Caenorhabditis elegans TBX-2 Directly Regulates Its Own Expression in a Negative Autoregulatory Loop

Abstract

T-box genes often exhibit dynamic expression patterns, and their expression levels can be crucial for normal function. Despite the importance of these genes, there is little known about T-box gene regulation. We have focused on the Caenorhabditis elegans gene tbx-2 to understand how T-box gene expression is regulated, and here we demonstrate TBX-2 itself directly represses its own expression in a negative autoregulatory loop. tbx-2 is essential for normal pharyngeal muscle development, and a tbx-2 promoter gfp fusion (Ptbx-2::gfp) is transiently expressed in the pharynx during embryogenesis and in a small number of head neurons in larvae and adults. Reduced tbx-2 function resulted in ectopic Ptbx-2::gfp expression in the seam cells and gut in larvae and adults. Mutation of potential T-box binding sites within the tbx-2 promoter resulted in a similar pattern of ectopic Ptbx-2::gfp expression, and chromatin immunoprecipitation analyses show TBX-2 binds these sites in vivo. This pattern of ectopic Ptbx-2::gfp expression in tbx-2 mutants was very similar to that observed in mutants affecting the NF-Y complex, and our results comparing tbx-2 and nfyb-1 single- and double mutants suggest TBX-2 and NF-Y function in a single pathway to repress the tbx-2 promoter. The tbx-2 promoter is the first direct target identified for TBX-2, and we used it to ask whether SUMOylation is essential for TBX-2 repression. RNAi knockdown of SUMOylation pathway components led to ectopic Ptbx-2::gfp expression in the seam cells and gut. Ectopic Ptbx-2::gfp also was observed in the syncytial hypodermis, suggesting either the tbx-2 promoter is repressed by other SUMOylation dependent mechanisms, or that decreased SUMOylation leads to stable changes in seam cell nuclei as they fuse with the syncytial hypodermis. We suggest negative autoregulation is an important mechanism that allows precise control of tbx-2 expression levels and may allow rapid changes in gene expression during development.

Keywords: C. elegans; NF-Y; SUMOylation; T-box; negative autoregulation.

Figure 1

Ptbx-2::gfp expression in embryos and late larvae. (A) Schematic diagram of the tbx-2 region contained in fosmid WRM063aG09 indicating the location of the 3.8-kb Ptbx-2 promoter fragment and the positions of consensus T-box binding sites within this fragment (red bars). (B) Diagram of a portion of the Ptbx-2 promoter indicating the positions of candidate T-box binding sites (red dots). For clarity the orientation of this fragment is reversed relative to (A) and only the region containing T-box sites is indicated. A promoter proximal site is located at −259 bp upstream of the tbx-2 ATG (AGGTGGCA, minus strand sequence), and a pair of closely spaced distal sites are located at -1135 bp (AGGTGGCA, plus strand) and −1156 bp (ATGTGTGA, plus strand) upstream of the tbx-2 ATG, respectively. (C, D) Green fluorescent protein (GFP; left) and differential interference contrast (DIC; right) images of Ptbx-2::gfp expression in the pharyngeal primordium in an embryo at the end of gastrulation (anterior to the left). (E, F) GFP (top) and DIC (bottom) images of an L4 animal expressing Ptbx-2::gfp in head neurons (arrows). Occasional GFP expression was also observed in tail neurons, but no expression was detected elsewhere. Gut cell cytoplasm contains autofluorescent gut granules (bracket).

Figure 2

Ptbx-2::gfp is ectopically expressed in animals with reduced tbx-2 expression. Green fluorescence protein (GFP) and differential interference contrast (DIC) images of L4 and young adult animals of the indicated genotypes expressing Ptbx-2::gfp. Ectopic Ptbx-2::gfp expression detectable in seam cells (arrowheads) and gut nuclei (arrows) are indicated in tbx-2(RNAi) (C, D) and tbx-2(bx59) mutants (E, F).

Figure 3

Ectopic Ptbx-2::gfp expression in tbx-2(ok529)/+ heterozygotes. Green fluorescence protein (left) and differential interference contrast (right) images of a young adult tbx-2(ok529)/+ heterozygote ectopically expressing Ptbx-2::gfp in seam cells (arrowheads). tbx-2(ok529) is a null allele containing a 1.1-kb deletion that removes sequences encoding the C-terminus of the T-box DNA-binding domain (Roy Chowdhuri et al. 2006).

Figure 4

Ectopic Ptbx-2::gfp expression detectable in the gut in tbx-2(bx59) and nfyb-1(cu13); tbx-2(bx59) L1 animals. Green fluorescence protein (GFP) and differential interference contrast (DIC) images of L1 animals of the indicated genotypes expressing Ptbx-2::gfp. GFP expressing head neurons (arrowheads), gut cells (arrows), hyp6 (lines), and tail neurons (t) are marked. The microscopy setup was identical for each of these images, and the exposure time for each fluorescence image is indicated in milliseconds (ms). Note that Ptbx-2::gfp is very highly expressed in head neurons in tbx-2 and nfyb-1 single and double mutants, and, when compared to wild-type animals, the exposure times are much shorter.

Figure 5

TBX-2 binds T-box sites in the tbx-2 promoter in vivo. Semiquantitative polymerase chain reaction analyses of representative chromatin immunoprecipitation from tbx-2(ok529); wgIs159 animals expressing a TBX-2::GFP fusion protein (A) and wild-type N2 animals lacking any GFP (B) immunoprecipitated with anti-GFP, nonspecific IgG or no antibody as indicated. 0.25% of the total protein lysate used in the immunoprecipitation reactions is shown as input. (C) Bar graph indicating the amount of product precipitated with anti-GFP relative to the amount precipitated with IgG. Data represents the average for 3 experiments with error bars indicating standard deviation. P < 0.03 (*) or P < 0.01 (**). GFP, green fluorescent protein.

Figure 6

Disruption of SUMOylation causes misexpression of Ptbx-2::gfp. Green fluorescence protein (GFP; left) and differential interference contrast (right) images of young adult ubc-9(RNAi) (A) and gei-17(RNAi) (B) animals ectopically expressing Ptbx-2::gfp ectopically in the indicated tissues. Nuclei of GFP-expressing seam cells, lateral hypodermis, and ventral hypodermis are marked with arrowheads, whereas nuclei of GFP expressing gut cells are marked with arrows. ‘v’ marks the ventral nerve cord, and ‘vul’ marks the vulva on the ventral surface of the animal.