Xenbase Image ID: 83477

Figure 6. HIRA Is Critical for H3.3 Assembly during Early Development (A) Nucleosome assembly independent of DNA synthesis requires HIRA in vitro. The scheme shows a nucleosome assembly assay independently of DNA synthesis using Xenopus high speed egg extracts (HSEs). We incubated circular DNA plasmids with HSE (5 μl) during the indicated times together with antibodies targeting xHIRA (αHIRA), PBS (CTL), or preimmune serum (not shown). After deproteinization, we analyzed plasmid DNA supercoiling by electrophoresis on agarose gel stained with ethidium bromide. Input DNA run in parallel (first lane on the left, 0 min) and migration positions of DNA plasmid form I (supercoiled), form II (nicked circular), form Ir (closed circular) are indicated. (B) HIRA downregulation affects the level of H3.3 in the chromatin. The scheme shows injections of indicated MO (18.4 ng for H3.3 and 138 ng for HIRA) in fertilized eggs. After incubation to reach stage 14 in controls (−), 40 embryos in each case were used to prepare soluble and chromatin fractions for western blot analysis. Results used a 2-fold dilution series (gradient bar) with the highest quantity being equivalent to three embryos, and detection with antibodies. Anti-β−actin and memcode staining served as loading controls. We note that H3.3 downregulation leads to overexpression of HIRA in soluble fractions. (C) MNase digestion profile of H3.3 and HIRA MO injected embryos. We injected 18.4 ng of H3.3 and 138 ng of HIRA MO in fertilized eggs. After incubation to reach stage 14 in noninjected controls (CTL1 and 2), 40 embryos in each case were used to prepare nuclei that we subjected to MNase digestion for increasing indicated incubation times. For each time point, we purified the resulting DNA fragments from the equivalent of 1.5 embryos to be analyzed by electrophoresis on agarose gel stained with ethidium bromide. Densitometric profiles of the 1 min digestion products are shown on the right. Image published in: Szenker E et al. (2012) Copyright © 2012. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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