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

Abstract

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

Phenotypic and Genotypic Variability in Four Males With MECP2 Gene Sequence Aberrations Including a Novel Deletion

“…Furthermore, MeCP2 recruits co-repressor complexes such as Sin3a and Ski, which remodel and repress chromatin and subsequently inhibit the transcription of certain neurodevelopmental genes (18). The mechanism of inhibition not only includes the transcription repression at gene promoters but also influences the chromatin structure through the formation of silent chromatin loops (19). The RTT neurobiology has been proven complicated as the expression of multiple genes is influenced in variable stages and at different dosages during the neuronal maturation (20).…”

“…Gene expression studies suggest that the R106W mutation has multiple impacts on the putative MeCp2 protein, inhibiting the initial binding to the heterochromatin via the MBD, and resulting in the compromise of the subsequent events of nucleosome-nucleosome interaction and final maximal chromatin condensation (19). Given the severe loss of function of MeCP2, early lethality or neonatal encephalopathy could be expected in a male proband.…”

DNA Binding Restricts the Intrinsic Conformational Flexibility of Methyl CpG Binding Protein 2 (MeCP2)

“…In terms of chromatin, we speculate that the MBD binds to linker DNA (4, 5, 9), whereas other DNA binding site(s) are used to interact with nucleosomal DNA and help MeCP2 to physically envelop the nucleosome (54). This in turn promotes compaction of MeCP2-bound chromatin fibers (8,9). In summary, our H/DX experiments allowed us to access site-specific backbone dynamic information that complements the earlier structural studies and significantly extends our understanding of the physical and chemical mechanisms of DNA binding of this highly abundant and functionally important component of neuronal chromatin.…”

“…Purified MeCP2 (155 l, 0.80 mg/ml) was added to either an unmethylated or methylated 198-bp DNA fragment (430 l, 0.2 mg/ml) in a total of 585 l of 10 mM Tris (pH 7.5), 10 mM NaCl buffer. MeCP2 minimally binds 11 bp of DNA (9). Under these conditions, unmethylated and methylated DNA binding sites were present in molar excess over MeCP2, and DNA concentrations were always above the K d (5) so that H/DX was being measured under saturated binding conditions and dual population (i.e.…”

“…4 -6, reviewed in Ref. 7), chromatin (4,(7)(8)(9), and RNA (10), MeCP2 interacts with many different nuclear proteins, including Sin3a (11), SUV39H1 (12), HP1 (13), DNMT1 (14), Ski and N-COR (15), PU.1 (16), BRM (17), and ATRX (18). Although originally hypothesized to be a specific repressor of methylated genes (11), MeCP2 is now recognized to be multifunctional, with roles in transcriptional activation and repression (19), RNA processing (20), and chromatin organization (8,3,21) (reviewed in Ref.…”