mxc mutant alleles: Lethal (L1-L2) Pharate lethal viable Drosophila spermatogenesis mxc mutations affect germ cell development mxc mutations causes loss of germline stem cells (GSCs), disruption of transit amplification divisions, and premature differentiation: mxc mutations affect histone biosynthesis mxc mutations affect the DNA repair response following replicative stress multi sex combs (mxc) controls histone biosynthesis and replicative stress response in Drosophila Severine Landais and Leanne Jones, Salk Institute, San Diego, CA, USA. H1 10 mxc G46 ;Nanos-Gal4>Gal4RNAi, 5do mxc G46 ;Nanos-Gal4>H3RNAi, 5do DAPI Tj Vasa DE-cad Hub Number of cell EdU+/testis Cyst cells Germ cells mxc G46 Control Number of GSCs EdU+/testis P<0.0001 mxc G46 testes U7 EY11305 testes Fold change relative to control lok (Chk2) mei-41 (ATR) tefu (ATM) mus304 (ATRIP) Control mxc G46 20 Control hydroxyurea mxc G46 mxc G46 ;Nanos-Gal4>H3RNAi DAPI p H2Av 10 Edu incorporation is affected in mxc mutant testes The number of cells EdU+ decreases in mxc mutant testes Phosphorylated H2Av accumulates in mxc mutant germ cells Genes that mediate the DNA repair response are downregulated in mxc mutant testes ABSTRACT The multi sex combs (mxc) gene as been characterized as a polycomb group gene more than 20 years ago. The strongest mutant alleles of mxc are lethal early in development, whereas the weakest, viable alleles (mxc G43 and mxc G46) are sterile. We characterized mxc mutant phenotype in the male gonad and found that spermatogenesis was disrupted at the earliest stages of germ cell development, with incomplete rounds of transit amplification divisionsand loss of germline stem cells (GSCs). We and others found that Mxc is a critical factor for the histone locus body (HLB) assembly, and proper HLB formation is impaired in mxc mutant cells, although all the core histone mRNA levels are not severely decreased in several mxc mutant alleles, prior to a general decrease of core histone mRNA. On the contrary, histone H3 mRNA is greatly increased in mxc G46. Interestingly, a specific depletion of H1 protein was detected in mxc mutant germ cells only, and H1 RNAi-mediated knockdown causes GSCs loss, but fail to recapitulate the overall germline defects characteristic of mxc mutation. On the other hand, H3 RNAi specifically expressed in the germline rescues 100% of mxc G46 germline phenotype. This suggests collaborative effects of impaired histones production on germ cells maintenance and proliferation. Accordingly, we observe that mxc mutant testes fail to incorporate EdU properly, which suggests replicative stress, a hallmark of histone level defects. A concomitant accumulation of phosphorylated gH2Av (pgH2Av) is observed in germ cells. However, pgH2Av persists in mxc mutant germ cells, suggesting a failure of the DNA repair response. We found that genes critical to this response were down regulated in mxc mutant testes, suggesting a role for Mxc in DNA repair. Therefore, mxc appears as a critical factor for chromatin maintenance involved in both the synthesis of basic packaging components and the DNA repair machinery. Mxc colocalizes with core components of the HLB HLB assembly is impaired In mxc mutants. (Described in White et al, JCB, Vol. 193 (4) 2011. Impaired histone mRNA levels in mxc mutants Decompaction of polytene chromosomes In mxc G43 salivary glands Germline specific H1 protein depletion in mxc mutants gonads Control larvae mxc G43 larvae Control adult mxc G43 adult Loss of GSCs after 10 days of H1 RNAi expression in the germline Rescue of mxc mutant germline phenotype with germline specific expression of H3 RNAi H1 RNAi expressed in the germ cells Fail to recapitulate mxc mutant phenotype Cell autonomous effect of mxc mutation on GSCs maintenance Control mxc G46 rescue Control larvae mxc G46 larvae mxc G43 larvae Control adult mxc G46 adult mxc G43 adult Vasa Tj
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