Control of a Novel Spermatocyte-Promoting Factor by the Male Germline Sex Determination Factor PHF7 of Drosophila melanogaster.

A key aspect of germ cell development is to establish germline sexual identity and initiate a sex-specific developmental program to promote spermatogenesis or oogenesis. Previously, we have identified the histone reader Plant Homeodomain Finger 7 (PHF7) as an important regulator of male germline identity. To understand how PHF7 directs sexual differentiation of the male germline, we investigated the downstream targets of PHF7 by combining transcriptome analyses, which reveal genes regulated by Phf7, with genomic profiling of histone H3K4me2, the chromatin mark that is bound by PHF7. Through these genomic experiments, we identify a novel spermatocyte factor Receptor Accessory Protein Like 1 (REEPL1) that can promote spermatogenesis and whose expression is kept off by PHF7 in the spermatogonial stage. Loss of Reepl1 significantly rescues the spermatogenesis defects in Phf7 mutants, indicating that regulation of Reepl1 is an essential aspect of PHF7 function. Further, increasing REEPL1 expression facilitates spermatogenic differentiation. These results indicate that PHF7 controls spermatogenesis by regulating the expression patterns of important male germline genes.

Phf7 controls male sex determination in the Drosophila germline.

Establishment of germline sexual identity is critical for production of male and female germline stem cells, as well as sperm versus eggs. Here we identify PHD Finger Protein 7 (PHF7) as an important factor for male germline sexual identity in Drosophila. PHF7 exhibits male-specific expression in early germ cells, germline stem cells, and spermatogonia. It is important for germline stem cell maintenance and gametogenesis in males, whereas ectopic expression in female germ cells ablates the germline. Strikingly, expression of PHF7 promotes spermatogenesis in XX germ cells when they are present in a male soma. PHF7 homologs are also specifically expressed in the mammalian testis, and human PHF7 rescues Drosophila Phf7 mutants. PHF7 associates with chromatin, and both the human and fly proteins bind histone H3 N-terminal tails with a preference for dimethyl lysine 4 (H3K4me2). We propose that PHF7 acts as a conserved epigenetic "reader" that activates the male germline sexual program.

The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin.

Activation and repression of transcription in eukaryotes involve changes in the chromatin fiber that can be accomplished by covalent modification of the histone tails or the replacement of the canonical histones with other variants. Here we show that the histone H2A variant of Drosophila melanogaster, H2Av, localizes to the centromeric heterochromatin, and it is recruited to an ectopic heterochromatin site formed by a transgene array. His2Av behaves genetically as a PcG gene and mutations in His2Av suppress position effect variegation (PEV), suggesting that this histone variant is required for euchromatic silencing and heterochromatin formation. His2Av mutants show reduced acetylation of histone H4 at Lys 12, decreased methylation of histone H3 at Lys 9, and a reduction in HP1 recruitment to the centromeric region. H2Av accumulation or histone H4 Lys 12 acetylation is not affected by mutations in Su(var)3-9 or Su(var)2-5. The results suggest an ordered cascade of events leading to the establishment of heterochromatin and requiring the recruitment of the histone H2Av variant followed by H4 Lys 12 acetylation as necessary steps before H3 Lys 9 methylation and HP1 recruitment can take place.

Trans-splicing as a novel mechanism to explain interallelic complementation in Drosophila.

Two mutant alleles of the same gene, each located in one of the two homologous chromosomes, may in some instances restore the wild-type function of the gene. This is the case with certain combinations of mutant alleles in the mod(mdg4) gene. This gene encodes several different proteins, including Mod(mdg4)2.2, a component of the gypsy insulator. This protein is encoded by two separate transcription units that can be combined in a trans-splicing reaction to form the mature Mod(mdg4)2.2-encoding RNA. Molecular characterization of complementing alleles shows that they affect the two different transcription units. Flies homozygous for each allele are missing the Mod(mdg4)2.2 protein, whereas wild-type trans-heterozygotes are able to synthesize almost normal levels of the Mod(mdg4)2.2 product. This protein is functional as judged by its ability to form a functional insulator complex. The results suggest that the interallelic complementation in the mod(mdg4) gene is a consequence of trans-splicing between two different mutant transcripts. A conclusion from this observation is that the trans-splicing reaction that takes place between transcripts produced on two different mutant chromosomes ensures wild-type levels of functional protein.