Reprogramming and differentiation in mammals: motifs and mechanisms.

The natural reprogramming of the mammalian egg and sperm genomes is an efficient process that takes place in less than 24 hours and gives rise to a totipotent zygote. Transfer of somatic nuclei to mammalian oocytes also leads to their reprogramming and formation of totipotent embryos, albeit very inefficiently and requiring an activation step. Reprogramming of differentiated cells to induced pluripotent stem (iPS) cells takes place during a period of time substantially longer than reprogramming of the egg and sperm nuclei and is significantly less efficient. The stochastic expression of endogenous proteins during this process would imply that controlled expression of specific proteins is crucial for reprogramming to take place. The fact that OCT4, NANOG, and SOX2 form the core components of the pluripotency circuitry would imply that control at the transcriptional level is important for reprogramming to iPS cells. In contradistinction, the much more efficient reprogramming of the mammalian egg and sperm genomes implies that other levels of control are necessary, such as chromatin remodeling, translational regulation, and efficient degradation of no longer needed proteins and RNAs.

Systems biology of the 2-cell mouse embryo.

The transcriptome of the 2-cell mouse embryo was analyzed to provide insight into the molecular networks at play during nuclear reprogramming and embryonic genome activation. Analysis of ESTs from a 2-cell cDNA library identified nearly 4,000 genes, over half of which have not been previously studied. Transcripts of mobile elements, especially those of LTR retrotransposons, are abundantly represented in 2-cell embryos, suggesting their possible role in introducing genomic variation, and epigenetic restructuring of the embryonic genome. Analysis of Gene Ontology of the 2-cell-stage expressed genes outlines the major biological processes that guide the oocyte-to-embryo transition. These results provide a foundation for understanding molecular control at the onset of mammalian development.

Characterization of a novel transcription factor binding to the regulatory regions of the human pro-alpha1(I) collagen gene.

We have identified a novel transcription factor TGP (TG Binding Protein) that binds to the consensus sequence 5'-TGTGGGGTGG-3' in the promoter and intron of the human pro-alpha1(I) collagen gene. This recognition sequence, or sequences closely resembling these sequences, was also identified in the pro-alpha1(I) and pro-alpha2(I) collagen genes of other species. Competition experiments revealed that TGP is related to but distinguishable from the Ap4/5 family of transcription factors and that it can be separated from Ap4/5 according to size.

Interaction of Ap1, Ap2, and Sp1 with the regulatory regions of the human pro-alpha1(I) collagen gene.

In the pro-alpha1(I) collagen gene a number of cis-regulatory elements, which interact with a variety of trans-acting factors, are present in the promoter and first intron. We have undertaken a comprehensive study of Sp1, Ap1, and Ap2 binding in the region spanning -442 to +1697 nt. DNase I footprinting analysis revealed these factors bind with varying affinities to some of the potential sites: Sp1 binds to 16 of 34 potential sites, Ap2 binds to 22 of 40 potential binding sites, and Ap1 binds to its only potential site. The Sp1 sites were mostly clustered in the intron region, while the Ap2 sites were clustered in the promoter region. Transmission electron microscopic analysis of DNA-protein complexes not only confirmed these results, but also clearly showed that heterologous and/or homologous protein-protein interactions between Sp1 and/or Ap2 bring the promoter and intron in contact with each other, with the resulting looping out of the intervening DNA. This strongly suggests that the DNA-looping model is an explanation for the orientation preference of the enhancing element in the first intron as these interactions possibly create an optimum environment for the binding of the rest of the transcriptional machinery.

A cysteine for glycine substitution at position 175 in an alpha 1 (I) chain of type I collagen produces a clinically heterogeneous form of osteogenesis imperfecta.

The molecular basis for Osteogenesis Imperfecta in a large kindred with a highly variable phenotype was identified by sequencing the mutant pro alpha 1 (I) protein, cDNA and genomic DNA from the proband. Fibroblasts from different affected individuals all synthesize both normal Type I procollagen molecules and abnormal Type I procollagen molecules in which one or both pro alpha 1 (I) chain(s) contain a cysteine residue within the triple helical domain. Protein studies of the proband localized the mutant cysteine residue to the alpha 1 (I) CB 8 peptide. We now report that cysteine has replaced glycine at triple helical residue 175 disrupting the invariant Gly-X-Y structural motif required for perfect triple helix formation. The consequences include post-translational overmodification, decreased thermal stability, and delayed secretion of mutant molecules. The highly variable phenotype in the present kindred cannot be explained solely on the basis of the cysteine for glycine substitution but will require further exploration.

Prenatal analysis in two suspected cases of glutathione synthetase deficiency.

Prenatal diagnosis was performed in a family affected by generalized glutathione synthetase deficiency. The disorder is transmitted by autosomal recessive inheritance. The first child born in this family died of the disorder at 6 weeks of age. Prenatal diagnosis was performed in two subsequent pregnancies. Amniotic fluid samples were collected by amniocentesis in the 16th and 17th weeks of pregnancy, respectively. In the case of the second pregnancy the concentration of 5-oxoproline in the amniotic fluid was measured by stable isotope dilution, while both stable isotope dilution and glutathione synthetase activity measurements were employed in the prenatal analysis of the third pregnancy. The 5-oxoproline concentration in the second pregnancy was even lower than that of the controls and in the case of the third pregnancy the results fell within the control range. The second pregnancy resulted in the birth of a clinically healthy girl, and the outcome of 5-oxoproline concentration in a urine sample taken just after birth confirmed the unaffected state. The third pregnancy resulted in the birth of a healthy boy at term, and the 5-oxoproline concentration in his urine and the glutathione synthetase activity in haemolysates were determined. The results confirmed that this infant was also unaffected and he apparently had two normal alleles for the enzyme.

The molecular defect in an autosomal dominant form of osteogenesis imperfecta. Synthesis of type I procollagen containing cysteine in the triple-helical domain of pro-alpha 1(I) chains.

Synthesis of procollagen was examined in skin fibroblasts from a patient with a moderately severe autosomal dominant form of osteogenesis imperfecta. Proteolytic removal of the propeptide regions of newly synthesized procollagen, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, revealed the presence of type I collagen in which two alpha 1(I) chains were linked through interchain disulfide bonds. Fragmentation of the disulfide-bonded alpha 1(I) dimers with vertebrate collagenase and cyanogen bromide demonstrated the presence of a cysteine residue in alpha 1(I)CB8, a fragment containing amino acid residues 124-402 of the alpha 1(I) collagen chain. Cysteine residues are not normally found in the triple-helical domain of type I collagen chains. The heterozygous nature of the molecular defect resulted in the formation of three kinds of type I trimers: a normal type with normal pro-alpha(I) chains, a type I trimer with one mutant pro-alpha 1(I) chain and two normal chains, and a type I trimer containing two mutant pro-alpha 1(I) chains and one normal pro-alpha 2(I) chain. The presence of one or two mutant pro-alpha 1(I) chains in trimers of type I procollagen was found to reduce the thermal stability of the protein by 2.5 and 1 degree C, respectively. In addition to post-translational overmodification, procollagen containing one mutant pro-alpha 1(I) chain was also cleared more slowly from cultured fibroblasts. The most likely explanation for these disruptive changes in the physical stability and secretion of the mutant procollagen is that a cysteine residue is substituted for a glycine in half of the pro-alpha 1(I) chains synthesized by the patient's fibroblasts.