22 research outputs found
Transcription factor expression landscape in Drosophila embryonic cell lines
Background: Transcription factor (TF) proteins are a key component of the gene regulatory networks that control cellular fates and function. TFs bind DNA regulatory elements in a sequence-specific manner and modulate target gene expression through combinatorial interactions with each other, cofactors, and chromatin-modifying proteins. Large-scale studies over the last two decades have helped shed light on the complex network of TFs that regulate development in Drosophila melanogaster. Results: Here, we present a detailed characterization of expression of all known and predicted Drosophila TFs in two well-established embryonic cell lines, Kc167 and S2 cells. Using deep coverage RNA sequencing approaches we investigate the transcriptional profile of all 707 TF coding genes in both cell types. Only 103 TFs have no detectable expression in either cell line and 493 TFs have a read count of 5 or greater in at least one of the cell lines. The 493 TFs belong to 54 different DNA-binding domain families, with significant enrichment of those in the zf-C2H2 family. We identified 123 differentially expressed genes, with 57 expressed at significantly higher levels in Kc167 cells than S2 cells, and 66 expressed at significantly lower levels in Kc167 cells than S2 cells. Network mapping reveals that many of these TFs are crucial components of regulatory networks involved in cell proliferation, cell–cell signaling pathways, and eye development. Conclusions: We produced a reference TF coding gene expression dataset in the extensively studied Drosophila Kc167 and S2 embryonic cell lines, and gained insight into the TF regulatory networks that control the activity of these cells. © The Author(s) 2024
Transcriptome profile in Drosophila Kc and S2 embryonic cell lines
Drosophila melanogaster cell lines are an important resource for a range of studies spanning genomics, molecular genetics, and cell biology. Amongst these valuable lines are Kc167 (Kc) and Schneider 2 (S2) cells, which were originally isolated in the late 1960s from embryonic sources and have been used extensively to investigate a broad spectrum of biological activities including cell-cell signaling and immune system function. Whole-genome tiling microarray analysis of total RNA from these two cell types was performed as part of the modENCODE project over a decade ago and revealed that they share a number of gene expression features. Here, we expand on these earlier studies by using deep-coverage RNA-sequencing approaches to investigate the transcriptional profile in Kc and S2 cells in detail. Comparison of the transcriptomes reveals that ∼75% of the 13,919 annotated genes are expressed at a detectable level in at least one of the cell lines, with the majority of these genes expressed at high levels in both cell lines. Despite the overall similarity of the transcriptional landscape in the two cell types, 2,588 differentially expressed genes are identified. Many of the genes with the largest fold change are known only by their CG designations, indicating that the molecular control of Kc and S2 cell identity may be regulated in part by a cohort of relatively uncharacterized genes. Our data also indicate that both cell lines have distinct hemocyte-like identities, but share active signaling pathways and express a number of genes in the network responsible for dorsal-ventral patterning of the early embryo. © The Author(s) 2023. Published by Oxford University Press on behalf of the Genetics Society of America
Investigating the sequence landscape in the Drosophila initiator core promoter element using an enhanced MARZ algorithm
The core promoter elements are important DNA sequences for the regulation of RNA polymerase II transcription in eukaryotic cells. Despite the broad evolutionary conservation of these elements, there is extensive variation in the nucleotide composition of the actual sequences. In this study, we aim to improve our understanding of the complexity of this sequence variation in the TATA box and initiator core promoter elements in Drosophila melanogaster. Using computational approaches, including an enhanced version of our previously developed MARZ algorithm that utilizes gapped nucleotide matrices, several sequence landscape features are uncovered, including an interdependency between the nucleotides in position 2 and 5 in the initiator. Incorporating this information in an expanded MARZ algorithm improves predictive performance for the identification of the initiator element. Overall our results demonstrate the need to carefully consider detailed sequence composition features in core promoter elements in order to make more robust and accurate bioinformatic predictions. Copyright 2023 Dresch et al
Disruption of the Abdominal-B Promoter Tethering Element Results in a Loss of Long-Range Enhancer-Directed Hox Gene Expression in Drosophila
There are many examples within gene complexes of transcriptional enhancers interacting with only a subset of target promoters. A number of molecular mechanisms including promoter competition, insulators and chromatin looping are thought to play a role in regulating these interactions. At the Drosophila bithorax complex (BX-C), the IAB5 enhancer specifically drives gene expression only from the Abdominal-B (Abd-B) promoter, even though the enhancer and promoter are 55 kb apart and are separated by at least three insulators. In previous studies, we discovered that a 255 bp cis-regulatory module, the promoter tethering element (PTE), located 5′ of the Abd-B transcriptional start site is able to tether IAB5 to the Abd-B promoter in transgenic embryo assays. In this study we examine the functional role of the PTE at the endogenous BX-C using transposon-mediated mutagenesis. Disruption of the PTE by P element insertion results in a loss of enhancer-directed Abd-B expression during embryonic development and a homeotic transformation of abdominal segments. A partial deletion of the PTE and neighboring upstream genomic sequences by imprecise excision of the P element also results in a similar loss of Abd-B expression in embryos. These results demonstrate that the PTE is an essential component of the regulatory network at the BX-C and is required in vivo to mediate specific long-range enhancer-promoter interactions
The Dictyostelium discoideum genome lacks significant DNA methylation and uncovers palindromic sequences as a source of false positives in bisulfite sequencing
DNA methylation, the addition of a methyl (CH3) group to a cytosine residue, is an evolutionarily conserved epigenetic mark involved in a number of different biological functions in eukaryotes, including transcriptional regulation, chromatin structural organization, cellular differentiation and development. In the social amoeba Dictyostelium, previous studies have shown the existence of a DNA methyltransferase (DNMA) belonging to the DNMT2 family, but the extent and function of 5-methylcytosine in the genome are unclear. Here, we present the whole genome DNA methylation profile of Dictyostelium discoideum using deep coverage replicate sequencing of bisulfite-converted gDNA extracted from post-starvation cells. We find an overall very low number of sites with any detectable level of DNA methylation, occurring at significant levels in only 303-3432 cytosines out of the ∼7.5 million total cytosines in the genome depending on the replicate. Furthermore, a knockout of the DNMA enzyme leads to no overall decrease in DNA methylation. Of the identified sites, significant methylation is only detected at 11 sites in all four of the methylomes analyzed. Targeted bisulfite PCR sequencing and computational analysis demonstrate that the methylation profile does not change during development and that these 11 cytosines are most likely false positives generated by protection from bisulfite conversion due to their location in hairpin-forming palindromic DNA sequences. Our data therefore provide evidence that there is no significant DNA methylation in Dictyostelium before fruiting body formation and identify a reproducible experimental artifact from bisulfite sequencing. © 2023 The Author(s). Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics
Recommended from our members
Functional Evolution of cis-Regulatory Modules at a Homeotic Gene in Drosophila
It is a long-held belief in evolutionary biology that the rate of molecular evolution for a given DNA sequence is inversely related to the level of functional constraint. This belief holds true for the protein-coding homeotic (Hox) genes originally discovered in Drosophila melanogaster. Expression of the Hox genes in Drosophila embryos is essential for body patterning and is controlled by an extensive array of cis-regulatory modules (CRMs). How the regulatory modules functionally evolve in different species is not clear. A comparison of the CRMs for the Abdominal-B gene from different Drosophila species reveals relatively low levels of overall sequence conservation. However, embryonic enhancer CRMs from other Drosophila species direct transgenic reporter gene expression in the same spatial and temporal patterns during development as their D. melanogaster orthologs. Bioinformatic analysis reveals the presence of short conserved sequences within defined CRMs, representing gap and pair-rule transcription factor binding sites. One predicted binding site for the gap transcription factor KRUPPEL in the IAB5 CRM was found to be altered in Superabdominal (Sab) mutations. In Sab mutant flies, the third abdominal segment is transformed into a copy of the fifth abdominal segment. A model for KRUPPEL-mediated repression at this binding site is presented. These findings challenge our current understanding of the relationship between sequence evolution at the molecular level and functional activity of a CRM. While the overall sequence conservation at Drosophila CRMs is not distinctive from neighboring genomic regions, functionally critical transcription factor binding sites within embryonic enhancer CRMs are highly conserved. These results have implications for understanding mechanisms of gene expression during embryonic development, enhancer function, and the molecular evolution of eukaryotic regulatory modules
Parent-of-origin effects on genome-wide DNA methylation in the Cape honey bee (Apis mellifera capensis) may be confounded by allele-specific methylation
Global sensitivity analysis of a dynamic model for gene expression in Drosophila embryos
It is well known that gene regulation is a tightly controlled process in early organismal development. However, the roles of key processes involved in this regulation, such as transcription and translation, are less well understood, and mathematical modeling approaches in this field are still in their infancy. In recent studies, biologists have taken precise measurements of protein and mRNA abundance to determine the relative contributions of key factors involved in regulating protein levels in mammalian cells. We now approach this question from a mathematical modeling perspective. In this study, we use a simple dynamic mathematical model that incorporates terms representing transcription, translation, mRNA and protein decay, and diffusion in an early Drosophila embryo. We perform global sensitivity analyses on this model using various different initial conditions and spatial and temporal outputs. Our results indicate that transcription and translation are often the key parameters to determine protein abundance. This observation is in close agreement with the experimental results from mammalian cells for various initial conditions at particular time points, suggesting that a simple dynamic model can capture the qualitative behavior of a gene. Additionally, we find that parameter sensitivites are temporally dynamic, illustrating the importance of conducting a thorough global sensitivity analysis across multiple time points when analyzing mathematical models of gene regulation