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Fruitless Recruits Two Antagonistic Chromatin Factors to Establish Single-Neuron Sexual Dimorphism

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Fruitless Recruits Two Antagonistic Chromatin Factors to Establish Single-Neuron Sexual Dimorphism Fruitless Recruits Two Antagonistic Chromatin Factors to Establish Single-Neuron Sexual Dimorphism Hiroki Ito,1 Kosei Sato,1 Masayuki Koganezawa,1,4 Manabu Ote,1,4 Ken Matsumoto,2 Chihiro Hama,3 and Daisuke Yamamoto1,* 1Division of Neurogenetics, Tohoku University Graduate School of Life Sciences, Sendai 980-8577, Japan 2Japan Advanced Institute of Science and Technology, Nomi, Ishikawa 923-1292, Japan 3Faculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japan 4These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.cell.2012.04.025 SUMMARY for phenotypic modifiers of fru in the compound eye yielded several factors involved in transcription machineries, including The Drosophila fruitless (fru) gene encodes a set of Bonus (Bon), a homolog of the mammalian TIF1 family of tran- putative transcription factors that promote male scriptional cofactors. Here, we demonstrate that Bon physically sexual behavior by controlling the development of associates with Fru in vivo, and the Fru-Bon complex recruits sexually dimorphic neuronal circuitry. However, the a chromatin regulator, Heterochromatin protein 1a (HP1a) or mechanism whereby fru establishes the sexual fate Histone deacetylase 1 (HDAC1), to discrete chromosomal sites. of neurons remains enigmatic. Here, we show that A reduction in HDAC1 activity enhanced the demasculinizing activity of fru mutations on the mAL neurons, whereas a reduction Fru forms a complex with the transcriptional cofactor in HP1a activity counteracted it. We propose that Fru bound to Bonus (Bon), which, in turn, recruits either of its target sites alters the chromatin configuration in collaboration two chromatin regulators, Histone deacetylase 1 with Bon, HP1a, and HDAC1, thereby orchestrating transcription (HDAC1), which masculinizes individual sexually of a series of downstream genes that function to determine the dimorphic neurons, or Heterochromatin protein 1a development of gender-typical neural circuitry. (HP1a), which demasculinizes them. Manipulations of HDAC1 or HP1a expression change the proportion RESULTS of male-typical neurons and female-typical neurons rather than producing neurons with intersexual char- bon as a fru Modifier The fru primary transcript from the distal-most promoter (P1) is acteristics, indicating that on a single neuron level, 0 0 this sexual switch operates in an all-or-none manner. subject to alternative splicing at the 5 and 3 portions (Figures 1A and 1B) and expressed primarily in the nervous system. The pattern of splicing at exon 2 is sex dependent, producing INTRODUCTION either disabled mRNAs in females or functional male-specific mRNAs (Usui-Aoki et al., 2000; Lee et al., 2000). The FruM Gender-related differences in a given behavior have as their proteins encoded by the male-specific mRNAs have an basis the sexual dimorphism in structure and function of the N-terminal extension of 101 amino acids in length (herein neural circuitry underlying that behavior. In Drosophila, salient referred to as ‘‘M’’ for male-specific), which is absent from the sexual dimorphism has been documented in identified inter- other Fru proteins (called FruCOM hereafter) encoded by non- neuron groups (Cachero et al., 2010; Kimura et al., 2005, 2008; sex-specific transcripts generated by other promoters (Ryner Ruta et al., 2010; Yu et al., 2010). The mAL neurons, for example, et al., 1996; Usui-Aoki et al., 2000)(Figures 1A and 1B). Approx- differ in number, projection, and dendritic field between the imately 2,000 neurons (2% of the entire neuronal population) in sexes. These sexual differences in mAL depend solely on the the male central nervous system (CNS) express FruM, whereas presence or absence of the protein products of fruitless (fru)(Ki- female CNS cells express neither FruM nor FruCOM (Figure S1 mura et al., 2005), the master regulator gene for male sexual available online). The fru gene has five alternative 30 exon usages behavior (Dickson, 2008). Despite its spectacular phenotypic each of which yields a different C terminus called A, B, C, D, or E, action on the brain and behavior, the mechanism of action of giving rise to variation in the number and sequence of Zn-finger the Fru protein in organizing the sex-specific neural circuitry motifs (Ryner et al., 1996; Usui-Aoki et al., 2000; Billeter et al., remains unclear. The Fru protein has an N-terminal BTB domain 2006)(Figures 1A and 1B). Thus, the type-BM Fru protein (FruBM) and two Zn-finger motifs in its C terminus, implying a role as carries the M sequence in its N terminus and the B sequence in a transcription factor (Ito et al., 1996; Ryner et al., 1996). Screens its C terminus. Cell 149, 1327–1338, June 8, 2012 ª2012 Elsevier Inc. 1327 Figure 1. bon Suppresses the Effect of Fru Overexpression (A) The fru locus. The P-insertions (triangles), promoters (P1–P4), exons (I–XI), and deletions (thick black [established] and dashed [ill-defined] lines) are indicated. Shown below are the transcripts with an alternative 30 end. The nomenclature of Fru isoforms in this study follows that originally proposed by Usui-Aoki et al. (2000). Types A, B, C, and E in our study correspond to isoforms A, C, D, and B of other research groups, respectively (Mellert et al., 2010; Billeter et al., 2006). (B) Exon-intron organization of types A, B, and E with different Zinc-finger motifs. The regions containing epitopes for the anti-Fru antibodies are indicated. (C) Endogenous Fru expression in the wild-type eye-antennal disc. (D) Strong expression of Fru in the postfurrow region (arrowhead) in the GMR-Gal4,UAS-fru-typeB+/+ disc. (E) A wild-type eye. (F) A GMR-Gal4,UAS-fru-typeB+/+ eye. (G) bon21B suppresses the phenotype in a GMR-Gal4,UAS-fru-typeB+/+; bon21B /+ fly. (H) The bon gene. The P-insertion (triangle) in bonS48 is indicated. Exon I is deleted (indicated by thick lines) in bon21B. The structure of the transcript and protein in bon is indicated below. Scale bars: 100 mm. See also Figure S1. Forced expression of fru in the developing eye disc driven by bon Modulates fru-Dependent Male Courtship GMR-Gal4 resulted in severe roughness (Figures 1C–1F). We To determine whether bon plays a role in mediating the sex- screened for genes that dominantly modify this gain-of-func- specific function of fru, the effect of bon21B on male courtship tion phenotype of fru (Goto et al., 2011) when introduced behavior was examined in the presence or absence of fru loss- into the fly as a heterozygous mutant allele or a UAS-fusion of-function mutations (Figure 2C). The level of male courtship transgene that drives expression of genomic sequence(s) via activity toward a target female was quantified by the courtship a Gene Search (GS) P-element insertion (Toba et al., 1999). index (CI), which represents the percent time spent by a male Screening of 1,364 fly lines yielded 40 suppressors of the fru on any one of the courtship steps before copulation in a 5 min gain-of-function phenotype. One of the strongest suppressors observation period. The male flies of fru hypomorphic mutants thus obtained was bon21B (Figure 1G), a loss-of-function allele (fru2/frusat) exhibited a reduced level of courtship compared of bon (Figure 1H). Bon encodes a well-characterized tran- with the control males (Figure 2C). The courtship activity of fru scription cofactor (Figure 1H), and is expressed in almost all mutant males further diminished when they were heterozygous neurons in the CNS, including fru-expressing neurons for bon21B (Figure 2C). Furthermore, this reduction in male court- (Figure S1). ship activity due to the presence of bon21B was counteracted by 1328 Cell 149, 1327–1338, June 8, 2012 ª2012 Elsevier Inc. The TIF1 family of cofactors, which are the vertebrate homo- logs of Bon (Beckstead et al., 2001, 2005), play a key role in histone modification leading to gene silencing, and HP1a (Fig- ure 2A) is a critical binding partner for TIF1 proteins in this process (Khetchoumian et al., 2004; Nielsen et al., 1999). HP1a is a chromodomain protein that binds to methylated histone H3 lysine 9, promoting further methylation of H3 lysine 9 in nearby nucleosomes (Bannister et al., 2001; Lachner et al., 2001). H3 lysine 9 methylation is a repressive event that is accomplished by concomitant lysine deacetylation through the action of histone deacetylases such as HDAC1 (Schreiber and Bernstein, 2002)(Figure 2B). To evaluate the possibility that Bon mediates Fru sex-specific functions by a mechanism analogous to that of TIF1, we exam- ined the effect of halving the gene dosage of HP1a and HDAC1 on male courtship activity in the presence or absence of fru loss-of-function mutations. In Drosophila, the locus Rpd3 encodes HDAC1 and Su(var)205 encodes HP1a. When heterozy- gous, both HDAC1 and HP1a null alleles had no effect on the courtship activity of males that are wild-type for fru (Figure 2C). In fru hypomorphic mutants (fru2/frusat), both HDAC1 and HP1a affected male courtship performance when their gene dosage was reduced to half (Figure 2C). Unexpectedly, mutation of HDAC1 and HP1a led to opposite phenotypic outcomes: a reduction in HDAC1 enhanced the fru mutant phenotype, Figure 2. Genetic Interactions among the fru, bon, HDAC1, and decreasing male courtship activity, whereas a reduction in HP1a Genes Observed in Male-to-Female Courtship Behavior (A) Structure of HP1a protein and a mutation in the Su(var)2055 allele. HP1a mitigated the fru defects, increasing male courtship (B) Schematic representation of the HDAC1 gene. The deleted region in activity. However, relief from courtship repression by an HP1a Rpd3def8 is indicated by a solid line above the exon-intron map (middle). mutant copy was not observed in fru hypomorphic males when Structure of HDAC1 protein (bottom).
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