A Drosophila melanogaster model of spinal muscular atrophy reveals a function for SMN in striated muscle

Mutations in human survival motor neurons 1 (SMN1) cause spinal muscular atrophy (SMA) and are associated with defects in assembly of small nuclear ribonucleoproteins (snRNPs) in vitro. However, the etiological link between snRNPs and SMA is unclear. We have developed a Drosophila melanogaster system to model SMA in vivo. Larval-lethal Smn-null mutations show no detectable snRNP reduction, making it unlikely that these animals die from global snRNP deprivation. Hypomorphic mutations in Smn reduce dSMN protein levels in the adult thorax, causing flightlessness and acute muscular atrophy. Mutant flight muscle motoneurons display pronounced axon routing and arborization defects. Moreover, Smn mutant myofibers fail to form thin filaments and phenocopy null mutations in Act88F, which is the flight muscle–specific actin isoform. In wild-type muscles, dSMN colocalizes with sarcomeric actin and forms a complex with α-actinin, the thin filament crosslinker. The sarcomeric localization of Smn is conserved in mouse myofibrils. These observations suggest a muscle-specific function for SMN and underline the importance of this tissue in modulating SMA severity

In vivo kinetics of Cajal body components

Cajal bodies (CBs) are subnuclear domains implicated in small nuclear ribonucleoprotein (snRNP) biogenesis. In most cell types, CBs coincide with nuclear gems, which contain the survival of motor neurons (SMN) complex, an essential snRNP assembly factor. Here, we analyze the exchange kinetics of multiple components of CBs and gems in living cells using photobleaching microscopy. We demonstrate differences in dissociation kinetics of CB constituents and relate them to their functions. Coilin and SMN complex members exhibit relatively long CB residence times, whereas components of snRNPs, small nucleolar RNPs, and factors shared with the nucleolus have significantly shorter residence times. Comparison of the dissociation kinetics of these shared proteins from either the nucleolus or the CB suggests the existence of compartment-specific retention mechanisms. The dynamic properties of several CB components do not depend on their interaction with coilin because their dissociation kinetics are unaltered in residual nuclear bodies of coilin knockout cells. Photobleaching and fluorescence resonance energy transfer experiments demonstrate that coilin and SMN can interact within CBs, but their interaction is not the major determinant of their residence times. These results suggest that CBs and gems are kinetically independent structures

Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product

Cajal bodies (CBs) are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin (Fb) and Nopp140 (Nopp), the survival motor neuron (SMN) protein complex, and the CB marker protein, p80 coilin. We report the generation of knockout mice lacking the COOH-terminal 487 amino acids of coilin. Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals. Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds. Analysis of tissues and cell lines from mutant animals reveals the presence of extranucleolar foci that contain Fb and Nopp but not other typical nucleolar markers. These so-called “residual” CBs neither condense Sm proteins nor recruit members of the SMN protein complex. Transient expression of wild-type mouse coilin in knockout cells results in formation of CBs and restores these missing epitopes. Our data demonstrate that full-length coilin is essential for proper formation and/or maintenance of CBs and that recruitment of snRNP and SMN complex proteins to these nuclear subdomains requires sequences within the coilin COOH terminus

T Follicular Helper Cell-Dependent Clearance of a Persistent Virus Infection Requires T Cell Expression of the Histone Demethylase UTX

Epigenetic changes, including histone methylation, control T cell differentiation and memory formation, though the enzymes that mediate these processes are not clear. We show that UTX, a histone H3 lysine 27 (H3K27) demethylase, supports T follicular helper (Tfh) cell responses that are essential for B cell antibody generation and the resolution of chronic viral infections. Mice with a T cell-specific UTX deletion had fewer Tfh cells, reduced germinal center responses, lacked virus-specific immunoglobulin G (IgG), and were unable to resolve chronic lymphocytic choriomeningitis virus infections. UTX-deficient T cells showed decreased expression of interleukin-6 receptor-α and other Tfh cell-related genes that were associated with increased H3K27 methylation. Additionally, Turner Syndrome subjects, who are predisposed to chronic ear infections, had reduced UTX expression in immune cells and decreased circulating CD4(+) CXCR5(+) T cell frequency. Thus, we identify a critical link between UTX in T cells and immunity to infection

A Noncoding Point Mutation of Zeb1 Causes Multiple Developmental Malformations and Obesity in Twirler Mice

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

Coilin forms the bridge between Cajal bodies and SMN, the Spinal Muscular Atrophy protein

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

UTX and UTY Demonstrate Histone Demethylase-Independent Function in Mouse Embryonic Development

<div><p>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, <em>in vitro</em> assays suggest that human UTY has lost enzymatic activity due to sequence divergence. We produced mouse mutations in both <em>Utx</em> and <em>Uty</em>. Homozygous <em>Utx</em> 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 <em>in vivo</em> demethylase activity, so hemizygous X<em>^Utx−</em> Y<em>⁺</em> mutant male embryos should phenocopy homozygous X<em>^Utx−</em> X<em>^Utx−</em> females. However, X<em>^Utx−</em> Y<em>⁺</em> mutant male embryos develop to term; although runted, approximately 25% survive postnatally reaching adulthood. Hemizygous X<em>⁺</em> Y<em>^Uty−</em> mutant males are viable. In contrast, compound hemizygous X<em>^Utx−</em> Y<em>^Uty−</em> males phenocopy homozygous X<em>^Utx−</em> X<em>^Utx−</em> 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.</p> </div

<i>Utx</i> mutant alleles.

<p>(A) Schematics of mouse mutations in <i>Utx</i>. Included are annotations and locations of where the protein would be mutated. Two <i>Utx</i> mutant alleles included a gene trap in intron 3 (X<i>^UtxGT1</i>) and a gene trap/floxed exon 3 (X<i>^UtxGT2fl</i>). A UTX protein annotation is illustrated at the top to indicate to positions of <i>Utx</i> alleles. A germline Cre recombinase deleted exon 3 in the X<i>^UtxGT2fl</i> background to create X<i>^UtxGT2Δ</i>. Additionally, the gene trap of X<i>^UtxGT2fl</i> was excised with Flp recombinase to create a standard floxed exon 3 (X<i>^Utxfl</i>) and Cre recombination created X<i>^UtxΔ</i>. (B) Western blotting of E18.5 liver demonstrates a complete loss of UTX in X<i>^UtxGT1</i> Y<i>^Uty+</i> lysates. RbBP5 was used as a loading control. (C) Western blotting of E10.5 whole embryo demonstrates a complete loss of UTX in X<i>^UtxΔ</i> Y<i>^Uty+</i> and X<i>^UtxΔ</i> X<i>^UtxΔ</i> lysates. RbBP5 was used as a loading control. (D) Western blotting of E12.5 primary MEFs demonstrates a reduction of UTX in X<i>^UtxGT2fl</i> Y<i>^Uty+</i> and X<i>^UtxGT2fl</i> X<i>^UtxGT2fl</i> lysates. RbBP5 was used as a loading control.</p

UTX and UTY redundancy is essential for progression of cardiac development.

<p>(A) Similar sized <i>Utx</i> heterozygous (i), <i>Utx</i> homozygous (ii), <i>Utx</i> hemizygous (iii), or <i>Utx/Uty</i> compound hemizygous (iv) embryos were analyzed in more detail for cardiac developmental abnormalities. Frontal views of the respective hearts of these embryos revealed that <i>Utx</i> homozygotes and <i>Utx/Uty</i> compound hemizygotes (A-vi, viii) have smaller hearts that have not completed looping relative to <i>Utx</i> heterozygotes (A-v) or hemizygotes (A-vii). A white dashed line was drawn at an identical angle in all panels to illustrate the failure of hearts to loop around in alignment with this appropriate plane. Only control and <i>Utx</i> hemizygous embryos (A-v, vii) have initiated the formation of the interventricular groove (white arrows), indicative of early interventricular septum development. (B) Transverse sections of E10.5 X<i>^UtxGT2Δ</i> X<i>^UtxGT2Δ</i> (B-ii) and X<i>^UtxGT2Δ</i> Y<i>^UtyGT</i> (B-iv) embryos reveal smaller heart size with defects in ventricular myocardial trabeculation and organization. The control and <i>Utx</i> hemizygous hearts initiated the formation of the interventricular septum (B-i, iii, IVS, black arrow), while other mutant combinations (B-ii, iv) have not (red asterisk). RA and LA = Right and Left Atrium, and RV and LV = Right and Left Ventricle. (C) More magnified images further illustrate the narrowing of the ventricular wall (red scale) and the lack of myocardial cells and structure in <i>Utx</i> homozygotes and <i>Utx/Uty</i> compound hemizygotes (Epi = epicardium, MC = myocardium, Endo = Endocardium).</p