Characterisation of the 3'-boundary of the beta-globin chromosomal domain in adult chicken erythrocytes
Daniel M. Staines
This Thesis describes experiments carried out to characterise the chromatin of a region downstream of the chicken beta-globin genes in adult erythrocytes, in search of the 3'-boundary of the beta-globin domain. Chapter 1 is a general discussion of chromatin and transcription. Details of the genomic clones used to generate DNA probes are given in Appendix A. Novel DNA sequence from the area under investigation is given in Appendix B.
Chapter 2 describes experiments to map the relative DNase I sensitivity of the chromatin from map position +21 to +40 kb (the globin genes lie between map positions +5 and +19 kb), which suggest that the 3'-boundary region is located at approximately +32.7 to +33.4 kb, giving an active chromatin domain of approximately 38 kb. The putative 3'-boundary region contains potential binding sites for multiple general and erythroid-specific transcription factors and an area of relative CpG enrichment.
The solubility of polynucleosomes from active chromatin in 150 mM NaCl is higher than that of bulk. Chapter 3 describes experiments to assay the salt-solubility of adult chicken erythrocyte chromatin using probes from around the putative 3'-boundary region. An overall drop in salt solubility occurs between map positions +32 to +36 kb, supporting the domain boundary proposed in Chapter 2. There is an anomalous region of high salt solubility located between map positions +33 and +35 kb, the significance of which is unknown.
In the work described in Chapter 4, Part A, indirect end-labelling experiments were used to locate DNase I-hypersensitive sites in the 3' region of the active chromatin domain. There are no hypersensitive sites present at the putative 3'-boundary region, but a hypersensitive site is located upstream, at map position +25 kb; its function and significance are unknown. Chapter 4, Part B, describes experiments to determine the methylation status of CpG dinucleotides in the region 3' of the bA-globin gene using the methylation-sensitive enzymes HpaII and HhaI. A transition from partial to full methylation occurs between +22 and +26 kb, and the putative 3'-boundary region appears fully methylated. This result is discussed in relation to the hypersensitive site at +25 kb.
To determine if nuclear factors bind potential factor binding sites from the putative boundary region in vitro, bandshift experiments were carried out and are described in Chapter 5. A 254 bp probe from this region binds multiple factors from an erythroid nuclear extract. At least one factor (absent in brain nuclei) binds a region containing a potential EKLF binding site (CACCC). Other factors appear to be present in both erythroid and brain nuclei, suggesting that the region may be bound by general and erythroid-specific factors. The factors responsible are present in a 500 mM NaCl fraction eluted from a DNA-cellulose column.
The analysis of sequence encompassing the putative 3'-boundary region is described in Chapter 6. The sequence contains a 945 bp open reading frame (ORF) characteristic of protein coding sequence, although there is no obvious promoter. It encodes a 315 residue protein with homology to the R7G family of membrane-bound receptors. The ORF and surrounding DNA have homology to two 'orphan' enhancer regions implicated in two forms of deletional hereditary persistence of foetal haemoglobin (HPFH) syndrome, which affects the developmental regulation of the human b-like globin genes. It is not known whether the chicken ORF has similar enhancer activity. All three DNA elements show a significant relative CpG enrichment.
In summary, the 3'-boundary of the active beta-globin chromatin domain in adult chicken erythrocytes is located at map position ~+33 kb and binds multiple unknown proteins from chicken erythroid and brain nuclei. Sequence from the boundary is homologous to sequence from HPFH-linked enhancers. There is no hypersensitive si
