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Characterization of a dominant negative C. elegans Twist mutant protein with implications for human Saethre-Chotzen syndrome

¹ Department of Biology, The Catholic University of America, Washington, DC 20064, USA
² Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD 20892-0510, USA
³ Department of Biology, University of Utah, Salt Lake City, UT 84112-0840, USA

View larger version (24K): [in a new window]   Fig. 1. Comparison of the basic domain amino acid sequence of CeTwist and several other bHLH homologs. The amino acids from the basic region and surrounding sequence are shown. Sequences were obtained from the following references: CeTwist, C. elegans (Harfe et al., 1998b); dTwist, D. melanogaster (Thisse et al., 1988); hTwist, H. sapiens (Wang et al., 1997); mTwist, M. musculus (Wolf et al., 1991); xTwist, X. laevis (Hopwood et al., 1989); CeMyoD (Krause et al., 1990); CeE/DA (Krause et al., 1997). Percent values of bHLH identity include the comparison of amino acids across the basic region that is shown and the HLH region that is not shown. Numbers surrounding the amino acids correspond to amino acid numbers in each Twist homolog. The location of the n2170sd point mutation is indicated in blue. The bottom three lines indicate the single amino acid changes made in the CeTwist protein (in Fig. 4) corresponding to known mutations found in hTwist of individuals with Saethre-Chotzen syndrome (Howard et al., 1997; Rose et al., 1997; Paznekas et al., 1998).

View larger version (60K): [in a new window]   Fig. 2. The SMs are underdivided in n2170sd animals. The M lineage for the hermaphrodite is shown in A [adapted from Sulston and Horvitz (Sulston and Horvitz, 1977)]. The time of postembryonic development is indicated on the left in hours and larval stages (L1-L4). The M blast cell divides in characteristic planes: d, dorsal; v, ventral; l, left; r, right; a, anterior; p, posterior. Subsequent divisions occur on the a-p axis. gfp reporters exist that mark each tissue type that differentiates from M and the green lines shown represent gfp expression from hlh-8::gfp in SM and its descendants. Vertical lines represent cells, and horizontal lines represent cell divisions. (B-E) Images are Nomarski/GFP merged micrographs of animals with integrated hlh-8::gfp. Dorsal is upwards, and anterior is leftwards. (B) Wild-type animals have 16 SM descendants (eight can be seen in this focal plane) surrounding the developing vulval opening (white arrowhead). The green on the dorsal side of the animal is due to intestinal autofluorescence and not GFP. (C) The SMs divide in nr2061 (–) animals and this image shows greater than the wild-type number of cells near the vulval opening. (D,E) The undivided SMs in n2170sd animals can be seen along the ventral length of the animal (white arrows). The animal in D is the same age as in B and C, based on vulval development, and has three undivided SMs. In older animals (E), the SMs still have not divided and continue to express hlh-8::gfp.

View larger version (50K): [in a new window]   Fig. 3. A CeTwist protein product is detectable in nuclei of n2170sd animals. The central region of a single n2170sd hermaphrodite is shown at two different magnifications. Dorsal is upwards. (A,B) CeTwist antibody was used to observe cells that are expressing mutant CeTwist E29K protein (yellow arrowheads). (C,D) Nuclei are observed with DAPI staining. (D) A merged image of DAPI (blue) and -Twist immunofluorescence (red) staining.

View larger version (69K): [in a new window]   Fig. 4. Overexpression of DNA-binding domain mutations of hlh-8 causes phenotypes in the C. elegans postembryonic mesoderm. Transgenic lines were made by injecting various hlh-8 expression constructs in addition to the dominant marker, rol-6 listed in A into animals that already contained the hlh-8::gfp integrated reporter construct. Stable lines were established before the phenotype of each hlh-8 expression construct was assayed. Transgenic animals from the number of independent lines indicated were observed at either the L3 larval stage (when wild-type animals have two SMs) or the L4 stage (when wild-type animals have 16 SM descendants). We scored animals as mutant at the L3 stage if they had greater than two SMs and at the L4 stage if they had undivided SMs, underdivided SMs or greater than 16 SM descendants. Examples of phenotypes seen in L4 animals are shown in B-I. (B,C) Animals with no additional CeTwist expression. (D,E) Animals expressing wild-type CeTwist. (F-I) Animals expressing the analogous human Q119P mutation in CeTwist. (B,D,F,H) Nomarski and GFP merged images (not from identical focal planes). (C,E,G,I) GFP only images. (H,I) GFP images are merged from two different focal planes with yellow SM descendants and a green undivided SM. (B-G) Ventral views. (H,I) Lateral views with dorsal upwards. In all images anterior is towards the left. The white arrowhead indicates developing vulval opening. The white arrows indicate the position of undivided SMs. All other gfp-expressing cells that are green or yellow are SM descendants.

View larger version (91K): [in a new window]   Fig. 5. The mutant CeTwist (E29K) protein binds to DNA as a homodimer and as a heterodimer with CeE/DA, whereas the human Saethre-Chotzen mutations in CeTwist bind DNA only as heterodimers with CeE/DA. In vitro gel shift assays were performed using primers containing an E box bHLH binding site. Bacterially expressed and purified proteins added to the assay are indicated across the top of the gel. DA, CeE/DA; Tw, CeTwist; B, CeTwist without the basic DNA-binding domain; E29K, CeTwist with the n2170sd point mutation causing the E29K amino acid change; hQ119P, hR118H and hR116W, CeTwist with the human Saethre-Chotzen amino acid substitutions. Protein/DNA complexes are indicated on the left of the gel based on shifts in molecular weight.

View larger version (38K): [in a new window]   Fig. 6. The mutant CeTwist E29K protein cannot cooperate in vivo with CeE/DA to activate a reporter construct. Animals that had an integrated egl-15::gfp reporter construct plus extragenic heat shock expression construct(s) indicated were subjected to heat shock and after a period of recovery were scored for the amount of GFP expression observed. Two lines each of the expression constructs were scored. Embryos expressing CeTwist and CeE/DA from the heat shock promoter are labeled Wild type CeTwist. Embryos expressing CeTwist E29K and CeE/DA from heat shock promoters are labeled CeTwist E29K. Cells where either CeTwist or CeTwist E29K plus CeE/DA were overexpressed from the heat shock promoter were observed by immunostaining embryos with either the CeTwist or CeE/DA antibody (B,F or D,H, respectively). Representative embryos are shown. (A,C) Expression of egl-15::gfp was observed only in cells where CeTwist and CeE/DA were ectopically expressed. (E,G) Only background levels of fluorescence can be seen when mutant CeTwist E29K and CeE/DA are overexpressed in egl-15::gfp-containing embryos.

View larger version (73K): [in a new window]   Fig. 7. Heterozygous n2170sd/+ animals do not turn on egl-15::gfp. All panels show the central region of adult hermaphrodites surrounding the vulval opening (indicated by white arrowheads). (A,C,E,G,I,K) Nomarski images. (B,D,F,H,J,L) GFP only images showing either the vulval muscles expressing egl-15::gfp or uterine muscles expressing rgs-2::gfp as indicated. (A,B,E,F,I,J) Lateral views. (C,D,G,H,K,L) Ventral views. (A-D) Wild type. (E-H,I-L) Heterozygous nr2061 (–)/+ and n2170sd/+ animals, respectively. Both wild-type and nr2061 (–)/+ animals express egl-15::gfp and rgs-2::gfp. (I,J) n2170sd/+ animals do not express egl-15::gfp in vulval muscles. (K,L) In these animals, uterine muscles are observed by cells expressing rgs-2::gfp and vulval muscles as observed by arg-1::gfp (not shown) are formed.

View larger version (32K): [in a new window]   Fig. 8. One possible model to explain the phenotypes observed with hlh-8 homozygous and heterozygous mutants. Red oval with protruding square, wild-type CeTwist. Orange oval with protruding triangle, mutant CeTwist E29K. Blue oval, CeTwist partner protein that could be CeE/DA and/or an unknown protein. Gray lines represent dsDNA from the promoter of a target gene (e.g. egl-15) and green arrows represent transcription proceeding from the egl-15::gfp promoter and other SM patterning and differentiation targets. For details of each panel, see Discussion.