Hawley Lab R. Scott Hawley, Ph.D. Investigator and American Cancer Society Research Professor rsh@stowers.org

     We focus on elucidating the mechanisms by which chromosomes pair, the mechanism and control of recombination, and the mechanisms by which chiasmata ensure segregation. All 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 work in our laboratory during the last eight years is divided into several basic areas (pairing and recombination, segregation, and control of meiotic progression), each of which is described briefly below.

Studies of Pairing and Recombination

The role of the replication protein Mcm5 in meiotic recombination (Cathy Lake)

     Members of the mini-chromosome maintenance (MCM) family have pivotal roles in many biological processes. Although originally studied for their role in DNA replication, it is becoming increasingly apparent that certain members of this family are multifunctional and also play roles in transcription, cohesion, condensation, and recombination. In a genetic screen engineered to identify mutations in essential genes we recovered a meiosis-specific allele of mcm5 which we demonstrated in Lake et al., 2007 an unexpected function for this protein. First, we showed that homozygotes for a null allele of mcm5 die as third instar larvae, apparently as a result of blocking those replication events that lead to mitotic divisions without impairing endo-reduplication. However, we have also recovered a viable and fertile allele of mcm5 (denoted mcm5[A7]) that specifically impairs the meiotic recombination process. We demonstrate that the decrease in recombination observed in females homozygous for mcm5[A7] is not due to a failure to create or repair meiotically induced double strand breaks (DSBs), but rather to a failure to resolve those DSBs into meiotic crossovers. Consistent with their ability to repair meiotically induced DSBs, flies homozygous for mcm5[A7] are fully proficient in somatic DNA repair. These results strengthen the observation that members of the pre-replicative complex have multiple functions and provide evidence that mcm5 plays a critical role in the meiotic recombination pathway. We are now trying to elucidate the mechanism by which Mcm5 acts during the process of crossingover.

The role of the zinc finger protein Trade Embargo in promoting meiotic recombination (Cathy Lake and Rachel Nielsen)

     An allele of trade embargo (CG4413) was uncovered in the course of a genetic screen engineered to identify meiosis-defective mutations in both essential and non-essential genes Page et al. 2007). This mutant, trem[F9], shows high levels of X chromosome nondisjunction as a consequence of the large decrease in recombination. InDel and deficiency mapping placed the mutation within the interval 92A2-93F4. A pBac insertion in the 5’ end of CG4413 failed to complement trem[F9]. Sequence analysis of the trem[F9] mutant reveals a single C-T transition when compared to the target chromosome. This mutation changes a proline to a leucine. trem is predicted to encode a zinc finger protein and is located in a cluster of four zinc finger protein encoding genes. Trem[F9] mutation lies in a conserved residue in the linker region between the first and second zinc finger domains. Oocytes homozygous for this mutation show normal localization of the synaptonemal complex protein C(3)G; however, trem[F9] is defective in both double strand break formation and recombination. We have analyzed five additional trem alleles that were obtained through TILLING. Two alleles which mutate critical residues in the first zinc finger domain also show high levels of X nondisjunction indicating that the first zinc finger and linker region are important for its function during the meiotic recombination process. We are currently in the process of determining the role(s) of the Trem protein in the meiotic recombination pathway.

Changes in the structure of the oocyte nucleus and function during Drosophila meiosis (Satomi Takeo)

     The oocyte nucleus undergoes dynamic structural changes during meiotic prophase I in Drosophila. At the earliest stage of prophase I, homologous chromosomes are paired, synapsed and recombined. The synaptonemal complex (SC) which physically holds homologs plays critical roles to ensure proper recombination and subsequent chromosome segregation. After recombination, the SC starts to dissemble and chromosomes separate from the nuclear envelope (NE) and become a compact structure, called the karyosome. The karyosome is thought to be required to build a single bipolar spindle at prometaphase I after nuclear envelope breakdown (NEB). Interestingly, there are particular stages at which the karyosome is transiently decondensed and then recondensed before NEB. Thus, it is likely that these changes in the structure of the oocyte nucleus are closely related to meiotic and developmental events.

     Our research is to understand the mechanism underlying these dynamic changes in the nucleus structure and their functional importance in cellular processes during oogenesis. We have performed careful evaluation of structure of oocyte nucleus at every stage by immunostaining for the SC, NE and chromosomes. Currently, we are focusing on how the events at the earliest prophase I stage such as pairing, synapsis and recombination can be achieved in terms of the nucleus structure including chromosome movement, as well as genetic analysis of some mutants. We are also interested in the stage where the karyosome is decondensed. Since it is known that a short burst of transcription activity occurs at this stage, this karyosome decondensation might be related to RNA synthesis in the oocyte nucleus. It would be worthwhile to identify genes that probably have to be produced transiently and specifically in the oocyte but not in nurse cells that can supply oocytes with large amount of RNA and proteins required for oocyte growth and differentiation.

The mechanisms of synapsis and synaptonemal complex (SC) formation and function during meiosis (Heather Hall and Justin Blumenstiel)

     The SC forms between homologous chromosomes during meiotic prophase and functions to regulate meiotic recombination and maintain chromosome pairing. The Drosophila C(3)G protein is structurally and functionally similar to ZIP1 and SCP1 of yeast and mammals, respectively, and is a component of the transverse filaments of the SC (Page et al., 2001; Anderson et al., 2005). The secondary structure prediction for C(3)G shows a central coiled coil-rich (CC) domain flanked by N- and C-terminal globular domains. To identify functional domains of C(3)G, Jennifer has designed a full length (FL) wild type C(3)G expression construct and a series of six constructs that express C(3)G with an in-frame deletion of either of the globular domains, or portions of the CC domain. Deletion of the N-terminus, or the N-terminal end of the CC domain ablates the ability for C(3)G to promote synapsis and recombination. C(3)G lacking the C-terminal domain failed to localize along chromosomes but rather concentrated within a polycomplex-like structure that we have further analyzed by electron microscopy. These data demonstrate that the N- and C-termini of C(3)G play important, but distinct roles in C(3)G function. The C-terminus may be necessary for connection with the lateral elements of the SC. The N-terminus and, in particular, a CC region adjacent to the N-terminus could be important for the dimerization of C(3)G or connecting C(3)G homodimers across the SC central element. We have recently identified a novel SC protein, Corona, that is essential for polymerization of C(3)G into transverse filaments (Page et al. 2008) perhaps by stabilizing the interactions between the N-termini of C(3)G.

     We are now focused on the effects of a series of deletions that remove the middle region of c(3)G, the coiled coil domain. Preliminary studies of these constructs have indicated that, while still ablating the ability of C(3)G to promote pairing and synapsis, in-frame deletions of the central coiled-coil domain still allow high to normal levels of recombination. Thus, these deletions may suggest a role for C(3)G in executing reciprocal recombination that is independent of its role in mediating pairing and synapsis. Efforts to further examine the phenotypes of these constructs are now underway in the laboratory. We are also designing both genetic and molecular screens to identify C(3)G interacting proteins.

     Perhaps the most important question we can ask about the SC concerns its function. We propose that the SC serves to communicate to two homologous centromeres that they are connected by a crossover and thus facilitate their co-orientation. Evidence that crossingover does indeed ensure proper centromere pairings has already been provided in yeast by the Dawson and Roeder labs. Youbin Xiang studies of the process of secondary nondisjunction in Drosophila, in which two nonexchange X chromosomes can segregate from a Y chromosome while exchange chromosomes segregate from each other (ignoring the Y), suggest that exchanges alter the spatial relationships between centromeric regions long before nuclear envelope breakdown, and in doing so set up centromere co-orientation. Therefore, we propose that the primary function of the SC is to communicate distal exchanges to the centromeres (Xiang and Hawley, 2006).

Studies of Achiasmate Segregation

Understanding the mechanisms controlling the segregation of achiasmate chromosomes using live-imaging techniques (Stacie Hughes)

     Live-imaging techniques have been used by the Hawley lab to visualize meiosis I in both wild-type oocytes. These studies have revealed that achiasmate homologs undergo unexpected dynamic movements upon the meiotic spindle during late prometaphase, including crossing the metaphase plate, the re-association of homologs, and the eventual congression of achiasmate chromosomes back to the metaphase plate. These movements are likely facilitated by heterochromatic threads that connect achiasmate chromosomes (see below). Live-imaging has also been used to examine the timing of GVBD, the segregation of achiasmate chromosomes, and the control of anaphase entry in several mutant backgrounds. Future live-imaging studies include examining oocytes with chromosome aberrations and defects in recombination.

Axs and the sheath-like structure that surrounds the meiotic spindle (Stacie Hughes)

     A dominant mutation in Aberrant X segregation (Axs^D) was isolated, which increased achiasmate chromosome nondisjunction during female fly meiosis without affecting the incidence of nondisjunction of chiasmate chromosomes. The Axs gene was found to encode a member of a new family of transmembrane proteins that includes members in all major kingdoms except Monera. The 8 transmembrane domain protein encoded by the Axs gene localizes to the endoplasmic reticulum until germinal vesicle breakdown. Axs then localizes to a novel membranous sheath encasing the meiotic spindle and chromosomes. The Axs^D mutation also causes defects in meiotic chromosome positioning, spindle structure and increases the incidence of anaphase. These phenotypes suggest that the membranous sheath is important for holding the chromosomes in place during meiosis, for focusing the microtubules on the developing spindle, and for controlling the meiotic cell cycle.

     Future research questions to be pursued include, “Where does the sheath surrounding the meiotic spindle come from and what are other components of this sheath?” Biochemical and candidate approaches should yield additional components of the sheath and suggest from where the components are recruited. By using electron microscopy we hope to determine the fine structure of the meiotic sheath, as well as get the first detailed picture of the metaphase spindle in Drosophila oocytes. An overexpression screen is being conducted for suppressors of the Axs^D overexpression phenotypes. Candidates identified in this screen will likely yield genes that interact functionally with Axs and potentially reveal new regulators in the control of spindle assembly, achiasmate chromosome segregation, and meiotic cell cycle control. Finally live-imaging is being used to investigate the potential function of Axs in calcium regulation during meiosis I.

Characterization of Nod, the Drosophila chromokinesin-like protein required for chromosome segregation (Kimberly Collins)

     During meiosis I, chromosomes that have undergone recombination are held together on the metaphase plate by physical linkages called chiasmata. This linkage prevents the aberrant and precocious segregation of chromosomes. In the absence of chiasmata, a mechanism called the distributive system functions to maintain achiasmate chromosomes on the metaphase plate until anaphase I. An essential component of the distributive system in Drosophila is a chromokinesin-like protein called Nod. Nod is conserved throughout higher eukaryotes and functions in the anti-poleward force that maintains achiasmate chromosomes on the metaphase plate. In Drosophila, the tiny fourth chromosomes are always achiasmate and the X chromosomes are achiasmate in 5-10% of meioses. Critically, the anti-poleward force, known as the polar ejection force, is essential to counteract the poleward force exerted on co-oriented chromosomes by kinetochore microtubules. The balance between the poleward and anti-poleward forces retains chromosomes on the metaphase plate.

     We are interested in identifying genes that regulate Nod function to better understand questions such as: how are the polar ejection forces at the metaphase to anaphase I transition regulated? To identify genes that regulate Nod function, we performed a screen looking for deficiencies which modify the reduced eye phenotype caused by an antimorphic allele, called nod^DTW. We have identified eight deficiencies which modify both the mitotic eye defect and the meiotic defect caused by nod^DTW and we are currently mapping the two strongest mutants. Also to identify genes which regulate Nod, we are conducting a screen for autosomal dominant suppressors of nod^DTW's cold sensitive phenotype.

Studies on the Control of Meiotic Progression

Roles of Ald/Mps1 in Drosophila female meiosis (Bill Gilliland)

     These projects are centered around the roles of altered disjunction (ald), which is the fly homolog of a conserved kinetochore-associated kinase called monopolar spindles 1 (mps1) that is involved in the spindle assembly checkpoint (Gilliland et al 2005). This gene is required for the maintenance of sister chromatid cohesion during prometaphase; without it, cohesion is lost soon after spindle formation, and sister chromatids separate precociously and appear to bypass metaphase arrest (Gilliland et al 2007). The ald mutant effects also include the observation of DAPI staining threads between homologs (Gilliland et al 2005). This observation has led to an answer for a long-standing problem in Drosophila female meiosis; it has been known for some time that heterochromatin is necessary and sufficient for achiasmate chromosomes to pair with and segregate from their homologs, but what is the mechanism that maintains that association? We propose that heterochromatic associations establish DNA-DNA linkages, which are exposed by the precocious separation in ald mutants. We also propose that these threads persist while achiasmate chromosomes are separated on the spindle, and allow homologs to successfully co-orient on the spindle (manuscript in preparation). In addition to its role as a kinetochore component, Ald protein was found to localize with another kinetochore component, Polo, to numerous long filaments throughout the oocyte cytoplasm (Gilliland et al 2007). We are working to identify what these filaments are composed of through immunoprecipitation and immunogold electron microscopy.

The mtrm gene encodes a protein that interacts with Polo and acts to maintain G2 arrest in female meiosis (Youbin Xiang)

     The Drosophila Matrimony (Mtrm) protein is expressed specifically in oocytes from the end of pachytene until the end of first meiotic prophase, an event defined by nuclear envelope breakdown (NEB). Loss-of-function matrimony (mtrm) mutants result in defects in achiasmate chromosome segregation and precocious NEB. Mutants in the mtrm gene exhibit dosage-sensitive effects on achiasmate segregation in Drosophila oocytes, as evidenced by high levels of achiasmate nondisjunction in mtrm heterozygotes. Fluorescent in-situ hybridization showed that achiasmate nondisjunction in mtrm heterozygotes results from a defect in the ability to ensure proper centromere co-orientation at metaphase. Consistent with multiple roles in the control of meiotic and mitotic cell cycles, Mtrm protein interacts with Polo kinase. The polo alleles strongly suppress the meiotic defects in mtrm/+ heterozygotes. Over-expression of Polo in mtrm heterozygotes leads to female sterility.

     Co-immunoprecipitation experiments reveal that Mtrm physically interacts with Polo kinase (Polo) in vivo, and multidimensional protein identification technology mass spectrometry analysis reveals that Mtrm binds to Polo with an approximate stoichiometry of 1:1. Mutation of a Polo-Box Domain (PBD) binding site in Mtrm ablates the function of Mtrm and the physical interaction of Mtrm with Polo. The meiotic defects observed in mtrm/+ heterozygotes are fully suppressed by reducing the dose of polo+, demonstrating that Mtrm acts as an inhibitor of Polo. Mtrm acts as a negative regulator of Polo during the later stages of G2 arrest. Therefore, both the repression of Polo expression until stage 11 and the inactivation of newly synthesized Polo by Mtrm until stage 13 play critical roles in maintaining and properly terminating G2 arrest. Exploring the mechanism by which Mtrm inhibits Polo kinase to control the timing of GVBD and it’s function with respect to achiasmate chromosome segregation are the major goals of Youbin’s research efforts.

Characterizing the mechanism by which Matrimony interacts with Polo kinase (Kendall Smith)

     Xiang and others in the lab have already shed considerable light on the Mtrm-Polo interaction (Xiang et al., 2007), but additionally, and perhaps more importantly, the work has generated many new questions and ideas regarding the precise mechanism by which Mtrm serves to inhibit Polo during Drosophila female meiosis. The purpose of this project is to follow up on several leads provided by Xiang et al. regarding the intricacies of the Mtrm-Polo kinase interaction. For example, in addition to showing that phosphorylation of MtrmT40 is critical for the Mtrm-Polo interaction, Xiang et al. demonstrated several other evolutionarily conserved sites on Mtrm to be reproducibly phosphorylated (MtrmS48 and MtrmS52) while in complex with Polo. The physiological significance of these phospho-serines, however, remains unclear. Interestingly, we found that mutating these residues to nonphosphorylateable alanine ablates the interaction of Mtrm and Polo kinase in a yeast two-hybrid system. In addition to independently confirming the critical role of these residues (and others) in the Mtrm-Polo interaction utilizing a baculovirus expression system, we are examining their functional significance in the context of the fly by observing the meiotic characteristics of oocytes from transgenic females expressing mutant versions of the Mtrm protein. One hypothesis that underscores our work is that differentially phosphorylated species of Mtrm regulate Polo kinase at multiple stages of oogenesis. This speculation could explain the seemingly functionally separable phenotypes observed (e.g., precocious nuclear envelope breakdown and achiasmate nondisjunction) when the balance between Mtrm and Polo is perturbed in the developing oocyte.

Finally, the very essence of our lab — screens for new meiotic mutants (everybody)

     Each of the epochs of our lab’s existence have been based on a screen for new meiotic mutants. At this time there are several new screens ongoing in the lab, including screens aimed at looking for genes whose protein products interact with Mtrm, Polo, Axs, and Nod. We are also characterizing a large collection of ts lethal mutants created by Krishna Bhat for meiotic defects.

Academic Appointments: Professor of Molecular and Integrative Physiology, School of Medicine, University of Kansas Medical Center; Adjunct Professor of Biological Sciences at the University of Missouri Kansas City; Adjunct Professor of Undergraduate Program in Biology, The University of Kansas