On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation.

Determining the mechanisms by which genes are switched on and off during development is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using an on-microscope analysis, we monitored mouse α-globin transcription in live cells throughout erythropoiesis. We find that changes in the overall levels of α-globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in transcriptional activity whereas at the peak of gene expression, in early erythroblasts, transcription is relatively stable. Later during differentiation as α-globin expression declines, we again observe more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of active transcriptional compartments as gene expression is regulated during differentiation.

Monosomy for the most telomeric, gene-rich region of the short arm of human chromosome 16 causes minimal phenotypic effects.

We have examined the phenotypic effects of 21 independent deletions from the fully sequenced and annotated 356 kb telomeric region of the short arm of chromosome 16 (16p13.3). Fifteen genes contained within this region have been highly conserved throughout evolution and encode proteins involved in important housekeeping functions, synthesis of haemoglobin, signalling pathways and critical developmental pathways. Although a priori many of these genes would be considered candidates for critical haploinsufficient genes, none of the deletions within the 356 kb interval cause any discernible phenotype other than alpha thalassaemia whether inherited via the maternal or paternal line. These findings contrast with previous observations on patients with larger (> 1 Mb) deletions from the 16p telomere and therefore address the mechanisms by which monosomy gives rise to human genetic disease.

alpha-thalassemia resulting from a negative chromosomal position effect.

To date, all of the chromosomal deletions that cause alpha-thalassemia remove the structural alpha genes and/or their regulatory element (HS -40). A unique deletion occurs in a single family that juxtaposes a region that normally lies approximately 18-kilobase downstream of the human alpha cluster, next to a structurally normal alpha-globin gene, and silences its expression. During development, the CpG island associated with the alpha-globin promoter in the rearranged chromosome becomes densely methylated and insensitive to endonucleases, demonstrating that the normal chromatin structure around the alpha-globin gene is perturbed by this mutation and that the gene is inactivated by a negative chromosomal position effect. These findings highlight the importance of the chromosomal environment in regulating globin gene expression.

Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation.

A goal of molecular genetics is to understand the relationship between basic nuclear processes, epigenetic changes and the numerous proteins that orchestrate these effects. One such protein, ATRX, contains a highly conserved plant homeodomain (PHD)-like domain, present in many chromatin-associated proteins, and a carboxy-terminal domain which identifies it as a member of the SNF2 family of helicase/ATPases. Mutations in ATRX give rise to characteristic developmental abnormalities including severe mental retardation, facial dysmorphism, urogenital abnormalities and alpha-thalassaemia. This circumstantial evidence suggests that ATRX may act as a transcriptional regulator through an effect on chromatin. We have recently shown that ATRX is localized to pericentromeric heterochromatin during interphase and mitosis, suggesting that ATRX might exert other chromatin-mediated effects in the nucleus. Moreover, at metaphase, some ATRX is localized at or close to the ribosomal DNA (rDNA) arrays on the short arms of human acrocentric chromosomes. Here we show that mutations in ATRX give rise to changes in the pattern of methylation of several highly repeated sequences including the rDNA arrays, a Y-specific satellite and subtelomeric repeats. Our findings provide a potential link between the processes of chromatin remodelling, DNA methylation and gene expression in mammalian development.

Chromosomal stabilisation by a subtelomeric rearrangement involving two closely related Alu elements.

We have characterised a subtelomeric rearrangement involving the short arm of chromosome 16 that gives rise to alpha-thalassaemia by deleting the major, remote regulatory element controlling alpha-globin expression. The chromosomal breakpoint lies in an Alu family repeat located only approximately 105 kb from the 16p subtelomeric region. The broken chromosome has been stabilised with a newly positioned telomere acquired by recombination between this 16p Alu element and a closely related subtelomeric Alu element of the Sx subfamily. It seems most likely that this abnormal chromosome has been rescued by the mechanism of telomere capture which may reflect a more general process by which subtelomeric sequences are normally dispersed between chromosomal ends.

Contrasting effects of alpha and beta globin regulatory elements on chromatin structure may be related to their different chromosomal environments.

Expression of the human alpha and beta globin gene clusters is regulated by remote sequences, referred to as HS -40 and the beta-locus control region (beta-LCR) that lie 5-40 kb upstream of the genes they activate. Because of their common ancestry, similar organization and coordinate expression it has often been assumed that regulation of the globin gene clusters by HS -40 and the beta-LCR occurs via similar mechanisms. Using interspecific hybrids containing chromosomes with naturally occurring deletions of HS -40 we have shown that, in contrast to the beta-LCR, this element exerts no discernible effect on long-range chromatin structure and in addition does not influence formation of DNase I hypersensitive sites at the alpha globin promoters. These differences in the behaviour of HS -40 and the beta-LCR may reflect their contrasting influence on gene expression in transgenic mice and may result from the differing requirements of these elements in their radically different, natural chromosomal environments; the alpha cluster lying within a region of constitutively 'open' chromatin and the beta cluster in a segment of chromatin which opens in a tissue-specific manner. Differences in the hierarchical control of the alpha and beta globin clusters may exemplify more general differences in the regulation of eukaryotic genes which lie in similar open or closed chromosomal regions.

Healing of broken human chromosomes by the addition of telomeric repeats.

We have characterized and compared a series of naturally occurring chromosomal truncations involving the terminal region of the short arm of human chromosome 16 (16p13.3). All six broken chromosomes appear to have been stabilized by the direct addition of telomeric repeats (TTAGGG)n to nontelomeric DNA. In five of the six chromosomes, sequence analysis shows that the three of four nucleotides preceding the point of telomere addition are complementary to and in phase with the putative RNA template of human telomerase. Otherwise we have found no common structural features around the breakpoint regions. These findings, together with previously reported in vitro data, suggest that chromosome-healing events in man can be mediated by telomerase and that a small region of complementarity to the RNA template of telomerase at the end of a broken chromosome may be sufficient to prime healing in vivo.

Analysis of the human alpha globin upstream regulatory element (HS-40) in transgenic mice.

We have analysed the effect of a 1.4 kb segment of DNA containing the upstream alpha globin regulatory element (HS-40) on human alpha globin gene expression in fetal mice and lines of transgenic mice. High levels of tissue-specific, human alpha mRNA expression were seen in all transgenic animals and in this sense expression was position independent. However, the level of human alpha mRNA expression per integrated gene copy decreased during development and was inversely related to copy number. The limitation in expression with increasing gene copy number was shown to be in cis since homozygotes for the transgene produced twice as much human alpha mRNA as hemizygotes. In many respects HS -40 appears similar to single elements within the previously described beta globin locus control region and in cross breeding experiments we have shown that HS -40 behaves in a similar manner to such elements in transgenic mice.

Cis-acting sequences regulating expression of the human alpha-globin cluster lie within constitutively open chromatin.

Current models suggest that tissue-specific genes are arranged in discrete, independently controlled segments of chromatin referred to as regulatory domains. Transition from a closed to open chromatin structure may be an important step in the regulation of gene expression. To determine whether the human alpha-globin cluster, like the beta-globin cluster, lies within a discrete, erythroid-specific domain, we have examined the long-range genomic organization and chromatin structure around this region. The alpha genes lie adjacent to at least four widely expressed genes. The major alpha-globin regulatory element lies 40 kb away from the cluster within an intron of one of these genes. Therefore, unlike the beta cluster, cis-acting sequences controlling alpha gene expression are dispersed within a region of chromatin that is open in both erythroid and nonerythroid cells. This implies a difference in the hierarchical control of alpha- and beta-globin expression.

Characterization of the major regulatory element upstream of the human alpha-globin gene cluster.

The major positive regulatory activity of the human alpha-globin gene complex has been localized to an element associated with a strong erythroid-specific DNase I hypersensitive site (HS -40) located 40 kb upstream of the zeta 2-globin mRNA cap site. Footprint and gel shift analyses of the element have demonstrated the presence of four binding sites for the nuclear factor GATA-1 and two sites corresponding to the AP-1 consensus binding sequence. This region resembles one of the major elements of the beta-globin locus control region in its constitution and characteristics; this together with evidence from expression studies suggests that HS -40 is a primary element controlling alpha-globin gene expression.

Inactivation of human alpha-globin gene expression by a de novo deletion located upstream of the alpha-globin gene cluster.

Synthesis of normal human hemoglobin A, alpha 2 beta 2, is based upon balanced expression of genes in the alpha-globin gene cluster on chromosome 16 and the beta-globin gene cluster on chromosome 11. Full levels of erythroid-specific activation of the beta-globin cluster depend on sequences located at a considerable distance 5' to the beta-globin gene, referred to as the locus-activating or dominant control region. The existence of an analogous element(s) upstream of the alpha-globin cluster has been suggested from observations on naturally occurring deletions and experimental studies. We have identified an individual with alpha-thalassemia in whom structurally normal alpha-globin genes have been inactivated in cis by a discrete de novo 35-kilobase deletion located approximately 30 kilobases 5' from the alpha-globin gene cluster. We conclude that this deletion inactivates expression of the alpha-globin genes by removing one or more of the previously identified upstream regulatory sequences that are critical to expression of the alpha-globin genes.

A major positive regulatory region located far upstream of the human alpha-globin gene locus.

We have identified a remote, tissue-specific, positive regulatory element that is of major importance in determining the level of human alpha-globin gene expression. Stable transformants containing this DNA segment linked to the alpha gene in mouse erythroleukemia cells expressed human alpha mRNA at levels that are indistinguishable from those seen in interspecific hybrids containing the human alpha genes in their normal context on chromosome 16. Furthermore, all transgenic mice containing the alpha genes linked to this region expressed alpha-globin mRNA at high levels in erythroid tissues; and in one such mouse, readily detectable levels of human alpha-globin chains could be demonstrated in the peripheral blood. There is considerable similarity in the position, structure, and function of this region upstream of the alpha-globin complex with previously described elements within the beta-globin dominant control region (DCR). This is m marked contrast to other structural and functional differences between the two gene clusters. It seems likely that these critical, positive regulatory regions might provide target sequences through which coordinate regulation of the alpha- and beta-like globin genes is achieved.

Alpha-thalassemia caused by a large (62 kb) deletion upstream of the human alpha globin gene cluster.

We describe a family in which alpha-thalassemia occurs in association with a deletion of 62 kilobases from a region upstream of the alpha globin genes. DNA sequence analysis has shown that the transcription units of both alpha genes downstream of this deletion are normal. Nevertheless, they fail to direct alpha globin synthesis in an interspecific hybrid containing the abnormal (alpha alpha)RA chromosome. It seems probable that previously unidentified positive regulatory sequences analogous to those detected in a corresponding position of the human beta globin cluster are removed by this deletion.

Alpha thalassaemia in two Spanish families.

Two Spanish families with alpha thalassaemia, including 4 individuals with Hb H disease, are described. DNA mapping shows that, in addition to the common alpha thalassaemia determinant (-alpha 3.7), a different and previously unreported allele is present in each family. In one, there is a deletion of 10.5-12 kb of DNA including both alpha genes (--SPAN). In the other, a deletion of more than 100 kb has removed the entire alpha globin gene complex (--BR).

Alpha-thalassaemia in the north west of England.

Although it is extremely rare amongst the indigenous population, we have previously identified several British individuals with alpha-thalassaemia. It was to be expected that the underlying molecular defect in these individuals would result from racial admixture; however, we found that many of them share a specific, previously reported determinant of alpha-thalassaemia, referred to as --BRIT (Higgs et al. 1985). Several of these individuals with the --BRIT determinant originated from the North West of England and therefore a more extensive survey of individuals from this area was undertaken. We identified 14 individuals with the --BRIT determinant out of 200,000 individuals studied. These, together with all other known examples bring the total number of cases identified to 36. It seems likely that this almost exclusively British determinant of alpha-thalassaemia (--BRIT) has become established through genetic drift in this population.

Alpha-thalassaemia in Nigeria: its interaction with sickle-cell disease.

We have determined the molecular basis and frequency of alpha-thalassaemia in Nigeria. The alpha-thalassaemia determinant in this population is caused by only one type of single alpha globin gene deletion (-alpha 3.7). Comparison of the haematological features of those patients who have sickle-cell disease with (-alpha/alpha alpha, -alpha/-alpha) or without (alpha alpha/alpha alpha) alpha-thalassaemia showed similar trends to those reported in Jamaican and U.S. patients with these interactions. However, in contrast to studies in some other African populations we have shown that the frequency of alpha-thalassaemia in Nigeria is the same (0.24) in patients with or without homozygous sickle-cell disease (AA, AS and AC genotypes).

Analysis of the human alpha-globin gene cluster reveals a highly informative genetic locus.

Extensive molecular studies have characterized 15 dimorphic and 2 multiallelic genetic markers within the human alpha-globin gene cluster. Analysis of these markers in 9 populations has shown that the alpha-globin locus is remarkably polymorphic and is therefore an ideal marker on chromosome 16 for the construction of a human genetic linkage map. The combined analysis of 9 polymorphic markers has established alpha-globin haplotypes that provide the means to study the molecular genetics and common mutants of this cluster. The novel association of a conventional restriction fragment length polymorphism haplotype and linked, hypervariable regions of DNA should allow a comparison of the rate of change of such markers.

Alpha thalassaemia in British people.

Although alpha thalassaemia is rare in north Europeans, it has been identified in British people with no known foreign ancestry. Twelve such patients were studied, of whom eight shared a distinctive molecular defect, which was clearly different from defects seen in subjects of Mediterranean or South East Asian origin. A rare but specific form of alpha thalassaemia is therefore present in the British population. In addition, two patients from families of mixed racial origin were encountered who had a moderately severe form of thalassaemia (HbH disease) due to the inheritance of one form of alpha thalassaemia from the British parent and another type from the foreign parent. This shows the importance of careful genetic counselling of British patients with haematological findings of thalassaemia.