[Show abstract][Hide abstract] ABSTRACT: Despite a vast body of literature linking chromatin structure to regulation of gene expression, the role of architectural proteins in higher order chromatin transitions required for transcription activation and repression has remained an under-studied field. To demonstrate the current knowledge of the role of such proteins, we have focused our attention on the methylated DNA binding and chromatin-associated protein MeCP2. Structural studies using chromatin assembled in vitro have revealed that MeCP2 can associate with nucleosomes in an N-terminus dependent manner and efficiently condense nucleosome arrays. The present review attempts to match MeCP2 structural domains, or lack thereof, and specific chromatin features needed for the proper recruitment of MeCP2 to its multiple functions as either activator or repressor. We specifically focused on MeCP2's role in Rett syndrome, a neurological disorder associated with specific MeCP2 mutations.
[Show abstract][Hide abstract] ABSTRACT: Recent studies of the mechanisms involved in the regulation of gene expression in eukaryotic organisms depict a highly complex process requiring a coordinated rearrangement of numerous molecules to mediate DNA accessibility. Silencing in Saccharomyces cerevisiae involves the Sir family of proteins. Sir3p, originally described as repressing key areas of the yeast genome through interactions with the tails of histones H3 and H4, appears to have additional roles in that process, including involvement with a DNA binding component. Our in vitro studies focused on the characterization of Sir3p-nucleic acid interactions and their biological functions in Sir3p-mediated silencing using binding assays, EM imaging, and theoretical modeling. Our results suggest that the initial Sir3p recruitment is partially DNA-driven, highly cooperative, and dependent on nucleosomal features other than histone tails. The initial step appears to be rapidly followed by the spreading of silencing using linker DNA as a track.
[Show abstract][Hide abstract] ABSTRACT: Recent biochemical studies evaluated the affinity of histones to DNA in the context of nucleosome core particle (NCP). These have indicated a concentration-dependence for nucleosome stability. However, when studying chromatin the preferred templates are nucleosome arrays (NA) and not the NCP. Biochemical methods are poorly suited for structural analysis of chromatin. To overcome that technical hindrance, and investigate the effect of concentration on stability of the histone-DNA interactions, we have applied the multigel Quantitative Agarose Gel Electrophoresis (QAGE) method to in vitro-assembled nucleosomal arrays. The results demonstrated the method to be extremely valuable for the evaluation of the effect of low concentration on NA. However, QAGE is a fairly time-demanding and complex method. To maximize the efficiency of use of this technology, we devised a protocol that allowed for multiple sets of templates to be analyzed simultaneously. Briefly, samples can be loaded at regular intervals and analyzed individually for their molecular composition. The technique presented in this study describes the calibration steps and proof of concept necessary to validate the use of multiple loading of multigel to evaluate the composition of nucleosomal arrays as a function of concentration.
Journal of Biochemical and Biophysical Methods 09/2007; 70(5):721-6. DOI:10.1016/j.jbbm.2007.03.006 · 1.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transition from transcription activation to repression is regulated at multiple levels by the DNA sequence and DNA modification to its compaction through chromatin packaging. The GAGA factor (GAF) is one of a few transcription factors that can regulate gene expression at multiple levels. It displays both activator/antirepressor and repressor activity, depending on its target genomic location. The GAF-mediated modulation of expression appears to be intimately linked with modifications of the chromatin structure. The GAF can associate with highly compacted heterochromatin, contributing to gene repression, or participate in nucleosome remodeling to activate specific genes. In this review, we are attempting to elucidate the contribution(s) of the various domains of the GAF to the recruitment of its functional partners, leading to seemingly opposite functions. We surveyed the current scientific literature for evidence of GAF involvement in regulatory events associated with changes of chromatin composition or conformation.
[Show abstract][Hide abstract] ABSTRACT: Although histone deacetylases (HDACs) are generally viewed as corepressors, we show that HDAC1 serves as a coactivator for the glucocorticoid receptor (GR). Furthermore, a subfraction of cellular HDAC1 is acetylated after association with the GR, and this acetylation event correlates with a decrease in promoter activity. HDAC1 in repressed chromatin is highly acetylated, while the deacetylase found on transcriptionally active chromatin manifests a low level of acetylation. Acetylation of purified HDAC1 inactivates its deacetylase activity, and mutation of the critical acetylation sites abrogates HDAC1 function in vivo. We propose that hormone activation of the receptor leads to progressive acetylation of HDAC1 in vivo, which in turn inhibits the deacetylase activity of the enzyme and prevents a deacetylation event that is required for promoter activation. These findings indicate that HDAC1 is required for the induction of some genes by the GR, and this activator function is dynamically modulated by acetylation.
[Show abstract][Hide abstract] ABSTRACT: Recent studies have focused attention on chromatin as both a negative and positive regulator of specific nuclear events. The vast majority of this research has been centered on chromatin remodeling and histone post-translational modifications over the regulatory regions of specific genes. However, due the technical difficulties of such studies, the contribution of the higher-order structure of chromatin on the regulation of gene expression has not been as thoroughly investigated and the majority of the initial studies have used biophysical methods or microscopy. Until recent technical developments, the main hindrance for these biophysical investigations of chromatin has been an almost absolute requirement for large amounts of highly purified material. The development of an agarose gel electrophoresis method (quantitative agarose gel electrophoresis), initially designed for the analysis of the three-dimensional structure of purified and in vivo-assembled chromatin over a promoter region, has been expanded to include studies of chromatin in the presence of a Drosophila crude extract. The technique presented in the study reported here will help in paving the way for subsequent analyses of structural modifications of chromatin that are linked with the recruitment of various chromatin-associated factors present in the provided extract(s).
Journal of Biochemical and Biophysical Methods 07/2006; 67(2-3):141-50. DOI:10.1016/j.jbbm.2006.02.004 · 1.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since the initial characterization of chromatin remodeling as an ATP-dependent process, many studies have given us insight into how nucleosome-remodeling complexes can affect various nuclear functions. However, the multistep DNA-histone remodeling process has not been completely elucidated. Although new studies are published on a nearly weekly basis, the nature and roles of interactions of the individual SWI/SNF- and ISWI-based remodeling complexes and DNA, core histones, and other chromatin-associated proteins are not fully understood. In addition, the potential changes associated with ATP recruitment and its subsequent hydrolysis have not been fully characterized. This review explores possible mechanisms by which chromatin-remodeling complexes are recruited to specific loci, use ATP hydrolysis to achieve actual remodeling through disruption of DNA-histone interactions, and are released from their chromatin template. We propose possible roles for ATP hydrolysis in a chromatin-release/target-scanning process that offer an alternative to or complement the often overlooked function of delivering the energy required for sliding or dislodging specific subsets of core histones.
[Show abstract][Hide abstract] ABSTRACT: Chromatin fibers are intrinsically dynamic macromolecular complexes whose biological functions are intimately linked with their structure and interactions with chromatin-associated proteins (CAPs). Three-dimensional architectural transitions between or within the two co-existing chromatin types referred to as euchromatin and heterochromatin have been associated with activation or repression of nuclear functions. The presence of specific subsets of chromosomal proteins co-existing with the different chromatin conformations suggests a functional significance for their co-localization. The major points of emphasis of this review will assess the structure, function and recently documented exchanges amongst various members of the CAP family.