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BMC Research Notes Sep 2023Investigating protein-DNA interactions is imperative to understanding fundamental concepts such as cell growth, differentiation, and cell development in many systems....
OBJECTIVES
Investigating protein-DNA interactions is imperative to understanding fundamental concepts such as cell growth, differentiation, and cell development in many systems. Sequencing techniques such as ChIP-seq can yield genome-wide DNA binding profiles of transcription factors; however this assay can be expensive, time-consuming, may not be informative for repetitive regions of the genome, and depend heavily upon antibody suitability. Combining DNA fluorescence in situ hybridization (FISH) with immunofluorescence (IF) is a quicker and inexpensive approach which has historically been used to investigate protein-DNA interactions in individual nuclei. However, these assays are sometimes incompatible due to the required denaturation step in DNA FISH that can alter protein epitopes, hindering primary antibody binding. Additionally, combining DNA FISH with IF may be challenging for less experienced trainees. Our goal was to develop an alternative technique to investigate protein-DNA interactions by combining RNA FISH with IF.
RESULTS
We developed a hybrid RNA FISH-IF protocol for use on Drosophila melanogaster polytene chromosome spreads in order to visualize colocalization of proteins and DNA loci. We demonstrate that this assay is sensitive enough to determine if our protein of interest, Multi sex combs (Mxc), localizes to single-copy target transgenes carrying histone genes. Overall, this study provides an alternative, accessible method for investigating protein-DNA interactions at the single gene level in Drosophila melanogaster polytene chromosomes.
Topics: Animals; Drosophila; Drosophila melanogaster; RNA; Polytene Chromosomes; In Situ Hybridization, Fluorescence; Fluorescent Antibody Technique; Tumor Suppressor Proteins; Drosophila Proteins
PubMed: 37679799
DOI: 10.1186/s13104-023-06482-0 -
Developmental Biology Dec 2023The transcription factor ZFH-2 has well-documented roles in Drosophila neurogenesis and other developmental processes. Here we provide the first evidence that ZFH-2 has...
The transcription factor ZFH-2 has well-documented roles in Drosophila neurogenesis and other developmental processes. Here we provide the first evidence that ZFH-2 has a role in oogenesis. We demonstrate that ZFH-2 is expressed in the wild-type ovary and that a loss of zfh-2 function produces a mutant ovary phenotype where egg chambers are reduced in number and fused. We also show that a loss of zfh-2 function can suppress a daughterless loss-of-function ovary phenotype suggesting a possible genetic relationship between these two genes in the ovary. We also show that ZFH-2 is located at the boundary between bands and interbands on polytene chromosomes and that at a subset of these sites ZFH-2 colocalizes with the insulator/promoter cofactor CP190.
Topics: Animals; Female; Chromosomes; Drosophila; Drosophila melanogaster; Drosophila Proteins; Microtubule-Associated Proteins; Nuclear Proteins; Ovarian Follicle; Ovary; Polytene Chromosomes
PubMed: 37666353
DOI: 10.1016/j.ydbio.2023.09.001 -
BioRxiv : the Preprint Server For... Jun 2023Investigating protein-DNA interactions is imperative to understanding fundamental concepts such as cell growth, differentiation, and cell development in many systems....
OBJECTIVES
Investigating protein-DNA interactions is imperative to understanding fundamental concepts such as cell growth, differentiation, and cell development in many systems. Sequencing techniques such as ChIP-seq can yield genome-wide DNA binding profiles of transcription factors; however this assay can be expensive, time-consuming, may not be informative for repetitive regions of the genome, and depend heavily upon antibody suitability. Combining DNA fluorescence in situ hybridization (FISH) with immunofluorescence (IF) is a quicker and inexpensive approach which has historically been used to investigate protein-DNA interactions in individual nuclei. However, these assays are sometimes incompatible due to the required denaturation step in DNA FISH that can alter protein epitopes, hindering primary antibody binding. Additionally, combining DNA FISH with IF may be challenging for less experienced trainees. Our goal was to develop an alternative technique to investigate protein-DNA interactions by combining RNA FISH with IF.
RESULTS
We developed a hybrid RNA FISH and IF protocol for use on polytene chromosome spreads in order to visualize colocalization of proteins and DNA loci. We demonstrate that this assay is sensitive enough to determine if our protein of interest, Multi-sex combs (Mxc), localizes to single-copy target transgenes carrying histone genes. Overall, this study provides an alternative, accessible method for investigating protein-DNA interactions at the single gene level in polytene chromosomes.
PubMed: 37398336
DOI: 10.1101/2023.06.12.544616 -
Doklady. Biochemistry and Biophysics Apr 2023The Drosophila transcription factor СР190 is one of the key proteins that determine the activity of housekeeping gene promoters and insulators. CP190 has an N-terminal...
The Drosophila transcription factor СР190 is one of the key proteins that determine the activity of housekeeping gene promoters and insulators. CP190 has an N-terminal BTB domain that allows for dimerization. Many of known Drosophila architectural proteins interact with the hydrophobic peptide-binding groove in the BTB domain, which is presumably a mechanisms for recruiting CP190 to regulatory elements. To study the role of the BTB domain in the interaction with architectural proteins, we obtained transgenic flies expressing CP190 variants with mutations in the peptide-binding groove, which disrupts their interaction with architectural proteins. As a result of the studies, it was found that mutations in the BTB domain do not affect binding of the CP190 protein to polytene chromosomes. Thus, our studies confirm the previously obtained data that CP190 is recruited to regulatory elements by several transcription factors interacting, in addition to BTB, with other CP190 domains.
Topics: Animals; Drosophila melanogaster; BTB-POZ Domain; Nuclear Proteins; Microtubule-Associated Proteins; Transcription Factors; Drosophila Proteins; Drosophila; Mutation; Peptides
PubMed: 37340291
DOI: 10.1134/S1607672922600208 -
Acta Tropica Sep 2023Simulium damnosum s.l., the most important vector of onchocerciasis in Africa, is a complex of sibling species described on the basis of differences in their larval... (Review)
Review
Simulium damnosum s.l., the most important vector of onchocerciasis in Africa, is a complex of sibling species described on the basis of differences in their larval polytene chromosomes. These (cyto) species differ in their geographical distributions, ecologies and epidemiological roles. In Togo and Benin, distributional changes have been recorded as a consequence of vector control and environmental changes (e.g. creation of dams, deforestation), with potential epidemiological consequences. We review the distribution of cytospecies in Togo and Benin and report changes observed from 1975 to 2018. The elimination of the Djodji form of S. sanctipauli in south-western Togo in 1988 seems to have had no long-term effects on the distribution of the other cytospecies, despite an initial surge by S. yahense. Although we report a general tendency for long-term stability in most cytospecies' distributions, we also assess how the cytospecies' geographical distributions have fluctuated and how they vary with the seasons. In addition to seasonal expansions of geographical ranges by all species except S. yahense, there are seasonal variations in the relative abundances of cytospecies within a year. In the lower Mono river, the Beffa form of S. soubrense predominates in the dry season but is replaced as the dominant taxon in the rainy season by S. damnosum s.str. Deforestation was previously implicated in an increase of savanna cytospecies in southern Togo (1975-1997), but our data had little power to support (or refute) suggestions of a continuing increase, partly because of a lack of recent sampling. In contrast, the construction of dams and other environmental changes including climate change seem to be leading to decreases in the populations of S. damnosum s.l. in Togo and Benin. If so, combined with the disappearance of the Djodji form of S. sanctipauli, a potent vector, plus historic vector control actions and community directed treatments with ivermectin, onchocerciasis transmission in Togo and Benin is much reduced compared with the situation in 1975.
Topics: Animals; Simuliidae; Seasons; Togo; Onchocerciasis; Benin; Insect Vectors
PubMed: 37339715
DOI: 10.1016/j.actatropica.2023.106970 -
Bulletin of Entomological Research Aug 2023The FARQ species complex consists of four highly destructive agricultural pests of Africa, namely , , , and . The members of the complex are considered very closely...
The FARQ species complex consists of four highly destructive agricultural pests of Africa, namely , , , and . The members of the complex are considered very closely related and the species limits among them are rather obscure. Their economic significance and the need for developing biological methods for their control makes species identification within the complex an important issue, which has become clear that can only be addressed by multidisciplinary approaches. Chromosomes, both mitotic and polytene, can provide a useful tool for species characterization and phylogenetic inference among closely related dipteran species. In the current study, we present the mitotic karyotype and the polytene chromosomes of and together with hybridization data. We performed a comparative cytogenetic analysis among the above two species and , the only other cytogenetically studied member of the FARQ complex, by comparing the mitotic complement and the banding pattern of the polytene chromosomes of each species to the others, as well as by studying the polytene chromosomes of hybrids between them. Our analysis revealed no detectable chromosomal rearrangements discriminating the three FARQ members studied, confirming their close phylogenetic relationships.
Topics: Animals; Tephritidae; Rosa; Phylogeny; Karyotyping; Karyotype
PubMed: 37325903
DOI: 10.1017/S0007485323000214 -
Cells Mar 2023Nrf2 is the dominant cellular stress response factor that protects cells through transcriptional responses to xenobiotic and oxidative stimuli. Nrf2 malfunction is...
Nrf2 is the dominant cellular stress response factor that protects cells through transcriptional responses to xenobiotic and oxidative stimuli. Nrf2 malfunction is highly correlated with many human diseases, but the underlying molecular mechanisms remain to be fully uncovered. GATA4 is a conserved GATA family transcription factor that is essential for cardiac and dorsal epidermal development. Here, we describe a novel interaction between Nrf2 and GATA4 proteins, i.e., cap'n'collar C (CncC) and Pannier (Pnr), respectively. Using the bimolecular fluorescence complementation (BiFC) assay-a unique imaging tool for probing protein complexes in living cells-we detected CncC-Pnr complexes in the nuclei of embryonic and salivary gland cells. Visualization of CncC-Pnr BiFC signals on the polytene chromosome revealed that CncC and Pnr tend to form complexes in euchromatic regions, with a preference for loci that are not highly occupied by CncC or Pnr alone. Most genes within these loci are activated by the CncC-Pnr BiFC, but not by individually expressed CncC or Pnr fusion proteins, indicating a novel mechanism whereby CncC and Pnr interact at specific genomic loci and coactivate genes at these loci. Finally, CncC-induced early lethality can be rescued by Pnr depletion, suggesting that CncC and Pnr function in the same genetic pathway during the early development of . Taken together, these results elucidate a novel crosstalk between the Nrf2 xenobiotic/oxidative response factor and GATA factors in the transcriptional regulation of development. This study also demonstrates that the polytene chromosome BiFC assay is a valuable tool for mapping genes that are targeted by specific transcription factor complexes.
Topics: Animals; Chromatin; Drosophila; Drosophila Proteins; GATA4 Transcription Factor; NF-E2-Related Factor 2; Polytene Chromosomes; Xenobiotics; Transcriptional Activation
PubMed: 36980279
DOI: 10.3390/cells12060938 -
RNA (New York, N.Y.) May 2023Eukaryotic mRNAs are modified at the 5' end with a methylated guanosine (mG) that is attached to the transcription start site (TSS) nucleotide. The TSS nucleotide is...
Eukaryotic mRNAs are modified at the 5' end with a methylated guanosine (mG) that is attached to the transcription start site (TSS) nucleotide. The TSS nucleotide is 2'--methylated (Nm) by CMTR1 in organisms ranging from insects to human. In mammals, the TSS adenosine can be further -methylated by RNA polymerase II phosphorylated CTD-interacting factor 1 (PCIF1) to create mAm. Curiously, the fly ortholog of mammalian PCIF1 is demonstrated to be catalytic-dead, and its functions are not known. Here, we show that mutant flies display a reduced fertility which is particularly marked in females. Deep sequencing analysis of mutant ovaries revealed transcriptome changes with a notable increase in expression of genes belonging to the mitochondrial ATP synthetase complex. Furthermore, the Pcif1 protein is distributed along euchromatic regions of polytene chromosomes, and the mutation behaved as a modifier of position-effect-variegation (PEV) suppressing the heterochromatin-dependent silencing of the gene. Similar or stronger changes in the transcriptome and PEV phenotype were observed in flies that expressed a cytosolic version of Pcif1. These results point to a nuclear cotranscriptional gene regulatory role for the catalytic-dead fly Pcif1 that is probably based on its conserved ability to interact with the RNA polymerase II carboxy-terminal domain.
Topics: Female; Animals; Humans; Drosophila; RNA Polymerase II; Fertility; Transcriptome; Nucleotides; Drosophila melanogaster; Mammals; Nuclear Proteins; Adaptor Proteins, Signal Transducing
PubMed: 36754578
DOI: 10.1261/rna.079192.122 -
Methods in Molecular Biology (Clifton,... 2023Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins that modulate multiple aspects of gene activity and RNA processing, including...
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins that modulate multiple aspects of gene activity and RNA processing, including transcription, splicing, localization, translation, and decay of RNA. Interaction of hnRNPs with RNA is a highly dynamic but regulated process. Poly(ADP-ribose) polymerase (PARP)-dependent PARylation of different hnRNPs is a well-known posttranslational modification that affects their interactions with RNA. Here, we described a protocol for in situ localization of RNA-binding proteins (RBPs) on giant polytene chromosomes in Drosophila larval salivary glands, which have been widely used to visualize the dynamic binding profiles of various RBPs and other transcription-related proteins at specific loci on chromosomes. This chapter also includes a stepwise description of RNA:RNA in situ hybridization, in conjunction with immunostaining, using polytene chromosome squashes or intact tissues. We also highlight advanced live cell imaging methods, including FRAP and FLIP, using transgenic lines that express fluorescent-tagged hnRNPs. These cytological approaches can be used to visualize the localization of RNA-binding proteins and their interacting RNAs under different cellular conditions.
Topics: Animals; Drosophila; RNA-Binding Proteins; Heterogeneous-Nuclear Ribonucleoproteins; Drosophila Proteins; Poly(ADP-ribose) Polymerases; Polytene Chromosomes; RNA
PubMed: 36515841
DOI: 10.1007/978-1-0716-2891-1_16 -
Cells Dec 2022The pericentromeric heterochromatin is largely composed of repetitive sequences, making it difficult to analyze with standard molecular biological methods. At the same...
The pericentromeric heterochromatin is largely composed of repetitive sequences, making it difficult to analyze with standard molecular biological methods. At the same time, it carries many functional elements with poorly understood mechanisms of action. The search for new experimental models for the analysis of heterochromatin is an urgent task. In this work, we used the mutation, which suppresses the underreplication of all types of repeated sequences, to analyze heterochromatin regions in polytene chromosomes of . In the background, we discovered and described in detail a new inversion, , which arose on a chromosome already carrying the inversion and transferred a large part of X chromosome heterochromatin, including the nucleolar organizer to a new euchromatic environment. Using nanopore sequencing and FISH, we have identified the eu- and heterochromatin breakpoints of . The combination of the new inversion and the mutation provides a promising tool for studies of X chromosome heterochromatin structure, nucleolar organization, and the nucleolar dominance phenomenon. In particular, we found that, with the complete polytenization of rDNA repeats, the nucleolus consists of a cloud-like structure corresponding to the classical nucleolus of polytene chromosomes, as well as an unusual intrachromosomal structure containing alternating transcriptionally active and inactive regions.
Topics: Animals; Drosophila melanogaster; Heterochromatin; X Chromosome; Repetitive Sequences, Nucleic Acid; Nucleolus Organizer Region; Carrier Proteins; Drosophila Proteins
PubMed: 36497131
DOI: 10.3390/cells11233872