Nuclear factors regulating erythroid progenitor cell differentiation and gene expression

Nuclear factors are effector molecules that directly regulate proliferation and differentiation of progenitor cells. Gene targeting experiments in mice have demonstrated that several nuclear factors are essential at different stages of erythroid development including cell cycling factors (Rb), and both ubiquitous (c-myb) and erythroid-specific (GA TA-1) DNA binding factors. In addition, DNA binding factors are required to establish the DNase I hypersensitive sites in the human β-globin locus control region (LCR). By 'opening' chromatin in the β-globin locus early in development, the LCR permits correct temporal and spatial expression of the globin genes in the maturing cells of the erythroid lineage.     

The Higher Structure of Chromatin in the LCR of the β-Globin Locus Changes during Development

The β-globin locus control region (LCR) is able to enhance the expression of all globin genes throughout the course of development. However, the chromatin structure of the LCR at the different developmental stages is not well defined. We report DNase I and micrococcal nuclease hypersensitivity, chromatin immunoprecipitation analyses for histones H2A, H2B, H3, and H4, and 3C (chromatin conformation capture) assays of the normal and mutant β-globin loci, which demonstrate that nucleosomes at the DNase I hypersensitive sites of the LCR could be either depleted or retained depending on the stages of development. Furthermore, MNase sensitivity and 3C assays suggest that the LCR chromatin is more open in embryonic erythroblasts than in definitive erythroblasts at the primary- and secondary-structure levels; however, the LCR chromatin is packaged more tightly in embryonic erythroblasts than in definitive erythroblasts at the tertiary chromatin level. Our study provides the first evidence that the occupancy of nucleosomes at a DNase I hypersensitive site is a developmental stage-related event and that embryonic and adult cells possess distinct chromatin structures of the LCR.     

Cohesin mediates chromatin interactions that regulate mammalian β-globin expression

The β-globin locus undergoes dynamic chromatin interaction changes in differentiating erythroid cells that are thought to be important for proper globin gene expression. However, the underlying mechanisms are unclear. The CCCTC-binding factor, CTCF, binds to the insulator elements at the 5' and 3' boundaries of the locus, but these sites were shown to be dispensable for globin gene activation. We found that, upon induction of differentiation, cohesin and the cohesin loading factor Nipped-B-like (Nipbl) bind to the locus control region (LCR) at the CTCF insulator and distal enhancer regions as well as at the specific target globin gene that undergoes activation upon differentiation. Nipbl-dependent cohesin binding is critical for long-range chromatin interactions, both between the CTCF insulator elements and between the LCR distal enhancer and the target gene. We show that the latter interaction is important for globin gene expression in vivo and in vitro. Furthermore, the results indicate that such cohesin-mediated chromatin interactions associated with gene regulation are sensitive to the partial reduction of Nipbl caused by heterozygous mutation. This provides the first direct evidence that Nipbl haploinsufficiency affects cohesin-mediated chromatin interactions and gene expression. Our results reveal that dynamic Nipbl/cohesin binding is critical for developmental chromatin organization and the gene activation function of the LCR in mammalian cells.     

A molecular description of telometic heterochromatin in secale species

We report the nucleotide sequence of a rabbit β-globin pseudogene, ψβ2. A comparison of the ψβ2 sequence with that of the rabbit adult β-globin gene, β1, reveals the presence of frameshift mutations and premature termination codons in the protein coding sequence which render ψβ2 unable to encode a functional β-globin polypeptide. ψβ2 contains two intervening sequences at the same locations in the globin protein coding sequence as β1 and all other sequenced β-globin genes. An examination of the DNA sequences at the intron/exon junctions suggests that a putative ψβ2 precursor mRNA could not be spliced normally. We compare the flanking and noncoding sequences of ψβ2 and β1 and discuss the evolutionary relationship between these two genes.