Tulle Hazelrigg

The Destinies and Destinations of RNAs

Thanks to Larry Etkin and Howard Lipshitz for organizing a very informative and rewarding meeting. Larry Etkin, Howard Lipshitz, and Rob Singer provided helpful comments on the entire manuscript, and several of the meeting participants provided helpful information for specific sections. Thanks to all of them, to Ava Brent for her help in preparing Figure 1Figure 1, and to the people who contributed images for this figure, including Malgosia Kloc and Larry Etkin, Elizabeth Gavis, Shailesh Shenoy and Rob Singer, Julie Zhu and Edward Kislauskis, Gavin Wilkie and Ilan Davis, and Maritza Martinez and Gary Bassell.

Histone methylation is required for oogenesis in Drosophila.

SET domain proteins are histone lysine methyltransferases (HMTs) that play essential roles in development. Here we show for the first time that histone methylation occurs in both the germ cells and somatic cells of the Drosophila ovary, and demonstrate in vivo that an HMT, the product of the eggless (egg) gene, is required for oogenesis. Egg is a SET domain protein that is similar to the human protein SETDB1 and its mouse ortholog ESET. These proteins are members of a small family of HMTs that contain bifurcated SET domains. Because depletion of SETDB1 in tissue culture cells is cell-lethal, and an ESET mutation causes very early periimplantation embryonic arrest, the role of SETDB1/ESET in development has proven difficult to address. We show that egg is required in the Drosophila ovary for trimethylation of histone H3 at its K9 residue. In females bearing an egg allele that deletes the SET domain, oogenesis arrests at early stages. This arrest is accompanied by reduced proliferation of somatic cells required for egg chamber formation, and by apoptosis in both germ and somatic cell populations. We propose that other closely related SET domain proteins may function similarly in gametogenesis in other species.

Domains of Heterochromatin Protein 1 Required for Drosophila melanogaster Heterochromatin Spreading

Centric regions of eukaryotic genomes are packaged into heterochromatin, which possesses the ability to spread along the chromosome and silence gene expression. The process of spreading has been challenging to study at the molecular level due to repetitious sequences within centric regions. A heterochromatin protein 1 (HP1) tethering system was developed that generates “ectopic heterochromatin” at sites within euchromatic regions of the Drosophila melanogaster genome. Using this system, we show that HP1 dimerization and the PxVxL interaction platform formed by dimerization of the HP1 chromo shadow domain are necessary for spreading to a downstream reporter gene located 3.7 kb away. Surprisingly, either the HP1 chromo domain or the chromo shadow domain alone is sufficient for spreading and silencing at a downstream reporter gene located 1.9 kb away. Spreading is dependent on at least two H3K9 methyltransferases, with SU(VAR)3-9 playing a greater role at the 3.7-kb reporter and dSETDB1 predominately acting at the 1.9 kb reporter. These data support a model whereby HP1 takes part in multiple mechanisms of silencing and spreading.

The Drosophila microtubule-associated protein mini spindles is required for cytoplasmic microtubules in oogenesis

The XMAP215/TOG family of proteins is a closely related set of MAPs (microtubule-associated proteins) found in animals, yeast, and plants . In yeast and animal cells, the XMAP215/TOG proteins are required for both mitosis and meiosis. Although effects of XMAP215/TOG proteins on cytoplasmic microtubules have not previously been shown in animal cells, in plants the Arabidopsis family member MOR1 is required for the organization of cortical microtubule arrays . The Drosophila family member, encoded by the mini spindles (msps) gene, is maternally expressed and loaded into the egg, where it is an essential component of meiotic and mitotic spindles . Here we show that msps is also required during oogenesis for the structure and function of cytoplasmic microtubules. Localization of bicoid (bcd) mRNA in the oocyte is a microtubule-mediated event . We show that bcd RNA localization is defective in msps mutants. We also identify defects in cytoplasmic microtubules in both the germ and follicle cells of mutant ovaries and determine the expression pattern of msps mRNA and protein in developing egg chambers. Our findings reveal a new role for msps in cell patterning and raise the possibility that other family members may perform similar functions.

Epigenetic regulation of oogenesis and germ stem cell maintenance by the Drosophila histone methyltransferase Eggless/dSetDB1

The Drosophila melanogaster histone lysine methyltransferase (HKMT) Eggless (Egg/dSETDB1) catalyzes methylation of Histone H3 lysine 9 (H3K9), a signature of repressive heterochromatin. Our previous studies showed that H3K9 methylation by Egg is required for oogenesis. Here we analyze a set of EMS-induced mutations in the egg gene, identify the molecular lesions of these mutations, and compare the effects on oogenesis of both strong loss-of-function and weak hypomorphic alleles. These studies show that H3K9 methylation by Egg is required for multiple stages of oogenesis. Mosaic expression experiments show that the egg gene is not required intrinsically in the germ cells for their early differentiation, but is required in the germ cells for their survival past stage 5 of oogenesis. egg is also required in germ stem cells for their maintenance, since egg- germ stem cells initially survive but are not maintained as females age. Mosaic analysis also reveals that the early egg chamber budding defects in egg- ovaries are due to an intrinsic requirement for egg in follicle stem cells and their descendents, and that egg plays a non-autonomous role in somatic cells in the germarium to influence the differentiation of early germ cells.

A male-specific 3′-UTR regulates the steady-state level of theexuperantia mRNA during spermatogenesis in Drosophila

TheDrosophila exuperantia gene (exu) functions in both oogenesis and spermatogenesis. Alternative RNA processing and promoter usage generates sex-specific transcripts which differ in their 5′ and 3′ untranslated regions, but encode the same predicted protein. We have sequenced the breakpoints of anexu allele which is defective in spermatogenesis but functions normally in oogenesis. This allele deletes most of the sequence specific to the male 3′-UTR, together with some flanking DNA, and causes a reduction in steady-state level ofexu mRNA in the testis. In addition, we find that a smaller deletion which removes only sequence within the male 3′-UTR reduces the steady-state level of the mRNA and prevents anexu transgene from rescuing male sterility. Males carrying multiple copies of this transgene are fertile, suggesting that the male-specific 3′-UTR functions to maintain a proper level ofexu product in the germline.

Lost in Translation Gets an oskar

Nuclear history affects the fates of mRNAs in the cytoplasm of cells. Proteins loaded onto mRNAs in the nucleus mark RNAs for subsequent translational regulation, stability, degradation, and subcellular RNA localization. New results show that the Drosophila heterogeneous nuclear ribonucleoprotein (hnRNP) Hrp48 contributes to coordinated RNA localization and translational control in oocytes.