Roles of Cofactors and Chromatin Accessibility in Hox Protein Target Specificity
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE69796 | GEO | 2015/12/07
SECONDARY ACCESSION(S): PRJNA286786
REPOSITORIES: GEO
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Project description:Regulation of specific target genes by transcription factors is central to gene network control in development. How target specificity is achieved in eukaryotic genomes is poorly understood, as exemplified by the Hox family, which show limited in vitro DNA-binding specificity but clear functional specificity in vivo. We generated genome-wide binding profiles for three Hox proteins, Ubx, Abd-A and Abd-B, in Drosophila Kc167 cells, revealing clear target specificity and a striking influence of chromatin accessibility. Ubx and Abd-A bind to similar target sites in accessible chromatin whereas Abd-B binds additional specific targets. Provision of the TALE class cofactors, Exd and Hth, alters the Ubx binding profile, enabling binding to additional targets in the genome. Both the Abd-B specific targets and the cofactor-dependent Ubx targets are in relatively DNase1 inaccessible chromatin, suggesting that competition with nucleosomes is a key factor determining Hox protein target specificity. This ChIP-Seq study performed on Kc167 cells involves two experiments and 6 ChIP samples. In Experiment 1, we generated genome-wide binding profiles for Ubx, Abd-A and Abd-B. An equal volume of input chromatin was retained from each of the Hox samples and combined to represent the input, which was purified alongside the ChIP samples. We performed two biological replicates for each sample. Sequencing was performed using the Illumina MiSeq platform. In Experiment 2, we generated genome-wide binding profiles for Ubx, mutant Ubx and Ubx in the presence of Hth. We performed two biological replicates for each sample except Ubx where we performed just one. Sequencing was performed using the Illumina HiSeq 2000 platform. For all samples, Experiment 1 input chromatin was used as the reference control to assay ChIP enrichment.
2015-12-07 | E-GEOD-69796 | biostudies-arrayexpress
Project description:Hox genes encode a family of transcription factors that are key developmental regulators with a highly conserved role in specifying segmental diversity along the metazoan body axis. Although they have been shown to regulate a wide variety of downstream processes, direct transcriptional targets have been difficult to identify and this has been a major obstacle to our understanding of Hox gene function. Here we report the use of a YFP-tagged Drosophila protein-trap line together with chromatin immunoprecipitation and microarray analysis to identify genome-wide binding sites for the Hox protein Ultrabithorax (Ubx). We find 1,147 genes bound by Ubx in chromatin from the haltere imaginal disc, a prominent site of Ubx function where it specifies haltere versus wing development. The functional relevance of these genes is supported by their overlap with genes showing differential expression between wing and haltere imaginal discs. The Ubx-bound genes are highly enriched in genes involved in developmental processes. They contain both high-level regulatory genes as well as genes involved in more basic cell functions supporting the idea that Hox genes regulate many levels of developmental pathways. Several signalling pathways are highly enriched and within these pathways Ubx target genes occur at multiple level from ligands to transcriptional effectors. We also performed genome-wide analysis of the binding sites for the Hox cofactor Homothorax (Hth), revealing a striking similarity in the binding profiles of Hth and Ubx. We suggest that these binding profiles may be strongly influenced by chromatin accessibility and we provide evidence of a clear link between Ubx/Hth binding and the chromatin state at genes regulated by Polycomb silencing. The role of chromatin accessibility has important implications for Hox gene function and for genomic target selection by transcription factors in general.
2011-04-14 | GSE23864 | GEO
Project description:Hox genes are early determinants of cell identity along the anterior-posterior body axis across bilaterians. Several late non-homeotic functions of Hox genes have emerged in a variety of processes involved in organogenesis in several organisms, including mammals. Several studies have reported the misexpression of Hox genes in a variety of malignancies including acute myeloid leukemia. The Hox genes Dfd, Ubx, abd-A and Abd-B were overexpressed via the UAS-Gal4 system using Cg-Gal4, Lsp2-Gal4, He-Gal4 and HmlD3-Gal4 as specific drivers. Genetic interaction was tested by bringing overexpression lines in heterozygous mutant backgrounds of Polycomb and trithorax group factors. Larvae were visually scored for melanized bodies. Circulating hemocytes were quantified and tested for differentiation. Pupal lethality was assessed. Expression of Dfd, Ubx and abd-A, but not Abd-B in the hematopoietic compartment of Drosophila led to the appearance of circulating melanized bodies, an increase in cell number, cell-autonomous proliferation, and differentiation of hemocytes. Pupal lethality and melanized pseudo-tumors were suppressed in Psc1 and esc2 backgrounds while polycomb group member mutations Pc1 and Su(z)123 and trithorax group member mutation TrlR85 enhanced the phenotype. Dfd, Ubx and abd-A are leukemogenic. Mutations in Polycomb and trithorax group members modulate the leukemogenic phenotype.
2020-12-30 | GSE163983 | GEO
Project description:Hox proteins are transcription factors and key regulators of segmental identity along the anterior posterior axis across all bilateral animals. Despite decades of research, mechanism by which Hox proteins select their targets and specify segmental identity remains elusive. To address this question we carried out whole genome ChIP-chip experiments to identify direct targets of Hox protein Ultrabithorax (Ubx) during haltere development in Drosophila. When mis-expressed in wing segment (T2) Ubx converts its identity to that of haltere segment (T3). We used CbxHm/+ wing discs ectopically expressing Ubx in the pouch region of discs to obtain chromatin. This helped us focus on targets of Ubx involved in pouch development without mixing with the targets involved in notum development. Polyclonal Ubx antibodies against N-terminal region (excluding homeodomain) were generated in our lab and used to pull down Ubx bound regions from CbxHm/+ wing discs. Mock DNA (No antibody) was used as control. Test Vs. Mock experiment. CbxHm/+ discs for Chromatin source. Biological replicates: 3 [Agilent two-color ChIP-on-Chip experiment,Organism: Drosophila melanogaster ,Drosophila Whole Genome ChIP-on-Chip Set 244K Microarray 1 of 2 (AMADID: 014816 and 014817)]
2011-12-22 | E-GEOD-28778 | biostudies-arrayexpress
Project description:Hox homeodomain transcription factors are key regulators of animal development. They specify the identity of segments along the anterior-posterior body axis in metazoans by controlling the expression of diverse downstream targets, including transcription factors and signaling pathway components. The Drosophila melanogaster Hox factor Ultrabithorax (Ubx) directs the development of thoracic and abdominal segments and appendages, and loss of Ubx function can lead for example to the transformation of third thoracic segment appendages (e.g. halters) into second thoracic segment appendages (e.g. wings), resulting in a characteristic four-wing phenotype. Here we present a Drosophila melanogaster strain with a V5-epitope tagged Ubx allele, which we employed to obtain a high quality genome-wide map of Ubx binding sites using ChIP-seq. We confirm the sensitivity of the V5 ChIP-seq by recovering 7/8 of well-studied Ubx-dependent cis-regulatory regions. Moreover, we show that Ubx binding is predictive of enhancer activity as suggested by comparison with a genome-scale resource of in vivo tested enhancer candidates. We observed densely clustered Ubx binding sites at 12 extended genomic loci that included ANTP-C, BX-C, Polycomb complex genes, and other regulators and the clustered binding sites were frequently active enhancers. Furthermore, Ubx binding was detected at known Polycomb response elements (PREs) and was associated with significant enrichments of Pc and Pho ChIP signals in contrast to binding sites of other developmental TFs. Together, our results show that Ubx targets developmental regulators via strongly clustered binding sites and allow us to hypothesize that regulation by Ubx might involve Polycomb group proteins to maintain specific regulatory states in cooperative or mutually exclusive fashion, an attractive model that combines two groups of proteins with prominent gene regulatory roles during animal development. Ubx was endogenously tagged with V5 epitope in Drosophila melanogaster (Ubx-V5). ChIP-seq was performed with chromatin from 0-16 hrs wild type and Ubx-V5 embryos. The samples were subjected to single-end sequencing in two replicates.
2016-07-03 | E-GEOD-64284 | biostudies-arrayexpress
Project description:Hox protein transcription factors specify segmental diversity along the anterior-posterior body axis in metazoans. Understanding the basis of Hox function has long faced the problem that, while the different members of the Hox family show clear functional specificity in vivo, they all show very similar binding specificity in vitro. Based on in vitro studies, Hox cofactors may increase Hox binding selectivity but a satisfactory understanding of in vivo Hox target selectivity is still lacking. We have carried out a systematic analysis of the in vivo genomic binding profiles of all eight Drosophila Hox proteins using transient transfection in Kc167 cells to examine Hox protein targeting. We find that Hox proteins show considerable binding selectivity in vivo in the absence of the canonical Hox cofactors Extradenticle and Homothorax. Hox binding selectivity is strongly associated with chromatin accessibility; binding sites in less accessible chromatin show the highest selectivity and the different Hox proteins exhibit different propensities to bind less accessible chromatin. High Hox binding selectivity is also associated with high affinity binding regions, leading to a model where Hox proteins derive binding selectivity through an affinity-based competition with nucleosomes. Provision of the Extradenticle/Homothorax cofactors generally leads to an increase in the number of Hox binding regions and promotes the binding to regions in less accessible chromatin, however the provision of these cofactors has little effect on the overall selectivity of Hox targeting. These studies indicate that chromatin accessibility plays a key role in Hox selectivity and we propose that relative chromatin accessibility provides a basis for subtle differences in binding specificity and affinity to generate significantly different sets of genomic targets for different Hox proteins. We suggest that this mechanism may also be relevant to other transcription factor families. This SuperSeries is composed of the SubSeries listed below.
2019-05-15 | GSE122575 | GEO
Project description:Amongst the various different insect groups, there is remarkable diversity in the number and size of wings. However the development of the basic body plan in insects is similar to a large extent. The genes of the hox complex regulate various pathways to bring about the development or modification of different organs. Ubx, a gene of the bithorax hox complex is expressed in the third thoracic segment of insects and is known to specify the fate of wing appendage in that segment.To understand the role of Ubx and how its regulatory mechanism has evolved through the course of evolution we have compared its genome wide targets in different insect orders. The identification of regulatory pathways and the key players Ubx regulates is crucial to understand how it has controlled wing development across insect orders. Our lab has previously identified direct targets of Ubx in Drosophila using ChIP-chip (Agrawal et al, 2011). To further our knowledge on the role of regulation in development and modification of hind wing appendage we have studied the targets in the hind wings of other insects (silk moth; Lepidoptera and honeybee; Hymenoptera) and performed a comparative analysis. We have employed ChIP followed by illumina sequencing to identify the targets of Ubx in developing hind and fore wing buds of Bombyx larvae. This is a first next generation sequencing study in Lepidoptera in an attempt to understand wing development. Chromatin Immunoprecipitation (ChIP) was used to identify genome wide targets bound by Ubx in Bombyx larval wing buds. The experiment to enrich Ubx bound regions was carried out using a Bombyx N terminal-Ubx specific poylclonal antibody raised in Rabbit and purified against a Protein A column to obtain IgG fraction. An Immunoprecipitation (IP) with Normal Rabbit IgG was used as a negative control to eliminate the regions that pertained to non specific binding to an Immunogloubulin. The normalization of both ChIP and IgG was done against sequenced input chromatin. Two replicates of single end 36 bp reads were sequenced using Ilumina for all the three conditions and for both fore and hind wing tissue samples.The peaks common to both the replicates were considered after applying a FDR cutoff.The fore wing target set was used for comparison with the hind wing targets.
2016-07-27 | E-GEOD-71990 | biostudies-arrayexpress
Project description:Hox proteins are transcription factors and key regulators of segmental identity along the anterior posterior axis across all bilateral animals. Despite decades of research, mechanism by which Hox proteins select their targets and specify segmental identity remains elusive. To address this question we carried out whole genome ChIP-chip experiments to identify direct targets of Hox protein Ultrabithorax (Ubx) during haltere development in Drosophila. When mis-expressed in wing segment (T2) Ubx converts its identity to that of haltere segment (T3). We used CbxHm/+ wing discs ectopically expressing Ubx in the pouch region of discs to obtain chromatin. This helped us focus on targets of Ubx involved in pouch development without mixing with the targets involved in notum development. Polyclonal Ubx antibodies against N-terminal region (excluding homeodomain) were generated in our lab and used to pull down Ubx bound regions from CbxHm/+ wing discs. Mock DNA (No antibody) was used as control.
2011-12-22 | GSE28778 | GEO
Project description:Cell type diversity is controlled by transcription factors (TFs), which regulate specific yet flexible gene expression programs depending on the cellular environment. However, how sets of TFs work together to promote such diversity with high precision is still unknown. We used the Hox TF Ultrabithorax (Ubx) as a model because of its essential functions in multiple cell types. Assuming that functional diversity emerges from specifc protein interactions in different cell types, we explored Ubx protein interactomes in three different tissue lineages in vivo. Using proximity dependent Biotin IDentification (BioID) in Drosophila embryos, we find that Ubx interacts with largely non-overlapping sets of proteins in each tissue lineage. Intriguingly, this specificity does not primarily result from Ubx binding to proteins with lineage-restricted functions. Instead, Ubx interacts in a lineage-specific manner with ubiquitously expressed subunits of proteins complexes controlling general aspects of gene expression, like chromatin remodelling or RNA processing. Thus, our work reveals that cell type-specific protein interaction networks not necessarily involve cell type-specific partners and that the function of key TFs may extend to the control over the entire realm of gene expression, including splicing and translation. These findings challenge two central dogmas in cell lineage specification, namely the strong reliance on differential RNA expression as a measure for specificity determinants and the enhancer-centric approach to understand gene regulation.
2020-04-22 | PXD014481 | Pride
Project description:We profiled the chromatin accessibility, transcription factor binding, and gene expression in the homologous wing and haltere imaginal disc of Drosophila melanogaster, which are distinguished by the expression of the Hox protein Ultrabithorax (Ubx). Through the analysis of chromatin changes, we show Ubx has widespread effects on the chromatin landscape to specify cell identity.
2021-05-18 | GSE166714 | GEO