7. DNA COMPACTION:
9.1. S TRUCTURE AND FUNCTION OF LINKER - HISTONES
Linker-histones are some of the most abundant proteins in cellular nuclei. The linker-histone family in higher eukaryotes is a heterogeneous family of highly tissue-specific, basic proteins such as the histone H5 from nucleated erythrocytes of birds267 and sperm PL-I proteins268 which exhibit significant variations in sequence. However, most of eukaryotic histones H1 share a similar tripartite structure consisting of a globular domain flanked by relatively unstructured lysine-rich domains, a shorter amino-terminal one and a longer, carboxyl-amino-terminal one.260 Henceforth, these are referred to as N-tail and C-tail (Figure 9-1a).
The structure of the globular domain (diameter 2.9nm),269 which is highly conserved throughout all types of eukaryotic linker-histones,260 has been solved by X-ray crystallography270 and NMR studies271 identifying it to be member of the winged helix class of DNA-binding domains exhibiting three α-helices (Figure 9-1a). The globular domain is the only H1 domain that is folded in solution.272 However, in contrast to other members of this class of proteins, the globular domain of linker-histones contains a distinct, additional cluster of positively-charged amino acids. These form a second DNA-binding location, on a side of the protein opposite to the primary DNA-binding site.273 Linker-histones are known to attach specifically close to the entry and exit sites of linker-DNA on the nucleosome core bringing together the two linker DNA segments.257, 264, 274 Studies have shown that the globular domain is responsible for this structure-specific recognition,275 most likely provided by the specific arrangement of the two binding sites.276 However, little is known about the specific details of this important interaction, and several models for the exact positioning of linker-histones on the nucleosomal core particle have been proposed.274, 277-279
Contrary to the evolutionary well conserved winged helix motif of the globular domain, both linker-histone tails are extremely heterogeneous in length as well as amino acid
9. DNA Compaction: Linker histones H1
Figure 9-1: (a) Schematic representation of H1 structure showing the globular domain flanked by the N- and the C-tail. Additionally, a ribbon model of the 3D structure of the globular domain is given.270 (b) Linker-histone H1/linker-DNA interactions (bottom) mediate chromatin transition into the 30nm fiber (right).
composition. The absence of an α-helical structure of the tails in aqueous solutions is attributed to electrostatic repulsion between highly charged lysine side chains.
However, it has been shown that both tails of several linker-histone H1 subtypes exhibit structure when exposed to helix-stabilizer (e.g. trifluoroethanol, HClO4) and attain optimum secondary and hence tertiary structure upon interaction with DNA.280-286
Despite positioning along the nucleosomal core particle, it is not the globular domain but rather the highly charged, C-tail that imparts to linker-histones their unique ability to bind to linker-DNA.274, 287, 288 Thus, the presence of the C-tail appears to be essential for processes of chromatin folding and DNA compaction.255, 288, 289 In vivo, the absence of this structural feature leads to greatly reduced chromatin binding.290
The functional and structural roles of the N-tail are still widely unclear.288 In many H1 subtypes, the N-tail presents two distinct subregions.291 The distal half is devoid of basic residues, whereas the half immediately adjacent to the globular domain contains a large amount of basic groups. The majority of studies available in literature agree that N-tails bind to DNA rather poorly.288, 292 Even though the N-tail does not seem to be essential for chromatin folding, this domain may be involved in the exact positioning and anchoring of the globular domain.288 Furthermore, there is a controversy in literature about in how far N-tails may mediate head-to-tail interactions of neighboring
9. DNA Compaction: Linker histones H1
linker-histones leading to a preferential H1-H1 association and therefore cooperative binding to DNA.293
It has long been believed that the function of linker-histones is primary an architectural one helping to create higher order structures of the chromatin fiber.24 Although a chromatin fiber lacking linker-histones is able to fold to a certain extent,263 there is abundant evidence that the highly ordered chromatin compaction of the 30nm fiber is only attained in the presence of linker-histones.20, 24, 264 From changes in chromatin conformation observed at different salt concentrations, the mechanism of chromatin compaction is assumed to be primarily electrostatic.19, 294 Contributing to the free energy of chromatin folding, linker-histones help to select a specific folding state from among the set of compact states reached in its absence.253
Moreover, being involved in the build-up of the 30nm fiber, which presumably limits the access of the transcriptional machinery, linker-histones may function as generalized repressors. Consistently, transcriptionally active chromatin is typically depleted in linker-histones compared with inactive chromatin.295-297 However, experiments performed in vivo indicate that linker-histones may regulate transcription at a finer level by contributing to chromatin architecture and by participating in complexes that either activate or repress specific genes.264, 298-301 Besides their architectural and gene-regulatory functions, recent reports indicate that linker-histones are involved in much more fundamental cellular processes. Linker-histones have been shown to play a prominent role in cell ageing,302 DNA repair,303 apoptosis,304 and muscle development.305 This raises the possibility that biological functions of linker-histones are more varied than previously imagined. However, all of these functions are believed to be highly depending on H1/DNA interaction mechanisms.
Histone H1 has also been shown to condense DNA in vitro.287, 306-314 It has been argued that H1/DNA complexes are a good model system for studying aspects of the interaction of H1 with chromatin. The observation, that salt concentration effects are remarkable in H1/DNA reactions, is suggestive of a nonspecific binding, mainly controlled by electrostatic interactions analog to processes in vivo.306, 314 H1/DNA complexes observed by electron microscopy posses a unique tramtrack-like conformation consisting of two DNA strands bridged by an array of protein molecules and multiples of these.308 This reflects the existence of the two distinct DNA binding sites on the globular domain, apparently mirroring the situation at the H1 binding site on the nucleosome. Although the molecular basis of H1/DNA condensation is not yet clear, it is generally believed that understanding the way in which these proteins bind DNA can help clarifying the still unresolved problem of how H1 bind to nucleosomal structures in chromatin.24. From a more general point of view, the H1/DNA system can be regarded as a model for non-specific DNA-protein interactions.
9. DNA Compaction: Linker histones H1
Figure 9-2: Real-time monitoring of linker-histone H1 induced DNA compaction in a hydrodynamic focusing device. (a) Simulation results (top) and birefringence data (bottom) close to the confluence region are contrasted (uDNA = 600µm.s-1). The product of the complexation reaction appears in the diffusion cone of side and main stream components due to its highly increased viscosity. (b) Simulated H1 concentration profile of the whole device (x = -200-12000µm).
In the following, the interaction of DNA with calf thymus linker-histone H1 (isolated lysine rich fraction,315 Sigma-Aldrich GmbH, Taufkirchen, Germany) with a molecular weight of Mw = 21.5kDa and 55 positive charges at physiological pH conditions314 is studied in hydrodynamic focusing devices. X-ray data are recorded at the beamline ID10b at the ESRF (chapter 2).