Chromatin fiber linker histones

Figure 1. Hierarchical model of chromosome structure, (a) In interphase cells, DNA is packed in a nucleus as forming nucleosome and chromatin, (b) DNA forms nucleosome structure together with core histone octamer, which is then folded up into 30nm fiber with a help of linker histone HI. This 30 nm fiber is further folded into 80 nm fiber and 300 nm loop structures in a nucleus. In mitosis, chromosome is highly condensed. Proteins which are involved in each folding step are indicated above and non-protein factors are indicated below, (c) The amino acid sequences of histone tails (H2A, H2B, H3 and H4) are shown to indicate acetylation, methylation and phosphorylation sites. (See Colour Plate 1.)...

Linker histones (HI, H5 and others) are also major components of metaphase chromosome, and occupy 5.8% of the total protein amount (Uchiyama et al, 2005). They play an important role in the formation of the 30 nm fiber (see also section 2.3). These linker histones carry more lysine residues ( 30% of the total amino acids) than the core histones and have a core domain in the middle part that binds to a nucleosome. The linker histones could be easily extracted from the chromatin with 0.5 M NaCl, whereas the core histone octamers need more than 0.8 M NaCl to dissociate from nucleosomes. 

Biochemical reconstitution of the 30 nm fiber has recently been succeeded by using a salt-dialysis procedure with a long DNA template (>100 kb) (Hizume et al, 2005). AFM imaging of the reconstituted chromatin has shown that the beads-on-a-string structure of the nucleosomes ( 400 nucleosomes on 100 kb DNA) are converted to a thicker fiber in the presence of histone HI. The thickness of the fiber changes reversibly between 20 nm and 30 nm, depending on the salt environment (in 50 mM and 100 mM NaCl, respectively) (Fig. 4) namely, the linker histone directly promotes a thicker fiber formation in a salt-dependent manner. 

Figure 4. In vitro reconstituted 30 nm chromatin fiber. Dynamic structural changes in the chromatin fiber in the absence (top) or presence (bottom) of linker histone HI with different NaCl concentration were observed by AFM. Nucleosomes were reconstituted on the 106 kb plasmid and then fixed in the buffer containing 50 mM (top left) or 100 mM NaCl (top right). Nucleosomes were well-spread in 50 mM NaCl but attached each other and partially aggregated in 100 mM NaCl. After the addition of histone HI, the thicker fibers were formed. The width of the fibers is 20nm in 50mM NaCl (bottom left) or 30 nm in lOOmM NaCl (bottom right)...

Hizume K, Yoshimura SH, Maruyama H, Kim J, Wada H, Takeyasu K (2002) Chromatin reconstitution development of a salt-dialysis method monitored by nano-technology. Arch Histol Cytol 65 405 13 Hizume K, Yoshimura SH, Takeyasu K (2004) Atomic force microscopy demonstrates a critical role of DNA superhelicity in nucleosome dynamics. Cell Biochem Biophys 40 249—262 Hizume K, Yoshimura SH, Takeyasu K (2005) Linker histone HI per se can induce three-dimensional folding of chromatin fiber. Biochemistry 44 12978-12989 Hofmann WA, de Lanerolle P (2006) Nuclear actin to polymerize or not to polymerize. J Cell Biol 172 495-496... 

Kepert J.F Mazurkiewicz J, Heuvelman GL, Toth KF, Rippe K (2005) NAPl modulates binding of linker histone HI to chromatin and induces an extended chromatin fiber conformation. J. Biol. Chem 280 34063-34072... 

These results raise the prospect of dynamics of nucleosomes in linker histone-free chromatin, that is, of a thermal fluctuation of nucleosomes between closed negative , open , and closed positive states identified in the minicircle system. If this equilibrium exists, an extra supercoiling constraint applied to the fiber should displace it in one direction or the other depending on the sign of that constraint, and this displacement should be reversible upon its removal. 

Also, local changes in the structural and chemical variation of DNA may have important effects on the overall extent of chromatin folding. For instance, transitions from the B to the Z form of DNA will result in nucleosome dissolution (as discussed earlier) and this could affect the folding of the fiber. As well, chemical modifications of the bases such as methylation have been shown to increase the folding of the chromatin fiber when linker histones are present [250] although the mechanism involved in this later case remains to be elucidated. 

Since linker histones are involved in compacting the chromatin fiber, it was of interest to see whether the methylation-mediated chromatin compaction could be explained by a higher affinity of linker histones for methylated DNA. The literature on this point is highly controversial some reports demonstrated strong preference of linker histones for methylated DNA [168,169] whereas others found that linker histone binding was indifferent to the methylation status of the DNA [164,170,171]. 

Fig. 5. SFM images of chicken erythrocyte chromatin fibers. (A) Untrypsinized, linker histone-containing control fibers, and (B) linker histone-stripped fibers. The stripping of linker histones destroys both the three-dimensional interactions of adjacent nucleosomes and the zig-zag arrangement of consecutive nucleosomes. Trypsinization of the N-terminal histone tails of the linker histones and core histone H3 result in the loss of the three-dimensional association of the consecutive nucleosomes, but does not destroy the zig-zag configuration. Imaging of fibers deposited onto mica was performed in air under conditions of ambient humidity and temperature (from Ref. [32]). Full width of each image corresponds to 500 nm.

Leuba, S.H., Bustamante, C., Zlatanova, J., and van Holde, K. (1998) Contributions of linker histones and histone H3 to chromatin structure scanning force microscopy studies on trypsinized fibers. Biophys. J. 74(6), 2823-2829. 

Fig. 12.2 DNA target models used for simulations. Gray DNA helix dark gray histone octamer black globular core of the linker histone H5. The chromatin fiber target model is represented in a simplified manner to improve the visualization of the structure. (According to Bernhardt et al. 2003, with permission)...

Nucleosomes are connected to one another by linker DNA of variable length and the linker-binding histone HI protein (Fig. 1) (7). These long arrays of nucleosomes spontaneously condense to form helical arrays of nucleosomes, termed the 30-nm fiber after its apparent diameter (Fig. 1) (8). Additional condensation and compaction of chromatin occur through intemucleosomal interactions. One important internucleosomal interaction required for chromatin fiber formation is the interaction of a highly acidic patch of histone H2A with the histone H4 tail (8). Ultimately, these internucleosomal interactions form interphase chromatin with an unknown architecture (Fig. 1) (9). 

Pairs of four different histones (H2A, H2B, H3, and H4) combine to form an eight-protein bead around which DNA is wound this bead-like structure is called a nucleosome (Figure 24-10). A nucleosome has a diameter of 10 nm and contains approximately 200 base pairs. Each nucleosome is linked to an adjacent one by a short segment of DNA (linker) and another histone (HI). The DNA in nucleosomes is further condensed by the formation of thicker structures called chromatin fibers, and ultimately DNA must be condensed to fit into the metaphase chromosome that is observed at mitosis (Figure 24-11). 

As the in vivo introduction of new methyl groups on DNA involves the inhibition of poly(ADP-ribosyl)ation, which in turn can be correlated to a process of chromatin remodeling, parallel experiments were carried out, the only difference being that—for the in vitro reconsti-mtion of chromatin fibers—DNA sequences involved were either unmethylated or methylated with bacterial 5 I methyltransferase. The reconstitution of chromatin fibers was performed by adding either only the core histones or additionally H1 linker histone. 

The same parameters were obtained by AFM imaging of chromatin fibers reconstituted in vitro with unmethylated and methylated DNA when reconstitution was performed in the absence of linker histone. After addition of H1 histone to chromatin fiber reconstituted around methylated DNA, the degree of compaction observed was different and was very close to that observed in vivo after induction ofDNA hypermethylation. The values obtained were 0.46 for the control vs. 0.70 for the hypermethylated chromatin fibers from the in vivo experiments and 0.48 for the control vs. 0.67 for the methylated HI-containing reconstituted ones. This leads to the conclusion that HI histone is required for DNA-methylation dependent compaction of chromatin structure. ... 

Kaiymov MA, Tomschik M, Leuba SH et al. DNA methylation-dependent chromatin fiber compaction in vivo and in vitro Requirement for linker histone. FASEB J 2001 15 2631-2641. 

The association of poly(ADP-ribose) polymerase with chromatin has been well described by Aubin et al. (1) and by Butt et al. (2) and we have found die enzyme to be associated mainly with tri- and tetranucleosomes (1). Poly(ADP-ribosyl)ation in vitro has been found to alter chromatin structure by decondensation of the 30 mn fiber (3). Poly(ADP-ribose) polymerase win alter chromatin structure by the modification of core histones, linker histone HI and also by the interaction of automodified enzyme with chromatin. In this paper, we will review the different mechanisms by which poly(ADP-ribose) polymerase could modify specific nuclear proteins and alter chromatin stmcture. We present a model for the interaction of the enzyme with the various subnuclear components. 

Nucleosome A union of eight core histones, one linker histone, and ca. 160 base pairs of DNA. The elementary building block of the chromatin fiber. 

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