Transcriptional Control of C. elegans Male Tail Tip Morphogenesis by DMD-3
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. Overall design: ChIP-seq of GFP::DMD-3 in C. elegans L4-stage males using a GFP antibody non-antibody treated input DNA samples were used as a control