Structural and dynamic properties of linker histone H1 binding to DNA.

Abstract

Found in all eukaryotic cells, linker histones H1 are known to bind to and rearrange nucleosomal linker DNA. In vitro, the fundamental nature of H1∕DNA interactions has attracted wide interest among research communities-from biologists to physicists. Hence, H1∕DNA binding processes and structural and dynamical information about these self-assemblies are of broad importance. Targeting a quantitative understanding of H1 induced DNA compaction mechanisms, our strategy is based on using small-angle x-ray microdiffraction in combination with microfluidics. The usage of microfluidic hydrodynamic focusing devices facilitates a microscale control of these self-assembly processes, which cannot be achieved using conventional bulk setups. In addition, the method enables time-resolved access to structure formation in situ, in particular, to transient intermediate states. The observed time dependent structure evolution shows that the H1∕DNA interaction can be described as a two-step process: an initial unspecific binding of H1 to DNA is followed by a rearrangement of molecules within the formed assemblies. The second step is most likely induced by interactions between the DNA and the H1's charged side chains. This leads to an increase in lattice spacing within the DNA∕protein assembly and induces a decrease in the correlation length of the mesophases, probably due to a local bending of the DNA.

Full text

Page 1

1


Structural and dynamic properties of linker histone H1 binding to
DNA


Rolf Dootz,1 Adriana Cristina Toma2 and Thomas Pfohl1,2*

1Max-Planck-Institute for Dynamics and Self-Organization, Bunsenstraße 10,
37073 Göttingen, Germany
2Chemistry Department, University of Basel, Klingelbergstrasse 80, 4056 Basel, Switzerland

Corresponding author: Thomas Pfohl
mail: thomas.pfohl@unibas.ch


Found in all eukaryotic cells, linker histones H1 are known to bind to and rearrange
nucleosomal linker DNA. In vitro, the fundamental nature of H1/DNA interactions has
attracted wide interest among research communities - for biologists from a chromatin
organization deciphering point of view, and for physicists from the study of
polyelectrolyte interactions point of view. Hence, H1/DNA binding processes, structural
and dynamical information about these self-assemblies is of broad importance.
Targeting a quantitative understanding of H1 induced DNA compaction mechanisms
our strategy is based on using small angle X-ray microdiffraction in combination with
microfluidics. The usage of microfluidic hydrodynamic focusing devices facilitate a
microscale control of these self-assembly processes. In addition, the method enables
time-resolved access to structure formation in situ, in particular to transient
intermediate states. The observed time dependent structure evolution shows that the
interaction of H1 with DNA can be described as a two step process: an initial unspecific
binding of H1 to DNA is followed by a rearrangement of molecules within the formed
assemblies. The second step is most likely induced by interactions between the charged
side chains of the protein and DNA. This leads to an increase in lattice spacing within
the DNA/protein assembly and induces a decrease in the correlation length of the
mesophases, probably due to a local bending of the DNA.

Page 2

2
\body
The linker-histone family is a heterogeneous family of highly tissue-specific, basic proteins,
which exhibit significant variations in sequence (1,2). However, most eukaryotic linker-
histones H1 share a similar, tripartite structure consisting of a globular domain flanked by two
lysine-rich tails, a shorter amino-terminal domain (N-tail) and a longer, carboxyl-terminal one
(C-tail) (3). Linker-histones H1 are known to attach close to the entry and exit sites of linker-
DNA on the nucleosome core bringing together two linker-DNA segments (4-6). Studies have
shown that the globular domain is responsible for this positioning (7,8). The globular domain
with a diameter of 2.9 nm is the only domain that is folded in solution exhibiting three -
helices (9-11). Its most noticeable structural features are the two DNA-binding sites situated
on opposite sides of the molecule (12), which with the help of the C-terminal domain render
H1-chromatin binding highly dynamic (13,14).
Despite positioning along the nucleosome core particle, it is not the globular domain but
rather the highly positively charged C-tail that imparts to linker-histones their unique ability
to bind to linker-DNA through non-specific electrostatic interactions (6,15,16). In vivo, the
absence of C-tails leads to greatly reduced chromatin binding (17). Binding of linker-histones
to the linker-DNA facilitates the shift of chromatin structure towards more condensed, higher
order forms (18) Although a chromatin fiber lacking linker-histones is able to fold to a certain
extent (19), there is abundant evidence that the highly ordered chromatin compaction of the
30 nm fiber is only attained in the presence of linker-histones (5,18,20). In vivo studies on H1
depleted chicken cells have shown an altered chromatin structured as well as chromosomal
aberrations and increased DNA damage during replication, suggesting that H1 was involved
in transcription regulation (21). Linker-histones help to select a specific folding state from
among the set of compact states reached in its absence by contributing to the free energy of
chromatin folding (22). This suggests that linker-histones are of central importance in genome
organization and regulation.
Since the linker histone’s position on the nucleosome is still a matter of debate, understanding
more closely its interaction with DNA upon condensing it is therefore essential for
understanding the role H1 plays in gene regulation and chromatin structure. It has been argued
that H1/DNA assemblies are an excellent model system for studying aspects of the interaction
of H1 with chromatin. H1/DNA interaction in vitro (15, 23-31) was reported to be dependent
on the ionic strength (23, 31-33), suggesting an interaction governed by electrostatics. At high
salt concentrations the screening of interactions was so significant that assembling was no
longer reported (34), denoting a rather weak binding in comparison to protamine-DNA

Page 3

3
assemblies that can still be formed even at monovalent salt concentrations as high as 1.3M
(35). Thus, reinforcing the belief that H1 is a highly mobile chromatin component compared
to the germinal chromatin which is made mechanically stable and transcriptionally inactive by
strong protamine binding. In a more general context, the H1/DNA system can be regarded as
a model for non-specific DNA-protein interactions. Herein, using the innovative junction
between X-ray scattering and microfluidics, we probed linker histone/DNA interaction
dynamics and structure formation in real-time. A microfluidic channel with a cross geometry
device provides means to control mixing of H1 and DNA uncovers a two-step assembly
process. The non-specific binding of linker histones to DNA lead to the formation of primary
assemblies with a columnar hexagonal structure governed by electrostatic interactions. As the
binding reaction evolves and the concentration of H1 histones increases, the columnar
structure rearrange to less organized assemblies with smaller correlation lengths. We attribute
the latter observation to an additional bending of DNA molecules induced by the interaction
of the linker histones tails with DNA.


Results and discussion.
Aside from advantages such as reduced sample volumes and the possibility of high
throughput and parallel operations, microfluidic techniques are particularly useful for
investigations of biomaterials. Importantly the combination of using microfluidics and X-rays,
significantly reduces radiation induced damage of the sample - an important experimental
consideration given the damage X-rays cause to protein solutions (36). The hydrodynamic
focusing device used here consists of two perpendicular microchannels in the form of a cross
with three inlets and one outlet. A semi-diluted aqueous DNA solution is injected in the
reaction channel and hydrodynamically focused by two side streams of aqueous H1 solutions.
Figure 1 gives a schematic representation of the experimental setup.
Laminar flow conditions due to microscale channel dimensions force mixing to occur purely
by diffusion. Following the confluence of the microchannels, H1 molecules diffuse into the
DNA stream establishing stable concentration gradients. Mixing and concentration
distributions in the reaction channel can be adjusted by controlling main and side channel
velocities. Flow velocities are chosen such that concentration gradients of reactants extend
along the measurable length of the device. It follows that the composition of the formed
assemblies varies at every accessible point along the reaction channel. This allows the access
to the dynamics of H1/DNA structure formation in situ, in particular to transient intermediate

Page 4

4
states. Thus, it is essential to first quantify concentrations and therefore the composition of the
aggregates. This is achieved by a detailed comparison of microscopic images and finite
element simulations of flow conditions in the microfluidic device.

Quantifying local conditions in microfluidic devices. Polarized light microscopy has been
used widely to study DNA assemblies, which are known to be highly birefringent (37-39).
The birefringence signal is increased upon self-assembly of DNA aggregates under conditions
of alignment and elongation which are imposed by using microfluidic devices. Concurrently,
combining microfluidics with polarized light microscopy provides a fast and easy access to
direct imaging of H1 induced DNA compaction. Data are acquired in the reaction channel
(main channel) at three different flow velocities uDNA = 60, 150, and 600 µm.s-1. The flow
velocities in the side channels are varied such that a flow velocity ratio uH1/uDNA = 1 is
maintained.
Finite element simulations of the flow profiles inside the microchannels are performed and
compared to experiments in order to elucidate the experimental parameters. In Figure 2a,
simulation data are shown for uDNA = 600 µm.s-1 and contrasted to corresponding
experimental results. Owing to the symmetry of the microfluidic device in two dimensions, it
is sufficient to simulate half of the device. Following the intersection of the two fluid flows,
H1 molecules diffuse into the DNA stream and the H1/DNA interactions can be observed
along the reaction channel. The optically birefringent pattern reflects that DNA chains in the
assemblies are orientationally ordered due to the superimposed flow. For direct comparison
with experimental results, the modeled velocity profile (white arrows in Figure 2a) in the
hydrodynamic focusing device is overlaid to the recorded birefringence image (Figure 2a
bottom). The strong increase in local viscosity connected to the compaction reaction can be
exploited to visualize H1 DNA assemblies in the simulations. Insignificant deviations of
simulation results from the experimentally recorded shape are observed in the crossing area,
where the center stream is slightly expanding into the side channels. These deviations result
from the fact that simulations are performed in 2D whereas the experimental system is
affected by additional walls at the top and the bottom of the device (40). Apart from this
detail, experiments and simulations show good agreement. The result of the corresponding
simulations over the whole range of the device is given in Figure 2b.
From simulations, local experimental parameters such as flow velocities and concentrations
can be obtained at each position. Local concentrations are translated into assembly
compositions given in terms of the relative charge ratio N/P. N is the total number of positive

Page 5

5
amine charges of H1 and P is the total number of negatively charged DNA phosphate groups.
In Figure 2c, N/P ratios are plotted for three different flow velocities as a function of the
position x along the center of the outlet channel (y = 0). Flow velocities result in final charge
ratios at the furthest measurable point of the device (x ≈ 12 mm) of N/P < 2.4, 3.3, and 5.1 for
uDNA = 60, 150, and 600 µm.s-1, respectively. Local flow velocities can be used to translate
positional changes along each streamline into corresponding reaction time coordinates t.
Figure 2d shows the t dependence of N/P represented as well for three different flow
velocities. For larger t, N/P ratios unite into o a single curve. Deviations at initial time states
reflect differences in the flow velocity depending strain rate
yu 
/
 
(41). Once the
information concerning the local concentrations and hence assemblies’ compositions is at
hand, it is possible to analyze H1/DNA structure formation in detail.

Small angle X-ray diffraction of H1/DNA assemblies. Spatially resolved small angle X-ray
(micro-)diffraction is employed as the principle method of analysis to access relevant
molecular length scales for the study of biomolecular interactions. Data are obtained at
different x-positions along the main channel for all three different flow velocities. In Figure 3,
characteristic 2D diffraction images are shown. The observed alignment is due to the
elongational flow at the confluence of the solution streams. Structural information can be
obtained by analyzing the radial integrated intensity profiles. The diffraction intensity is
plotted as a function of the scattering vector q, which is inversely proportional to the periodic
distance between reticular planes d (42). Since DNA has a significantly higher electron
density than H1 proteins, the DNA self-assembly promotes or leads to the formation of
mesophases that dominate the scattering profile. At positions close to the middle of the cross
channels, the diffraction intensity curve is composed of a broad peak with a shoulder in the
low q values region. The best decomposition of this broad peak is successfully made by fitting
two Lorentzian functions yielding peak positions q1 and q2. In Figure 3, this is shown for the
scattered intensity at x = 100 µm.
In Figure 4a, the dependence of the peak positions q1 and q2 on the channel position x is given
for the data set recorded at uDNA = 150 µm.s-1. To elucidate their dependence on assembly
composition, it is reasonable to plot quantities of interest versus N/P obtained from
simulations. This is shown for q1 and q2 in Figure 4b.
Plotting quantities extracted from X-ray diffraction data obtained at different flow velocities
against N/P allows for superposition of all data onto a single plot showing their dependence
on composition. In Figure 5a this is demonstrated for peak positions q1 (lower curve) and q2