During red blood cell development, differentiation and cell cycle progression are intimately and uniquely linked through interdependent mechanisms involving the erythroid transcriptional suppressor PU.1 and the cyclin-dependent kinase inhibitor p57KIP2.

In the mammalian genome, 5’-CpG-3’ dinucleotides are frequently methylated, correlating with transcriptional silencing. Genome-wide demethylation is thought to occur only twice during development, in primordial germ cells and in the pre-implantation embryo. These demethylation events are followed by de novo methylation, setting up a pattern inherited throughout development and modified only at tissue-specific loci. Here we studied DNA methylation in differentiating mouse erythroblasts in vivo using genomic-scale reduced representation bisulfite sequencing (RRBS). Demethylation at the erythroid-specific β-globin locus was coincident with global DNA demethylation at most genomic elements. Global demethylation was continuous throughout differentiation and required rapid DNA replication. Hence, DNA demethylation can occur globally during somatic cell differentiation, providing an experimental model for its study in development and disease.

Objective. To determine whether biopsy specimens obtained from systemic sclerosis (SSc) lesions show a distinctive gene profile, whether that gene profile is maintained in fibroblasts cultured from SSc skin biopsy specimens, and whether results from tissue obtained from multiple clinical centers can be combined to yield useful observations in this rare disease.Methods. Biopsy samples and passaged fibroblasts were stored in RNAlater solution prior to processing for RNA. RNA from SSc and control skin biopsy specimens, as well as SSc and control explanted passage 4 fibroblasts, from 9 patients and 9 controls was hybridized to Affymetrix HG-U133A arrays. Data were analyzed using the BRB ArrayTools system. When appropriate, findings were followed up with immunohistochemical analysis or TaqMan studies.Results. Biopsy samples obtained from patients with SSc had a robust and distinctive gene profile, with ϳ1,800 qualifiers distinguishing normal skin from SSc skin at a significant level. The SSc phenotype was the major driver of sample clusters, independent of origin. Alterations in transforming growth factor ␤ and Wnt pathways, extracellular matrix proteins, and the CCN family were prominent. Explanted fibroblasts from SSc biopsy samples showed a far smaller subset of changes that were relatively variable between samples, suggesting that either nonfibroblast cell types or other aspects of the dermal milieu are required for full expression of the SSc phenotype.Conclusion. SSc has a distinct gene profile that is not confounded by geographic location, indicating that extended multicenter studies may be worthwhile to identify distinct subsets of disease by transcript profiling. Explanted SSc fibroblasts show an incomplete reflection of the SSc phenotype.

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Escherichia coli IbpB was overexpressed in a strain carrying a deletion in the chromosomal ibp operon and purified by refolding. Under our experimental conditions, IbpB exhibited pronounced size heterogeneity. Basic oligomers, roughly spherical and approximately 15 nm in diameter, interacted to form larger particles in the 100 -200-nm range, which themselves associated to yield loose aggregates of micrometer size. IbpB suppressed the thermal aggregation of model proteins in a concentration-dependent manner, and its CD spectrum was consistent with a mostly ␤-pleated secondary structure. Incubation at high temperatures led to a partial loss of secondary structure, the progressive exposure of tryptophan residues to the solvent, the dissociation of high molecular mass aggregates into Ϸ600-kDa oligomers, and an increase in surface hydrophobicity. Structural changes were reversible between 37 and 55°C, and, up to 55°C, hydrophobic sites were reburied upon cooling. IbpB exhibited a biphasic unfolding trend upon guanidine hydrochloride (GdnHCl) treatment and underwent comparable conformational changes upon melting and during the first GdnHCl-induced transition. However, hydrophobicity decreased with increasing GdnHCl concentrations, suggesting that efficient exposure of structured hydrophobic sites involves denaturant-sensitive structural features. By contrast, IbpB hydrophobicity rose at high NaCl concentrations and increased further at high temperatures. Our results support a model in which temperature-driven conformational changes lead to the reversible exposure of normally shielded binding sites for nonnative proteins and suggest that both hydrophobicity and charge context may determine substrate binding to IbpB.