Rachel A. Horowitz-Scherer

Structural analysis of the yeast SWI/SNF chromatin remodeling complex

Elucidating the mechanism of ATP-dependent chromatin remodeling is one of the largest challenges in the field of gene regulation. One of the missing pieces in understanding this process is detailed structural information on the enzymes that catalyze the remodeling reactions. Here we use a combination of subunit radio-iodination and scanning transmission electron microscopy to determine the subunit stoichiometry and native molecular weight of the yeast SWI/SNF complex. We also report a three-dimensional reconstruction of yeast SWI/SNF derived from electron micrographs.

Multiple modes of interaction between the methylated DNA binding protein MeCP2 and chromatin.

ABSTRACT 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.

Architecture of the SWI/SNF-nucleosome complex.

ABSTRACT The SWI/SNF complex disrupts and mobilizes chromatin in an ATP-dependent manner. SWI/SNF interactions with nucleosomes were mapped by DNA footprinting and site-directed DNA and protein cross-linking when SWI/SNF was recruited by a transcription activator. SWI/SNF was found by DNA footprinting to contact tightly around one gyre of DNA spanning ∼50 bp from the nucleosomal entry site to near the dyad axis. The DNA footprint is consistent with nucleosomes binding to an asymmetric trough of SWI/SNF that was revealed by the improved imaging of free SWI/SNF. The DNA site-directed cross-linking revealed that the catalytic subunit Swi2/Snf2 is associated with nucleosomes two helical turns from the dyad axis and that the Snf6 subunit is proximal to the transcription factor recruiting SWI/SNF. The highly conserved Snf5 subunit associates with the histone octamer and not with nucleosomal DNA. The model of the binding trough of SWI/SNF illustrates how nucleosomal DNA can be mobilized while SWI/SNF remains bound.

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

hMeCP2 (human methylated DNA-binding protein 2), mutations of which cause most cases of Rett syndrome (RTT), is involved in the transmission of repressive epigenetic signals encoded by DNA methylation. The present work focuses on the modifications of chromatin architecture induced by MeCP2 and the effects of RTT-causing mutants. hMeCP2 binds to nucleosomes close to the linker DNA entry-exit site and protects ∼11 bp of linker DNA from micrococcal nuclease. MeCP2 mutants differ in this property; the R106W mutant gives very little extra protection beyond the ∼146-bp nucleosome core, whereas the large C-terminal truncation R294X reveals wild type behavior. Gel mobility assays show that linker DNA is essential for proper MeCP2 binding to nucleosomes, and electron microscopy visualization shows that the protein induces distinct conformational changes in the linker DNA. When bound to nucleosomes, MeCP2 is in close proximity to histone H3, which exits the nucleosome core close to the proposed MeCP2-binding site. These findings firmly establish nucleosomal linker DNA as a crucial binding partner of MeCP2 and show that different RTT-causing mutations of MeCP2 are correspondingly defective in different aspects of the interactions that alter chromatin architecture.

MeCP2 Binds Cooperatively to Its Substrate and Competes with Histone H1 for Chromatin Binding Sites

ABSTRACT 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.

Unique physical properties and interactions of the domains of methylated DNA binding protein 2.

Methylated DNA binding protein 2 (MeCP2) is a methyl CpG binding protein whose key role is the recognition of epigenetic information encoded in DNA methylation patterns. Mutation or misregulation 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 approximately 60% unstructured, with nine interspersed alpha-molecular recognition features (alpha-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 that 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 that 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 led to new mechanistic and biochemical insights regarding the conformational modulations of this intrinsically disordered protein, and its context-dependent in vivo roles.

Organization of interphase chromatin

The organization of interphase chromatin spans many topics, ranging in scale from the molecular level to the whole nucleus, and its study requires a concomitant range of experimental approaches. In this review, we examine these approaches, the results they have generated, and the interfaces between them. The greatest challenge appears to be the integration of information on whole nuclei obtained by light microscopy with data on nucleosome–nucleosome interactions and chromatin higher-order structures, obtained in vitro using biophysical characterization, atomic force microscopy, and electron microscopy. We consider strategies that may assist in the integration process, and we review emerging technologies that promise to reduce the “resolution gap.”

Rett Syndrome-causing Mutations in Human MeCP2 Result in Diverse Structural Changes That Impact Folding and DNA Interactions

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 α-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.

Visualization and 3D Structure Determination of Defined Sequence Chromatin and Chromatin Remodeling Complexes

Publisher Summary The purpose of this chapter is to understand the different states of chromatin at an atomic level of resolution, but the range of chromatin structures accessible to X-ray crystallography is very limited. This limitation can, in principle, be overcome using lower-resolution 3Dvolumes generated by an electron microscope (EM) into which atomic level structures can be “docked.” The range of transmission EM-based technologies available for visualizing chromatin and chromatin remodeling complexes is listed in a table in the chapter. Electron cryomicroscopy (ECM) permits the observation of specimens in their solution conformation, without stains, shadowing, or substrate adhesion and the accompanying flattening and drying artifacts. In most instances, chemical fixation is unnecessary. Recent progress in preparing and purifying defined sequence chromatin and chromatin remodeling complexes provides an opportunity to determine the three-dimensional (3D) changes that accompany major remodeling events, such as histone modification, the binding of transcriptional repressors and activators, and the action of ATP-dependent remodeling complexes.