Seiji Hira

Genetically encoded system to track histone modification in vivo

Post-translational histone modifications play key roles in gene regulation, development, and differentiation, but their dynamics in living organisms remain almost completely unknown. To address this problem, we developed a genetically encoded system for tracking histone modifications by generating fluorescent modification-specific intracellular antibodies (mintbodies) that can be expressed in vivo. To demonstrate, an H3 lysine 9 acetylation specific mintbody (H3K9ac-mintbody) was engineered and stably expressed in human cells. In good agreement with the localization of its target acetylation, H3K9ac-mintbody was enriched in euchromatin, and its kinetics measurably changed upon treatment with a histone deacetylase inhibitor. We also generated transgenic fruit fly and zebrafish stably expressing H3K9ac-mintbody for in vivo tracking. Dramatic changes in H3K9ac-mintbody localization during Drosophila embryogenesis could highlight enhanced acetylation at the start of zygotic transcription around mitotic cycle 7. Together, this work demonstrates the broad potential of mintbody and lays the foundation for epigenetic analysis in vivo.

Innexin2 gap junctions in somatic support cells are required for cyst formation and for egg chamber formation in Drosophila

Germ cells require intimate associations with surrounding somatic cells during gametogenesis. During oogenesis, gap junctions mediate communication between germ cells and somatic support cells. However, the molecular mechanisms by which gap junctions regulate the developmental processes during oogenesis are poorly understood. We have identified a female sterile allele of innexin2 (inx2), which encodes a gap junction protein in Drosophila. In females bearing this inx2 allele, cyst formation and egg chamber formation are impaired. In wild-type germaria, Inx2 is strongly expressed in escort cells and follicle cells, both of which make close contact with germline cells. We show that inx2 function in germarial somatic cells is required for the survival of early germ cells and promotes cyst formation, probably downstream of EGFR pathway, and that inx2 function in follicle cells promotes egg chamber formation through the regulation of DE-cadherin and Bazooka (Baz) at the boundary between germ cells and follicle cells. Furthermore, genetic experiments demonstrate that inx2 interacts with the zero population growth (zpg) gene, which encodes a germline-specific gap junction protein. These results indicate a multifunctional role for Inx2 gap junctions in somatic support cells in the regulation of early germ cell survival, cyst formation and egg chamber formation. Inx2 gap junctions may mediate the transfer of nutrients and signal molecules between germ cells and somatic support cells, as well as play a role in the regulation of cell adhesion.

H3K36 Trimethylation-Mediated Epigenetic Regulation is Activated by Bam and Promotes Germ Cell Differentiation During Early Oogenesis in Drosophila

ABSTRACT Epigenetic silencing is critical for maintaining germline stem cells in Drosophila ovaries. However, it remains unclear how the differentiation factor, Bag-of-marbles (Bam), counteracts transcriptional silencing. We found that the trimethylation of lysine 36 on histone H3 (H3K36me3), a modification that is associated with gene activation, is enhanced in Bam-expressing cells. H3K36me3 levels were reduced in flies deficient in Bam. Inactivation of the Set2 methyltransferase, which confers the H3K36me3 modification, in germline cells markedly reduced H3K36me3 and impaired differentiation. Genetic analyses revealed that Set2 acts downstream of Bam. Furthermore, orb expression, which is required for germ cell differentiation, was activated by Set2, probably through direct H3K36me3 modification of the orb locus. Our data indicate that H3K36me3-mediated epigenetic regulation is activated by bam, and that this modification facilitates germ cell differentiation, probably through transcriptional activation. This work provides a novel link between Bam and epigenetic transcriptional control.

Binding of Drosophila maternal Mamo protein to chromatin and specific DNA sequences

Alterations in chromatin structure dynamically occur during germline development in Drosophila and are essential for the production of functional gametes. We had previously reported that the maternal factor Mamo, which contains both a BTB/POZ domain and C₂H₂ zinc-finger domains and is enriched in primordial germ cells (PGCs), is required for the regulation of meiotic chromatin structure and the production of functional gametes. However, the molecular mechanisms by which Mamo regulates germline development remained unclear. To evaluate the molecular function of Mamo protein, we have investigated the binding of Mamo to chromatin and DNA sequences. Our data show that Mamo binds to chromatin and specific DNA sequences, particularly the polytene chromosomes of salivary gland cells. Overexpression of Mamo affected the organization of polytene chromosomes. Reduction in maternal Mamo levels impaired the formation of germline-specific chromatin structures in PGCs. Furthermore, we found that the zinc-finger domains of Mamo directly bind to specific DNA sequences. Our results suggest that Mamo plays a role in regulating chromatin structure in PGCs.