by ~1 residue (~100°) relative to the rest of the GD as compared to the other structures for H1.0. This indicates a degree of plasticity inherent in the H1.0GD that would help foster the variety of different modes displayed by this variant.\"\nThe notion that the register of helix α3 for this structure (PDB 7xx6) is shifted by 1 residue relative to other H1.0 structures is quite an extraordinary claim that would require strong justification. In actual fact, helix α3 for PDB 7xx6 is in perfect register with helix α3 of a previously reported human H1.0 structure (PDB 7k5x) and with the available AlphaFold model. Rather, it is helix α3 of the authors' H1.0-169an structure (PDB 7xvl) that is out of register with these other models. Of the 5 structures reported in this manuscript, 7xvl has the lowest resolution (3.5 Å). The authors appear to have introduced an error while refining this structure, since the quality and low resolution of the electron density does not justify rotating the helix by 100°.\n\n4. Section entitled \"Tails Provide Electrostatic Glue\", p.15. Given the comments above regarding the inappropriate modeling of the N- and C-terminal domains of H1.x, most of the statements made in this section are completely untethered to any observational data.\n\n5. Regarding the binding data shown in Fig. S16:\n(i) Why does panel (d) only show the molar input ratios 5.25-6 for H1x and not 1-6 as in panel (c) ?\n(ii) The standards shown on the right-hand side of panel (c) cover only the low end of the range of band intensities observed on the left-hand part of the gel. Was the standard curve used in Table S4 generated from only this limited set of data ? (Notably, the condensate binding curve in Figure 7a shows a conspicuous jump between rH1 = 3.5 and 4 for H1.0. Might this reflect a quantitation error based on an extrapolated standard curve?)\n\n6. Many of the structural figures are somewhat difficult to interpret. In certain cases, it was necessary to view the atomic coordinates of the deposited PDB file in order to better understand the figure. A small inset showing a global view of the dinucleosome might help orient the reader."}, "author_response": "We thank the reviewers wholeheartedly for their valuable time and input on our\nmanuscript. We have carefully considered the recommendations of the reviewers, whose\ninsightful comments have been very helpful in compiling the revised version. Below we\noutline the revisions implemented for each of the points raised by the reviewers.\nIn response to comments by Reviewer #1:\n1. The manuscript by Adhireksan et al. is an extension of their earlier work published in Nat.\nCommun. 2020 & Nucleic Acids Res. 2021. The current study aimed to study the association\nof nucleosome and linker histone subtypes (Human H1x, H1.0, H1.3 and Chicken H5) in oligo-\nnucleosome chromatin fibers at an atomic level using X-ray crystallography. The authors found\nthat LHs bind in ‘non-canonical’ off-dyad modes, associating with the inter-nucleosomal linker\nDNA and other positions on the nucleosome. They find that in many cases LHs engage in\nmultiple bridging interactions between DNA segments nucleosomes, with some variation\nbetween the LH subtypes studied. The study reports the first structural visualization of\nalternative H1 binding modes (beyond the canonical on-dyad mode) in a chromatin complex,\nextending our knowledge of how LH subtypes interact in non-canonical ways in chromatin,\nespecially in situations where there is stoichiometric excess of LH over nucleosomes. However,\nenthusiasm is somewhat damped by the low resolution for two of the four structures, some\nissues with the way the data is analyzed and presented, some lack of connection to prior work,\nand discussion of limits of the current study.\nResponse: We appreciate the reviewer’s comments. We have addressed the general\ncriticisms raised here by responding to the specific comments outlined below.\nComments:\n2. In the abstract and other places the authors state that the commonly accepted mode of LH\nbinding is based on studies of individual nucleosomes. However, the paper ignores studies of\nH1-di- and tri-nucleosome complexes (Syed et al., 2010 PNAS) and the crystal structure of a\n6-nucleosome array bound to histone H1 (Garcia-Saez, 2018 Mol. Cell), both showing on-dyad\nbinding to oligonucleosomes is identical to full-length H1 binding to mononucleosomes.\nResponse: We have modified the abstract and incorporated these studies into the\ndescription within the Introduction section (currently, references 16 and 17).\n3. Relatedly, the ‘closed-loop’ dinucleosomes structures appear to force a full cross-over of\nlinker DNA, perhaps interfering with some LH binding to the canonical on-dyad site. Is this\nthe case, i.e. are there steric hinderances or distortions that would inhibit LHG domain fitting\nin at the canonical on-dyad position? This should be discussed/presented in the MS.\nResponse: We have incorporated a discussion of this issue at the end of the Linker\nHistones Bind Multiple Nucleosomes in Diverse Modes section: “The ability of H5 to associate\nwith the 169a N dimer via the on-dyad mode notably indicates that this binding site is available\nto the LH in the 169 crystals, in spite of structural constraints that may be imposed by the\nGD\nnature of N pairing. Nonetheless, the absence of on-dyad H5 binding in the second, opposing,\nN of the dimer may be a consequence of either a special favorability associated with the\nobserved H5 occupancy at the non-dyad binding site or an on-dyad-disfavoring/incompatible\nN conformation (in any case, a significant degree of plasticity is inherent in the nature of on-\ndyad LH -N interactions; vide infra).”\nGD\n4. Results: The authors show an ‘on-dyad’ mode of binding for H5, but there is no comparison\nto previous reports of on-dyad binding modes for this or other LHs (Zhou et al., 2015 Mol.\nCell; Bednar et al., 2017 Mol. Cell; Zhou et al., 2021 Mol. Cell).\nResponse: We have included a discussion of these works together with a structural\ncomparison (Supplementary Fig. 18) in the first paragraph of the Discussion section.\n5. Page 7, Authors statement that “compaction of Ns within the crystal is fostered by DNA\nbinding of LH, N- and C-terminal tails of the core histones and Ca2+ ions, which function in\nlarge part to provide electrostatic counterparts for the double helix”. The authors should show\nor document a comparison of structures in absence and presence of LH to support this\nconclusion.\nResponse: We have further supported this statement by including (in the Nucleosome-\nLinker Histone Assembly Structures section) a comparison of the counterbalancing charges in\nthe structure, also with reference to previous works (refs. 24 & 25) on LH-free nucleosome\nstructure.\n6. Page 7, Author’s mentioned that based on crystallization outcome LHs present between the\nlinker DNA or within the roughly cube shaped cavity. Please label this cavity in Fig. 1. In\naddition, it would be helpful to the reader to see an animation/video of the different LHs\npositioning between the linker DNAs and in the cavity to support Fig. 2 (besides Ref. 20&21),\nand furthermore tabulate the distances/interactions between LHs and nucleosomes/DNA for\nFig. 3, 4, and 5b to provide better understanding of the interacting residues at an atomic level\nbeside their surface accessibility as mentioned in Table S2.\nResponse: The cube-shaped cavity has been indicated in Fig. 1b. We have included an\nanimation to accompany Figure 2b (Supplementary Movie 1). In addition, we have included a\ntabulation, as Supplementary Data 1, of the LH-nucleosome H-bonding interactions to support\nthe figures.\n7. Figure S3, Some of the electron density for LHNT and LHCT has been found and the region\nhas been modelled and marked to interact with the distal nucleosome. It would be interesting\nto show a pictorial view of the interaction and tabulate the distances of the interacting residues\nas well. Similarly, indicate the LHNT interactions for Figure S11.\nFigure 8 should be extended by showing individual orientation with interacting residues\nfollowed by a combined model, it’s always nice to show the interacting partners/resides in\ntabulated form.\nResponse: A panel (c) has been added to Supplementary Figure 3 to illustrate the 3D\nH1.0 tail structure and nucleosome interactions, which are also tabulated in Supplementary\nData 1. Figure 9 (formerly Figure 8) has been extended by showing the five structures\nindividually together with interacting residues (in stereo view); panels a through e of\nSupplementary Figure 17.\n8. The authors report extremely tight binding for H1-N interactions. Given that the authors\ndetail multiple alternative binding modes for these LHs, how well does the 1:1 kinetic binding\nmodel fit the data? If >1 H1 binding sites exist per nucleosome, how does this affect the\ncalculation of overall affinity?\nResponse: The 1:1 LH:N binding model resulted in a conclusive fit to the sensorgram\nprofiles for both of the replicates (Supplementary Fig. 14). In any case, the density of the\nsurface-immobilized nucleosomes is sufficiently sparse so as not to permit interaction of one\nLH molecule with more than one nucleosome. The opposing scenario, whereby a single\nnucleosome interacts with multiple LH molecules is neither expected nor observed. For\ninstance, in the native-PAGE analysis of Supplementary Fig. 10, LH titration to the 169a\nnucleosome (the same used in the kinetic/BLI measurements) yields only 1:1 complex up to\nhigher stoichiometry, at which point large aggregates form. The 15–45 pM range of K values\nD\nfound for the LH-169a system is in close agreement with solution-state fluorescence assay (HI-\nFI FRET) measurements on the binding of H1 to a variety of nucleosomal constructs (White et\nal., 2016; PMID: 26750377).\n9. Discussion: Is there potential for numerous additional alternative H1-binding modes in\nchromatin beyond what is reported here?\nResponse: We have added a comment in this regard in the second paragraph of the\nDiscussion section: “In fact, the heterogenous nature of local N organization observed in\ncellular chromatin30,31 might suggest the existence of many distinct linker histonophores, and\nthus ‘non-dyad’ modes (that is, potentially well beyond what we have observed so far).”\nMinor comments:\n10. Results, p. 5 bottom: The authors state: “Various constructs were also co-crystallized with\ndifferent LH variants, which included human H1x, H1.0, H1.1–H1.5 and chicken H5 (a.k.a.\navian H1.0)”. I found this presentation to be confusing when no results are presented for most\nof these proteins. Simply say that ‘similar attempts were made with LHs X, Y, and Z, but\ninsufficient density was detected to allow analysis for these proteins’?\nResponse: This sentence and the corresponding first paragraph of the Results section\nhave been reworded.\n11. Fig. 1A. What does the cloud around the LH cartoon structures indicate? This should be\nnoted in the figure legend.\nResponse: The Figure 1 legend has been amended to include a description of this\nfeature (which was added to emphasize the LHs is this first depiction).\n12. Fig. 2. The current representation of Fig. 2a-b is somewhat crowded and its hard to the\nlayering. Again, a video or animation would help the reader here.\nResponse: We have included an animation to accompany Figure 2b (Supplementary\nMovie 1).\n13. Similarly, the authors state: “With one particular type of construct, which consists of a\n165 bp double helix having 4-nucleotide overhangs at either terminus, we were able to solve\nmany structures with the four different LH variants.” Again, confusing. What is meant by\n“many structures”? Is the linker DNA evenly distributed about the nucleosome core binding\nsite? Shouldn’t “either terminus” be “both termini”?\nResponse: This description (in the second paragraph of the Results section) has been\nreworded.\n14. Results, p. 6, top: Related, the authors state “However, the best diffracting crystals were\ntypically the ones co-crystallized with H1.0 or H1x, and these two variants together with H5,\nand to a lesser extent H1.3, were the only LH types for which the associated electron density\nwas sufficiently clear to permit explicit modeling of at least the LHGD (Table S1 & Figs. S1–\nS8). This is a long and confusing sentence.\nResponse: This sentence and the corresponding first paragraph of the Results section\nhave been reworded.\n15. Related, p. 7, middle para: “A total of 11 LHGD of H1x, H1.0, H1.3 and H5 across 7\ndifferent N-LH structures, encompassing nearly identical N packing (lattice) configurations,\nall adopt the winged helix fold and are overall similar to each other (Fig. 2c). Again, long,\ncumbersome, confusing to the reader.\nResponse: This sentence has been split and reworded.\n16. What are the DNA sequences for the templates, and specifically what do a, ak, and an\nmean in terms of sequence?\nResponse: We have added a brief description of these DNA fragments in the first\nparagraph of the Methods section. Further details are available in Adhireksan et al., 2021 (ref.\n20).\n17. Fig. S1-S8: Choose contrast color and bolding for numeric labelling of residues for better\nvisualization.\nResponse: Supplementary Figures 1 through 8 have all been revised so that the residue\nnumbers stand out.\n18. Page 7, in my opinion the statement “Electron density maps stemming from H1x-169a\nassembly crystals are especially well defined” should be specified: ... .well defined H1x\nglobular domain.\nResponse: We appreciate the reviewer’s point here, but our intension in this opening\nstatement is to make an overall qualification of this data set (or crystal type). The electron\ndensity clarity allowed us to model other features as well, including solvent species (Ca2+ ions,\netc.) and core histone tails, which was generally not feasible with the other data sets. In any\ncase, the quality of the electron density associated with the globular versus the tail domains is\ndescribed/presented in detail later in the manuscript.\n19. Figure S9, please mark the interface with a dotted line and labelled the respective\nnucleosomes as shown in S9a.\nResponse: We have demarcated the nucleosome-nucleosome interface in\nSupplementary Figure 9a and signified the distinct nucleosomal components in Supplementary\nFigures 9b and 9c.\n20. Please expand what the author means in the header of Table S1. I understand it makes\nmore sense to show the data set of H1x-169aP, H1.0-169aN1, H1.0-169anR, H1.3-169aF, H5-\n169aD/N2 but what about the data set with similar nucleosome conditions (mono or di)?\nResponse: To Supplementary Table 1, we have added a note, which explains that the\nH1.0P-169a and H1xP-338b data/refinement were described earlier in Adhireksan et al., 2021\n(ref. 20).\nIn response to comments by Reviewer #2:\n21. The manuscript by Adhireksen et al. builds on previous work by the authors reported\nin ref. 18, in which they engineered a number of nucleosomal constructs with cohesive-ended\nDNA that allowed them to crystallize nucleosomes in a circular or continuous fibre\nconfiguration. The current manuscript reports five additional such structures at resolutions\nranging from 2.7 to 3.5 Å. These describe nucleosomes in complex with four different linker\nhistone (LHs) - the human histones H1.0 (two structures), H1x and H1.3 as well as chicken\nhistone H5. The new structures all comprise a circular configuration of a nucleosome dimer,\nin which the entry and exit linker arms of one nucleosome connect to the exit and entry arms\nof the other, respectively. These structures show similar crystal packing and contain the same\npalindromic DNA sequence (169a), except for an H1.0 structure that contains a non-\npalindromic sequence (169an). The crystal structures capture the LHs in three previously\nobserved configurations (on-dyad, off-dyad and peripheral) as well as three new\nconfigurations that the authors term reverse peripheral, non-dyad(1) and non-dyad(2).\nThe authors also report complementary data that include: bio-layer interferometry data\nshowing that the four LHs bind the 169a mononucleosome with similar affinity; a hydroxyl\nradical footprint showing that H1x binds to the dyad and linker DNA of the 169a nucleosome;\nfluorescence microscopy data showing that the addition of H1x or H1.0 alters the appearance\nof nucleosomal condensates; and binding data indicating that H1x and H1.0 can associate with\ncondensates in a molar ratio exceeding 1 LH per nucleosome.\nOverall, the reported structures represent an impressive technical achievement, with\nthe new observed LH configurations providing novelty. However, to what extent the\nnucleosomal packing in these engineered crystal forms is relevant to the cellular chromatin\ncontext is unclear, as is the physiological relevance of the novel observed LH configurations.\nAlso, the authors observe certain LH configurations associated with specific H1 variants.\nHowever, whether this H1 variant dependence sheds light on the functional diversity of H1\nvariants in the cell or is merely a consequence of the specific crystal packing constraints\nassociated with the engineered nucleosome constructs is an open question. The link between\nthe different reported LH configurations and the condensate binding data reported in Figure\n7 is also somewhat tenuous.\nResponse: We appreciate the reviewer’s comments. Regarding the H1 variant\ndependence, our inferences are also supported by the fact that the 169a, 169an, and 338b\ncrystals, which encompass the H1x, H1.0, H1.3, and H5 structures, are either nearly\nisomorphous, or at least have very similar overall nucleosome packing. Therefore, it can be\nassumed that differences observed between the LH variants within this set of crystals, grown\nand stabilized under identical conditions, are a reflection of distinct nucleosome binding\npredispositions. We also note that in most instances, we had collected and inspected multiple\nX-ray data sets for a given compound/crystal type, thus screening for the existence of potential\ncrystal polyforms that could display distinct LH binding. Different crystals/batches with H1x,\nH5, and H1.3 all display the same binding mode(s) within the LH variant set, whereas for H1.0,\ndifferent modes are seen between crystals of even the exact same composition. Moreover,\nsince H5 is reproducibly seen to associate on-dyad (in the one copy per nucleosome dimer),\nwhile other variants are not, this also speaks for a variant-dependent predisposition with respect\nto this particular ‘canonical’ binding mode. In fact, the LH-condensate binding data also\nreveals variant-dependent behavior.\nWith respect to the link between the crystal structure and condensate binding data, one\ncould envision an alternative scenario, in which LH titration of the condensates would show\nbinding saturation around 1:1 H1:nucleosome stoichiometry and moreover not show linking\ntogether/aggregation of the individual condensates. In this hypothetical situation, we would\nnot be able to argue that there must be alternative/additional binding modes, since a valid\nassumption could be that the single binding site occupied per nucleosome is the on-dyad mode\n(or a mix of on- and off-dyad modes). Furthermore, based on that hypothetical data, we could\nalso not argue that H1 is active in linking multiple nucleosomes, that is, multiple sections of\nnucleosome fiber, together. Therefore, we argue that the condensate data decisively\nstrengthens the crystallographic/structural observations.\n22. Most remarkably, the authors make the claim that they could trace the entire N- and C-\nterminal domains of H1.x. If true, this would be a highly significant and original result.\nUnfortunately, 2Fo-Fc and omit maps calculated from the data deposited by the authors (PDB\n7xx7) reveal that the claim is unfounded. The density for residues 1-35 and 120-213 is\nuninterpretably poor. The atomic model for these regions contains long stretches of residues\ndevoid of any secondary structure or of any contacts with neighbouring molecules, rendering\na unique stable conformation highly improbable. The geometry for these regions is also very\npoor. In short, the atomic model proposed for the H1.x N- and C-terminal domains appears\ninconsistent with the diffraction data and with stereochemical considerations.\nResponse: We do not agree with the premise that the H1x N- and C-terminal tail\nstructures are not supported by the data. We went to great lengths to carefully model the tail\nstructures and also to document the support of the modeled conformations with omit electron\ndensity maps (Supplementary Figures 2 and 3). As we noted above and in the manuscript, the\nelectron density quality over the tail regions fluctuates significantly, but there is information\ncontent (supported by the omit maps), which importantly illustrates the character of the H1\ntails. In any case, however, we want to make it clear that there are apparently alternative tail\nconformations sampled in the H1x-169a crystals and most likely multiple, if not many, distinct\nconformations available to the tails in other nucleosomal contexts (i.e., in chromatin). As such,\nwe have added a further discussion of the tails following the second paragraph of the\nDiscussion section.\nBy analogy to the core histone tails, the H1x tails conformational behavior is consistent\nwith observations that the N- and C-terminal linker histone tails are unstructured in solution\n(reviewed in McBryant and Hansen, 2012, for instance). In fact, in the H1x-169a structure, the\nlinker histone tails display structural and electron density characteristics that are fully\nanalogous to that seen previously with the core histone tails at high and lower resolutions (i.e.,\n~2–3 Å; for example, Davey et al., 2002), whereby the tails undergo multiple disorder-to-order\ntransitions as they ‘meander’ through the lattice. In particular, the most ordered regions of the\ntails coincide with contacts to other protein or DNA elements. For the tail regions that are the\nmost disordered, there are likely to be alternative (multiple) conformations that connect to the\nbetter ordered, or interacting, regions. Although the H1x tail regions are almost completely\ndevoid of regular secondary structural features, this would be consistent with their richness in\nand regular intervals of proline and glycine residues.\nSpecific points.\n23. 1. On p.7 the authors state: \"A total of 11 LHGD copies of H1x, H1.0, H1.3 and H5 across\n7 different N-LH structures, encompassing nearly identical N packing (lattice) configurations,\nall adopt the winged helix fold and are overall similar to each other (Fig. 2c).\" To which 11\nLHGD copies and 7 N-LH structures are the authors referring? Table S1 lists only 5 PDB\nstructures and inspection of these reveals a total of only 8 LH polypeptide chains.\nResponse: The legend for Figure 2 has been amended to specify the structures in this\nanalysis: “(c) Superimposition of the LH regions from the 169a (pdb codes 7XX7 [H1x] ,\nGD 2\n6LAB [H1.0] [ref. 20], 7XX6 [H1.0] , 7XX5 [H1.3], 7XVM [H5] ), 169an (pdb code 7XVL\n2 2 2\n[H1.0]), and 338b (pdb code 6L9Z [H1x] [ref. 20]) crystal structures (11 LH molecules in total;\ncolored by variant type).” The text has been further amended elsewhere to specify the\nrespective pdb codes involved in the analyses.\n24. 2. Related to the previous point, Fig. 2c shows H1.0 as adopting three configurations: non-\ndyad, peripheral and reverse-peripheral. However, the two corresponding PDB structures\n(7xx6 and 7xvl) only show the non-dyad configuration (occuring twice) and the reverse\nperipheral configuration. Which structure shows H1.0 as adopting a peripheral configuration?\n(Is it 6lab from ref.18 ? If so, this should be indicated.)\nResponse: Yes, the H1.0-169a structure coinciding with the peripheral mode is 6LAB,\nwhich was originally disclosed in Adhireksan et al., 2021 (therein Table 2, “169a”). The text\nhas been amended to reflect this and otherwise elsewhere to specify the respective pdb codes\ninvolved in the analyses.\n25. 3. On p.10, the authors state \"Nonetheless, the α1/α3 motif is in fact employed in a distinct\nfashion, whereby the association is with an SHL 4 minor groove and coincides with remodeling\nof the H1.0GD conformation to accommodate the distinct binding context of this mode (Figs.\n3, 4 & 5a). Specifically, the register of α3 is rotated by ~1 residue (~100°) relative to the rest\nof the GD as compared to the other structures for H1.0. This indicates a degree of plasticity\ninherent in the H1.0GD that would help foster the variety of different modes displayed by this\nvariant.\" The notion that the register of helix α3 for this structure (PDB 7xx6) is shifted by 1\nresidue relative to other H1.0 structures is quite an extraordinary claim that would require\nstrong justification. In actual fact, helix α3 for PDB 7xx6 is in perfect register with helix α3 of\na previously reported human H1.0 structure (PDB 7k5x) and with the available AlphaFold\nmodel. Rather, it is helix α3 of the authors' H1.0-169an structure (PDB 7xvl) that is out of\nregister with these other models. Of the 5 structures reported in this manuscript, 7xvl has the\nlowest resolution (3.5 Å). The authors appear to have introduced an error while refining this\nstructure, since the quality and low resolution of the electron density does not justify rotating\nthe helix by 100°.\nResponse: We thank the reviewer for pointing this out, and while it is not an error in\nour modeling, we have added an analysis/discussion of this issue in the Linker Histones Bind\nMultiple Nucleosomes in Diverse Modes section: “…[regarding the H1.0-169a non-dyad\npolymorph,] Specifically, the register of α3 is rotated by ~1 residue (~100°) relative to the rest\nof the GD as compared to that in the other H1.0-169a structure (showing the peripheral mode)\nand the H1.0-169an structure (coinciding with the reverse-peripheral mode). However, the two\nperipheral-type structures are in fact the exceptions, as the α3 register seen in this H1.0-169a\nnon-dyad polymorph is apparently the common, ‘consensus/canonical’, H1.0 helical register\nGD\nthat is seen in all of our other H1.0 on-dyad/non-dyad structures20,21 (Fig. 4: H1.0D*,\nH1.0N*,\nH1.0N1; Fig. 9) in addition to that also observed in a recent cryo-EM structure of the H1.0-\nchromatosome26. In any case, the structural polymorphism indicates a degree of plasticity\ninherent in the H1.0 that would help foster the variety of different modes displayed by this\nGD\nvariant.” In addition, we also note that the peripheral-mode H1.0-169a structure, at 3.2 Å\nresolution (originally disclosed in Adhireksan et al., 2021), displays the same register as the\n3.5 Å-resolution reverse peripheral-mode H1.0-169an structure. The omit electron density\nmaps support the modeled configurations, and the distinctions in register of the α3 helix also\ncoincide with concerted changes in neighboring GD elements that help rationalize the overall\nconformations that are adopted.\n26. 4. Section entitled \"Tails Provide Electrostatic Glue\", p.15. Given the comments above\nregarding the inappropriate modeling of the N- and C-terminal domains of H1.x, most of the\nstatements made in this section are completely untethered to any observational data.\nResponse: We do not agree with the premise that the H1x N- and C-terminal tail\nstructures are not supported by the data. We went to great lengths to carefully model the tail\nstructures and also to document the support of the modeled conformations with omit electron\ndensity maps (Supplementary Figures 2 and 3). As we noted above and in the manuscript, the\nelectron density quality over the tail regions fluctuates significantly, but there is information\ncontent (supported by the omit maps), which importantly illustrates the character of the H1\ntails. In any case, however, we want to ensure a clear perspective for the reader. As such,\nwe have added a further discussion of the tails following the second paragraph of the\nDiscussion section: “Indeed, the LH tails display conformational and electron density characteristics\nclosely analogous to that of the core histone tails24, which is consistent with a general activity\nof the histone tails in electrostatically-driven stabilization of nucleosome binding and\nchromatin compaction. The richness in secondary structure-disrupting, proline/glycine,\nresidues appear to foster the conformational character of the H1x tails, which are largely\ncomposed of random coil structural elements in the H1x-169a crystals. While the two copies\nof the H1x structures are overall highly distinct, the H1x displays molten globule-like\nNT/CT CT\nconformations that are consistent with the LH being an intrinsically disordered domain that\nCT\nundergoes a disorder-to-order transition or condensation upon binding nucleosomes\n(characterized for H1.0)32. In any case, there are apparently alternative tail structures sampled\nin the H1x-169a crystals and most likely multiple, if not many, distinct conformations that\nwould be available to the tails in other nucleosomal contexts (i.e., in chromatin).”\n27. 5. Regarding the binding data shown in Fig. S16:\n(i) Why does panel (d) only show the molar input ratios 5.25-6 for H1x and not 1-6 as in panel\n(c) ?\n(ii) The standards shown on the right-hand side of panel (c) cover only the low end of the range\nof band intensities observed on the left-hand part of the gel. Was the standard curve used in\nTable S4 generated from only this limited set of data ? (Notably, the condensate binding curve\nin Figure 7a shows a conspicuous jump between rH1 = 3.5 and 4 for H1.0. Might this reflect\na quantitation error based on an extrapolated standard curve?)\nResponse: In order to allow for complete and redundant coverage of all the samples,\nwe had to run many gels, from which standard slopes were calculated and applied to the\ncondensate samples. Given the number of data points required to explore a finely sampled\nsaturation curve, it was not possible to obtain a full range of samples and standards within a\nsingle gel. We therefore analyzed many gels and fit condensate samples to the most proximal\nstandard slopes. To establish further confidence in our findings, we went carefully back\nthrough all of the condensate experiments and were able to locate additional gels that we have\nnow included in the full analysis. While this has not resulted in any significant changes in the\nfinal values, it further confirms the accuracy of the experimental approach. Nonetheless, we\nhave accordingly updated panel a of Figure 8 and Supplementary Tables 4-7. In addition, we\nare providing the collection of gels used in the analysis as Supplementary Data 2. Notably, the\nmost conspicuous feature, that of the H1x ‘jump’ between 3.5 to 4.0, is still apparent. It is even\nmore pronounced when looking only at data points within a single gel (15951; Supplementary\nTable 5), whereby the two 3.5 data points are 1.2 and 1.5 (average=1.4), while that of the 4\ndata point is 3.2. This represents a slightly greater jump (1.8) than for the whole data set\naverage (1.5).\n28. 6. Many of the structural figures are somewhat difficult to interpret. In certain cases, it\nwas necessary to view the atomic coordinates of the deposited PDB file in order to better\nunderstand the figure. A small inset showing a global view of the dinucleosome might help\norient the reader.\nResponse: We have revised Figure 5 to include a panel (5a) to orient the viewer with\nrespect to the H1 binding context that is detailed. In addition, an animation to support Figure\n2b (Supplementary Movie 1) has been included that should provide further orientational value\noverall as well as for Figure 5 (also referred to in the Fig. 5 legend)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed all of our queries and comments in the revised MS. However I would suggest the authors include a table of all 10 constructs that yielded well-diffracting crystals to complement the initial part of the Results, providing general information about each. This would help readers keep straight the PBD identifiers for each, which contained H1s, what H1 they contained, which H1s had sufficient densities for modeling and which did not, what H1 binding modes were detected in each structure, and overall resolutions for each of the 10. It would be helpful to also list the 14 constructs that did not yield well-diffracting crystals, perhaps in the table legend.\n\nI will also note that H1s forming general bridging interactions between multiple DNA segments was first elucidated by Jean Thomas and David Clark (cf JMB 1986 187:569; EJB 1988 178:225). Given the multiple DNA bridging modes by H1s described in the current MS, it seems appropriate to cite this work.", "reviewer_2": "My comments are in the accompanying PDF file."}, "author_response": "RESPONSES TO REVIEWER COMMENTS (Manuscript NCOMMS-23-39237A)\nWe owe substantial gratitude to the reviewers, who went to great lengths to evaluate our work\nand recommend improvements. The actions undertaken and the resulting current manuscript\naddress all the concerns and incorporate all the suggested revisions of both reviewers.\nMoreover, the manuscript has been reconfigured from scratch to include additional biophysical\nexperiments and an exhaustive re-analysis, yielding a streamlined presentation with decisive\nconclusions. The structural issues that were outlined by reviewer #2 are all rectified, and the\ndata associated with the five unpublished X-ray crystal structures, which are part of the\nsubmission, are freely accessible at the Protein Data Bank platform (PDB codes and structural\nsummary now provided in Table 1). Below we outline the revisions implemented for each of\nthe points raised by the reviewers.\nIn response to comments by Reviewer #1:\n1. The authors have adequately addressed all of our queries and comments in the revised MS.\nHowever, I would suggest the authors include a table of all 10 constructs that yielded well-\ndiffracting crystals to complement the initial part of the Results, providing general information\nabout each. This would help readers keep straight the PBD identifiers for each, which\ncontained H1s, what H1 they contained, which H1s had sufficient densities for modeling and\nwhich did not, what H1 binding modes were detected in each structure, and overall resolutions\nfor each of the 10. It would be helpful to also list the 14 constructs that did not yield well-\ndiffracting crystals, perhaps in the table legend.\nTable 1 has been added to the manuscript. It summarizes all (10) of the linker histone-\nnucleosome assembly crystal structures (encompassing 6 different DNA constructs), where one\nor more linker histone molecules were explicitly modeled. The other constructs, which are\ndescribed in reference 21, are referred to in the table legend.\n2. I will also note that H1s forming general bridging interactions between multiple DNA\nsegments was first elucidated by Jean Thomas and David Clark (cf JMB 1986 187:569; EJB\n1988 178:225). Given the multiple DNA bridging modes by H1s described in the current MS,\nit seems appropriate to cite this work.\nThese two works have been incorporated into the discussion and cited as references 36\nand 37.\nIn response to comments by Reviewer #2:\nSynopsis:\nMoving forward with this manuscript necessitates the following steps.\ni) The authors should correct their H1.0-169an structure (pdb 7xvl) by repositioning helix a3\nin the same register as that of Zhou et al (pdb 7k5x).\nii) They should correct their H1x-169a structure (pdb 7xx7) by removing the NTD and CTD,\nand by removing all core histone tail residues that are dubious according to current best\ncrystallographic practice.\niii) They should obtain new PDB validation reports for the updated structures and provide\nthese together with the updated coordinates upon manuscript resubmission.\niv) They should revise their manuscript accordingly by removing or modifying any text, tables\nor figures that refer to the H1 helix a3, to the H1 NTD and CTD, or to the core histone tails.\nThis includes deleting most if not all of the \"Tails Provide Electrostatic Glue\" section.\n3. i) The authors should correct their H1.0-169an structure (pdb 7xvl) by repositioning helix\na3 in the same register as that of Zhou et al (pdb 7k5x).\nThe H1.0-169an structure (7XVL), as well as the H1.0-169a structure (6LAB), have\nbeen amended and re-refined so that the α3 helix register is identical to that of the published\nH1.0/H5 ‘consensus’ structures and all our other H1.0 and H5 structures.\n4. ii) They should correct their H1x-169a structure (pdb 7xx7) by removing the NTD and CTD,\nand by removing all core histone tail residues that are dubious according to current best\ncrystallographic practice.\nThe H1x-169a structure (formerly 7XX7) was amended and re-refined, and the H1x-\n169a X-ray data was extended to include all the recorded low-resolution terms (2.7–92 Å; the\nresolution range was originally 2.7–20 Å). A total of 587 protein residues were removed from\nthe model, including nearly all of the core histone and linker histone N- and C-terminal tail\nregions. Only short motifs of the H1x NTD (34-44, chains U and V) and CTD (117-120, chain\nU; 117-127, chain V), as the emerge from the globular domain (GD, residues 45-116), remain\nin the final model. All of the H2A C-terminal tails have also been removed the model, and 7 of\nthe 8 core histone N-terminal tails have been removed from the model. The final model and\nrevised X-ray data set were deposited at the PDB and released under the new entry code, 8YTI.\n5. iii) They should obtain new PDB validation reports for the updated structures and provide\nthese together with the updated coordinates upon manuscript resubmission.\nThe three revised structures (8YTI, 7XVL, and 6LAB) as well as all the other structures\nfrom this work are freely accessible on the PDB platform. The accessible information includes\nvalidation reports in addition to a variety of available data and assessment tools associated with\neach of the depositions.\n6. iv) They should revise their manuscript accordingly by removing or modifying any text,\ntables or figures that refer to the H1 helix a3, to the H1 NTD and CTD, or to the core histone\ntails. This includes deleting most if not all of the \"Tails Provide Electrostatic Glue\" section.\nAll of the data and the structures have been analyzed de novo, and the manuscript was\nrewritten accordingly. All of the former references that were made to the structural elements,\nwhich are now removed from or revised in the models, are absent in the current manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_2": "My evaluation of the initial version of this manuscript submitted in 2023 raised two broad categories of concerns: (i) issues regarding the validity of the crystallographic models and the scientific claims derived from them, and (ii) issues concerning the biological relevance of the linker-histone binding modes observed in the authors’ engineered crystal systems. As the concerns in category (i) were not adequately addressed in the authors' response letter, my subsequent evaluation focused exclusively on these deficiencies and deferred any assessment of how they had addressed the concerns in category (ii). The authors have now satisfactorily resolved the first category: the revised structural models have been corrected for the major errors identified previously, and the crystallographic foundations of the study are substantially improved. However, the second category of concerns remains unresolved, as the manuscript still does not establish that the structural observations made in this highly engineered system have meaningful implications for how linker histones function in vivo.\n\nThe central finding of this study is that H1 globular domains can bridge distinct DNA segments through various canonical and non-canonical modes. While the authors' analyses of on- and off-dyad binding modes add only incrementally to earlier structural studies from several groups, their detailed examination of the linking modes (L1, L1b, L2 and L2b) extends and elaborates on the non-dyad (L2) binding mode they previously reported using a related construct (Adhireksan et al., Nat Commun 2020), thereby expanding the catalogue of H1-DNA interaction modes observed in vitro. Thus, there is some structural novelty here, although the conceptual advance that a non-dyad binding mode is possible was already established in their 2020 study.\n\nImportantly, however, these findings do not demonstrate that the linking modes observed in the crystal structures are used, or are even accessible, in physiological chromatin. The data and arguments presented in support of biological relevance are not sufficiently persuasive. In particular, the observation that condensates can bind linker histones at stoichiometries above 1 is of uncertain biological significance. (The authors point to the existence of cell types with H1:nucleosome ratios above 1 as supporting evidence, but the cited reference [Woodcock et al., 2006] reports H1:nucleosome ratios below 1 in most systems, with values slightly above 1 observed only in highly specialized contexts). More importantly, the array experiments do not show that H1 within the condensates adopts the L1 or L2 binding modes reported in the crystal structures, raising the possibility that these modes may be in vitro phenomena specific to their crystallization system.\n\nIn conclusion, while the structural analyses have some interest, the study does not appreciably advance our understanding of linker histone function in vivo, which limits its overall impact for the chromatin field.", "reviewer_3": "The issues are:\n(1) Do the new structural models represent an important contribution to the chromatin literature? There were a surprising number of errors in the structures described in the first version of this paper. However, the structures have been corrected. Although they are variants on the authors' previous interdigitated fibre model (Adhireksan et al. ref. 22), it is important to understand the various binding modes of H1 variants within nucleosomal fibres. This paper describes differences between binding modes in structures containing H1x, H10 or H5 and is therefore an important addition to the literature.\n(2) Do the various linker histone binding modes observed have any biological significance? The large majority of the literature supports a stoichiometry of up to one H1 molecule per nucleosome. Consequently, chromatin structures with more than one H1 per nucleosome, such as those presented in this paper, might not be biologically important. However, there is one clear and relevant example of chromatin with more than one H1 molecule per nucleosome: transcriptionally inert chicken erythrocyte chromatin has ~0.9 molecules of H5 and ~0.4 molecules of H1 (Bates & Thomas, 1981; PMID 7312631). In that paper, the stoichiometry of linker histone to core histones was also measured very carefully for lymphocyte and glial chromatin, finding one H1 per nucleosome in both cases. This paper could be cited as support for the biological relevance of models with more than one linker histone per nucleosome (at least for those including H5). In their Discussion, the authors note that even if the average stoichiometry is one linker histone per nucleosome, H1 molecules might not be distributed evenly in chromatin, resulting in some regions with two linker histones per nucleosome and others with none. This suggestion is supported by refs. 34 and 35.\n(3) The use of 'N' for 'nucleosome' does not seem necessary."}, "author_response": "RESPONSES TO REVIEWER COMMENTS (Manuscript NCOMMS-23-39237B-Z)\nWe thank the reviewers for their valuable time and input on our manuscript.\nIn response to comments by Reviewer #2:\n1. However, the second category of concerns remains unresolved, as the manuscript still does not establish that the structural observations made in this highly engineered system have meaningful implications for how linker histones function in vivo.\nImportantly, however, these findings do not demonstrate that the linking modes observed in the crystal structures are used, or are even accessible, in physiological chromatin. The data and arguments presented in support of biological relevance are not sufficiently persuasive. In particular, the observation that condensates can bind linker histones at stoichiometries above 1 is of uncertain biological significance. (The authors point to the existence of cell types with H1:nucleosome ratios above 1 as supporting evidence, but the cited reference [Woodcock et al., 2006] reports H1:nucleosome ratios below 1 in most systems, with values slightly above 1 observed only in highly specialized contexts). More importantly, the array experiments do not show that H1 within the condensates adopts the L1 or L2 binding modes reported in the crystal structures, raising the possibility that these modes may be in vitro phenomena specific to their crystallization system.\n\nIn conclusion, while the structural analyses have some interest, the study does not appreciably advance our understanding of linker histone function in vivo, which limits its overall impact for the chromatin field.\n\nWe agree that these are crucial considerations. We should note, however, that a linker\nhistone:nucleosome stoichiometry greater than unity is not, a priori, a prerequisite for the in\nvivo relevance of the linking modes. In fact, a recent cryo-EM analysis of tetra-nucleosome\narrays, having different and physiologically relevant nucleosome repeat lengths (NRLs),\nshowed that certain NRLs are refractory to H1 binding, even under saturating conditions\n(H1:nucleosome molar stoichiometry of 1.2; Dombrowski et al., 2022). This suggests that a\nsubset of nucleosomes in the cell do not allow on/off-dyad H1 binding, which would effectively\nincrease the pool of available H1. In any case, we mean to argue that a >1 bound\nH1:nucleosome stoichiometry would necessitate additional linker histone-nucleosome\ninteraction types (i.e., linking modes) beyond the conventional on/off-dyad modes. The\nquantification of isolated cellular chromatin studies, which have shown that certain cell types\ndisplay >1 chromatin-bound linker histone stoichiometry (Bates & Thomas, 1981; Woodcock\net al., 2006), would mean that the alternative linker histone binding modes would have to be\nrelevant within at least certain cellular contexts. Our array condensate data, showing that all\nthe somatic linker histone variants are capable of high stoichiometry nucleosome binding in\nvitro, lend support to this H1 activity and demonstrate potential structural (chromatin\ncompacting/condensing) consequences that moreover support the X-ray structures, and vice\nversa. Since we are concerned that we may not have made this main argument based on the >1\nstoichiometry clear, and in conjunction with the related comments by Reviewer #3, we have\nadded a clarification paragraph to the Discussion.\nRegarding the main aspect as to the biological relevance of the linking modes, and more\nspecifically whether they may coincide with crystalline-state artifacts, it is a reasonable concern\nthat we also shared, at least initially. However, as we have tried to emphasize in the manuscript,\nthe nucleosomal crystals are analogous to the crowded molecular environment and ionic\n(especially divalent metal cation) conditions within the nucleus. By analogy, one may consider\nthe first structure of a nucleosome, a crystal structure of the linker DNA-lacking core particle\n(NCP; Luger et al., 1997, and further details with Davey et al., 2002), which displayed\nassociation of the H4 tail with the H2A/H2B-acidic patch and non-specific DNA binding of\nthe disordered histone tails, both presenting as crystal (internucleosomal) contacts. Moreover,\nDNA twist defects (aka, DNA stretching) were also observed as a phenomenon presumably\nstabilized, if not induced, by crystal contacts. However, all three of these crystal structure\nobservations have ultimately been established as biologically relevant. In our engineered\nsystems here, most importantly, the crystalline context allows for the possibility to both support\nand observe multi-nucleosome binding by an individual linker histone molecule. In contrast,\ncryo-EM structures of linker histone-nucleosome assemblies are obtained by using very ionic\nstrength and divalent metal-free conditions in order to prevent nucleosome aggregation on the\ngrid, which correspondingly facilitates the single-particle reconstructions. In this manner, it is\nnot possible to observe the linking modes since the conditions select against this. Along the\nsame lines, short (3-, 4-, 6-) nucleosome arrays or 30 nm fiber structures in isolation would not\nbe amenable to linking mode linker histone binding.\nIn terms of crystallographic approaches, only two nucleosome-linker histone structures\nhave been reported outside of our work (Zhou et al., 2015 and Bednar et al., 2017). We infer\nthat the scarcity of crystal structures for this key system is a reflection of the prevalence of\nlinker histone binding diversity, which tends to thwart crystallization efforts. This was initially\nour motivation for engineering nucleosomal constructs that would promote lattice order by\nreplacing the loosely stacking blunt-ended DNA termini with annealing single-stranded\noverhangs. In this sense, we believe that examples of the linking mode had not been reported\nearlier as an in vitro system was lacking, which was capable of both supporting and revealing\nthis type of linker histone association.\nIn response to comments by Reviewer #3:\n2. Do the various linker histone binding modes observed have any biological significance?\nThe large majority of the literature supports a stoichiometry of up to one H1 molecule per\nnucleosome. Consequently, chromatin structures with more than one H1 per nucleosome, such\nas those presented in this paper, might not be biologically important. However, there is\none clear and relevant example of chromatin with more than one H1 molecule per nucleosome:\ntranscriptionally inert chicken erythrocyte chromatin has ~0.9 molecules of H5 and ~0.4\nmolecules of H1 (Bates & Thomas, 1981; PMID 7312631). In that paper, the stoichiometry of\nlinker histone to core histones was also measured very carefully for lymphocyte and glial\nchromatin, finding one H1 per nucleosome in both cases. This paper could be cited as support\nfor the biological relevance of models with more than one linker histone per nucleosome (at\nleast for those including H5). In their Discussion, the authors note that even if the average\nstoichiometry is one linker histone per nucleosome, H1 molecules might not be distributed\nevenly in chromatin, resulting in some regions with two linker histones per nucleosome and\nothers with none. This suggestion is supported by refs. 34 and 35.\nThank you for pointing out the underlying reference, which provides more detail to\nthese important observations. The Bates & Thomas, 1981 study has been added to the\ndiscussion as well as a further elaboration to ensure clarity in the arguments surrounding\nstoichiometry and linking mode association.\n3. The use of 'N' for 'nucleosome' does not seem necessary.\n“N” has been replaced with “nucleosome” throughout the text. We have left it in place\nin figure labelling (and references thereto) where it is necessary because of space limitations."}]

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