WDR55 is a nucleolar modulator of ribosomal RNA synthesis, cell cycle progression, and teleost organ development

The thymus is a vertebrate-specific organ where T lymphocytes are generated. Genetic programs that lead to thymus development are incompletely understood. We previously screened ethylnitrosourea-induced medaka mutants for recessive defects in thymus development. Here we report that one of those mutants is caused by a missense mutation in a gene encoding the previously uncharacterized protein WDR55 carrying the tryptophan-aspartate-repeat motif. We find that WDR55 is a novel nucleolar protein involved in the production of ribosomal RNA (rRNA). Defects in WDR55 cause aberrant accumulation of rRNA intermediates and cell cycle arrest. A mutation in WDR55 in zebrafish also leads to analogous defects in thymus development, whereas WDR55-null mice are lethal before implantation. These results indicate that WDR55 is a nuclear modulator of rRNA synthesis, cell cycle progression, and embryonic organogenesis including teleost thymus development.     

The interaction of the human MutL homologues in hereditary nonpolyposis colon cancer

Germline mutations in two human mismatch repair (MMR) genes, hMSH2 and hMLH1, appear to account for approximately 70% of the common cancer susceptibility syndrome hereditary nonpolyposis colorectal cancer (HNPCC). Although the hMLH1 protein has been found to copurify with another MMR protein hPMS2 as a heterodimer, their function in MMR is unknown. In this study, we have identified the physical interaction regions of both hMLH1 with hPMS2. We then examined the effects of hMLH1 missense alterations found in HNPCC kindreds for their interaction with hPMS2. Four of these missense alterations (L574P, K616Delta, R659P, and A681T) displayed >95% reduction in binding to hPMS2. Two additional missense alterations (K618A and K618T) displayed a >85% reduction in binding to hPMS2, whereas three missense alterations (S44F, V506A, and E578G) displayed 25-65% reduction in binding to hPMS2. Interestingly, two HNPCC missense alterations (Q542L and L582V) contained within the consensus interaction region displayed no effect on interaction with hPMS2, suggesting that they may affect other functions of hMLH1. These data confirm that functional deficiencies in the interaction of hMLH1 with hPMS2 are associated with HNPCC as well as suggest that other unknown functional alteration of the human MutL homologues may lead to tumorigenesis in HNPCC kindreds.     

WD repeat-containing protein 5, a ubiquitously expressed histone methyltransferase adaptor protein, regulates smooth muscle cell-selective gene activation through interaction with pituitary homeobox 2

WD repeat-containing protein 5 (WDR5) is a common component of mammalian mixed lineage leukemia methyltransferase family members and is important for histone H3 lysine 4 methylation (H3K4me), which has been implicated in control of activation of cell lineage genes during embryogenesis. However, WDR5 has not been considered to play a specific regulatory role in epigenetic programming of cell lineage because it is ubiquitously expressed. Previous work from our laboratory showed the appearance of histone H3K4me within smooth muscle cell (SMC)-marker gene promoters during the early stages of development of SMC from multipotential embryonic cells but did not elucidate the underlying mechanisms that mediate SMC-specific and locus-selective H3K4me. Results presented herein show that knockdown of WDR5 significantly decreased SMC-marker gene expression in cultured SMC differentiation systems and in Xenopus laevis embryos in vivo. In addition, we showed that WDR5 complexes within SMC progenitor cells contained H3K4 methyltransferase enzymatic activity and that knockdown of WDR5 selectively decreased H3K4me1 and H3K4me3 enrichment within SMC-marker gene promoter loci. Moreover, we present evidence that it is recruited to these gene promoter loci through interaction with a SMC-selective pituitary homeobox 2 (Pitx2). Taken together, studies provide evidence for a novel mechanism for epigenetic control of SMC-marker gene expression during development through interaction of WDR5, homeodomain proteins, and chromatin remodeling enzymes.     

Ciliopathies with skeletal anomalies and renal insufficiency due to mutations in the IFT-A gene WDR19

A subset of ciliopathies, including Sensenbrenner, Jeune, and short-rib polydactyly syndromes are characterized by skeletal anomalies accompanied by multiorgan defects such as chronic renal failure and retinitis pigmentosa. Through exome sequencing we identified compound heterozygous mutations in WDR19 in a Norwegian family with Sensenbrenner syndrome. In a Dutch family with the clinically overlapping Jeune syndrome, a homozygous missense mutation in the same gene was found. Both families displayed a nephronophthisis-like nephropathy. Independently, we also identified compound heterozygous WDR19 mutations by exome sequencing in a Moroccan family with isolated nephronophthisis. WDR19 encodes IFT144, a member of the intraflagellar transport (IFT) complex A that drives retrograde ciliary transport. We show that IFT144 is absent from the cilia of fibroblasts from one of the Sensenbrenner patients and that ciliary abundance and morphology is perturbed, demonstrating the ciliary pathogenesis. Our results suggest that isolated nephronophthisis, Jeune, and Sensenbrenner syndromes are clinically overlapping disorders that can result from a similar molecular cause.     

Niemann-Pick C1 disease: correlations between NPC1 mutations, levels of NPC1 protein, and phenotypes emphasize the functional significance of the putative sterol-sensing domain and of the cysteine-rich luminal loop

To obtain more information of the functional domains of the NPC1 protein, the mutational spectrum and the level of immunoreactive protein were investigated in skin fibroblasts from 30 unrelated patients with Niemann-Pick C1 disease. Nine of them were characterized by mild alterations of cellular cholesterol transport (the "variant" biochemical phenotype). The mutations showed a wide distribution to nearly all NPC1 domains, with a cluster (11/32) in a conserved NPC1 cysteine-rich luminal loop. Homozygous mutations in 14 patients and a phenotypically defined allele, combined with a new mutation, in a further 10 patients allowed genotype/phenotype correlations. Premature-termination-codon mutations, the three missense mutations in the sterol-sensing domain (SSD), and A1054T in the cysteine-rich luminal loop all occurred in patients with infantile neurological onset and "classic" (severe) cholesterol-trafficking alterations. By western blot, NPC1 protein was undetectable in the SSD missense mutations studied (L724P and Q775P) and essentially was absent in the A1054T missense allele. Our results thus enhance the functional significance of the SSD and demonstrate a correlation between the absence of NPC1 protein and the most severe neurological form. In the remaining missense mutations studied, corresponding to other disease presentations (including two adults with nonneurological disease), NPC1 protein was present in significant amounts of normal size, without clear-cut correlation with either the clinical phenotype or the "classic"/"variant" biochemical phenotype. Missense mutations in the cysteine-rich luminal loop resulted in a wide array of clinical and biochemical phenotypes. Remarkably, all five mutant alleles (I943M, V950M, G986S, G992R, and the recurrent P1007A) definitively correlated with the "variant" phenotype clustered within this loop, providing new insight on the functional complexity of the latter domain.     

Cranioectodermal Dysplasia, Sensenbrenner Syndrome, Is a Ciliopathy Caused by Mutations in the IFT122 Gene

Cranioectodermal dysplasia (CED) is a disorder characterized by craniofacial, skeletal, and ectodermal abnormalities. Most cases reported to date are sporadic, but a few familial cases support an autosomal-recessive inheritance pattern. Aiming at the elucidation of the genetic basis of CED, we collected 13 patients with CED symptoms from 12 independent families. In one family with consanguineous parents two siblings were affected, permitting linkage analysis and homozygosity mapping. This revealed a single region of homozygosity with a significant LOD score (3.57) on chromosome 3q21-3q24. By sequencing candidate genes from this interval we found a homozygous missense mutation in the IFT122 (WDR10) gene that cosegregated with the disease. Examination of IFT122 in our patient cohort revealed one additional homozygous missense change in the patient from a second consanguineous family. In addition, we found compound heterozygosity for a donor splice-site change and a missense change in one sporadic patient. All mutations were absent in 340 control chromosomes. Because IFT122 plays an important role in the assembly and maintenance of eukaryotic cilia, we investigated patient fibroblasts and found significantly reduced frequency and length of primary cilia as compared to controls. Furthermore, we transiently knocked down ift122 in zebrafish embryos and observed the typical phenotype found in other models of ciliopathies. Because not all of our patients harbored mutations in IFT122, CED seems to be genetically heterogeneous. Still, by identifying CED as a ciliary disorder, our study suggests that the causative mutations in the unresolved cases most likely affect primary cilia function too.     

Human and mouse mutations in WDR35 cause short-rib polydactyly syndromes due to abnormal ciliogenesis

Defects in cilia formation and function result in a range of human skeletal and visceral abnormalities. Mutations in several genes have been identified to cause a proportion of these disorders, some of which display genetic (locus) heterogeneity. Mouse models are valuable for dissecting the function of these genes, as well as for more detailed analysis of the underlying developmental defects. The short-rib polydactyly (SRP) group of disorders are among the most severe human phenotypes caused by cilia dysfunction. We mapped the disease locus from two siblings affected by a severe form of SRP to 2p24, where we identified an in-frame homozygous deletion of exon 5 in WDR35. We subsequently found compound heterozygous missense and nonsense mutations in WDR35 in an independent second case with a similar, severe SRP phenotype. In a mouse mutation screen for developmental phenotypes, we identified a mutation in Wdr35 as the cause of midgestation lethality, with abnormalities characteristic of defects in the Hedgehog signaling pathway. We show that endogenous WDR35 localizes to cilia and centrosomes throughout the developing embryo and that human and mouse fibroblasts lacking the protein fail to produce cilia. Through structural modeling, we show that WDR35 has strong homology to the COPI coatamers involved in vesicular trafficking and that human SRP mutations affect key structural elements in WDR35. Our report expands, and sheds new light on, the pathogenesis of the SRP spectrum of ciliopathies.     

hMRE11 deficiency leads to microsatellite instability and defective DNA mismatch repair

DNA mismatch repair (MMR) is essential in the surveillance of accurate transmission of genetic information, and defects in this pathway lead to microsatellite instability and hereditary nonpolyposis colorectal cancer (HNPCC). Our previous study raised the possibility that hMRE11 might be involved in MMR through physical interaction with hMLH1. Here, we show that hMRE11 deficiency leads to significant increase in MSI for both mono- and dinucleotide sequences. Furthermore, RNA-interference-mediated hMRE11-knockdown in HeLa cells results in MMR deficiency. Analysis of seven HNPCC-associated hMLH1 missense mutations located within the hMRE11-interacting domain shows that four mutations (L574P, K618T, R659P and A681T) cause near-complete disruption of the interaction between hMRE11 and hMLH1, and two mutations (Q542L and L582V) cause a 30% reduction of protein interaction. These findings indicate that hMRE11 represents a functional component of the MMR pathway and the disruption of hMLH1-hMRE11 interaction could be an alternative molecular explanation for hMLH1 mutations in a subset of HNPCC tumours.     

A cluster of missense mutations at Arg356 of human steroid 21-hydroxylase may impair redox partner interaction

Lesions in the gene encoding steroid 21-hydroxylase result in congenital adrenal hyperplasia, with impaired secretion of cortisol and aldosterone from the adrenal cortex and overproduction of androgens. A limited number of mutations account for the majority of mutated alleles, but additional rare mutations are responsible for the symptoms in some patients. A total of 11 missense mutations has previously been implicated in this enzyme deficiency. We describe two novel missense mutations, both affecting the same amino acid residue, Arg356. The two mutations, R356P and R356Q, were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells, and enzyme activity towards the two natural substrates, 17-hydroxyprogesterone and progesterone, was determined. The R356P mutant reduced enzyme activity to 0.15% towards both substrates, whereas the R356Q mutant exhibited 0.65% of normal activity towards 17-hydroxyprogesterone, and 1.1% of normal activity towards progesterone. These activities correspond to the degrees of disease manifestation of the patients in whom they were found. Arg356 is located in a region which recently has been implicated in redox partner interaction, by modelling the structure of two other members of the cytochrome P450 superfamily. Of the 11 previously described missense mutations, three affect arginine residues within this protein domain. With the addition of R356P and R356Q, there is a clear clustering of five mutations to three closely located basic amino acids. This supports the model in which this protein domain is involved in redox partner interaction, which takes places through electrostatic interactions between charged amino acid residues. Received:17 December 1996 / Revised: 28 January 1997     

Structure of WDR5 bound to mixed lineage leukemia protein-1 peptide

The mixed lineage leukemia protein-1 (MLL1) catalyzes histone H3 lysine 4 methylation and is regulated by interaction with WDR5 (WD-repeat protein-5), RbBP5 (retinoblastoma-binding protein-5), and the Ash2L (absent, small, homeotic discs-2-like) oncoprotein. In the accompanying investigation, we describe the identification of a conserved arginine containing motif, called the "Win" or WDR5 interaction motif, that is essential for the assembly and H3K4 dimethylation activity of the MLL1 core complex. Here we present a 1.7-A crystal structure of WDR5 bound to a peptide derived from the MLL1 Win motif. Our results show that Arg-3765 of MLL1 is bound in the same arginine binding pocket on WDR5 that was previously suggested to bind histone H3. Thermodynamic binding experiments show that the MLL1 Win peptide is preferentially recognized by WDR5. These results are consistent with a model in which WDR5 recognizes Arg-3765 of MLL1, which is essential for the assembly and enzymatic activity of the MLL1 core complex.     

The Role of WD40-Repeat Protein 62 (MCPH2) in Brain Growth: Diverse Molecular and Cellular Mechanisms Required for Cortical Development

Genetic disruptions of spindle/centrosome-associated WD40-repeat protein 62 (WDR62) are causative for autosomal recessive primary microcephaly (MCPH) and a broader range of cortical malformations. Since the identification of WDR62 as encoded by the MCPH2 locus in 2010, recent studies that have deleted/depleted WDR62 in various animal models of cortical development have highlighted conserved functions in brain growth. Here, we provide a timely review of our current understanding of WDR62 contributions in the self-renewal, expansion and fate specification of neural stem and progenitor cells that are critical for neocortical development. Recent studies have revealed multiple functions for WDR62 in the regulation of spindle organization, mitotic progression and the duplication and biased inheritance of centrosomes during asymmetric divisions. We also discuss recently elaborated WDR62 interaction partners that include Aurora and c-Jun N-terminal kinases as part of complex signalling mechanisms that may define its neural functions. These studies provide new insights into the molecular and cellular processes that are required for brain formation and implicated in the genesis of primary microcephaly.     

Autoinduction of the trefoil factor 2 (TFF2) promoter requires an upstream cis-acting element

Most benign brain tumors are associated with loss of the Nf2 gene tumor suppressor product schwannomin/merlin. Interactions between schwannomin fragments have given rise to hypotheses of in vivo schwannomin folding and dimerization. Previously, we showed that schwannomin with missense mutations L360P, L535P, and Q538P alters interaction with betaII-spectrin and Hrs. Using yeast two-hybrid tests of interaction, we now show the effects of 11 Nf2 missense mutations on schwannomin self-interaction as well as schwannomin interaction with Hrs isoforms 1 and 2, betaII-spectrin, and p110. Missense mutations L46R and K364I significantly decreased affinity of schwannomin for binding all interacting proteins. The schwannomin L46R mutation may result in a complex conformational change that alters folding and denies betaII-spectrin access to an intact binding site in the C-terminal half of schwannomin. We show that unique inter- and intramolecular interactions occur for schwannomin isoform 2, suggesting that this schwannomin isoform has unique functional properties compared to schwannomin isoform 1.     

Late-onset Stargardt disease is associated with missense mutations that map outside known functional regions of ABCR (ABCA4)

Based on recent studies of the photoreceptor-specific ABC transporter gene ABCR (ABCA4) in Stargardt disease (STGD1) and other retinal dystrophies, we and others have developed a model in which the severity of retinal disease correlates inversely with residual ABCR activity. This model predicts that patients with late-onset STGDI may retain partial ABCR activity attributable to mild missense alleles. To test this hypothesis, we used late-onset STGDI patients (onset: > or =35 years) to provide an in vivo functional analysis of various combinations of mutant alleles. We sequenced directly the entire coding region of ABCR and detected mutations in 33/50 (66%) disease chromosomes, but surprisingly, 11/33 (33%) were truncating alleles. Importantly, all 22 missense mutations were located outside the known functional domains of ABCR (ATP-binding or transmembrane), whereas in our general cohort of STGDI subjects, alterations occurred with equal frequency across the entire protein. We suggest that these missense mutations in regions of unknown function are milder alleles and more susceptible to modifier effects. Thus, we have corroborated a prediction from the model of ABCR pathogenicity that (1) one mutant ABCR allele is always missense in late-onset STGD1 patients, and (2) the age-of-onset is correlated with the amount of ABCR activity of this allele. In addition, we report three new pseudodominant families that now comprise eight of 178 outbred STGD1 families and suggest a carrier frequency of STGD1-associated ABCR mutations of about 4.5% (approximately 1/22).     

Structure-function analysis of Friedreich's ataxia mutants reveals determinants of frataxin binding and activation of the Fe-S assembly complex

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-S cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a k(cat)/K(M) higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest k(cat)/K(M) of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe-S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.