The Drosophila Gene brinker Reveals a Novel Mechanism of Dpp Target Gene Regulation

Abstractdecapentaplegic (dpp), a Drosophila member of the TGFβ family of secreted molecules, functions as a long-range morphogen in patterning of the embryo and the adult appendages. Dpp signals via the SMAD proteins Mad and Medea. Here we show that in the absence of brinker (brk), Mad is not required for the activation of Dpp target genes that depend on low levels of Dpp. brk encodes a novel protein with features of a transcriptional repressor. brk itself is negatively regulated by Dpp. Dpp signaling might relieve brk’s repression of low-level target genes either by transcriptional repression of brk or by antagonizing a repressor function of brk at the target gene promoters

Characterisation of retinoblastomas without RB1 mutations: genomic, gene expression, and clinical studies

SummaryBackgroundRetinoblastoma is the childhood retinal cancer that defined tumour-suppressor genes. Previous work shows that mutation of both alleles of the RB1 retinoblastoma suppressor gene initiates disease. We aimed to characterise non-familial retinoblastoma tumours with no detectable RB1 mutations.MethodsOf 1068 unilateral non-familial retinoblastoma tumours, we compared those with no evidence of RB1 mutations (RB1+/+) with tumours carrying a mutation in both alleles (RB1−/−). We analysed genomic copy number, RB1 gene expression and protein function, retinal gene expression, histological features, and clinical data.FindingsNo RB1 mutations (RB1+/+) were reported in 29 (2·7%) of 1068 unilateral retinoblastoma tumours. 15 of the 29 RB1+/+ tumours had high-level MYCN oncogene amplification (28–121 copies; RB1+/+MYCNA), whereas none of 93 RB1−/− primary tumours tested showed MYCN amplification (p<0·0001). RB1+/+MYCNA tumours expressed functional RB1 protein, had fewer overall genomic copy-number changes in genes characteristic of retinoblastoma than did RB1−/− tumours, and showed distinct aggressive histological features. MYCN amplification was the sole copy-number change in one RB1+/+MYCNA retinoblastoma. One additional MYCNA tumour was discovered after the initial frequencies were determined, and this is included in further analyses. Median age at diagnosis of the 17 children with RB1+/+MYCNA tumours was 4·5 months (IQR 3·5–10), compared with 24 months (15–37) for 79 children with non-familial unilateral RB1−/− retinoblastoma.InterpretationAmplification of the MYCN oncogene might initiate retinoblastoma in the presence of non-mutated RB1 genes. These unilateral RB1+/+MYCNA retinoblastomas are characterised by distinct histological features, only a few of the genomic copy-number changes that are characteristic of retinoblastoma, and very early age of diagnosis.FundingNational Cancer Institute–National Institutes of Health, Canadian Institutes of Health Research, German Research Foundation, Canadian Retinoblastoma Society, Hyland Foundation, Toronto Netralaya and Doctors Lions Clubs, Ontario Ministry of Health and Long Term Care, UK-Essen, and Foundations Avanti-STR and KiKa

Temporal Coordination of Gene Networks by Zelda in the Early Drosophila Embryo

In past years, much attention has focused on the gene networks that regulate early developmental processes, but less attention has been paid to how multiple networks and processes are temporally coordinated. Recently the discovery of the transcriptional activator Zelda (Zld), which binds to CAGGTAG and related sequences present in the enhancers of many early-activated genes in Drosophila, hinted at a mechanism for how batteries of genes could be simultaneously activated. Here we use genome-wide binding and expression assays to identify Zld target genes in the early embryo with the goal of unraveling the gene circuitry regulated by Zld. We found that Zld binds to genes involved in early developmental processes such as cellularization, sex determination, neurogenesis, and pattern formation. In the absence of Zld, many target genes failed to be activated, while others, particularly the patterning genes, exhibited delayed transcriptional activation, some of which also showed weak and/or sporadic expression. These effects disrupted the normal sequence of patterning-gene interactions and resulted in highly altered spatial expression patterns, demonstrating the significance of a timing mechanism in early development. In addition, we observed prevalent overlap between Zld-bound regions and genomic “hotspot” regions, which are bound by many developmental transcription factors, especially the patterning factors. This, along with the finding that the most over-represented motif in hotspots, CAGGTA, is the Zld binding site, implicates Zld in promoting hotspot formation. We propose that Zld promotes timely and robust transcriptional activation of early-gene networks so that developmental events are coordinated and cell fates are established properly in the cellular blastoderm embryo

The Drosophila Pioneer Factor Zelda Modulates the Nuclear Microenvironment of a Dorsal Target Enhancer to Potentiate Transcriptional Output.

Connecting the developmental patterning of tissues to the mechanistic control of RNA polymerase II remains a long-term goal of developmental biology. Many key elements have been identified in the establishment of spatial-temporal control of transcription in the early Drosophila embryo, a model system for transcriptional regulation. The dorsal-ventral axis of the Drosophila embryo is determined by the graded distribution of Dorsal (Dl), a homolog of the nuclear factor κB (NF-κB) family of transcriptional activators found in humans [1, 2]. A second maternally deposited factor, Zelda (Zld), is uniformly distributed in the embryo and is thought to act as a pioneer factor, increasing enhancer accessibility for transcription factors, such as Dl [3-9]. Here, we utilized the MS2 live imaging system to evaluate the expression of the Dl target gene short gastrulation (sog) to better understand how a pioneer factor affects the kinetic parameters of transcription. Our experiments indicate that Zld modifies probability of activation, the timing of this activation, and the rate at which transcription occurs. Our results further show that this effective rate increase is due to an increased accumulation of Dl at the site of transcription, suggesting that transcription factor hubs induced by Zld [10] functionally regulate transcription

The Drosophila Pioneer Factor Zelda Modulates the Nuclear Microenvironment of a Dorsal Target Enhancer to Potentiate Transcriptional Output.

Connecting the developmental patterning of tissues to the mechanistic control of RNA polymerase II remains a long-term goal of developmental biology. Many key elements have been identified in the establishment of spatial-temporal control of transcription in the early Drosophila embryo, a model system for transcriptional regulation. The dorsal-ventral axis of the Drosophila embryo is determined by the graded distribution of Dorsal (Dl), a homolog of the nuclear factor κB (NF-κB) family of transcriptional activators found in humans [1, 2]. A second maternally deposited factor, Zelda (Zld), is uniformly distributed in the embryo and is thought to act as a pioneer factor, increasing enhancer accessibility for transcription factors, such as Dl [3-9]. Here, we utilized the MS2 live imaging system to evaluate the expression of the Dl target gene short gastrulation (sog) to better understand how a pioneer factor affects the kinetic parameters of transcription. Our experiments indicate that Zld modifies probability of activation, the timing of this activation, and the rate at which transcription occurs. Our results further show that this effective rate increase is due to an increased accumulation of Dl at the site of transcription, suggesting that transcription factor "hubs" induced by Zld [10] functionally regulate transcription

A Systematic Ensemble Approach to Thermodynamic Modeling of Gene Expression from Sequence Data

Summary To understand the relationship between an enhancer DNA sequence and quantitative gene expression, thermodynamics-driven mathematical models of transcription are often employed. These "sequence-to-expression" models can describe an incomplete or even incorrect set of regulatory relationships if the parameter space is not searched systematically. Here, we focus on an enhancer of the Drosophila gene ind and demonstrate how a systematic search of parameter space can reveal a more comprehensive picture of a gene's regulatory mechanisms, resolve outstanding ambiguities, and suggest testable hypotheses. We describe an approach that generates an ensemble of ind models; all of these models are technically acceptable solutions to the sequence-to-expression problem in light of wild-type data, and some represent mechanistically distinct hypotheses about the regulation of ind. This ensemble can be restricted to biologically plausible models using requirements gleaned from in vivo perturbation experiments. Biologically plausible models make unique predictions about how specific ind enhancer sequences affect ind expression; we validate these predictions in vivo through site mutagenesis in transgenic Drosophila embryos.This work was supported by NSF grant EFRI 1136913, MICINN grant BFU2011-23611, NIH grant R01GM086537, and NIH grant R01 5R01GM114341.Peer Reviewe

Zelda Potentiates Morphogen Activity by Increasing Chromatin Accessibility

SummaryZygotic genome activation (ZGA) is a major genome programming event whereby the cells of the embryo begin to adopt specified fates. Experiments in Drosophila and zebrafish have revealed that ZGA depends on transcription factors that provide large-scale control of gene expression by direct and specific binding to gene regulatory sequences [1–5]. Zelda (Zld) plays such a role in the Drosophila embryo, where it has been shown to control the action of patterning signals [1, 2]; however, the mechanisms underlying this effect remain largely unclear. A recent model proposed that Zld binding sites act as quantitative regulators of the spatiotemporal expression of genes activated by Dorsal (Dl), the morphogen that patterns the dorsoventral axis [6]. Here we tested this model experimentally, using enhancers of brinker (brk) and short gastrulation (sog), both of which are directly activated by Dl, but at different concentration thresholds [7–9]. In agreement with the model, we show that there is a clear positive correlation between the number of Zld binding sites and the spatial domain of enhancer activity. Likewise, the timing of expression could be advanced or delayed. We present evidence that Zld facilitates binding of Dl to regulatory DNA, and that this is associated with increased chromatin accessibility. Importantly, the change in chromatin accessibility is strongly correlated with the change in Zld binding, but not Dl. We propose that the ability of genome activators to facilitate readout of transcriptional input is key to widespread transcriptional induction during ZGA