Wilhelm bernhard workshop on the cell nucleus

MALE CHROMATIN REMODELING AFTER FERTILIZATION IN SEA URCHINS: THE ROLE OFA THIOL

Concha C¹, Morín V¹., Even Y²., Oliver M.I¹., Gutiérrez S¹., Puchi M¹., Genevière A.M²., and Imschenetzky M¹.

¹Department of Molecular Biology, University of Concepción, Concepción, Chile, and ²Laboratoire Arago, Banyuls-sur-Mer, France.
In sea urchins male chromatin remodeling involves the replacement ofsperm specific histones (SpH) by maternal histones (CS variants) thatoccurs concomitantly with a step wise SpH degradation. At intermediatestages of male chromatin remodeling hybrid nucleosomes formed byphosphorylated Sp-H2B and Sp-H1 and a subset of poly(ADP-ribosylated) CSvariants were found, whereas after amphimixis the chromatin is organizedexclusively by poly(ADP-ribosylated) CS variants. We have investigatedthe events associated to male chromatin remodeling. Among the moleculesthat were accumulated into male pronucleus we have found a thiol-proteaseand the regulators of the S-phase CdK2 and cyclines E and A. The zymogenof this thiol protease was primarily localized in the nuclei ofunfertilized eggs and found to be activated shortly after fertilization. This enzyme selectively degrades the SpH leaving the maternal CS variantsunaffected. The activity of this enzyme was modulated by the post-translational modification status of its substrates: poly(ADPribosylation) and phosphorylation protects modified histones fromdegradation. When the activity of the thiol-protease was inhibited invivo with E 64-d, the typical degradation of SpH was blocked, thefusion of both pronuclei remained unaffected and the initial S phase wasabolished. The thiol-protease inhibitor E.64-d did not modify the initialS phase in eggs activated with Ca(II) ionophore A-23187, indicating that this compound does not affect per se the initial S phase. Consistently, it was found that nuclear accumulation of the criticalS-phase regulators CdK2/cyclines A and E were not modified by E 64-d.Similarly to normal zygotes, CdK2 was active all through the initial cellcycle in zygotes treated with E 64-d. Taken together these resultsindicate that SpH degradation is a fundamental events for thereestablishment of the diploid condition of the zygotes and has an impacton the license to replicate its DNA. 

Grants: Universidad de Concepción (DIUC) 200.031.088-10, FONDECYT 1011073,  and ECOS France /CONICYT Chile