Localized Ca2+ uncaging reveals polarized distribution of Ca2+-sensitive Ca2+ release sites: mechanism of unidirectional Ca2+ waves

Ca2+-induced Ca2+ release (CICR) plays an important role in the generation of cytosolic Ca2+ signals in many cell types. However, it is inherently difficult to distinguish experimentally between the contributions of messenger-induced Ca2+ release and CICR. We have directly tested the CICR sensitivity of different regions of intact pancreatic acinar cells using local uncaging of caged Ca2+. In the apical region, local uncaging of Ca2+ was able to trigger a CICR wave, which propagated toward the base. CICR could not be triggered in the basal region, despite the known presence of ryanodine receptors. The triggering of CICR from the apical region was inhibited by a pharmacological block of ryanodine or inositol trisphosphate receptors, indicating that global signals require coordinated Ca2+ release. Subthreshold agonist stimulation increased the probability of triggering CICR by apical uncaging, and uncaging-induced CICR could activate long-lasting Ca2+ oscillations. However, with subthreshold stimulation, CICR could still not be initiated in the basal region. CICR is the major process responsible for global Ca2+ transients, and intracellular variations in sensitivity to CICR predetermine the activation pattern of Ca2+ waves

An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition

<div><p>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 <i>Drosophila melanogaster</i> chromatin and a <i>D</i>. <i>melanogaster-</i>specific antibody into standard ChIP reactions. Specifically, the use of an antibody that exclusively recognizes the <i>D</i>. <i>melanogaster</i> histone variant H2Av enables precipitation of <i>D</i>. <i>melanogaster</i> chromatin as a minor fraction of the total ChIP DNA. The <i>D</i>. <i>melanogaster</i> 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.</p></div

Reduced H3K27me3 binding is detected by ChIP-qPCR.

<p><b>(A)</b> ChIP was performed using chromatin from KARPAS-422 cells treated with the EZH2 inhibitor CPI-360. qPCR using the positive control primer <i>MYT1</i> showed reduced H3K27me3 occupancy in the presence of the inhibitor. <b>(B)</b> ChIP was performed using chromatin from PC9 cells treated with the EZH2 inhibitor GSK126. qPCR using the positive control primer <i>MYT1</i> showed reduced H3K27me3 occupancy in cells treated with the inhibitor. (<b>C</b>) Libraries were generated from KARPAS-422 cells using 15 cycles of PCR amplification. Library DNA was diluted and qPCR was performed using positive control primers for <i>MYT1</i> and <i>CCND2</i>. (<b>D</b>) Libraries were generated from PC9 cells as described in (C) and library DNA was used for qPCR using positive control primers for <i>MYT1</i> and <i>CCND2</i>. All experiments are represented as the mean of two independent experiments with qPCRs performed in triplicate ±SD. The <i>ACTB</i> promoter served as a negative control for all experiments.</p