GENETIC ANALYSIS OF THE FUNCTION OF THE DROSOPHILA DOUBLESEX-RELATED FACTOR dmrt93B

DMRT (Doublesex and Mab-3 related transcription factor) proteins generally associated with sexual differentiation in many organisms share a common DNA binding domain and are often expressed in reproductive tissues. Aside from doublesex, which is a central factor in the regulation of sex determination, Drosophila possesses three different dmrt genes that are of unknown function. Because the association with sexual differentiation and reproduction is not universal and some DMRT proteins have been found to play other developmental roles we chose to further characterize one of these Drosophila genes. We carried out genetic analysis of dmrt93B, which was previously found to be expressed sex-specifically in the developing somatic gonad and to affect testis morphogenesis in RNAi knockdowns. In order to disrupt this gene, the GAL4 yeast transcriptional activator followed by a polyadenylation signal was inserted after the dmrt93B start codon and introduced into the genome by homologous recombination. Analysis of the knock-in mutation as well as a small deletion removing all dmrt93B sequence demonstrate that loss of function causes partial lethality at the late pupal stage. Surprisingly, these mutations have no significant effect on gonad formation or male fertility. Analysis of GAL4-driven GFP reporter expression indicates that the dmrt93B promoter activity is highly specific to neurons in the suboesophageal and proventricular ganglion in larva and adult of both sexes suggesting a possible role in digestive tract function. Using the Capillary Feeder (CAFÉ) assay to measure daily food intake we find that reduction in this gene’s function leads to an increase in food consumption. These results suggest dmrt93 plays an important role in the formation or maintenance of neurons that affect feeding and support the idea that dmrt genes may not be restricted to roles in sexual differentiation.

Negative regulation of Drosophila Transformer2 by SR splicing factors

The Drosophila Transformer-2 (Tra2) protein activates the splicing of doublesex and fruitless pre-mRNA and represses M1 intron splicing in its own RNA in male germline. The M1 retention is part of negative feedback mechanism that controls Tra2 protein synthesis. However it is not known how the M1 intron is repressed or why Tra2 activates splicing of some RNAs while repressing splicing in others. Here we show that Tra2 and SR protein Rbp1 function together to specifically repress M1 splicing in vitro through the same intronic silencer by binding independently to distinct sites. The role of Rbp1 in M1 repression in vivo was validated by the finding that increased expression of Rbp1 in S2 cells promotes M1 retention. Furthermore, Tra2 blocks prespliceosomal A complex formation, a step corresponding to U2 snRNP recruitment to the branchpoint. High levels of Tra2 repression require an upstream enhancer. Together, we propose that the complex formed by Tra2 and Rbp1 on the silencer achieves splicing repression by blocking the recognition of the branchpoint or antagonizing enhancer function. ^ In addition, both splicing regulatory activities of Tra2 are essential developmental events, doublesex splicing is the key for Drosophila sex determination in the soma, while M1 retention occurs in the male germline and is necessary for spermatogenesis. However, active Tra2 is expressed ubiquitously. So another issue we have studied is how Tra2 accomplishes negative and positive splicing regulation in a tissue-specific fashion. Surprisingly, we found that nuclear extract from somatically-derived S2 cells support M1 repression in vitro. This led us to hypothesize that no germline specific factor is required and that high levels of Tra2 expression in the male germline is sufficient to trigger M1 retention. To test it, I examined whether increased expression of Tra2 could promote M1 retention in cells outside male germline. My results show that increased Tra2 expression promotes M1 retention in somatically-derived S2 cells as well as in the somatic tissues of living flies. These results show that somatic tissues are capable of supporting M1 repression but do not normally do so because the low levels of Tra2 do not trigger negative feedback regulation. ^

A genetic characterization of {\it runt\/} activity during {\it Drosophila\/} embryogenesis

The Drosophila melanogaster gene runt encodes a novel transcriptional regulator that was originally identified on the basis of its key role in embryonic pattern formation. For my thesis I undertook a genetic analysis of runt activity to identify loci that interact with this unique transcriptional regulator. Specifically, I screened the genome with deficiencies for loci that interact with runt in a dose-dependent fashion during early embryogenesis. From this screen I discovered a vital dose-dependent interaction between runt and the achaete-scute complex (AS-C). The characterization of this interaction led to the exciting discovery of important roles for runt in sex determination and neurogenesis (Duffy and Gergen 1991, Duffy et al. 1991). I demonstrated that in sex determination runt is necessary for the normal transcriptional activation of the master sex-determining gene Sx1 and has all the properties of an X:A numerator element. I also showed that runt is required during the early stages of neurogenesis for the normal development of a subset of CNS ganglion mother cells and neurons. In addition, the screen, which focused on the identification and characterization of maternal loci that influence the activity of runt during segmentation, identified several new maternal loci, one of which affects the activity of the maternal posterior group genes on embryonic pattern formation. ^

Autoregulation of TRA-2 pre -mRNA splicing

One of the most elegant and tightly regulated mechanisms for control of gene expression is alternative pre-mRNA splicing. Despite the importance of regulated splicing in a variety of biological processes relatively little is understood about the mechanisms by which specific alternative splice choices are made and regulated. The transformer-2 (tra-2) gene encodes a splicing regulator that controls the use of alternative splicing pathways in the sex determination cascade of D. melanogaster and is particularly interesting because it directs the splicing of several distinct pre-mRNAs in different manners. The tra-2 protein positively regulates the splicing of both doublesex (dsx) and fruitless (fru) pre-mRNAs. Additionally tra-2 controls exuperantia (exu) by directing the choices between splicing and cleavage/polyadenylation and autoregulates the tra-2 pre-mRNA processing by repressing the removal of a specific intron (called M1). The goal of this study is to identify the molecular mechanisms by which TRA-2 protein affects the alternative splicing of pre-mRNA deriving from the tra-2 gene itself.^ The autoregulation of M1 splicing plays a key role in regulation of the relative levels of two functionally distinct TRA-2 protein isoforms expressed in the male germline. We have examined whether the structure, function, and regulation of tra-2 are conserved in Drosophila virilis, a species diverged from D. melanogaster by over 60 million years. We find that the D. virilis homolog of tra-2 produces alternatively spliced RNAs encoding a set of protein isoforms analogous to those found in D. melanogaster. When introduced into the genome of D. melanogaster, this homolog can functionally replace the endogenous tra-2 gene for both normal female sexual differentiation and spermatogenesis. Examination of alternative pre-mRNAs produced in D. virilis testes suggests that the germline-specific autoregulation of tra-2 function is accomplished by a strategy similar to that used in D. melanogaster.^ To identify elements necessary for regulation of tra-2 M1 splicing, we mutagenized evolutionarily conserved sequences within the tra-2 M1 intron and flanking exons. Constructs containing these mutations were used to generate transgenic fly lines that have been tested for their ability to carry out autoregulation. These transgenic fly experiments elucidated several elements that are necessary for setting up a context under which tissue-specific regulation of M1 splicing can occur. These elements include a suboptimal 3$\sp\prime$ splice site, an element that has been conserved between D. virilis and D. melanogaster, and an element that resembles the 3$\sp\prime$ portion of a dsx repeat and other splicing enhancers.^ Although important contextual features of the tra-2 M1 intron have been delineated in the transgenic fly experiments, the specific RNA sequences that interact directly with the TRA-2 protein were not identified. Using Drosophila nuclear extracts from Schneider cells, we have shown that recombinant TRA-2 protein represses M1 splicing in vitro. UV crosslinking analysis suggests that the TRA-2 protein binds to several different sites within and near the M1 intron. ^

Hsp110 chaperones control client fate determination in the hsp70-Hsp90 chaperone system.

Heat shock protein 70 (Hsp70) plays a central role in protein homeostasis and quality control in conjunction with other chaperone machines, including Hsp90. The Hsp110 chaperone Sse1 promotes Hsp90 activity in yeast, and functions as a nucleotide exchange factor (NEF) for cytosolic Hsp70, but the precise roles Sse1 plays in client maturation through the Hsp70-Hsp90 chaperone system are not fully understood. We find that upon pharmacological inhibition of Hsp90, a model protein kinase, Ste11DeltaN, is rapidly degraded, whereas heterologously expressed glucocorticoid receptor (GR) remains stable. Hsp70 binding and nucleotide exchange by Sse1 was required for GR maturation and signaling through endogenous Ste11, as well as to promote Ste11DeltaN degradation. Overexpression of another functional NEF partially compensated for loss of Sse1, whereas the paralog Sse2 fully restored GR maturation and Ste11DeltaN degradation. Sse1 was required for ubiquitinylation of Ste11DeltaN upon Hsp90 inhibition, providing a mechanistic explanation for its role in substrate degradation. Sse1/2 copurified with Hsp70 and other proteins comprising the "early-stage" Hsp90 complex, and was absent from "late-stage" Hsp90 complexes characterized by the presence of Sba1/p23. These findings support a model in which Hsp110 chaperones contribute significantly to the decision made by Hsp70 to fold or degrade a client protein.

Cytochrome P450 in mammalian brain: Purification, anatomical distribution and regulation of the cytochrome P450 monooxygenase system in rat brain

The cytochrome P450 (P450) monooxygenase system plays a major role in metabolizing a wide variety of xenobiotic as well as endogenous compounds. In performing this function, it serves to protect the body from foreign substances. However, in a number of cases, P450 activates procarcinogens to cause harm. In most animals, the highest level of activity is found in the liver. Virtually all tissues demonstrate P450 activity, though, and the role of the P450 monooxygenase system in these other organs is not well understood. In this project I have studied the P450 system in rat brain; purifying NADPH-cytochrome P450 reductase (reductase) from that tissue. In addition, I have examined the distribution and regulation of expression of reductase and P450 in various anatomical regions of the rat brain.^ NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by SDS-PAGE and Western blot techniques. Kinetic studies utilizing cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P4501A1 as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. These results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.^ Since the brain is not a homogeneous organ, dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monooxygenase system in brain is important in elucidating its function in that organ. Related to this is the regulation of P450 expression in brain. In order to study these issues female rats--both ovariectomized and not--were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and reductase determined using polymerase chain reaction. Results of this study indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR has allowed the determination of factors affecting the expression of message for these enzymes. ^