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Kabuki syndrome, a congenital craniofacial disorder, manifests from mutations in an X-linked histone H3 lysine 27 demethylase (UTX/KDM6A) or a H3 lysine 4 methylase (KMT2D). However, the cellular and molecular etiology of histone-modifying enzymes in craniofacial disorders is unknown. We now establish Kabuki syndrome as a neurocristopathy, whereby the majority of clinical features are modeled in mice carrying neural crest (NC) deletion of UTX, including craniofacial dysmorphism, cardiac defects, and postnatal growth retardation. Female UTX NC knockout (FKO) demonstrates enhanced phenotypic severity over males (MKOs), due to partial redundancy with UTY, a Y-chromosome demethylase-dead homolog. Thus, NC cells may require demethylase-independent UTX activity. Consistently, Kabuki causative point mutations upstream of the JmjC domain do not disrupt UTX demethylation. We have isolated primary NC cells at a phenocritical postmigratory timepoint in both FKO and MKO mice, and genome-wide expression and histone profiling have revealed UTX molecular function in establishing appropriate chromatin structure to regulate crucial NC stem-cell signaling pathways. However, the majority of UTX regulated genes do not experience aberrations in H3K27me3 or H3K4me3, implicating alternative roles for UTX in transcriptional control. These findings are substantiated through demethylase-dead knockin mutation of UTX, which supports appropriate facial development.

Cajal bodies (CBs) are nuclear subdomains involved in the biogenesis of several classes of small ribonucleoproteins (RNPs). A number of recent advances highlight progress in the understanding of the organization and dynamics of CB components. For example, a class of small Cajal body-specific (sca) RNPs has been discovered. Localization of scaRNPs to CBs was shown to depend on a conserved RNA motif. Intriguingly, this motif is also present in mammalian telomerase RNA and the evidence suggests that assembly of the active form of telomerase RNP occurs in and around CBs during S phase. Important steps in the assembly and modification of spliceosomal RNPs have also been shown to take place in CBs. Additional experiments have revealed the existence of kinetically distinct subclasses of CB components. Finally, the recent identification of novel markers for CBs in both Drosophila and Arabidopsis not only lays to rest questions about the evolutionary conservation of these nuclear suborganelles, but also should enable forward genetic screens for the identification of new components and pathways involved in their assembly, maintenance and function.

We report that mutation of COL11A2 causes deafness previously mapped to the DFNA13 locus on chromosome 6p. We found two families (one American and one Dutch) with autosomal dominant, non-syndromic hearing loss to have mutations in COL11A2 that are predicted to affect the triple-helix domain of the collagen protein. In both families, deafness is non-progressive and predominantly affects middle frequencies. Mice with a targeted disruption of Col11a2 also were shown to have hearing loss. Electron microscopy of the tectorial membrane of these mice revealed loss of organization of the collagen fibrils. Our findings revealed a unique ultrastructural malformation of inner-ear architecture associated with non-syndromic hearing loss, and suggest that tectorial membrane abnormalities may be one aetiology of sensorineural hearing loss primarily affecting the mid-frequencies.

The Sonic hedgehog (Shh) signalling pathway plays important roles during development and in cancer. Here we report a Shh-induced epigenetic switch that cooperates with Gli to control transcription outcomes. Before induction, poised Shh target genes are marked by a bivalent chromatin domain containing a repressive histone H3K27me3 mark and an active H3K4me3 mark. Shh activation induces a local switch of epigenetic cofactors from the H3K27 methyltransferase polycomb repressive complex 2 (PRC2) to an H3K27me3 demethylase Jmjd3/Kdm6b-centred coactivator complex. We also find that non-enzymatic activities of Jmjd3 are important and that Jmjd3 recruits the Set1/MLL H3K4 methyltransferase complexes in a Shh-dependent manner to resolve the bivalent domain. In vivo, changes of the bivalent domain accompanied Shh-activated cerebellar progenitor proliferation. Overall, our results reveal a regulatory mechanism that underlies the activation of Shh target genes and provides insight into the causes of various diseases and cancers exhibiting altered Shh signalling.

The early mammalian embryo utilizes histone H3 lysine 27 trimethylation (H3K27me3) to maintain essential developmental genes in a repressive chromatin state. As differentiation progresses, H3K27me3 is removed in a distinct fashion to activate lineage specific patterns of developmental gene expression. These rapid changes in early embryonic chromatin environment are thought to be dependent on H3K27 demethylases. We have taken a mouse genetics approach to remove activity of both H3K27 demethylases of the Kdm6 gene family, Utx (Kdm6a, X-linked gene) and Jmjd3 (Kdm6b, autosomal gene). Male embryos null for active H3K27 demethylation by the Kdm6 gene family survive to term. At mid-gestation, embryos demonstrate proper patterning and activation of Hox genes. These male embryos retain the Y-chromosome UTX homolog, UTY, which cannot demethylate H3K27me3 due to mutations in catalytic site of the Jumonji-C domain. Embryonic stem (ES) cells lacking all enzymatic KDM6 demethylation exhibit a typical decrease in global H3K27me3 levels with differentiation. Retinoic acid differentiations of these ES cells demonstrate loss of H3K27me3 and gain of H3K4me3 to Hox promoters and other transcription factors, and induce expression similar to control cells. A small subset of genes exhibit decreased expression associated with reduction of promoter H3K4me3 and some low-level accumulation of H3K27me3. Finally, Utx and Jmjd3 mutant mouse embryonic fibroblasts (MEFs) demonstrate dramatic loss of H3K27me3 from promoters of several Hox genes and transcription factors. Our results indicate that early embryonic H3K27me3 repression can be alleviated in the absence of active demethylation by the Kdm6 gene family., Author Summary H3K27me3 represses developmental genes at initial embryonic stages. The KDM6 family, comprised of UTX and JMJD3, are the only known proteins that demethylate H3K27me3 and they are hypothesized to catalyze the rapid removal of repressive chromatin in early mammalian development. However, we report that male embryos carrying mutations in both Utx and Jmjd3 survive to term and appear phenotypically normal at mid-gestation. We utilize several cell culture models to demonstrate that H3K27me3 is lost from repressed promoters in the absence of active KDM6 demethylation. Our data indicate that KDM6 H3K27me3 demethylation is not essential in the early embryo and that H3K27me3 loss from developmental genes occurs via novel mechanisms.

UTX (KDM6A) and UTY are homologous X and Y chromosome members of the Histone H3 Lysine 27 (H3K27) demethylase gene family. UTX can demethylate H3K27; however, in vitro assays suggest that human UTY has lost enzymatic activity due to sequence divergence. We produced mouse mutations in both Utx and Uty. Homozygous Utx mutant female embryos are mid-gestational lethal with defects in neural tube, yolk sac, and cardiac development. We demonstrate that mouse UTY is devoid of in vivo demethylase activity, so hemizygous XUtx− Y+ mutant male embryos should phenocopy homozygous XUtx− XUtx− females. However, XUtx− Y+ mutant male embryos develop to term; although runted, approximately 25% survive postnatally reaching adulthood. Hemizygous X+ YUty− mutant males are viable. In contrast, compound hemizygous XUtx− YUty− males phenocopy homozygous XUtx− XUtx− females. Therefore, despite divergence of UTX and UTY in catalyzing H3K27 demethylation, they maintain functional redundancy during embryonic development. Our data suggest that UTX and UTY are able to regulate gene activity through demethylase independent mechanisms. We conclude that UTX H3K27 demethylation is non-essential for embryonic viability., Author Summary Trimethylation at Lysine 27 of histone H3 (H3K27me3) establishes a repressive chromatin state in silencing an array of crucial developmental genes. Polycomb repressive complex 2 (PRC2) catalyzes this precise posttranslational modification and is required in several critical aspects of development including Hox gene repression, gastrulation, X-chromosome inactivation, mono-allelic gene expression and imprinting, stem cell maintenance, and oncogenesis. Removal of H3K27 trimethylation has been proposed to be a mechanistic switch to activate large sets of genes in differentiating cells. Mouse Utx is an X-linked H3K27 demethylase that is essential for embryonic development. We now demonstrate that Uty, the Y-chromosome homolog of Utx, has overlapping redundancy with Utx in embryonic development. Mouse UTY has a polymorphism in the JmjC demethylase domain that renders the protein incapable of H3K27 demethylation. Therefore, the overlapping function of UTX and UTY in embryonic development is due to H3K27 demethylase independent mechanism. Moreover, the presence of UTY allows UTX-deficient mouse embryos to survive until birth. Thus, UTX H3K27 demethylation is not essential for embryonic viability. These intriguing results raise new questions on how H3K27me3 repression is removed in the early embryo.

The assembly of metazoan Sm-class small nuclear ribonucleoproteins (snRNPs) is an elaborate, step-wise process that takes place in multiple subcellular compartments. The initial steps, including formation of the core RNP, are mediated by the survival motor neuron (SMN) protein complex. Loss-of-function mutations in human SMN1 result in a neuromuscular disease called spinal muscular atrophy. The SMN complex is comprised of SMN and a number of tightly associated proteins, collectively called Gemins. In this report, we identify and characterize the fruitfly ortholog of the DEAD box protein, Gemin3. Drosophila Gemin3 (dGem3) colocalizes and interacts with dSMN in vitro and in vivo. RNA interference for dGem3 codepletes dSMN and inhibits efficient Sm core assembly in vitro. Transposon insertion mutations in Gemin3 are larval lethals and also codeplete dSMN. Transgenic overexpression of dGem3 rescues lethality, but overexpression of dSMN does not, indicating that loss of dSMN is not the primary cause of death. Gemin3 mutant larvae exhibit motor defects similar to previously characterized Smn alleles. Remarkably, appreciable numbers of Gemin3 mutants (along with one previously undescribed Smn allele) survive as larvae for several weeks without pupating. Our results demonstrate the conservation of Gemin3 protein function in metazoan snRNP assembly and reveal that loss of either Smn or Gemin3 can contribute to neuromuscular dysfunction.

Coilin is the signature protein of the Cajal body (CB), a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Newly imported Sm-class snRNPs are thought to traffic through CBs before proceeding to their final nuclear destinations. Loss of coilin function in mice leads to significant viability and fertility problems. Coilin interacts directly with the spinal muscular atrophy (SMA) protein via dimethylarginine residues in its C-terminal domain. Although coilin hypomethylation results in delocalization of survival of motor neurons (SMN) from CBs, high concentrations of snRNPs remain within these structures. Thus, CBs appear to be involved in snRNP maturation, but factors that tether snRNPs to CBs have not been described. In this report, we demonstrate that the coilin C-terminal domain binds directly to various Sm and Lsm proteins via their Sm motifs. We show that the region of coilin responsible for this binding activity is separable from that which binds to SMN. Interestingly, U2, U4, U5, and U6 snRNPs interact with the coilin C-terminal domain in a glutathione S-transferase (GST)-pulldown assay, whereas U1 and U7 snRNPs do not. Thus, the ability to interact with free Sm (and Lsm) proteins as well as with intact snRNPs, indicates that coilin and CBs may facilitate the modification of newly formed snRNPs, the regeneration of 'mature' snRNPs, or the reclamation of unassembled snRNP components.

Spinal muscular atrophy (SMA) is a genetic disorder caused by mutations in the human survival of motor neuron 1 gene,SMN1. SMN protein is part of a large complex that is required for biogenesis of various small nuclear ribonucleoproteins (snRNPs). Here, we report that SMN interacts directly with the Cajal body signature protein, coilin, and that this interaction mediates recruitment of the SMN complex to Cajal bodies. Mutation or deletion of specific RG dipeptide residues within coilin inhibits the interaction both in vivo and in vitro. Interestingly, GST-pulldown experiments show that coilin also binds directly to SmB‘. Competition studies show that coilin competes with SmB‘ for binding sites on SMN. Ectopic expression of SMN and coilin constructs in mouse embryonic fibroblasts lacking endogenous coilin confirms that recruitment of SMN and splicing snRNPs to Cajal bodies depends on the coilin C-terminal RG motif. A cardinal feature of SMA patient cells is a defect in the targeting of SMN to nuclear foci; our results uncover a role for coilin in this process.

Heterozygous Twirler (Tw) mice develop obesity and circling behavior associated with malformations of the inner ear, whereas homozygous Tw mice have cleft palate and die shortly after birth. Zeb1 is a zinc finger protein that contributes to mesenchymal cell fate by repression of genes whose expression defines epithelial cell identity. This developmental pathway is disrupted in inner ears of Tw/Tw mice. The purpose of our study was to comprehensively characterize the Twirler phenotype and to identify the causative mutation. The Tw/+ inner ear phenotype includes irregularities of the semicircular canals, abnormal utricular otoconia, a shortened cochlear duct, and hearing loss, whereas Tw/Tw ears are severely malformed with barely recognizable anatomy. Tw/+ mice have obesity associated with insulin-resistance and have lymphoid organ hypoplasia. We identified a noncoding nucleotide substitution, c.58+181G>A, in the first intron of the Tw allele of Zeb1 (Zeb1Tw). A knockin mouse model of c.58+181G>A recapitulated the Tw phenotype, whereas a wild-type knockin control did not, confirming the mutation as pathogenic. c.58+181G>A does not affect splicing but disrupts a predicted site for Myb protein binding, which we confirmed in vitro. In comparison, homozygosity for a targeted deletion of exon 1 of mouse Zeb1, Zeb1ΔEx1, is associated with a subtle abnormality of the lateral semicircular canal that is different than those in Tw mice. Expression analyses of E13.5 Twirler and Zeb1ΔEx1 ears confirm that Zeb1ΔEx1 is a null allele, whereas Zeb1Tw RNA is expressed at increased levels in comparison to wild-type Zeb1. We conclude that a noncoding point mutation of Zeb1 acts via a gain-of-function to disrupt regulation of Zeb1Tw expression, epithelial-mesenchymal cell fate or interactions, and structural development of the inner ear in Twirler mice. This is a novel mechanism underlying disorders of hearing or balance., Author Summary Twirler (Tw) mice have a combination of abnormalities that includes cleft palate, malformations of the inner ear, hearing loss, vestibular dysfunction, obesity, and lymphoid hypoplasia. In this study, we show that the underlying mutation affects the Zeb1 gene. Zeb1 was already known to encode a protein normally expressed in mesenchymal cells, where it represses expression of genes that are uniquely expressed in epithelial cells. The Tw mutation is a rare example of a single-nucleotide substitution in a region of a gene that does not encode protein, promoter, or splice sites, so we engineered a mouse model with the mutation that confirmed its causative role. The Tw mutation disrupts a consensus DNA binding site sequence for the Myb family of regulatory proteins. We conclude that this mutation leads to abnormal expression of Zeb1, structural malformations of the inner ear, and a loss of hearing and balance function. A similar mechanism may underlie other features of Twirler, such as obesity and cleft palate.