The primary goal of our research is to understand the mechanisms that control meiotic chromosome behavior at the molecular level.
We focus on elucidating the mechanisms by which chromosomes pair, the mechanism and control of recombination, and the mechanisms that facilitate the faithful segregation of homologous chromosomes. Most of our studies begin with genetic screens to identify genes and chromosomal sites required for the meiotic process. Indeed, a reviewer of an American Cancer Society grant proposal once stated that we "have raised the genetic analysis of meiosis in Drosophila to an art form". The studies continue by analyzing the phenotypes of those mutants by both genetic and cytological methods (most notably characterizing meiosis in living oocytes). We then characterize the genes at the molecular level with the goal of elucidating the function and location of their protein products.
In addition to the analysis of Drosophila, we have also begun the characterization of meiosis in planaria. But for both organisms our goal remains the same, to understand how the molecular biology of meiosis explains Mendelian inheritance.
The work in our laboratory during the last decade is divided into several basic areas (pairing and recombination, segregation, and control of meiotic progression), each of which is described briefly below.
The molecular genetics of pairing and synapsis:
The role of centromere movement in the initiation of meiotic pairing and synapsis
Recent work in our lab suggests that the initiation of meiotic pairing and synapsis may be intimately associated with events that involve the centromeres. Moreover, these centromeric movements appear to be connected to the initiation of the assembly of the synaptonemal complex (SC). Our goal is to understand the mechanisms by which synapsis is initiated and the role of centromeres and their flanking heterochromatic regions in this process.
The role of the SC protein Cona in the assembly of the synaptonemal complex
The SC remains one of the most beautiful and enigmatic structures in the meiotic nucleus. While much is known about the proteins that comprise the SC, much remains to be learned about its function. Several years ago we identified a protein called Cona that comprises a critical part of a region of the SC known as the central region. Cona co-localizes with the SC transverse filament protein C(3)G and is required to bind transverse filaments together. We are using multiple approaches to determine just how Cona interacts with the transverse filaments and with other central element proteins. These approaches include a structure/function analysis of the Cona protein, genetic and biochemical screens for Cona-interactors, and an effort to purify intact synaptonemal complexes from Drosophila oocytes.
The molecular genetics of recombination:
The zinc finger protein Trem executes the first known step in promoting meiotic recombination
Although genetic recombination was discovered in Drosophila, and genetic mapping has been extensively done in this organism, very little is known about the mechanisms by which recombination is initiated or exchanges are distributed along the arms of chromosomes. We have recently identified a protein Trem that defines the first known function in the initiation of meiotic recombination. The Trem protein contains multiple zinc fingers and is associated with chromatin during the earliest stages of meiosis. Our long-term goal is to use Trem, and the proteins with which it interacts, to elucidate the mechanisms by which meiotic recombination is initiated in Drosophila.
The analysis of meiotic recombination and gene conversion by whole genome sequencing
As noted above, Drosophila has long been a powerful tool for classical genetic mapping. However, the precision of that mapping has not allowed us to identify those sequence motifs or chromatin structures that control the location of recombination. In addition, the analysis of gene conversion in Drosophila has been virtually limited to studies at one gene. We have performed crosses between highly inbred strains that differ by a very large number of single nucleotide polymorphisms and performed analysis of the progeny of those crosses by whole genome sequencing. These data have extended our understanding of gene conversion as well as precisely mapped recombination (crossover) sites to small physical intervals. We are currently conducting an analysis of the sequences that flank these sites with the hope of identifying sequence motifs that target the initiation of recombination.
The molecular biology of the homologous segregation process:
The mechanisms controlling chromosome movement during prometaphase I
We have previously shown that chromosomes that fail to undergo recombination (achiasmate chromosomes) display dynamic movements on the meiotic spindle during prometaphase I and that achiasmate X and 4th chromosomes are connected by novel heterochromatic threads. We are endeavoring to better understand the mechanisms controlling these chromosome movements as well as to elucidate the mechanisms that allow DNA threads to be formed, resolved and function. We are also investigating whether thread formation or maintenance is altered in known meiotic mutants. Additionally, we are examining the structure of these threads by looking for the presence of specific chromatin modifications that associate with them.
Genetic studies of the role of the Axs ion-channel protein in mediating the progression of prometaphase I
The Axs gene encodes an ion channel protein that localizes to a novel meiotic sheath that envelopes the first meiotic spindle. Dominant mutations in the Axs gene cause the missegregation of achiasmate chromosomes as well as abnormal spindles and improper chromosome alignment during prometaphase I. However, and quite surprisingly, a mutation that eliminates the Axs gene causes no discernable phenotype, possibly due to redundancy with another gene. To identify genes are functionally redundant with Axs we initiated a forward genetic EMS screen for mutations that cause phenotypes that differ in severity depending on whether the functional Axs protein is present. Two of these mutations show a strong genetic interaction with a null allele of the Axs gene. The genes defined by these mutations are now being identified, with the goal of determining and localizing their protein products.
Analyzing the function and regulation of Calcineurin in controlling the completion of female meiosis
Calcineurin, a Ca2+/calmodulin-dependent phosphatase, and its regulator Sra/RCAN (regulator of calcineurin) play an essential role in controlling the completion of the first meiotic division. Oocytes lacking these proteins are unable to complete meiosis and laid eggs show a meiotic arrested chromosome configuration at anaphase I. We are currently studying how calcineurin controls completion of meiosis by using genetic, biochemical and cytological approaches. After showing that calcineurin is positively regulated by a Sra phosphorylation-dependent mechanism, we are currently studying how calcineurin controls completion of meiosis by using genetic, biochemical and cytological approaches.
This work not only provides insights into a role for calcineurin signaling that appears to be evolutionarily conserved from flies to vertebrates, but also contributes to understanding the mechanism by which the activity of calcineurin is regulated by Sra/RCAN.
The role of the Mtrm protein in the meiotic regulation of Polo kinase
The Mtrm protein was initially identified on the basis of its dosage-dependent role in mediating the segregation of achiasmate chromosomes. We have subsequently shown that Mtrm acts by binding to Polo kinase in a fashion that appears to antagonize or delay Polo function. We have also shown that the binding of Mtrm to Polo occurs by a non-canonical mechanism. We are now focused on both purifying the Mtrm::Polo complex and on understanding precisely why the achiasmate segregation system is specifically sensitive to decreasing the dose of Mtrm.
Continuing the search for meiotic mutants:
A germline clone screen to identify new meiotic mutants
We are performing a large-scale germline clone screen to obtain new X chromosomal meiotic mutants. The advantage of doing a germline clone screen is that it allows recovery of sterile or lethal mutants that cannot be isolated from traditional meiotic mutant screens that require females to be both viable and fertile. We screen for mutants that create a severe defect in meiotic chromosome segregation by crossing them to males that carry a compound autosome. Only mutants strong enough to generate high levels of autosomal missegregation willproduce progeny in this cross. The first phase of the screen will be completed in summer 2011 and the analysis of new mutants will begin in earnest in the fall.
Adding a new experimental system:
The genetic and cytological analysis of meiosis in planaria
Although planaria are extensively used in regeneration studies, we have recently begun to use them to study meiosis. The ease with which RNAi can be used to disrupt the function of meiotic genes and the extraordinary cytology that is possible in both male and female gametogenesis make this organism an excellent system in which to explore the biology of meiosis. Our work with planaria is focused on the analysis of three meiotic events: the role of telomere clustering in the initiation of pairing and synapsis, the control of the initiation of meiotic recombination, and the regulation of the position of recombination events.