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Tissue-specific RNA-seq defines genes governing male tail tip morphogenesis in C. elegans.

ABSTRACT: Caenorhabditis elegans males undergo sex-specific tail tip morphogenesis (TTM) under the control of the transcription factor DMD-3. To find genes regulated by DMD-3, We performed RNA-seq of laser-dissected tail tips. We identified 564 genes differentially expressed (DE) in wild-type males vs. dmd-3(-) males and hermaphrodites. The transcription profile of dmd-3(-) tail tips is similar to that in hermaphrodites. For validation, we analyzed transcriptional reporters for 49 genes and found male-specific or male-biased expression for 26 genes. Only 11 DE genes overlapped with genes found in a previous RNAi screen for defective TTM. GO enrichment analysis of DE genes finds upregulation of genes within the UPR (unfolded protein response) pathway and downregulation of genes involved in cuticle maintenance. Of the DE genes, 40 are transcription factors, indicating that the gene network downstream of DMD-3 is complex and potentially modular. We propose modules of genes that act together in TTM and are coregulated by DMD-3, among them the chondroitin synthesis pathway and the hypertonic stress response.

SUBMITTER: Kiontke K

PROVIDER: S-EPMC10802606 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Tissue-specific RNA-seq defines genes governing male tail tip morphogenesis in C. elegans.

Kiontke Karin K Herrera R Antonio RA Mason D Adam DA Woronik Alyssa A Vernooy Stephanie S Patel Yash Y Fitch David H A DHA

bioRxiv : the preprint server for biology 20240112

Caenorhabditis elegans males undergo sex-specific tail tip morphogenesis (TTM) under the control of the transcription factor DMD-3. To find genes regulated by DMD-3, We performed RNA-seq of laser-dissected tail tips. We identified 564 genes differentially expressed (DE) in wild-type males vs. dmd-3(-) males and hermaphrodites. The transcription profile of dmd-3(-) tail tips is similar to that in hermaphrodites. For validation, we analyzed transcriptional report ...[more]

PMID: 38260477

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Project description:How transcriptional regulators are linked to cell biological effectors of morphogenesis is not fully understood. To elucidate this linkage, we use the C. elegans male tail-tip as a model. The tail-tip is made of four cells that, in males, undergo changes in shape and position during the L4 stage. The transcription factor DMD-3 is the master regulator of this Tail-Tip Morphogenesis (TTM). To identify other genes involved in TTM, the lab has previously performed a genome-wide RNAi screen and conducted an RNA-seq differential expression (DE) analysis using tail tips isolated from L4 wild-type and dmd-3 mutant males. This yielded candidates for genes downstream of and regulated by DMD-3 directly or indirectly. To identify the direct targets of DMD-3, we conducted male-specific DMD-3 ChIP-seq on whole worms during TTM. We identified 1755 DMD-3 binding peaks representing 6061 genes and a de novo DMD-3-associated binding motif, sharing similarity to the chromatin binding factor, EOR-1. Integrating this data with the previous RNAi screen and the DE analysis, we found 270 candidates for direct DMD-3 targets in tail-tip cells. To validate these candidates, we inserted GFP into the endogenous loci using CRISPR and evaluated the expression of the fusion proteins. We then deleted the sequences associated with DMD-3 ChIP peaks or the DMD-3-associated binding motif and assessed if this resulted in a protein expression change or defective TTM. We found four genes where deletion of a peak had an effect: The transcription factor FOS-1 is expressed in nuclei of male tail tips during TTM and in the vulva. Deletion of a DMD-3 peak or DMD-3-associated motif sites abolishes expression in male tail-tip nuclei. NMY-2 is localized as a “cap” at the tip of the tail during TTM. Removal of the peak reduces the level of tail tip NMY-2 expression. The pan-1 locus has two peaks. PAN-1::GFP is first seen in puncta at the cell surface, then at basal lateral membranes. Removal of the distal peak causes TTM defects in 10% of screened males, while removal of both peaks leads to global, previously reported, pan-1 phenotypes. HMR-1 is expressed at adherens junctions at the onset of TTM and later forms puncta at the cell surface. Deleting the peak has little effect on expression but results in epidermal bulges in both sexes, including the mail tail region. Because of the global phenotypes observed in peak mutants, we hypothesize the peak regions contain binding sites for other factors. Additionally, using extrachromosomal arrays, we tested and confirmed the DMD-3-associated motif’s contribution to wild-type TTM.

Dataset Information

Tissue-specific RNA-seq defines genes governing male tail tip morphogenesis in C. elegans.

Publications

Tissue-specific RNA-seq defines genes governing male tail tip morphogenesis in <i>C. elegans</i>.

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