Sally H. Cross

Purification of CpG islands using a methylated DNA binding column

CpG islands are short stretches of DNA containing a high density of non–methylated CpG dinucleotides, predominantly associated with coding regions. We have constructed an affinity matrix that contains the methyl–CpG binding domain from the rat chromosomal protein MeCP2, attached to a solid support. A column containing the matrix fractionates DNA according to its degree of CpG methylation, strongly retaining those sequences that are highly methylated. Using this column, we have developed a procedure for bulk isolation of CpG islands from human genomic DNA. As CpG islands overlap with approximately 60% of human genes, the resulting CpG island library can be used to isolate full–length cDNAs and to place genes on genomic maps.

Genomic structure and chromosomal mapping of the murine and human Mbd1, Mbd2, Mbd3, and Mbd4 genes

Abstract. DNA methylation is essential for murine development and is implicated in the control of gene expression. MeCP2, MBD1, MBD2, MBD3, and MBD4 comprise a family of mammalian, nuclear proteins related by the presence in each of an amino acid motif called the methyl-CpG binding domain (MBD). Each of these proteins, with the exception of MBD3, is capable of binding specifically to methylated DNA. MeCP2, MBD1 and MBD2 can also repress transcription. We describe the genomic structure and chromosomal localization of the human and murine Mbd1, Mbd2, Mbd3, and Mbd4 genes. We find that the highly similar MBD2 and MBD3 proteins are encoded by genes that map to different chromosomes in humans and mice but show a similar genomic structure. The Mbd1 and Mbd2 genes, in contrast, map together to murine and human Chromosomes (Chrs)18. The Mbd3 and Mbd4 genes map to murine Chrs 10 and 6, respectively, while the human MBD3 and MBD4 genes map to Chrs 19 and 3, respectively.

Transcriptional repression by methylation of CpG

Summary Methylated DNA in mammals is associated with transcriptional repression and nuclease resistant chromatin. In this review we discuss how these effects may be mediated by proteins that bind to methylated DNA.

Wa5 is a novel ENU-induced antimorphic allele of the epidermal growth factor receptor

Mice heterozygous for the N-ethyl-N-nitrosourea-induced Waved-5 (Wa5) mutation, isolated in a screen for dominant, visible mutations, exhibit a wavy coat similar to mice homozygous for the recessive Tgfawa1 or Egfrwa2 alleles. In this study, we show that Wa5 is a new allele of Egfr (EgfrWa5) containing a missense mutation within the coding region for the highly conserved DFG motif of the tyrosine kinase domain. In vivo analysis of placental development, modification of ApcMin tumorigenesis, and levels of EGF-dependent EGFR phosphorylation demonstrates that EgfrWa5 functions as an antimorphic allele, recapitulating many abnormalities associated with reduced EGFR activity. Furthermore, Egfrwa5 enhances EgfrWa2 compound or Tgfatm1Dcl double mutants exposing additional EGFR-dependent phenotypes. In vitro characterization shows that the antimorphic property of EgfrWa5 is caused by a kinase-dead receptor acting as a dominant negative.

A meckelin–filamin A interaction mediates ciliogenesis

MKS3, encoding the transmembrane receptor meckelin, is mutated in Meckel-Gruber syndrome (MKS), an autosomal-recessive ciliopathy. Meckelin localizes to the primary cilium, basal body and elsewhere within the cell. Here, we found that the cytoplasmic domain of meckelin directly interacts with the actin-binding protein filamin A, potentially at the apical cell surface associated with the basal body. Mutations in FLNA, the gene for filamin A, cause periventricular heterotopias. We identified a single consanguineous patient with an MKS-like ciliopathy that presented with both MKS and cerebellar heterotopia, caused by an unusual in-frame deletion mutation in the meckelin C-terminus at the region of interaction with filamin A. We modelled this mutation and found it to abrogate the meckelin-filamin A interaction. Furthermore, we found that loss of filamin A by siRNA knockdown, in patient cells, and in tissues from Flna(Dilp2) null mouse embryos results in cellular phenotypes identical to those caused by meckelin loss, namely basal body positioning and ciliogenesis defects. In addition, morpholino knockdown of flna in zebrafish embryos significantly increases the frequency of dysmorphology and severity of ciliopathy developmental defects caused by mks3 knockdown. Our results suggest that meckelin forms a functional complex with filamin A that is disrupted in MKS and causes defects in neuronal migration and Wnt signalling. Furthermore, filamin A has a crucial role in the normal processes of ciliogenesis and basal body positioning. Concurrent with these processes, the meckelin-filamin A signalling axis may be a key regulator in maintaining correct, normal levels of Wnt signalling.

Studies of DNA méthylation in animals

SUMMARY We have been studying the evolution and function of DNA methylation in vertebrate animals using three related approaches. The first is to further characterise proteins that bind to methylated DNA. Such proteins can be viewed as ‘receptors’ of the methyl-CpG ‘ligand’ that mediate downstream consequences of DNA modification. The second approach involves CpG islands. These patches of non-methylated DNA coincide with most gene promoters, but their origin and functional significance have only recently become the subject of intensive study. The third approach is to trace the evolution of DNA methylation. Genomic methylation patterns of vertebrates are strikingly different from those of invertebrates. By studying methylation in animals that diverged from common ancestors near to the invertebrate/vertebrate boundary, we will assess the possibility that changes in DNA methylation contributed causally to the evolution of the complex vertebrate lineage.

Filamin A Regulates Neural Progenitor Proliferation and Cortical Size through Wee1-Dependent Cdk1 Phosphorylation

Cytoskeleton-associated proteins play key roles not only in regulating cell morphology and migration but also in proliferation. Mutations in the cytoskeleton-associated gene filamin A (FlnA) cause the human disorder periventricular heterotopia (PH). PH is a disorder of neural stem cell development that is characterized by disruption of progenitors along the ventricular epithelium and subsequent formation of ectopic neuronal nodules. FlnA-dependent regulation of cytoskeletal dynamics is thought to direct neural progenitor migration and proliferation. Here we show that embryonic FlnA-null mice exhibited a reduction in brain size and decline in neural progenitor numbers over time. The drop in the progenitor population was not attributable to cell death or changes in premature differentiation, but to prolonged cell cycle duration. Suppression of FlnA led to prolongation of the entire cell cycle length, principally in M phase. FlnA loss impaired degradation of cyclin B1-related proteins, thereby delaying the onset and progression through mitosis. We found that the cdk1 kinase Wee1 bound FlnA, demonstrated increased expression levels after loss of FlnA function, and was associated with increased phosphorylation of cdk1. Phosphorylation of cdk1 inhibited activation of the anaphase promoting complex degradation system, which was responsible for cyclin B1 degradation and progression through mitosis. Collectively, our results demonstrate a molecular mechanism whereby FlnA loss impaired G2 to M phase entry, leading to cell cycle prolongation, compromised neural progenitor proliferation, and reduced brain size.

CpG island libraries from human chromosomes 18 and 22: landmarks for novel genes.

Abstract. CpG islands are found at the 5′ end of approximately 60% of human genes and so are important genomic landmarks. They are concentrated in early-replicating, highly acetylated gene-rich regions. With respect to CpG island content, human Chrs 18 and 22 are very different from each other: Chr 18 appears to be CpG island poor, whereas Chr 22 appears to be CpG island rich. We have constructed and validated CpG island libraries from flow-sorted Chrs 18 and 22 and used these to estimate the difference in number of CpG islands found on these two chromosomes. These libraries contain normalized collections of sequences from the 5′ end of genes. Clones from the libraries were sequenced and compared with the sequence databases; one third matched ESTs, thus anchoring these ESTs at the 5′ end of their gene. However, it was striking that many clones either had no match or matched only existing CpG island clones. This suggests that a significant proportion of 5′ gene sequences are absent from databases, presumably either because they are difficult to clone or the gene is poorly expressed and/or has a restricted expression pattern. This point should be taken into consideration if the currently available libraries are those used for the elucidation of complete, as opposed to partial, gene sequences. The Chr 18 and 22 CpG island libraries are a sequence resource for the isolation of such 5′ gene sequences from specific human chromosomes.

Diphthamide modification of eEF2 requires a J-domain protein and is essential for normal development.

The intracellular target of diphtheria toxin is a modified histidine residue, diphthamide, in the translation elongation factor, eEF2 (also known as EFT1). This enigmatic modification occurs in all eukaryotes and is produced in yeast by the action of five gene products, DPH1 to DPH5. Sequence homologues of these genes are present in all sequenced eukaryotic genomes and, in higher eukaryotes, there is functional evidence for DPH1, DPH2, DPH3 and DPH5 acting in diphthamide biosynthesis. We identified a mouse that was mutant for the remaining gene, Dph4. Cells derived from homozygous mutant embryos lacked the diphthamide modification of eEF2 and were resistant to killing by diphtheria toxin. Reporter-tagged DPH4 protein localized to the cytoskeleton, in contrast to the localization of DPH1 and consistent with evidence that DPH4 is not part of a proposed complex containing DPH1, DPH2 and DPH3. Mice that were homozygous for the mutation were retarded in growth and development, and almost always die before birth. Those that survive long enough had preaxial polydactyly, a duplication of digit 1 of the hind foot. This same defect has been seen in embryos that were homozygous for mutation of DPH1, suggesting that lack of diphthamide on eEF2 could result in translational failure of specific proteins, rather than a generalized translation downregulation.

Identification and characterization of a novel murine beta-defensin-related gene

Beta-defensins comprise a family of cationic peptides, which are predominately expressed at epithelial surfaces and have a broad-range antimicrobial activity. We have assembled two BAC-based contigs from the chromosomal region 8A4 that contain the murine defensins, and we have mapped six reported beta-defensin genes. In addition, we have isolated and functionally characterized a novel beta-defensin gene that deviates from the canonical six cysteine motif present in the mature functional peptide of all other beta-defensins. This defensin-related gene (Defr1) is most highly expressed in testis and heart. The genomic organization is highly similar to Defb3, 4, 5, and 6, and the exon 1 sequence is very highly conserved. A synthetic Defr1 peptide displayed antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Burkholderia cepacia. The antimicrobial activity of Defr1 against S. aureus, E.coli, and B. cepacia was found to be reduced in raised concentration of NaCl, but its action against P. aeruginosa was independent of NaCl concentration. This is the first report of a functional beta defensin that lacks one of the conserved cysteine residues in its predicted mature peptide. This study has major implications for the structure and functions of these important host defense molecules.