Nikolai Kirov

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

decapentaplegic (dpp), a Drosophila member of the TGFbeta 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 brks 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.

The zinc-finger protein Zelda is a key activator of the early zygotic genome in Drosophila

In all animals, the initial events of embryogenesis are controlled by maternal gene products that are deposited into the developing oocyte. At some point after fertilization, control of embryogenesis is transferred to the zygotic genome in a process called the maternal-to-zygotic transition. During this time, many maternal RNAs are degraded and transcription of zygotic RNAs ensues. There is a long-standing question as to which factors regulate these events. The recent findings that microRNAs and Smaug mediate maternal transcript degradation have shed new light on this aspect of the problem. However, the transcription factor(s) that activate the zygotic genome remain elusive. The discovery that many of the early transcribed genes in Drosophila share a cis-regulatory heptamer motif, CAGGTAG and related sequences, collectively referred to as TAGteam sites raised the possibility that a dedicated transcription factor could interact with these sites to activate transcription. Here we report that the zinc-finger protein Zelda (Zld; Zinc-finger early Drosophila activator) binds specifically to these sites and is capable of activating transcription in transient transfection assays. Mutant embryos lacking zld are defective in cellular blastoderm formation, and fail to activate many genes essential for cellularization, sex determination and pattern formation. Global expression profiling confirmed that Zld has an important role in the activation of the early zygotic genome and suggests that Zld may also regulate maternal RNA degradation during the maternal-to-zygotic transition.

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.

Conversion of a silencer into an enhancer: evidence for a co-repressor in dorsal-mediated repression in Drosophila.

The dorsal (dl) protein gradient determines patterns of gene expression along the dorsal‐ventral axis of the Drosophila embryo. dl protein is at peak levels in ventral nuclei of the embryo where it activates some genes (twist and snail) and represses others [zerknullt (zen), decapentaplegic and tolloid]. It is a member of the rel family of transcription factors and interacts with specific DNA sequences in the regulatory regions of its target genes. These sequences (dl binding sites), when taken from the context of either an activated or repressed promoter, mediate transcriptional activation of a heterologous promoter, but not repression. We found that T‐rich sequences close to the dl binding sites in the silencer region of the zen promoter are conserved between three Drosophila species. Using this sequence information we defined a minimal element that can mediate repression of a heterologous promoter. This element interacts with at least two factors present in embryonic extracts, one of which is dl protein. The other factor binds to the T‐rich site. Point mutations in either site abolish ventral repression in vivo. In addition, mutations in the T‐rich site cause ectopic expression in ventral regions indicating that the minimal silencer was converted into an enhancer.

Zelda Potentiates Morphogen Activity by Increasing Chromatin Accessibility

Zygotic 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. Zelda (Zld) plays such a role in the Drosophila embryo, where it has been shown to control the action of patterning signals; 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. 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. 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.

Peak levels of BMP in the Drosophila embryo control target genes by a feed-forward mechanism

Gradients of morphogens determine cell fates by specifying discrete thresholds of gene activities. In the Drosophila embryo, a BMP gradient subdivides the dorsal ectoderm into amnioserosa and dorsal epidermis, and also inhibits neuroectoderm formation. A number of genes are differentially expressed in response to the gradient, but how their borders of expression are established is not well understood. We present evidence that the BMP gradient, via the Smads, provides a two-fold input in regulating the amnioserosa-specific target genes such as Race. Peak levels of Smads in the presumptive amnioserosa set the expression domain of zen, and then Smads act in combination with Zen to directly activate Race. This situation resembles a feed-forward mechanism of transcriptional regulation. In addition, we demonstrate that ectopically expressed Zen can activate targets like Race in the presence of low level Smads, indicating that the role of the highest activity of the BMP gradient is to activate zen.

The transcriptional corepressor DSP1 inhibits activated transcription by disrupting TFIIA-TBP complex formation.

Transcriptional repression of eukaryotic genes is essential for many cellular and developmental processes, yet the precise mechanisms of repression remain poorly understood. The Dorsal Switch Protein (DSP1) was identified in a genetic screen for activities which convert Dorsal into a transcriptional repressor. DSP1 shares structural homology with the HMG‐1/2 family and inhibits activation by the rel transcription factors Dorsal and NF‐kappaB in transfection studies. Here we investigate the mechanism of transcriptional repression by DSP1. We found that DSP1 protein can act as a potent transcriptional repressor for multiple activator families in vitro and in transfection studies. DSP1 bound directly to the TATA binding protein (TBP), and formed a stable ternary complex with TBP bound to DNA. DSP1 preferentially disrupted the DNA binding of TBP complexes containing TFIIA and displaced TFIIA from binding to TBP. Consistent with the inhibition of TFIIA‐bound complexes, DSP1 was shown to inhibit activated but not basal transcription reactions in vitro. The ability of DSP1 to interact with TBP and to repress transcription was mapped to the carboxy‐terminal domain which contains two HMG boxes. Our results support the model that DSP1 represses activated transcription by interfering with the binding of TFIIA, a general transcription factor implicated in activated transcription pathways.

In vitro transcription through nucleosomes by T7 RNA polymerase.

The present work examines the fate of nucleosomes after in vitro transcription of a 1400 bp DNA template containing the mouse alpha‐globin sequences and the promoter of T7 RNA polymerase. Naked and nucleosome‐bearing templates (containing about four or seven histone H1‐lacking particles per template) have been studied by sedimentation, gel electrophoresis, digestion with restriction nucleases and electron microscopy. Both naked and nucleosome‐organized templates could be transcribed in vitro by the T7 polymerase. With all types of templates, both full length and shorter transcripts were obtained. The incomplete transcripts were represented by many distinct bands, pointing to the presence of multiple stops in the process of elongation. The electrophoretic pattern of the transcripts was identical in naked and in nucleosome‐containing templates, showing that the stops depended on some particular DNA sequences and not on the presence of nucleosomes. The efficiency of transcription in the presence of nucleosomes was decreased owing to three different factors: (i) blocked initiation in a fraction of the templates which had their promoters occupied by a nucleosome; (ii) a decreased rate of elongation and (iii) a lag period of initiation. Sedimentation velocity, electrophoretic mobility and protection of four different restriction sites of the templates demonstrated that T7 polymerase transcribed through nucleosomes without their displacement.

Threshold response of C15 to the Dpp gradient in Drosophila is established by the cumulative effect of Smad and Zen activators and negative cues

Morphogen gradients determine a range of cell fates by specifying multiple transcriptional threshold responses. In the dorsal ectoderm of the Drosophila embryo, a BMP gradient is translated into an activated Smad transcription factor gradient, which elicits at least three threshold responses - high, intermediate and low. However, the mechanism underlying differential response to Dpp is poorly understood, due in part to the insufficient number of well-studied target genes. We analyzed the regulation of the C15 gene, which can be activated in cells containing intermediate levels of Dpp. We show that C15 expression requires both dpp and zen, thus forming a genetic feed-forward loop. The C15 regulatory element contains clusters of Smad- and Zen-binding sites in close proximity. Mutational analysis shows that the number of intact Smad- and Zen-binding sites is essential for the C15 transcriptional response, and that the spatial limits of C15 expression are established through a repression mechanism in the dorsolateral cells of the embryo. Thus, the combinatorial action of Smad and Zen activators bound to a number of adjacent sites, and competing negative cues allows for proper gene response to lower than peak levels of the Dpp morphogen.