Linker histone H1 plays an important role in chromatin folding in vitro. To study the role of H1 in vivo, mouse embryonic stem cells null for three H1 genes were derived and were found to have 50% of the normal level of H1. H1 depletion caused dramatic chromatin structure changes, including decreased global nucleosome spacing, reduced local chromatin compaction, and decreases in certain core histone modifications. Surprisingly, however, microarray analysis revealed that expression of only a small number of genes is affected. Many of the affected genes are imprinted or are on the X chromosome and are therefore normally regulated by DNA methylation. Although global DNA methylation is not changed, methylation of specific CpGs within the regulatory regions of some of the H1 regulated genes is reduced. These results indicate that linker histones can participate in epigenetic regulation of gene expression by contributing to the maintenance or establishment of specific DNA methylation patterns.

Most eukaryotic cells contain nearly equimolar amounts of nucleosomes and H1 linker histones. Despite their abundance and the potential functional specialization of H1 subtypes in multicellular organisms, gene inactivation studies have failed to reveal essential functions for linker histones in vivo. Moreover, in vitro studies suggest that H1 subtypes may not be absolutely required for assembly of chromosomes or nuclei. By sequentially inactivating the genes for three mouse H1 subtypes (H1c, H1d, and H1e), we showed that linker histones are essential for mammalian development. Embryos lacking the three H1 subtypes die by midgestation with a broad range of defects. Triple-H1-null embryos have about 50% of the normal ratio of H1 to nucleosomes. Mice null for five of these six H1 alleles are viable but are underrepresented in litters and are much smaller than their littermates. Marked reductions in H1 content were found in certain tissues of these mice and in another compound H1 mutant. These results demonstrate that the total amount of H1 is crucial for proper embryonic development. Extensive reduction of H1 in certain tissues did not lead to changes in nuclear size, but it did result in global shortening of the spacing between nucleosomes.DNA in the eukaryotic nucleus is organized into a highly compact nucleoprotein complex referred to as chromatin (48,53). The histones constitute a family of proteins that are intimately involved in organizing chromatin structure. The nucleosome core particle, the highly conserved unit of chromatin organization in all eukaryotes, consists of an octamer of four core histones (H2A, H2B, H3, and H4) around which about 145 bp of DNA is wrapped. The chromatin fiber also contains a fifth histone, the linker histone (usually referred to as H1), which can bind to core particles and protect an additional ϳ20 bp of DNA (linker DNA) from nuclease digestion. The precise location and stoichiometry of H1 within the chromatin fiber are uncertain (45,46,49), but in higher eukaryotes, there is, on average, nearly one H1 molecule for each core particle (48). Most of our knowledge about the role of H1 in chromatin structure is based on in vitro experiments. These studies indicate that two principal functions of linker histones are to organize and stabilize the DNA as it enters and exits the core particle and to facilitate the folding of nucleosome arrays into more compact structures (19,38). Despite the presumed fundamental role of linker histones in chromatin structure, elimination of H1 in Tetrahymena, Saccharomyces cerevisiae, and Aspergillus nidulans and silencing of H1 in Ascobolus immersus showed that H1 is not essential in these unicellular eukaryotes (3,34,39,41,47).In higher organisms, additional levels of control on chromatin organization and function are available because of the existence of multiple nonallelic linker histone variants or subtypes (6, 33). In mice, there are at least eight H1 subtypes, including the widely expressed subtypes H1a through H1e, the testis-specific subtype H1...

Mutations of the methylated DNA binding protein MeCP2, a multifunctional protein that is thought to transmit epigenetic information encoded as methylated CpG dinucleotides to the transcriptional machinery, give rise to the debilitating neurodevelopmental disease Rett syndrome (RTT). In this in vitro study, the methylation-dependent and -independent interactions of wild-type and mutant human MeCP2 with defined DNA and chromatin substrates were investigated. A combination of electrophoretic mobility shift assays and visualization by electron microscopy made it possible to understand the different conformational changes underlying the gel shifts. MeCP2 is shown to have, in addition to its well-established methylated DNA binding domain, a methylation-independent DNA binding site (or sites) in the first 294 residues, while the C-terminal portion of MeCP2 (residues 295 to 486) contains one or more essential chromatin interaction regions. All of the RTT-inducing mutants tested were quantitatively bound to chromatin under our conditions, but those that tend to be associated with the more severe RTT symptoms failed to induce the extensive compaction observed with wild-type MeCP2. Two modes of MeCP2-driven compaction were observed, one promoting nucleosome clustering and the other forming DNA-MeCP2-DNA complexes. MeCP2 binding to DNA and chromatin involves a number of different molecular interactions, some of which result in compaction and oligomerization. The multifunctional roles of MeCP2 may be reflected in these different interactions.It is now well established that the severe neurodevelopmental Rett syndrome (RTT) is caused primarily by mutations in the X-linked MeCP2 gene (1). MeCP2 (Fig. 1A) is a member of the family of related proteins that bind specifically to symmetrically methylated CpG dinucleotides via a conserved methyl binding domain (MBD) (17,38,44). The binding of MeCP2 to methylated DNA has been shown to lead to transcriptional repression in a variety of experimental contexts (see, for example, references 13, 35, 44, 45, and 65), a property conferred by a transcriptional repression domain (TRD). Evidence suggests that repression occurs when Sin3A and histone deacetylases (HDACs) are recruited to the TRD, resulting in the deacetylation of nearby nucleosomes (reviewed in reference 48). Additional "AT hook" and "WW" motifs have been identified in MeCP2 (9, 34), as well as a nuclear localization signal (NLS). MeCP2 is widespread and highly conserved in vertebrates, and mice lacking MeCP2 or with a major C-terminal truncation exhibit neurological dysfunctions with remarkable parallels in human RTT patients (12,22,49).Analysis of RTT patients has revealed a small number of single amino acid changes at mutational "hot spots" in MeCP2, many of which are located in the MBD or TRD, as well as a series of C-terminal truncations. In addition to the hot spots, there are a large number of low-frequency mutations that lead to RTT (see the IRSA database at http://mecp2.chw.edu.au /mecp2/). There is growing evidence that...

MeCP2 is a methyl CpG binding protein whose key role is the recognition of epigenetic information encoded in DNA methylation patterns. Mutation or mis-regulation of MeCP2 function leads to Rett syndrome as well as a variety of other Autism Spectrum Disorders. Here, we have analyzed in detail the properties of six individually expressed human MeCP2 domains spanning the entire protein with emphasis on their interactions with each other, with DNA, and with nucleosomal arrays. Each domain contributes uniquely to the structure and function of the full-length protein. MeCP2 is ~60% unstructured, with nine interspersed α-Molecular Recognition Features (α-MoRFs), which are polypeptide segments predicted to acquire secondary structure upon forming complexes with binding partners. Large increases in secondary structure content are induced in some of the isolated MeCP2 domains and in the full-length protein by binding to DNA. Interactions between some MeCP2 domains in cis and trans seen in our assays, likely contribute to the structure and function of the intact protein. We also show that MeCP2 has two functional halves. The N-terminal portion contains the methylated DNA binding domain (MBD) and two highly disordered flanking domains which modulate MBD-mediated DNA binding. One of these flanking domains is also capable of autonomous DNA binding. In contrast, the C-terminal portion of the protein which harbors at least two independent DNA binding domains and a chromatin specific binding domain is largely responsible for mediating nucleosomal array compaction and oligomerization. These findings lead to new mechanistic and biochemical insights regarding the conformational modulations of this intrinsically disordered protein, and its context-dependent in vivo roles.

Sporadic mutations in the hMeCP2 gene, coding for a protein that preferentially binds symmetrically methylated CpGs, result in the severe neurological disorder Rett syndrome (RTT). In the present work, employing a wide range of experimental approaches, we shed new light on the many levels of MeCP2 interaction with DNA and chromatin. We show that strong methylation-independent as well as methylation-dependent binding by MeCP2 is influenced by DNA length. Although MeCP2 is strictly monomeric in solution, its binding to DNA is cooperative, with dimeric binding strongly correlated with methylation density, and strengthened by nearby A/T repeats. Dimeric binding is abolished in the F155S and R294X severe RTT mutants. MeCP2 also binds chromatin in vitro, resulting in compaction-related changes in nucleosome architecture that resemble the classical zigzag motif induced by histone H1 and considered important for 30-nm-fiber formation. In vivo chromatin binding kinetics and in vitro steady-state nucleosome binding of both MeCP2 and H1 provide strong evidence for competition between MeCP2 and H1 for common binding sites. This suggests that chromatin binding by MeCP2 and H1 in vivo should be viewed in the context of competitive multifactorial regulation.DNA methylation constitutes an important epigenetic component in transcriptional regulation, with methylation generally leading to repression of nearby genes (6). However, the mechanism by which the epigenetic signal is passed to the regulatory machinery is not well understood. Research in this area has been focused on a small family of methyl-CpG binding proteins, best characterized by MeCP2 (19), mutations in which result in Rett syndrome (RTT), a debilitating neurodevelopmental disease in humans (2).A mechanism of MeCP2-mediated gene silencing may involve recruitment of histone deacetylases upon methyl-specific binding (57). However, other mechanisms, which are not necessarily mutually exclusive, such as stabilization of large chromatin loops (29) and promotion of chromatin compaction (51), have also been suggested (14). Studies on in vivo distribution of MeCP2 in nuclei have revealed that, in addition to the expected occupancy of sites of CpG methylation, MeCP2 shows significant binding to unmethylated DNA (71). However, a recent analysis of MeCP2 occupancy has revealed that the genomic distribution of MeCP2 in mammalian neurons closely tracks methyl-CpG density (60). These results highlight our current lack of understanding of key questions pertinent to the binding of MeCP2 to DNA and chromatin. It is especially important, for example, to quantitate the modulation of binding by factors such as methylation density (8,37,48,60) and the presence of adjacent A/T-rich sequences (31) that are reported to influence binding. In the present work, we have used a variety of quantitative approaches to show that, when bound to DNA, MeCP2 exhibits a cooperative monomerdimer equilibrium, which is influenced by DNA length, methylation density, and the presence of nearby A/T repeats.Th...

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

The termini of eukaryotic chromosomes contain specialized protective structures, the telomeres, composed of TTAGGG repeats and associated proteins which, together with telomerase, control telomere length. Telomere shortening is associated with senescence and inappropriate telomerase activity may lead to cancer. Little is known about the chromatin context of telomeres, because, in most cells, telomere chromatin is tightly anchored within the nucleus. We now report the successful release of telomere chromatin from chicken erythrocyte and mouse lymphocyte nuclei, both of which have a reduced karyoskeleton. Electron microscopy reveals telomere chromatin fibers in the form of closed terminal loops, which correspond to the “t-loop” structures adopted by telomere DNA. The ability to recognize isolated telomeres in their native chromatin conformation opens the way for detailed structural and compositional studies.

Most cases of Rett syndrome (RTT) are caused by mutations in the methylated DNA-binding protein, MeCP2. Here, we have shown that frequent RTT-causing missense mutations (R106W, R133C, F155S, T158M) located in the methylated DNA-binding domain (MBD) of MeCP2 have profound and diverse effects on its structure, stability, and DNA-binding properties. Fluorescence spectroscopy, which reports on the single tryptophan in the MBD, indicated that this residue is strongly protected from the aqueous environment in the wild type but is more exposed in the R133C and F155S mutations. In the mutant proteins R133C, F155S, and T158M, the thermal stability of the domain was strongly reduced. Thermal stability of the wild-type protein was increased in the presence of unmethylated DNA and was further enhanced by DNA methylation. DNA-induced thermal stability was also seen, but to a lesser extent, in each of the mutant proteins. Circular dichroism (CD) of the MBD revealed differences in the secondary structure of the four mutants. Upon binding to methylated DNA, the wild type showed a subtle but reproducible increase in ␣-helical structure, whereas the F155S and R106W did not acquire secondary structure with DNA. Each of the mutant proteins studied is unique in terms of the properties of the MBD and the structural changes induced by DNA binding. For each mutation, we examined the extent to which the magnitude of these differences correlated with the severity of RTT patient symptoms.A key epigenetic signal in vertebrates is the symmetrical methylation of CpG dinucleotides, which may be passed on to subsequent generations by the action of hemi-methylases on newly replicated DNA (reviewed in Ref. 1). Screening for proteins that bind preferentially to methylated CpGs has revealed a family of methylated DNA-binding proteins, the founding member of which is the conserved and highly basic 52-kDa methylated DNA-binding protein 2, MeCP2 2 (reviewed in Ref.2). The portion of MeCP2 responsible for binding methylated DNA is known as the MBD (methylated DNA-binding domain), which extends from residues ϳ75 to ϳ164 (3). NMR and x-ray studies (4, 5) have shown the MBD to be ϳ60% structured, with segments of ␣-helix, ␤-strand, and ␤-turn forming a wedge-shaped structure (Fig. 1b). In contrast, the N-and C-terminal portions of MeCP2 are predicted to be largely unstructured (6). Signals encoded in methylated CpGs frequently lead to transcriptional repression, which appears to be a prominent consequence of MeCP2 binding (7). One model of the mechanism that leads from MeCP2 binding to transcriptional repression involves the recruitment of Sin 3A and histone deacetylase followed by local histone modification (8, 9). However, recent evidence suggests that MeCP2 locations in chromatin are not confined to sites of methylated DNA and that MeCP2 occupancy does not necessarily lead to transcriptional repression (10). Moreover, it is now clear that MeCP2 has a wide range of potential functions (reviewed in Refs. 11 and 12), including an involvement in RNA proce...