Duncan B. Sparrow

SmcHD1, containing a structural-maintenance-of-chromosomes hinge domain, has a critical role in X inactivation

X-chromosome inactivation is the mammalian dosage compensation mechanism by which transcription of X-linked genes is equalized between females and males. In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen on mice for modifiers of epigenetic reprogramming, we identified the MommeD1 (modifier of murine metastable epialleles) mutation as a semidominant suppressor of variegation. MommeD1 shows homozygous female-specific mid-gestation lethality and hypomethylation of the X-linked gene Hprt1, suggestive of a defect in X inactivation. Here we report that the causative point mutation lies in a previously uncharacterized gene, Smchd1 (structural maintenance of chromosomes hinge domain containing 1). We find that SmcHD1 is not required for correct Xist expression, but localizes to the inactive X and has a role in the maintenance of X inactivation and the hypermethylation of CpG islands associated with the inactive X. This finding links a group of proteins normally associated with structural aspects of chromosome biology with epigenetic gene silencing.

MEF‐2 function is modified by a novel co‐repressor, MITR

The MEF‐2 proteins are a family of transcriptional activators that have been detected in a wide variety of cell types. In skeletal muscle cells, MEF‐2 proteins interact with members of the MyoD family of transcriptional activators to synergistically activate gene expression. Similar interactions with tissue or lineage‐specific cofactors may also underlie MEF‐2 function in other cell types. In order to screen for such cofactors, we have used a transcriptionally inactive mutant of Xenopus MEF2D in a yeast two‐hybrid screen. This approach has identified a novel protein expressed in the early embryo that binds to XMEF2D and XMEF2A. The MEF‐2 interacting transcription repressor (MITR) protein binds to the N‐terminal MADS/MEF‐2 region of the MEF‐2 proteins but does not bind to the related Xenopus MADS protein serum response factor. In the early embryo, MITR expression commences at the neurula stage within the mature somites and is subsequently restricted to the myotomal muscle. In functional assays, MITR negatively regulates MEF‐2‐dependent transcription and we show that this repression is mediated by direct binding of MITR to the histone deacetylase HDAC1. Thus, we propose that MITR acts as a co‐repressor, recruiting a specific deacetylase to downregulate MEF‐2 activity.

Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2.

The yeast UBC9 gene encodes a protein with homology to the E2 ubiquitin-conjugating enzymes that mediate the attachment of ubiquitin to substrate proteins [1]. Depletion of Ubc9p arrests cells in G2 or early M phase and stabilizes B-type cyclins [1]. p18(Ubc9), the Xenopus homolog of Ubc9p, associates specifically with p88(RanGAP1) and p340(RanBP2) [2]. Ran-binding protein 2 (p340(RanBP2)) is a nuclear pore protein [3] [4], and p88(RanGAP1) is a modified form of RanGAP1, a GTPase-activating protein for the small GTPase Ran [2]. It has recently been shown that mammalian RanGAP1 can be conjugated with SUMO-1, a small ubiquitin-related modifier [5-7], and that SUMO-1 conjugation promotes RanGAP1s interaction with RanBP2 [2,5,6]. Here we show that p18(Ubc9) acts as an E2-like enzyme for SUMO-1 conjugation, but not for ubiquitin conjugation. This suggests that the SUMO-1 conjugation pathway is biochemically similar to the ubiquitin conjugation pathway but uses a distinct set of enzymes and regulatory mechanisms. We also show that p18(Ubc9) interacts specifically with the internal repeat domain of RanBP2, which is a substrate for SUMO-1 conjugation in Xenopus egg extracts.

Mutation of the LUNATIC FRINGE Gene in Humans Causes Spondylocostal Dysostosis with a Severe Vertebral Phenotype

The spondylocostal dysostoses (SCDs) are a heterogeneous group of vertebral malsegmentation disorders that arise during embryonic development by a disruption of somitogenesis. Previously, we had identified two genes that cause a subset of autosomal recessive forms of this disease: DLL3 (SCD1) and MESP2 (SCD2). These genes are important components of the Notch signaling pathway, which has multiple roles in development and disease. Here, we have used a candidate-gene approach to identify a mutation in a third Notch pathway gene, LUNATIC FRINGE (LFNG), in a family with autosomal recessive SCD. LFNG encodes a glycosyltransferase that modifies the Notch family of cell-surface receptors, a key step in the regulation of this signaling pathway. A missense mutation was identified in a highly conserved phenylalanine close to the active site of the enzyme. Functional analysis revealed that the mutant LFNG was not localized to the correct compartment of the cell, was unable to modulate Notch signaling in a cell-based assay, and was enzymatically inactive. This represents the first known mutation in the human LFNG gene and reinforces the hypothesis that proper regulation of the Notch signaling pathway is an absolute requirement for the correct patterning of the axial skeleton.

Mutated MESP2 Causes Spondylocostal Dysostosis in Humans

Spondylocostal dysostosis (SCD) is a term given to a heterogeneous group of disorders characterized by abnormal vertebral segmentation (AVS). We have previously identified mutations in the Delta-like 3 (DLL3) gene as a major cause of autosomal recessive spondylocostal dysostosis. DLL3 encodes a ligand for the Notch receptor and, when mutated, defective somitogenesis occurs resulting in a consistent and distinctive pattern of AVS affecting the entire spine. From our study cohort of cases of AVS, we have identified individuals and families with abnormal segmentation of the entire spine but no mutations in DLL3, and, in some of these, linkage to the DLL3 locus at 19q13.1 has been excluded. Within this group, the radiological phenotype differs mildly from that of DLL3 mutation-positive SCD and is variable, suggesting further heterogeneity. Using a genomewide scanning strategy in one consanguineous family with two affected children, we demonstrated linkage to 15q21.3-15q26.1 and furthermore identified a 4-bp duplication mutation in the human MESP2 gene that codes for a basic helix-loop-helix transcription factor. No MESP2 mutations were found in a further 7 patients with related radiological phenotypes in whom abnormal segmentation affected all vertebrae, nor in a further 12 patients with diverse phenotypes.

A Mechanism for Gene-Environment Interaction in the Etiology of Congenital Scoliosis

Congenital scoliosis, a lateral curvature of the spine caused by vertebral defects, occurs in approximately 1 in 1,000 live births. Here we demonstrate that haploinsufficiency of Notch signaling pathway genes in humans can cause this congenital abnormality. We also show that in a mouse model, the combination of this genetic risk factor with an environmental condition (short-term gestational hypoxia) significantly increases the penetrance and severity of vertebral defects. We demonstrate that hypoxia disrupts FGF signaling, leading to a temporary failure of embryonic somitogenesis. Our results potentially provide a mechanism for the genesis of a host of common sporadic congenital abnormalities through gene-environment interaction.

Mutation of HAIRY-AND-ENHANCER-OF-SPLIT-7 in humans causes spondylocostal dysostosis

Spondylocostal dysostosis (SCD) is an inherited disorder that is characterized by the presence of extensive hemivertebrae, truncal shortening and abnormally aligned ribs. It arises during embryonic development by a disruption of formation of somites (the precursor tissue of the vertebrae, ribs and associated tendons and muscles). Previously, three genes causing a subset of autosomal recessive forms of this disease have been identified: DLL3 (SCDO1: MIM 277300), MESP2 (SCDO2: MIM 608681) and LFNG (SCDO3: MIM609813). These genes are all important components of the Notch signaling pathway, which has multiple roles in development and disease. Here we have used autozygosity mapping to identify a mutation in a fourth Notch pathway gene, Hairy-and-Enhancer-of-Split-7 (HES7), in an autosomal recessive SCD family. HES7 encodes a bHLH-Orange domain transcriptional repressor protein that is both a direct target of the Notch signaling pathway, and part of a negative feedback mechanism required to attenuate Notch signaling. A missense mutation was identified in the DNA-binding domain of the HES7 protein. Functional analysis revealed that the mutant HES7 was not able to repress gene expression by DNA binding or protein heterodimerization. This is the first report of mutation in the human HES7 gene, and provides further evidence for the importance of the Notch signaling pathway in the correct patterning of the axial skeleton.

Notch inhibition by the ligand Delta-Like 3 defines the mechanism of abnormal vertebral segmentation in spondylocostal dysostosis

Mutations in the DELTA-LIKE 3 (DLL3) gene cause the congenital abnormal vertebral segmentation syndrome, spondylocostal dysostosis (SCD). DLL3 is a divergent member of the DSL family of Notch ligands that does not activate signalling in adjacent cells, but instead inhibits signalling when expressed in the same cell as the Notch receptor. Targeted deletion of Dll3 in the mouse causes a developmental defect in somite segmentation, and consequently vertebral formation is severely disrupted, closely resembling human SCD. In contrast to the canonical Notch signalling pathway, very little is known about the mechanism of cis-inhibition by DSL ligands. Here, we report that Dll3 is not presented on the surface of presomitic mesoderm (PSM) cells in vivo, but instead interacts with Notch1 in the late endocytic compartment. This suggests for the first time a mechanism for Dll3-mediated cis-inhibition of Notch signalling, with Dll3 targeting newly synthesized Notch1 for lysosomal degradation prior to post-translational processing and cell surface presentation of the receptor. An inhibitory role for Dll3 in vivo is further supported by the juxtaposition of Dll3 protein and Notch1 signalling in the PSM. Defining a mechanism for cis-inhibition of Notch signalling by Dll3 not only contributes greatly to our understanding of this ligands function during the formation of the vertebral column, but also provides a paradigm for understanding how other ligands of Notch cis-inhibit signalling.

Evolution of distinct EGF domains with specific functions

EGF domains are extracellular protein modules cross‐linked by three intradomain disulfides. Past studies suggest the existence of two types of EGF domain with three‐disulfides, human EGF‐like (hEGF) domains and complement C1r‐like (cEGF) domains, but to date no functional information has been related to the two different types, and they are not differentiated in sequence or structure databases. We have developed new sequence patterns based on the different C‐termini to search specifically for the two types of EGF domains in sequence databases. The exhibited sensitivity and specificity of the new pattern‐based method represents a significant advancement over the currently available sequence detection techniques. We re‐annotated EGF sequences in the latest release of Swiss‐Prot looking for functional relationships that might correlate with EGF type. We show that important post‐translational modifications of three‐disulfide EGFs, including unusual forms of glycosylation and post‐translational proteolytic processing, are dependent on EGF subtype. For example, EGF domains that are shed from the cell surface and mediate intercellular signaling are all hEGFs, as are all human EGF receptor family ligands. Additional experimental data suggest that functional specialization has accompanied subtype divergence. Based on our structural analysis of EGF domains with three‐disulfide bonds and comparison to laminin and integrin‐like EGF domains with an additional inter‐domain disulfide, we propose that these hEGF and cEGF domains may have arisen from a four‐disulfide ancestor by selective loss of different cysteine residues.

Cited1 is required in trophoblasts for placental development and for embryo growth and survival

ABSTRACT Cited1 is a transcriptional cofactor that interacts with Smad4, estrogen receptors α and β, TFAP2, and CBP/p300. It is expressed in a restricted manner in the embryo as well as in extraembryonic tissues during embryonic development. In this study we report the engineering of a loss-of-function Cited1 mutation in the mouse. Cited1 null mutants show growth restriction at 18.5 days postcoitum, and most of them die shortly after birth. Half the heterozygous females, i.e., those that carry a paternally inherited wild-type Cited1 allele, are similarly affected. Cited1 is normally expressed in trophectoderm-derived cells of the placenta; however, in these heterozygous females, Cited1 is not expressed in these cells. This occurs because Cited1 is located on the X chromosome, and thus the wild-type Cited1 allele is not expressed because the paternal X chromosome is preferentially inactivated. Loss of Cited1 resulted in abnormal placental development. In mutants, the spongiotrophoblast layer is irregular in shape and enlarged while the labyrinthine layer is reduced in size. In addition, the blood spaces within the labyrinthine layer are disrupted; the maternal sinusoids are considerably larger in mutants, leading to a reduction in the surface area available for nutrient exchange. We conclude that Cited1 is required in trophoblasts for normal placental development and subsequently for embryo viability.