Yutaka Nibu

Exploiting transcription factor binding site clustering to identify cis-regulatory modules involved in pattern formation in the Drosophila genome

A major challenge in interpreting genome sequences is understanding how the genome encodes the information that specifies when and where a gene will be expressed. The first step in this process is the identification of regions of the genome that contain regulatory information. In higher eukaryotes, this cis-regulatory information is organized into modular units [cis-regulatory modules (CRMs)] of a few hundred base pairs. A common feature of these cis-regulatory modules is the presence of multiple binding sites for multiple transcription factors. Here, we evaluate the extent to which the tendency for transcription factor binding sites to be clustered can be used as the basis for the computational identification of cis-regulatory modules. By using published DNA binding specificity data for five transcription factors active in the early Drosophila embryo, we identified genomic regions containing unusually high concentrations of predicted binding sites for these factors. A significant fraction of these binding site clusters overlap known CRMs that are regulated by these factors. In addition, many of the remaining clusters are adjacent to genes expressed in a pattern characteristic of genes regulated by these factors. We tested one of the newly identified clusters, mapping upstream of the gap gene giant (gt), and show that it acts as an enhancer that recapitulates the posterior expression pattern of gt.

dCtBP mediates transcriptional repression by Knirps, Krüppel and Snail in the Drosophila embryo

The pre‐cellular Drosophila embryo contains 10 well characterized sequence‐specific transcriptional repressors, which represent a broad spectrum of DNA‐binding proteins. Previous studies have shown that two of the repressors, Hairy and Dorsal, recruit a common co‐repressor protein, Groucho. Here we present evidence that three different repressors, Knirps, Krüppel and Snail, recruit a different co‐repressor, dCtBP. Mutant embryos containing diminished levels of maternal dCtBP products exhibit both segmentation and dorsoventral patterning defects, which can be attributed to loss of Krüppel, Knirps and Snail activity. In contrast, the Dorsal and Hairy repressors retain at least some activity in dCtBP mutant embryos. dCtBP interacts with Krüppel, Knirps and Snail through a related sequence motif, PXDLSXK/H. This motif is essential for the repression activity of these proteins in transgenic embryos. We propose that dCtBP represents a major form of transcriptional repression in development, and that the Groucho and dCtBP co‐repressors mediate separate pathways of repression.

The Nutrient-Responsive Hormone CCHamide-2 Controls Growth by Regulating Insulin-like Peptides in the Brain of Drosophila melanogaster

The coordination of growth with nutritional status is essential for proper development and physiology. Nutritional information is mostly perceived by peripheral organs before being relayed to the brain, which modulates physiological responses. Hormonal signaling ensures this organ-to-organ communication, and the failure of endocrine regulation in humans can cause diseases including obesity and diabetes. In Drosophila melanogaster, the fat body (adipose tissue) has been suggested to play an important role in coupling growth with nutritional status. Here, we show that the peripheral tissue-derived peptide hormone CCHamide-2 (CCHa2) acts as a nutrient-dependent regulator of Drosophila insulin-like peptides (Dilps). A BAC-based transgenic reporter revealed strong expression of CCHa2 receptor (CCHa2-R) in insulin-producing cells (IPCs) in the brain. Calcium imaging of brain explants and IPC-specific CCHa2-R knockdown demonstrated that peripheral-tissue derived CCHa2 directly activates IPCs. Interestingly, genetic disruption of either CCHa2 or CCHa2-R caused almost identical defects in larval growth and developmental timing. Consistent with these phenotypes, the expression of dilp5, and the release of both Dilp2 and Dilp5, were severely reduced. Furthermore, transcription of CCHa2 is altered in response to nutritional levels, particularly of glucose. These findings demonstrate that CCHa2 and CCHa2-R form a direct link between peripheral tissues and the brain, and that this pathway is essential for the coordination of systemic growth with nutritional availability. A mammalian homologue of CCHa2-R, Bombesin receptor subtype-3 (Brs3), is an orphan receptor that is expressed in the islet β-cells; however, the role of Brs3 in insulin regulation remains elusive. Our genetic approach in Drosophila melanogaster provides the first evidence, to our knowledge, that bombesin receptor signaling with its endogenous ligand promotes insulin production.

From Notochord Formation to Hereditary Chordoma: The Many Roles of Brachyury

Chordoma is a rare, but often malignant, bone cancer that preferentially affects the axial skeleton and the skull base. These tumors are both sporadic and hereditary and appear to occur more frequently after the fourth decade of life; however, modern technologies have increased the detection of pediatric chordomas. Chordomas originate from remnants of the notochord, the main embryonic axial structure that precedes the backbone, and share with notochord cells both histological features and the expression of characteristic genes. One such gene is Brachyury, which encodes for a sequence-specific transcription factor. Known for decades as a main regulator of notochord formation, Brachyury has recently gained interest as a biomarker and causative agent of chordoma, and therefore as a promising therapeutic target. Here, we review the main characteristics of chordoma, the molecular markers, and the clinical approaches currently available for the early detection and possible treatment of this cancer. In particular, we report on the current knowledge of the role of Brachyury and of its possible mechanisms of action in both notochord formation and chordoma etiogenesis.

Akirin links twist-regulated transcription with the Brahma chromatin remodeling complex during embryogenesis.

The activities of developmentally critical transcription factors are regulated via interactions with cofactors. Such interactions influence transcription factor activity either directly through protein–protein interactions or indirectly by altering the local chromatin environment. Using a yeast double-interaction screen, we identified a highly conserved nuclear protein, Akirin, as a novel cofactor of the key Drosophila melanogaster mesoderm and muscle transcription factor Twist. We find that Akirin interacts genetically and physically with Twist to facilitate expression of some, but not all, Twist-regulated genes during embryonic myogenesis. akirin mutant embryos have muscle defects consistent with altered regulation of a subset of Twist-regulated genes. To regulate transcription, Akirin colocalizes and genetically interacts with subunits of the Brahma SWI/SNF-class chromatin remodeling complex. Our results suggest that, mechanistically, Akirin mediates a novel connection between Twist and a chromatin remodeling complex to facilitate changes in the chromatin environment, leading to the optimal expression of some Twist-regulated genes during Drosophila myogenesis. We propose that this Akirin-mediated link between transcription factors and the Brahma complex represents a novel paradigm for providing tissue and target specificity for transcription factor interactions with the chromatin remodeling machinery.

Local action of long‐range repressors in the Drosophila embryo

Previous studies have identified two corepressors in the early Drosophila embryo: Groucho and dCtBP. Both proteins are recruited to the DNA template by interacting with short peptide motifs conserved in a variety of sequence‐specific transcriptional repressors. Once bound to DNA, Groucho appears to mediate long‐range repression, while dCtBP directs short‐range repression. The short‐range Krüppel repressor was converted into a long‐range repressor by replacing the dCtBP interaction motif (PxDLSxH) with a Groucho motif (WRPW). The resulting chimeric repressor causes a different mutant phenotype from that of the native Krüppel protein when misexpressed in transgenic embryos. The different patterning activities can be explained on the basis of long‐range silencing within the hairy 5′ regulatory region. The analysis of a variety of synthetic transgenes provides evidence that Groucho‐dependent long‐range repressors do not always cause the dominant silencing of linked enhancers within a complex cis‐regulatory region. We suggest a ‘hot chromatin’ model, whereby repressors require activators to bind DNA.

Drosophila Ebi mediates Snail-dependent transcriptional repression through HDAC3-induced histone deacetylation

The Drosophila Snail protein is a transcriptional repressor that is necessary for mesoderm formation. Here, we identify the Ebi protein as an essential Snail co‐repressor. In ebi mutant embryos, Snail target genes are derepressed in the presumptive mesoderm. Ebi and Snail interact both genetically and physically. We identify a Snail domain that is sufficient for Ebi binding, and which functions independently of another Snail co‐repressor, Drosophila CtBP. This Ebi interaction domain is conserved among all insect Snail‐related proteins, is a potent repression domain and is required for Snail function in transgenic embryos. In mammalian cells, the Ebi homologue TBL1 is part of the NCoR/SMRT–HDAC3 (histone deacetylase 3) co‐repressor complex. We found that Ebi interacts with Drosophila HDAC3, and that HDAC3 knockdown or addition of a HDAC inhibitor impairs Snail‐mediated repression in cells. In the early embryo, Ebi is recruited to a Snail target gene in a Snail‐dependent manner, which coincides with histone hypoacetylation. Our results demonstrate that Snail requires the combined activities of Ebi and CtBP, and indicate that histone deacetylation is a repression mechanism in early Drosophila development.

CtBP-dependent activities of the short-range Giant repressor in the Drosophila embryo

There are at least three short-range gap repressors in the precellular Drosophila embryo: Krüppel, Knirps, and Giant. Krüppel and Knirps contain related repression motifs, PxDLSxH and PxDLSxK, respectively, which mediate interactions with the dCtBP corepressor protein. Here, we present evidence that Giant might also interact with dCtBP. The misexpression of Giant in ventral regions of transgenic embryos results in the selective repression of eve stripe 5. A stripe5-lacZ transgene exhibits an abnormal staining pattern in dCtBP mutants that is consistent with attenuated repression by Giant. The analysis of Gal4-Giant fusion proteins identified a minimal repression domain that contains a sequence motif, VLDLS, which is conserved in at least two other sequence-specific repressors. Removal of this sequence from the native Giant protein does not impair its repression activity in transgenic embryos. We propose that Giant-dCtBP interactions might be indirect and mediated by an unknown bZIP subunit that forms a heteromeric complex with Giant. We also suggest that the VLDLS motif recruits an as yet unidentified corepressor protein.

CtBP-Independent Repression in the Drosophila Embryo

ABSTRACT There are three mechanisms of transcriptional repression in eukaryotes. The first is quenching, whereby repressors and activators co-occupy closely linked sites and then the repressor inhibits adjacent activators. The second is direct repression, in which repressors block the function of the core transcription complex. The third is competition, in which repressors compete with activators for a common DNA-binding site. Previous studies have shown that the Drosophila melanogaster CtBP corepressor (dCtBP) is essential for the quenching activity of three short-range sequence-specific repressors in the early Drosophila embryo: Krüppel, Knirps, and Snail. Here we demonstrate that dCtBP is dispensable for target enhancers that contain overlapping activator and repressor binding sites. However, it is essential when Krüppel and Knirps repressor sites do not overlap activator sites but are instead located adjacent to either activators or the core promoter. These findings provide evidence that competition is distinct from quenching and direct repression. Quenching and direct repression depend on dCtBP, whereas competition does not.

Functional Brachyury Binding Sites Establish a Temporal Read-out of Gene Expression in the Ciona Notochord

During notochord formation in chordate embryos, the transcription factor Brachyury employs different regulatory strategies to ensure the sequential activation of downstream genes and thereby the deployment of a specific developmental program at the right time and place.