The functional organization of eukaryotic DNA into chromatin uses histones as components of its building block, the nucleosome. Histone chaperones, which are proteins that escort histones throughout their cellular life, are key actors in all facets of histone metabolism; they regulate the supply and dynamics of histones at chromatin for its assembly and disassembly. Histone chaperones can also participate in the distribution of histone variants, thereby defining distinct chromatin landscapes of importance for genome function, stability, and cell identity. Here, we discuss our current knowledge of the known histone chaperones and their histone partners, focusing on histone H3 and its variants. We then place them into an escort network that distributes these histones in various deposition pathways. Through their distinct interfaces, we show how they affect dynamics during DNA replication, DNA damage, and transcription, and how they maintain genome integrity. Finally, we discuss the importance of histone chaperones during development and describe how misregulation of the histone flow can link to disease.

Proper genome packaging requires coordination of both DNA and histone metabolism. While histone gene transcription and RNA processing adequately provide for scheduled needs, how histone supply adjusts to unexpected changes in demand remains unknown. Here, we reveal that the histone chaperone nuclear autoantigenic sperm protein (NASP) protects a reservoir of soluble histones H3-H4. The importance of NASP is revealed upon histone overload, engagement of the reservoir during acute replication stress, and perturbation of Asf1 activity. The reservoir can be fine-tuned, increasing or decreasing depending on the level of NASP. Our data suggest that NASP does so by balancing the activity of the heat shock proteins Hsc70 and Hsp90 to direct H3-H4 for degradation by chaperone-mediated autophagy. These insights into NASP function and the existence of a tunable reservoir in mammalian cells demonstrate that contingency is integrated into the histone supply chain to respond to unexpected changes in demand.

This paper introduces the use of mass spectrometry to analyze peptide arrays for applications in profiling enzyme specificity. The strategy is illustrated with arrays containing 361 acetylated peptides to profile the activities of several histone deacetylases (HDACs). The arrays reveal distinct substrates that are preferred by members of the HDAC family. This example is particularly relevant because the label-dependent assays now used for these enzymes constrain the range of substrates that can be assayed and can perturb the intrinsic activities of the enzymes. KeywordsEnzymes; Post-translational modifications; SAMDI; Screening The application of peptide arrays for profiling biochemical activities has expanded since Frank and colleagues first reported the SPOT synthesis methodology, wherein peptides are synthesized directly on a cellulose membrane in a cost-efficient manner.[1] These arrays can be treated with enzymes and then analyzed using absorbance, fluorescence, or radio-isotopic assays to rank the activities of the peptides.[2] The requirement for labels can make it challenging to develop assays for certain enzymes and can also lead to false-positive and false-negative results. These limitations have motivated the development of label free formats based on optical methods [3] and mass spectrometry,[4] including our strategy to combine matrix-assisted laser desorption-ionization mass spectrometry with self-assembled monolayer substrates (i.e. the SAMDI method). [5] In this paper we demonstrate the combination of SAMDI-MS with peptide arrays, with an emphasis on the profiling of substrate specificities of several members of the HDAC family, which play a primary role in the regulation of gene expression.The assays now used to measure HDAC activity have been important for mechanistic studies and for identifying hit compounds in high throughput screens, but they have limitations when applied to studies of the substrate specificities. One group of assays identifies active substrates by labeling the deacetylated lysine with haptens that allow the isolation and subsequent sequencing of the peptide substrate. Hence, the original peptide NIH Public Access Author ManuscriptChembiochem. Author manuscript; available in PMC 2010 March 4. substrates must exclude residues susceptible to false positive labeling such as natural lysine, arginine, methionine, and cysteine.[6] A second group of assays conjugates a fluorophore to the carboxy-side of the acetylated lysine. Following HDAC treatment, the chromophore can be proteolytically released only in the deacetylated peptides, providing a convenient assay for HDAC activity compatible with microtiter plates. [7] With this format, the peptide sequence cannot be varied at the carboxy-side of the acetylated lysine and interactions between the fluorophore and enzyme can contribute to activity, making it difficult to understand the intrinsic enzyme specificities. In a clear example of this limitation, the assay revealed that resveratrol activates the SIRT1 deacetylase,[8] ...