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.
The work in our laboratory during the last decade is divided into several basic areas (pairing and synapsis, recombination, and segregation), each of which is described briefly below.
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 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 role of the protein Corolla in the central region of the SC
In a screen to isolate novel SC components in Drosophila, we recently identified the protein Corolla, which exhibits one of the strongest chromosome missegregation phenotypes isolated to date. The corolla gene is located on the X chromosome, and corolla mutants are defective in SC assembly, as assayed by localization of the transverse filament protein C(3)G and the central element component Cona. Specifically, mutants in Corolla are unable to build full-length SC, and the aberrant SC structure precociously disassembles. While Cona is required to localize Corolla to the SC, surprisingly, some Corolla localization persists in c(3)G mutants. Similar to other SC mutants, corolla mutants exhibit defective centromere clustering and a severe defect in recombination. Using structured illumination microscopy we determined that Corolla is a component of the central region of the SC, and a yeast two-hybrid assay indicates that Corolla strongly interacts with Cona. This demonstrates the first direct interaction between two inner-SC proteins in Drosophila. We are continuing to characterize Corolla and its role in SC structure and function.
The role of the SC protein C(2)M in the structure and function of the lateral elements
C(2)M has been described as a lateral element (LE) component of the SC. In female c(2)M mutants, meiosis does not progress properly and such individuals exhibit severe crossover and segregation defects. We are currently working by various approaches to elucidate the exact role of C(2)M protein in SC assembly and organization, as well as its precise localization pattern onto chromosomes.
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
Although Drosophila has long been a powerful tool for classical genetic mapping, 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, rosy. 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 and have precisely mapped recombination (crossover) sites to small physical intervals. Using this novel method we were also able to identify a 7-bp sequence motif, GTGGAAA, enriched near crossovers but not near gene conversions. We are currently expanding the scope of this work to identify more recombination and gene conversion locations as well as attempting to identify proteins that may have an affinity for this motif.
The molecular biology of the homologous segregation process:
The mechanisms of 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 and resolved and to function. Specifically we are driving to understand the role topoisomerases play in resolving heterochromatic associations. We are also investigating whether thread formation or maintenance is altered in known meiotic mutants.
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 understanding precisely why the achiasmate segregation system is specifically sensitive to decreasing the dose of mtrm and how Mtrm and Polo physically interact in vivo.
The role of the SC in chromosome segregation in XXY female Drosophila
Nonhomologous heterochromatic associations that take place during early meiosis need to be disrupted in order for proper chromosome segregation to take place. One method of disrupting these associations is through the formation of crossovers. XXY female Drosophila provide an excellent model system in which to study both X<–>X and XX<–>Y chromosome segregation in the absence of recombination, which is mediated by heterochromatic associations. Previously, we found that these heterochromatic associations are present in both recombinant and non-recombinant genotypes in early prophase, but persist until late prophase only when recombination does not take place. This suggests that the presence of a crossover can disrupt heterochromatic associations. We are investigating the role of the synaptonemal complex in mediating heterochromatic associations. In preliminary data, we have found that the lasting heterochromatic associations in non-recombinant genotypes no longer occur in the absence of the SC. This suggests that the synaptonemal complex is important for promoting lasting heterochromatic associations, particularly in late prophase.
Discovery of B chromosomes in Drosophila melanogaster
B chromosomes are small, nonrecombinant chromosomes that are not required for the viability of a species. We have identified a strain of Drosophila melanogaster containing B chromosomes that can be maintained in a wild-type background, despite being mitotically unstable. Fluorescent in situ hybridization reveals that these chromosomes are largely, if not entirely, composed of 4th chromosome heterochromatic sequences, and indirect immunofluorescence indicates the presence of centromeres. The further investigation of B chromosomes in a highly tractable genetic system like that of D. melanogaster could provide insight into chromosome formation and segregation, as well as shed light on issues that arise when extra or unstable chromosomes are present.
Continuing the search for meiotic mutants:
A germline clone screen to identify new meiotic mutants
We recently performed 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, which 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 will produce progeny in this cross. We are currently characterizing mutants from this screen—including the corolla mutant described above—and plan to expand the screen to identify new meiotic mutants on chromosomes 2R and 3L.
Adding a new experimental system:
The genetic and cytological analysis of meiosis in planarians
Although planarians are extensively used in regeneration studies, we have 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.