Differential regulation of transcription preinitiation complex assembly by activator and repressor homeo domain proteins (original) (raw)
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Transcriptional activation and repression by Ultrabithorax proteins in cultured Drosophila cells
Cell, 1989
Homeotic genes of Drosophila melanogaster such as Ultrabithorax (Ubx) and Antennapedia (Antp) have long been thought to select metameric identity during development by controlling the expression of various target genes. Here we describe a cotransfection assay in cultured D. melanogaster cells that is used to demonstrate that Ubx proteins (UBX) can repress an Antp promoter fusion and activate a Ubx promoter fusion, activities predicted from genetic studies. We show (a) that UBX proteins regulated the level of accurately initiated Antp P1 and Ubx transcripts, (b) that activation of the Ubx promoter required a downstream cluster of UBX bindhg sites, and (c) that binding site sequences were sufficient to confer regulation on a heterologous promoter, regardless of their orientation or precise position. We conclude that UBX proteins are transcriptional repressors and activators, and that their actions are mediated by binding to promoter region sequences. Each member of the UBX protein family has similar regulatory abilities, but the properties of synthetic mutant forms suggest that UBX proteins may have a medular design similar to other transcriptional regulators.
Proceedings of the National Academy of Sciences, 2003
The interaction of activators with mediator has been proposed to stimulate the assembly of RNA polymerase II (Pol II) preinitiation complexes, but there have been few tests of this model. The finding that the major adenovirus E1A and mitogen-activated protein kinase-phosphorylated Elk1 activation domains bind to Sur2 uniquely among the metazoan mediator subunits and the development of transcriptionally active nuclear extracts from WT and sur2 –/– embryonic stem cells, reported here, allowed a direct test of the model. We found that whereas VP16, E1A, and phosphorylated Elk1 activation domains each stimulate binding of mediator, Pol II, and general transcription factors to promoter DNA in extracts from WT cells, only VP16 stimulated their binding in extracts from sur2 –/– cells. This stimulation of mediator, Pol II, and general transcription factor binding to promoter DNA correlated with transcriptional activation by these activators in WT and mutant extracts. Because the mutant medi...
Journal of Biological Chemistry, 2015
Regulation of RNA polymerase II transcription is critical for the proper development, differentiation, and growth of an organism. The RNA polymerase II core promoter is the ultimate target of a multitude of transcription factors that control transcription initiation. Core promoters encompass the RNA start site and consist of functional elements such as the TATA box, initiator, and downstream core promoter element (DPE), which confer specific properties to the core promoter. We have previously discovered that Drosophila Caudal, which is a master regulator of genes involved in development and differentiation, is a DPE-specific transcriptional activator. Here, we show that the mouse Caudal-related homeobox (Cdx) proteins (mCdx1, mCdx2, and mCdx4) are also preferential core promoter transcriptional activators. To elucidate the mechanism that enables Caudal to preferentially activate DPE transcription, we performed structure-function analysis. Using a systematic series of deletion mutants (all containing the intact DNA-binding homeodomain) we discovered that the C-terminal region of Caudal contributes to the preferential activation of the fushi tarazu (ftz) Caudal target gene. Furthermore, the region containing both the homeodomain and the C terminus of Caudal was sufficient to confer core promoter-preferential activation to the heterologous GAL4 DNA-binding domain. Importantly, we discovered that Drosophila CREB-binding protein (dCBP) is a co-activator for Caudal-regulated activation of ftz. Strikingly, dCBP conferred the ability to preferentially activate the DPE-dependent ftz reporter to mini-Caudal proteins that were unable to preferentially activate ftz transcription themselves. Taken together, it is the unique combination of dCBP and Caudal that enables the co-activation of ftz in a core promoter-preferential manner.
Stimulation of transcription by an Ultrabithorax protein in vitro
Genes & Development, 1990
The Ultrabithorax (Ubx) gene of Drosophila melanogaster encodes a family of UBX proteins that are thought to specify the developmental fates of segments in the posterior thorax and anterior abdomen by controlling the expression of a set of target genes. UBX proteins bind DNA in vitro, and they activate or repress different natural and synthetic target promoters in cultured cells. Here it is shown that a purified UBX protein can stimulate transcription of a synthetic target gene in extracts of cultured D. melanogaster cells. Stimulation is dependent on the presence of upstream, promoter-region binding sites but is independent of binding site orientation. A naturally occurring binding site cluster and a binding site consensus sequence consisting of TAA trinucleotide repeats can mediate this activation. A minimal promoter fused to such sites is activated by UBX, suggesting that transcriptional stimulation could result from an interaction between the promoter-bound protein and the gener...
Developmental Biology, 2011
At the Drosophila melanogaster bithorax complex (BX-C) over 330kb of intergenic DNA is responsible for directing the transcription of just three homeotic (Hox) genes during embryonic development. A number of distinct enhancer cis-regulatory modules (CRMs) are responsible for controlling the specific expression patterns of the Hox genes in the BX-C. While it is has proven possible to identify orthologs of known BX-C CRMs in different Drosophila species using overall sequence conservation, this approach has not proven sufficiently effective for identifying novel CRMs or defining the key functional sequences within enhancer CRMs. Here we demonstrate that the specific spatial clustering of transcription factor (TF) binding sites is important for BX-C enhancer activity. A bioinformatic search for combinations of putative TF binding sites in the BX-C suggests that simple clustering of binding sites is frequently not indicative of enhancer activity. However, through molecular dissection and evolutionary comparison across the Drosophila genus we discovered that specific TF binding site clustering patterns are an important feature of three known BX-C enhancers. Sub-regions of the defined IAB5 and IAB7b enhancers were both found to contain an evolutionarily conserved signature motif of clustered TF binding sites which is critical for the functional activity of the enhancers. Together, these results indicate that the spatial organization of specific activator and repressor binding sites within BX-C enhancers is of greater importance than overall sequence conservation and is indicative of enhancer functional activity.
Background: High-resolution transcription start site (TSS) mapping in D. melanogaster embryos and cell lines has revealed a rich and detailed landscape of both cis-and trans-regulatory elements and factors. However, TSS profiling has not been investigated in an orthogonal in vivo setting. Here, we present a comprehensive dataset that links TSS dynamics with nucleosome occupancy and gene expression in the wandering third instar larva, a developmental stage characterized by large-scale shifts in transcriptional programs in preparation for metamorphosis. Results: The data recapitulate major regulatory classes of TSSs, based on peak width, promoter-proximal polymerase pausing, and cis-regulatory element density. We confirm the paucity of divergent transcription units in D. melanogaster, but also identify notable exceptions. Furthermore, we identify thousands of novel initiation events occurring at unannotated TSSs that can be classified into functional categories by their local density of histone modifications. Interestingly, a sub-class of these unannotated TSSs overlaps with functionally validated enhancer elements, consistent with a regulatory role for " enhancer RNAs " (eRNAs) in defining developmental transcription programs.
Proceedings of the National Academy of Sciences, 1996
We have used alanine scanning to analyze protein-protein interactions by human TATA-element binding protein (TBP) within the transcription preinitiation complex. The results indicate that TBP interacts with RNA polymerase II and general transcription factors IIA, IIB, and IIF within the functional transcription preinitiation complex and define the determinants of TBP for each of these interactions. The results permit construction of a model for the structure of the preinitiation complex.