Mouse Strains with an Active H2-Ea Meiotic Recombination Hot Spot Exhibit Increased Levels of H2-Ea-Specific DNA Breaks in Testicular Germ Cells
Journal - Molecular and Cellular Biology
We devised a sensitive method for the site-specific detection of rare meiotic DNA strand breaks in germ cell-enriched testicular cell populations from mice that possess or lack an active recombination hot spot at the H2-Ea gene. Using germ cells from adult animals, we found an excellent correlation between the frequency of DNA breaks in the 418-bp H2-Ea hot spot and crossover activity. The temporal appearance of DNA breaks was also studied in 7- to 18-day-old mice with an active hot spot during the first waves of spermatogenesis. The number of DNA breaks detected rose as leptotene and zygotene spermatocytes populate the testis with a peak at day 14 postpartum, when leptotene, zygotene, and early pachytene spermatocytes are the most common meiotic prophase I cell types. The number of DNA breaks drops precipitously 1 day later, when middle to late pachytene spermatocytes become the dominant subtype. The recombination-related breaks in the hot spot likely reflect SPO11-induced double-strand breaks and/or recombination intermediates containing free 3' hydroxyl groups.
Reductions in Linker Histone Levels Are Tolerated in Developing Spermatocytes but Cause Changes in Specific Gene Expression*
Journal - Journal of Biological Chemistry
H1 linker histones are involved in packaging chromatin into30-nm fibers and higher order structures. Most eukaryotic cellscontain nearly one H1 molecule for each nucleosome core particle.Male germ cells in mammals contain large amounts of a germ cell-specificlinker histone, HIST1HT, herein denoted H1t, which is particularlyabundant in pachytene spermatocytes. Despite its abundance inmale germ cells and significant divergence in primary sequencefrom other H1 subtypes, inactivation of the H1t gene in miceshowed that it is not required for spermatogenesis. Analysisof germ cell chromatin from H1t null mice showed that otherH1 subtypes, especially the testis-enriched HIST1H1A, hereindenoted as the H1a subtype, were able to compensate for theabsence of H1t to maintain a normal total H1 to nucleosome coreratio. To disrupt the compensation, we generated H1t and H1adouble null mice by two sequential gene-targeting steps in embryonicstem cells. Elimination of both H1t and H1a led to a 25% decreasein the ratio of H1 to nucleosome cores in double null germ cells.Surprisingly, the reduction in H1 did not perturb spermatogenesisor produce detectable defects in meiotic processes. Microarrayanalysis of gene expression showed that the reduced linker histonelevels did not affect global gene expression, but it did causechanges in expression of specific genes. Our results indicatethat a partial reduction in linker histone-nucleosome core particlestoichiometry is tolerated in developing male germ cells.* This work was supported by National Institutes of Health GrantsCA79057 (to A. I. S.) and HD33816 (to M. A. H.). The costs ofpublication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked"advertisement" in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. Current address: Harvard Partners Center for Genetics and Genomics,Massachusetts General Hospital, Harvard Medical School, 149,13th St., 149-4325, Charlestown, MA 02129.|| Current address: Laboratory for Reproductive and DevelopmentalToxicology, NIEHS, National Institutes of Health, Research TrianglePark, NC 27709.** Current address: The Jackson Laboratory, 600 Main St., Bar Harbor,ME 04609.