Immunostaining of Drosophila Polytene Chromosomes to Investigate Recruitment of Chromatin-Binding Proteins
Institut für Molekularbiologie und Tumorforschung (IMT), Philipps-Universität Marburg, Marburg, Germany. Methods in molecular biology (Clifton, N.J.)
(Impact Factor: 1.29).
01/2012; 809:267-77. DOI: 10.1007/978-1-61779-376-9_18
Gene transcription is a complex process that involves a large number of proteins. These proteins can be brought to their target genes by a variety of different mechanisms: many transcription factors interact with specific DNA sequences in promoters or enhancers, several epigenetic regulators bind histones bearing specific modifications, elongation factors and some RNA processing factors bind to the transcribing RNA polymerase, and other factors interact directly with nascent transcripts or noncoding RNA. Immunostaining of Drosophila polytene chromosomes allows the genome-wide localization of factors involved at different stages of transcriptional regulation. In this chapter, we present protocols that adapt the general technique to probe different recruitment mechanisms employed by these factors, including specific interactions with phosphorylated RNA polymerase II and RNA-mediated chromatin associations.
Available from: Ronit Wilk
- ") (Yip et al., 1997), which is similar to the organization of the ZNFs in human and chicken RREB-1 proteins (Murawska and Brehm, 2012; Rubin et al., 2000) (see also Fig. 9A). To determine which fingers bind to HBS-A and HBS-B, seven GST-fusion proteins (Fig. 2A) were made and tested by gelshift analysis. "
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ABSTRACT: The Drosophila Hindsight (hnt) gene encodes a C2H2-type Zinc-finger protein, HNT, that plays multiple developmental roles including control of embryonic germ band retraction and regulation of retinal cell fate and morphogenesis. While the developmental functions of the human HNT homolog, RREB-1, are unknown, it has been shown to function as a transcriptional modulator of several tumor suppressor genes. Here we investigate HNT's functional motifs, target genes and its regulatory abilities. We show that the C-terminal region of HNT, containing the last five of its 14 Zinc fingers, binds in vitro to DNA elements very similar to those identified for RREB-1. We map HNT's in vivo binding sites on salivary gland polytene chromosomes and define, at high resolution, where HNT is bound to two target genes, hnt itself and nervy (nvy). Data from both loss-of-function and over-expression experiments show that HNT attenuates the transcription of these two targets in a tissue-specific manner. RREB-1, when expressed in Drosophila, binds to the same polytene chromosome sites as HNT, attenuates expression of the hnt and nvy genes, and rescues the germ band retraction phenotype. HNT's ninth Zinc finger has degenerated or been lost in the vertebrate lineage. We show that a HNT protein mutant for this finger can also attenuate target gene expression and rescue germ band retraction. Thus HNT and RREB-1 are functional homologs at the level of DNA binding, transcriptional regulation and developmental control.
Available from: Bridlin Barckmann
- "TUNEL staining was done essentially as described in Rathke et al., 2007. Polytene chromosomes were prepared and stained as described in (Murawska and Brehm, 2012). The GFP-antibody (rabbit, Rockland Inc.) was applied in a 1:500 dilution. "
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ABSTRACT: By a conserved cellular differentiation process, spermatogenesis leads to formation of haploid sperm for successful reproduction. In Drosophila and in mammals, post-meiotic spermatid differentiation depends on several translationally repressed and stored mRNAs that are often expressed exclusively in the testis through a cell type specific transcriptional program. In Drosophila, the mRNAs of proteins required for post-meiotic chromatin reorganisation, like ProtB and Mst77F, are transcribed in meiotic spermatocytes and subjected to translational repression for days. Transcription of many of these translationally repressed mRNAs depends on testis-specific homologs of TATA box binding protein-associated factors (tTAFs). Here, we identified the testis-specific bromodomain protein, tBRD-1, that is only expressed in primary spermatocytes. Bromodomain proteins are able to recognise and bind acetylated histones and non-histone proteins. We generated tbrd-1 mutant flies and observed that function of tBRD-1 is required for male fertility. tBRD-1 partially colocalised with tTAFs, TAF1 and Polycomb to a Fibrillarin-deficient region within the spermatocyte nucleolus. The nucleolar localisation of tBRD-1 depended on tTAF function but not the other way round. Further, we could show that ectopically expressed tBRD-1-eGFP is able to bind to the interbands of polytene chromosomes. By inhibitor treatment of cultured testis we observed that sub-cellular localisation of tBRD-1 may depend on the acetylation status of primary spermatocytes.
Available from: Jun-Yuan Ji
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ABSTRACT: The steroid hormone ecdysone and its receptor (EcR) play critical roles in orchestrating developmental transitions in arthropods. However, the mechanism by which EcR integrates nutritional and developmental cues to correctly activate transcription remains poorly understood. Here, we show that EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. We observed that cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval-pupal transition.
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