Hemizygous deletion of chromosome 22q11 (del22q11) causes thymic, parathyroid, craniofacial and life-threatening cardiovascular birth defects in 1 in 4,000 infants. The del22q11 syndrome is likely caused by haploinsufficiency of TBX1, but its variable expressivity indicates the involvement of additional modifiers. Here, we report that absence of the Vegf164 isoform caused birth defects in mice, reminiscent of those found in del22q11 patients. The close correlation of birth and vascular defects indicated that vascular dysgenesis may pathogenetically contribute to the birth defects. Vegf interacted with Tbx1, as Tbx1 expression was reduced in Vegf164-deficient embryos and knocked-down vegf levels enhanced the pharyngeal arch artery defects induced by tbx1 knockdown in zebrafish. Moreover, initial evidence suggested that a VEGF promoter haplotype was associated with an increased risk for cardiovascular birth defects in del22q11 individuals. These genetic data in mouse, fish and human indicate that VEGF is a modifier of cardiovascular birth defects in the del22q11 syndrome.

PKC betaII autophosphorylates at its conserved carboxy-terminal hydrophobic phosphorylation site by an apparently intramolecular mechanism. Expression studies with kinase-inactive mutants revealed that this mechanism is the only one responsible for phosphorylating this motif in vivo. Thus, conventional PKC autoregulates the carboxy-terminal phosphorylation switch following phosphorylation by another kinase at the activation loop switch.

Autonomous expansions of trinucleotide repeats with the general structure 5'-d(CNG)n-3' are associated with several human genetic diseases. We have characterized nuclear proteins binding to the unstable 5'-d(CGG)n-3' repeat. Its expansion in the human FMR1 gene leads to the fragile X syndrome, one of the most frequent causes of mental retardation in human males. Electrophoretic mobility shift assays using nuclear extracts from several human and other mammalian cell lines and from primary human cells demonstrated specific binding to double-stranded DNA fragments containing only a 5'-d(CGG)17-3' repeat or the repeat and flanking genomic sequences of the human FMR1 gene. Protein binding was inhibited by complete methylation of the trinucleotide repeat. The complex formed with crude nuclear extract apparently did not contain the human transcription factor Sp1 that binds to a characteristic GC-rich sequence. A 20-kDa protein involved in specific binding to the double-stranded 5'-d(CGG)17-3' repeat was purified from HeLa nuclear extracts by DNA affinity chromatography.

Regulatory elements interacting with DNA-binding proteins have been investigated in the promoter sequence of the early PE38 gene in the Autographa californica nuclear polyhedrosis virus (AcNPV). A GATA motif located 50 nucleotides upstream of the PE38 transcriptional start site is recognized differentially in the course of infection. As demonstrated by footprint and gel mobility shift assays, the GATA sequences TTATCT are protected by nuclear extracts from uninfected Spodoptera frugiperda cells and from S. frugiperda cells early postinfection (p.i.) but not by S. frugiperda cell extracts isolated 40 h p.i. We have compared the binding capacity of the insect GATA-like protein with that of the vertebrate GATA-1 factor identified as erythroidspecific factor. Our results indicate that a factor present in mouse erythroleukemia cells, presumably GATA-1, can bind to the insect GATA motif and vice versa. Evidence from transient expression studies suggests that the mutated GATA sequences do not influence PE38 promoter activity in cell culture.

Patterns of DNA methylation in complex genomes like those of mammalian cells have been viewed as indicators of different levels of genetic activities. It is as yet unknown how these complicated patterns are generated and maintained during cell replication. There is evidence from many different biological systems that the sequence‐specific methylation of promoters in higher eukaryotes is one of the important factors in controlling gene activity at a long‐term level. In general, the fifth nucleotide 5‐methyldeoxycytidine can be considered as a modulator of protein‐DNA interactions. The degree and direction of this modulation has to be assessed experimentally in each individual instance. The establishment of de novo patterns of DNA methylation is characterized by the gradual non‐random spreading of DNA methylation by an essentially unknown mechanism. In this review, some of the general concepts of DNA methylation in mammalian systems are presented, and research currently performed in the authors' laboratory has been summarized.