Site-directed mutagenesis studies on the binding of the globular domain of linker histone H5 to the nucleosome

Buckle, Robin; Maman, Joseph D.; Allan, James

doi:10.1016/0022-2836(92)90981-o

Cited by 23 publications

(21 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3C). Thus, Lys-85 shows a unique placement, consistent with its key role (20,21). In these 27 solutions, GH5 helix H3 side chains Lys-69, Arg-73, and Arg-74 contact Ϸ15 bp of one DNA arm, whereas the loop that includes Lys-40 and Arg-42 and the side chains of Arg-94 and Lys-97 contact Ϸ5 bp of the second DNA arm.…”

Section: Resultsmentioning

confidence: 56%

“…GH5 side chains that have been implicated in nucleosome binding by radiolabeling (20), mutagenesis (21,22), and crosslinking experiments (8,23) are widely dispersed over the protein's surface, as are the positively charged side chains conserved in the H1 family (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

“…The wing conformation of molecule B is anchored by two hydrogen bonds between the Gln-83 side chain (at the base of the wing) and the main chain of Gly-88 at the top of the wing. Because the molecule B structure is less perturbed by crystal contacts, this molecule was used in our structure analysis and docking studies.GH5 side chains that have been implicated in nucleosome binding by radiolabeling (20), mutagenesis (21,22), and crosslinking experiments (8, 23) are widely dispersed over the protein's surface, as are the positively charged side chains conserved in the H1 family (Fig. 1A).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Complex of linker histone H5 with the nucleosome and its implications for chromatin packing

Фан

Roberts

2006

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Linker histones are essential for chromatin filament formation, and they play key roles in the regulation of gene expression. Despite the determination of structures of the nucleosome and linker histones, the location of the linker histone on the nucleosome is still a matter of debate. Here we show by computational docking that the globular domain of linker histone variant H5 (GH5) has three distinct DNA-binding sites, through which GH5 contacts the DNA at the nucleosome dyad and the linker DNA strands entering and exiting the nucleosome. Our results explain the extensive mutagenesis and crosslinking data showing that side chains spread throughout the GH5 surface interact with nucleosomal DNA. The nucleosome DNA contacts positively charged side chains that are conserved within the linker histone family, indicating that our model extends to linker histone-nucleosome interactions in general. Furthermore, our model provides a structural mechanism for formation of a dinucleosome complex specific to the linker histone H5, explaining its efficiency in chromatin compaction and transcription regulation. Thus, this work provides a basis for understanding how structural differences within the linker histone family result in functional differences, which in turn are important for gene regulation.computational docking ͉ DNA-protein interactions ͉ DOT ͉ linker DNA ͉ winged-helix protein T he interaction of the linker histone with the nucleosome is an ongoing controversial issue (1-3). Histone H5 has been the focus of linker-histone-related studies in recent years. H5 consists of a central globular domain (GH5) that is essential for nucleosome binding and is flanked by basic N-and C-terminal tails (4). Binding of either H5 or GH5 to nucleosomes protects an additional 20 bp of linker DNA from micrococcal nuclease digestion (5). Early studies based on micrococcal nuclease digestion and DNase I footprinting proposed a symmetrical model in which GH5 contacts the dyad of the nucleosome and both the entering and exiting DNA duplexes (also called DNA arms) (6, 7). A ''bridging'' model was later proposed based on experiments that mapped the binding site of GH5 on mixedsequence chicken chromatosomes by conjugating a crosslinking reagent to specific Ser-to-Cys substitutions (8). In this model, GH5 forms a bridge between one DNA arm and the dyad. A radically different ''off-axis'' model was developed from studies on a DNA fragment containing the Xenopus borealis somatic 5S RNA gene (9). Based on this model, GH5 is positioned Ϸ65 bp away from the dyad and is bound inside the DNA superhelix. The bridging and off-axis models imply there may be two equivalent linker histone-binding sites per nucleosome (1), but a 1:1 ratio of linker histone and nucleosome is observed (10, 11).There have been multiple proposals for defining the DNAbinding sites on the linker histone. GH5 has a winged-helix fold (12), consisting of a three-helix bundle in which helices H2 and H3 are part of a helix-turn-helix motif that is followed by a ␤-hairpin, termed the ...

show abstract

Section: Resultsmentioning

confidence: 56%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Complex of linker histone H5 with the nucleosome and its implications for chromatin packing

Фан

Roberts

2006

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Several conserved lysines within or in the vicinity of helix III are relatively protected from reductive methylation by association with the nucleosome core (36), and mutation of one of these residues (Lys-85) reduces the ability of the protein to generate a chromatosome stop (37). However, little is known about the relevance of basic residues at positions 40, 42, and 94, which comprise a second putative DNA binding surface for the specific binding of linker histones in chromatin.…”

Section: Resultsmentioning

confidence: 99%

A Putative DNA Binding Surface in the Globular Domain of a Linker Histone Is Not Essential for Specific Binding to the Nucleosome

Hayes

Kaplan

Ura

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

A fundamental step in the assembly of native chromatin is the specific recognition and binding of linker histones to the nucleoprotein subunit known as the nucleosome. A first step in defining this important interaction is the determination of residues within linker histones that are important for the structure-specific recognition of the nucleosome core. By combining in vitro assays for the native binding activity of linker histones and sitedirected mutagenesis, we have examined a cluster of basic residues within the globular domain of H1 0 , a somatic linker histone variant from Xenopus laevis. We show that these residues, which comprise a putative DNA binding surface within the globular domain, do not play an essential role in the structure-specific binding of a linker histone to the nucleosome.Chromatin is the true substrate in all biological processes involving DNA within the eukaryotic cell nucleus. Recent work has clearly demonstrated that the organization of DNA within the chromatin complex can exert both positive and negative influences on these processes (1-3). In order to fully understand how chromatin complexes have been integrated into gene control mechanisms, it will be necessary first to fully characterize the protein and DNA structures within chromatin.The primary unit of structure in chromatin, the nucleosome core, is comprised of two turns of DNA wound onto a spool of core histone proteins. The association of a single molecule of H1-type histone (i.e. linker histone) with the nucleosome core completes the formation of the nucleosome proper and stabilizes condensed forms of chromatin (4 -7). Posttranslational modifications of linker histones have been proposed to have important roles in chromosome condensation, replication, and transcription (3,8,9). Biophysical studies have led to the proposal that linker histones bind somewhere on the exterior surface of the nucleosome core (6). Linker histones bind specifically and preferentially to nucleosomes over naked DNA (10).We have developed a simple in vitro assay for detecting binding of linker histones to a model chromatin complex. This complex is based on a DNA fragment that contains a Xenopus borealis 5 S rRNA gene. This fragment yields "positioned" nucleosomes upon reconstitution with purified core histone proteins in vitro (11,12). Protein-DNA contacts and the details of the DNA structure have been precisely mapped within the 5 S nucleosome (11,13,14). Moreover, the binding of linker histones to reconstituted 5 S nucleosomes fulfills such classical criteria for the correct incorporation of these proteins into chromatin as the requirement for linker DNA flanking the nucleosome core and the generation of chromatosome-length DNA during micrococcal nuclease digestion of reconstituted complexes (10, 15). Thus the native interaction between linker histones and a nucleosome can be recapitulated in vitro (10,15).A protease-resistant ϳ80-amino acid residue globular domain within linker histones alone is responsible for specific recognition of the nucleos...

show abstract

“…Interestingly, with both H1 (30) and MeCP2, linker protection is retained after removal of a large C-terminal region.…”

Section: Discussionmentioning

confidence: 99%

MeCP2-Chromatin Interactions Include the Formation of Chromatosome-like Structures and Are Altered in Mutations Causing Rett Syndrome

Nikitina

Ghosh

Horowitz-Scherer

et al. 2007

Journal of Biological Chemistry

103

112

View full text Add to dashboard Cite

Sporadic mutations in hMeCP2 (human methylated DNAbinding protein 2) cause the majority of cases of the X-linked neurodevelopmental disease known as Rett syndrome (RTT), 2 a severe autism spectrum disorder (reviewed in Refs. 1-3). The disease results in a diverse range of debilitating physical and neurological symptoms that typically make their initial appearance in the first 6 -18 months of life in affected girls. Although most RTT cases result from a loss of function effect, mutations that increase the MeCP2 dose may also give rise to similar symptoms, a finding seen also in mice engineered to synthesize additional MeCP2 (4). Extensive research has suggested that MeCP2 acts as a transmitter of epigenetic information by binding to symmetrically methylated CpG dinucleotides, recruiting complexes that include histone deacetylase and methyltransferase, and leading to local transcriptional repression. However, there is also considerable evidence that MeCP2 governs additional processes such as chromatin condensation (5, 6) and that its function is not restricted to transcriptional repression (3).To better understand MeCP2 function, we are studying the basic interactions between MeCP2 and its DNA and chromatin substrates. In addition to a methylation-dependent interaction with DNA that is mediated by the methylated DNA-binding domain (MBD), MeCP2 has a C-terminal chromatin-binding region as well as additional DNA-binding regions (5, 6). MeCP2 binding contributes to the formation in nucleosomal arrays of a distinctive structural motif in which the entering and exiting linker DNA segments are brought into close juxtaposition forming a "stem." The stem motif appears very similar to structures induced by histone H1 on mono-nucleosomes and polynucleosomes (7,8) and is thought to initiate the zigzag conformation and compaction of H1-containing chromatin. MeCP2 shares with H1 the ability to induce chromatin compaction, but the multiple chromatin-binding regions of MeCP2 lead to a higher level of condensation (9).A distinctive property of H1-containing chromatin is the protection from micrococcal nuclease (Mnase) digestion of ϳ20 bp of DNA beyond the ϳ146 bp of the nucleosome core particle (NCP). There is considerable evidence that the globular domain of H1 binds near the linker entry-exit site of the nucleosome (reviewed in Ref. 10). However, the location of the ϳ20 bp of protected DNA with respect to the parent nucleosome is influenced by the underlying DNA sequence (11), and the detailed molecular structure of the H1-containing unit, termed the chromatosome (12), remains controversial (10).In the present study we show that hMeCP2, like H1, provides specific protection of linker DNA. However, the two proteins differ significantly in the length of protected DNA. With the

show abstract

Site-directed mutagenesis studies on the binding of the globular domain of linker histone H5 to the nucleosome

Cited by 23 publications

References 19 publications

Complex of linker histone H5 with the nucleosome and its implications for chromatin packing

Complex of linker histone H5 with the nucleosome and its implications for chromatin packing

A Putative DNA Binding Surface in the Globular Domain of a Linker Histone Is Not Essential for Specific Binding to the Nucleosome

MeCP2-Chromatin Interactions Include the Formation of Chromatosome-like Structures and Are Altered in Mutations Causing Rett Syndrome

Contact Info

Product

Resources

About