Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) has been instrumental in inferring the roles of histone post-translational modifications in the regulation of transcription, chromatin compaction and other cellular processes that require modulation of chromatin structure. However, analysis of ChIP-seq data is challenging when the manipulation of a chromatin-modifying enzyme significantly affects global levels of histone post-translational modifications. For example, small molecule inhibition of the methyltransferase EZH2 reduces global levels of histone H3 lysine 27 trimethylation (H3K27me3). However, standard ChIP-seq normalization and analysis methods fail to detect a decrease upon EZH2 inhibitor treatment. We overcome this challenge by employing an alternative normalization approach that is based on the addition of Drosophila melanogaster chromatin and a D. melanogaster-specific antibody into standard ChIP reactions. Specifically, the use of an antibody that exclusively recognizes the D. melanogaster histone variant H2Av enables precipitation of D. melanogaster chromatin as a minor fraction of the total ChIP DNA. The D. melanogaster ChIP-seq tags are used to normalize the human ChIP-seq data from DMSO and EZH2 inhibitor-treated samples. Employing this strategy, a substantial reduction in H3K27me3 signal is now observed in ChIP-seq data from EZH2 inhibitor treated samples.
Disruption of intramolecular interactions, translocation from one intracellular compartment to another, and binding to isozyme-specific anchoring proteins termed RACKs, accompany protein kinase C (PKC) activation. We hypothesized that in inactive ⑀PKC, the RACK-binding site is engaged in an intramolecular interaction with a sequence resembling its RACK, termed⑀RACK. An amino acid difference between the ⑀RACK sequence in ⑀PKC and its homologous sequence in ⑀RACK constitutes a change from a polar non-charged amino acid (asparagine) in ⑀RACK to a polar charged amino acid (aspartate) in ⑀PKC. Here we show that mutating the aspartate to asparagine in ⑀PKC increased intramolecular interaction as indicated by increased resistance to proteolysis, and slower hormone-or PMAinduced translocation in cells. Substituting aspartate for a non-polar amino acid (alanine) resulted in binding to ⑀RACK without activators, in vitro, and increased translocation rate upon activation in cells. Mathematical modeling suggests that translocation is at least a two-step process. Together our data suggest that intramolecular interaction between the ⑀RACK site and RACK-binding site within ⑀PKC is critical and rate limiting in the process of PKC translocation.The protein kinase C (PKC) 1 family of phospholipid (PL) -dependent serine/threonine kinases undergoes a conformational change and translocation, or movement, from the cytosolic to the cell particulate fraction upon activation (1, 2). Conformational changes in PKC from an inactive to an active state results in exposure of domains required for PKC anchoring to the particulate fraction and in increased sensitivity of the enzyme to proteases (1-3, 41). Therefore, the inactive state exists in a closed conformation, with the proteolytic sites protected, whereas the active state is in an open conformation with exposed proteolytic sites. Structural alterations from the closed to open states involve disruption of intramolecular interactions within the enzyme.An intramolecular interaction in inactive PKC between the catalytic site and a site in the regulatory domain that resembles a substrate phosphorylation site but lacks a serine or threonine phosphoacceptor (pseudosubstrate site) has been previously identified (3, 6). Deletion of the pseudosubstrate ( -substrate) site generated a constitutively active enzyme (6) and mutations of the basic residues in the -substrate site reduced the affinity of the catalytic site to the -substrate site generating a constitutively active enzyme, preferentially localized to the cell particulate fraction (6). Furthermore, conversion of the alanine in the -substrate site to a glutamic acid, mimicking a phosphorylated amino acid, resulted in loss of binding of the -substrate site to the catalytic site, creating a constitutively active enzyme (6). Finally, a peptide corresponding to the -substrate site is a competitive inhibitor of PKC catalytic activity (6).We previously demonstrated that translocation of PKC is associated with binding of each activated PKC isozyme to...
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