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Histone methylation is involved in the regulation of many cellular processes. In the past 2 years, several histone demethylases including BHC110/LSD1 have been characterized. BHC110, the first known histone lysine demethylase, removes methyl groups from methylated histone H3 lysine 4 and has been found in many multi-protein complexes. Using one-step affinity purification, we have isolated enzymatically...
The continuing identification of new histone post-translational modifications and ongoing discovery of their roles in nuclear processes has increased the demand for quick, efficient, and precise methods for their analysis. In the budding yeast Saccharomyces cerevisiae, a variety of methods exist for the characterization of histone modifications on a global scale. However, a wide gap in preparation...
Mass spectrometry-based approaches are commonly used to identify proteins from multiprotein complexes, typically with the goal of identifying new complex members or identifying post-translational modifications. However, with the recent demonstration that spectral counting is a powerful quantitative proteomic approach, the analysis of multiprotein complexes by mass spectrometry can be reconsidered...
The use of the budding yeast Saccharomyces cerevisiae as a simple eukaryotic model system for the study of chromatin assembly and regulation has allowed rapid discovery of genes that influence this complex process. The functions of many of the proteins encoded by these genes have not yet been fully characterized. Here, we describe a high-throughput methodology that can be used to illuminate gene function...
Gene expression in eukaryotes is largely controlled at the level of transcriptional initiation by gene-specific activators. Transcriptional activators stimulate the assembly of general transcription factors on the promoter to form a preinitiation complex (PIC). Such a stimulated assembly of PIC is believed to result from a direct interaction between the activator and one or more components of the...
Post-translational modifications of histones have been correlated with virtually all chromatin-templated processes, including gene expression regulation, DNA replication, mitosis and meiosis, and DNA repair. In order to better understand the mechanistic basis by which histone modifications participate in the control of cellular processes, it is essential to identify and characterize downstream effector...
Post-translational modifications to histone proteins regulate the packaging of genomic DNA into chromatin, gene activity and other functions of the genome. They are understood to play key roles in embryonic development and disease pathogenesis. Recent advances in technology have made it possible to analyze chromatin structure genome-wide in mammalian cells. Global patterns of histone modifications...
Chromatin modifying and remodeling enzymes play critical roles in many aspects of chromosome biology including transcription, replication, recombination, and repair. Our laboratory recently identified and characterized two multisubunit human chromatin remodeling enzymes designated the INO80 and SRCAP complexes. Mechanistic studies revealed that the human INO80 complex catalyzes nucleosome sliding...
Posttranslational histone modifications play an important role in regulating chromatin dynamics and function. One of the modifications, methylation, occurs on both lysine and arginine residues and participates in diverse range of biological processes including heterochromatin formation, X-chromosome inactivation, and transcriptional regulation. While acetylation, phosphorylation, and ubiquitylation...
Histone lysine methylation plays an important role in the regulation of gene expression and impacts many fundamental biological processes, such as cellular identity. Despite great efforts, the mechanisms behind the downstream consequences of histone methyl-recognition remain poorly understood. Here, we describe various methods to investigate specific histone lysine-methyl recognition, including the...
Knowing the genomic distribution of chromosomal proteins and of histone modifications provides essential insight into function. The giant polytene chromosomes of the Drosophila larval salivary glands provide a high-resolution genomic map with a resolution exceeded only by chromatin immunoprecipitation. Immunofluorescence localization of chromosomal proteins and specific post-translational modifications...
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