Evolutionarily assembled cis-regulatory module at a human ciliopathy locus

Abstract

Neighboring genes are often coordinately expressed within cis-regulatory modules, but evidence that nonparalogous genes share functions in mammals is lacking. Here, we report that mutation of either TMEM138 or TMEM216 causes a phenotypically indistinguishable human ciliopathy, Joubert syndrome. Despite a lack of sequence homology, the genes are aligned in a head-to-tail configuration and joined by chromosomal rearrangement at the amphibian-to-reptile evolutionary transition. Expression of the two genes is mediated by a conserved regulatory element in the noncoding intergenic region. Coordinated expression is important for their interdependent cellular role in vesicular transport to primary cilia. Hence, during vertebrate evolution of genes involved in ciliogenesis, nonparalogous genes were arranged to a functional gene cluster with shared regulatory elements.

Fig. 1

Genetic heterogeneity at JBTS2 locus and the evolutionary location of the TMEM138 and TMEM216 genes mutated in JBTS2-linked families. (A) JBTS2 locus (red box) on chromosome 11. TMEM138 encoding a trispan membrane protein is aligned in a head-to-tail configuration with TMEM216 encoding a tetraspan membrane protein. All missense and splicing mutations of TMEM138 and TMEM216 found in JBTS2-linked families are indicated on the predicted transcript and protein. (B) Schematic synteny representation of TMEM138, TMEM216, and the intergenic region showing that the head-to-tail configuration of TMEM138 and TMEM216 on the same chromosome is conserved from reptiles to humans, but not in lower vertebrates, in which the two TMEMs are located on different chromosomes (table S2). Conserved regions have >50% sequence similarity to humans. UTR, untranslated region.

Fig. 2

Coordinated expression of adjacent TMEM138 and TMEM216 mediated by the noncoding intergenic region. (A) Similar expression patterns of TMEM138 and TMEM216 based on in situ hybridization at 8 gw in human embryonic tissues. TMEM138 antisense (a and b), TMEM216 antisense (c and d), and sense control probes (TMEM138) (e and f) are shown. TMEM138 and TMEM216 are strongly expressed in kidney, gonad (go), and adrenal gland (ad) as well as in the central nervous system, in particular in the cerebellar bud (cb), telencephalon (tel), rhombencephalon (rh), and cranial nerve ganglia such as trigeminal (V). (B) Real-time qPCR of TMEM138 and TMEM216 in selected tissues indicates tightly coordinated mRNA expressions in mouse tissues (having head-to-tail configuration), but not zebrafish (having two genes on different chromosomes). Housekeeping genes 36B4 (mouse) and Rpl13a (zebrafish) are used for normalization.

Fig. 3

Tethered vesicular trafficking of TMEM138 and TMEM216 to primary cilia is required for ciliogenesis. (A) In patient fibroblasts under 48-hour serum starvation, TMEM138 p.A126T (MTI-656) mutations caused short cilia, and TMEM216 p.R73L mutations disrupt ciliogenesis (defined as having cilia <1 μm long).*P < 0.05, **P < 0.01 [versus wild type (WT) by one-way analysis of variance (ANOVA) with Bonferroni posttest, n = 40 to 50 cells]. Error bars indicate SEM. (B) In IMCD3 cells, high-resolution images of endogenous TMEM138/216 staining show that TMEM138 localized to ciliary axonemes and the base of cilia, whereas TMEM216 localized primarily to basal bodies. Both TMEMs also show closely adjacent vesicular patterns around the base of cilia (also see fig. S9C). Anti-Arl13b (cilia), anti-Glu/γ tubulin (cilia plus centrosome), polyclonal mouse anti-TMEM138 (fig. S9, A and B), and rabbit anti-TMEM216 antibodies were used. A, ciliary axoneme; T, transition zone; B, basal body. Scale bars, 5 μm.

Fig. 4

Functional relatedness of TMEM138 and TMEM216. (A) Live-cell imaging shows that knockdown of TRAPPC9, a major subunit of the TRAPPII complex, detached the tethered TMEM138 and TMEM216 vesicles (movies S7 and S8). Defects in tethering were largely rescued by myc-TRAPPC9* (9). ***P < 0.001 (by one-way ANOVA with Bonferroni posttest, n = 90 to 110). Scale bars, 5 μm. Error bars indicate SEM. (B) Injection of translation-blocking antisense morpholinos (MOs) to tmem138 (6 ng), tmem216 (4 ng), or double MOs in WT (AB) zebrafish embryos leads to ciliary phenotypes of curved or kinked tail (arrows) and heart edema (arrowheads) at 3 days post-fertilization in a synergistic and dose-dependent manner (fig. S18A). Only tmem216 morphants present hydrocephalus (*) (>50 embryos for each condition) (fig. S18B).