Developmental Specificity of the Interaction between the Locus Control Region and Embryonic or Fetal Globin Genes in Transgenic Mice with an HS3 Core Deletion

Abstract

The human ␤-globin locus contains five actively transcribed genes that are arranged in their developmental order of expression. High-level expression of the ␤-globin gene cluster is dependent on the presence of the locus control region (LCR) (18), an element characterized by a series of five DNase I-hypersensitive sites (HSs) located 6 to 22 kb upstream of the ε-globin gene (9,10,18,44). Naturally occurring deletions of this element result in changes in chromatin structure that extend at least 200 kb 3Ј of the deletion, transcriptional silencing of the ␤-globin locus, and a phenotype of ␤ thalassemia (4,5,12,22). Functional properties of the LCR include activation of the ␤-globin locus (10, 18), restriction of globin gene expression to cells of the erythroid cell lineage (18, 45), enhancement of globin gene expression (11,18,39), and protection from position effects of globin genes transferred in transgenic mice (13,18,25,41).Transgenic mice have been extensively used to study the developmental control of the ␤-globin genes, the function of the LCR, and the role of individual HSs in ␤-globin gene regulation. Linkage of individual HSs to individual globin genes have shown that HS2, HS3, and HS4 are capable of conferring position-independent expression of globin genes, with stronger activation of expression at a specific stage of development (14, 25). Several observations have led to a model suggesting that the HSs form a complex that directly interacts with globin gene promoters by looping of the intervening DNA (7, 28, 46). HS2, HS3, and HS4 have 200-to 400-bp core regions that are able to provide position-independent expression in transgenic mice (27,34,35,37,42). These HS core regions may be indispensable components of the LCR complex; deletions of the HS3 or HS4 core elements result in disruption of HS function and reduction of globin gene expression (3).Discernment of the function of individual HSs and analysis of how the LCR interacts with individual genes during development require studies in the context of intact, native ␤-globin loci. Entire ␤-globin loci have been used to generate transgenic mice, by ligating two cosmids to produce a 70-kb fragment (40) or by using 248-kb (30) or 150-kb (15, 36) yeast artificial chromosomes harboring the ␤-globin locus (␤-YACs). Mice carrying ␤-YACs show correct regulation of the human globin genes, presumably because all the human cis-regulatory elements are present in the transferred sequences of the ␤-globin locus and are properly recognized by the murine transacting environment. In ␤-YAC transgenic mice, the ε-globin gene is expressed during the embryonic stage of development and is confined to primitive erythropoiesis in the yolk sac. The ␥-globin genes are also expressed in the embryonic yolk sac, but unlike their murine homologous gene, ␤h1, ␥-globin gene expression continues in the fetal liver stage of erythropoiesis. Human ␤-globin gene expression occurs only in the cells of definitive erythropoiesis.To delineate the role of HS3 in LCR function and globin gene ...

Promoters of the murine embryonic β-like globin genes Ey and β h1 do not compete for interaction with the β-globin locus control region

“…Although the results of some studies of deletions of individual LCR elements in transgenic human ␤-globin loci have led to the suggestion of stage-specific roles for these elements (23), this conclusion is not true for the murine locus (24)(25)(26) and is unclear for human locus transgenes in general. Deletions of different HSs from human transgenes have parallel effects on developmental regulation, with the greatest decreases in expression occurring at the embryonic and fetal stages (9,(27)(28)(29). These results suggest that any given HS deletion is not associated with a defect in switching but that there is a general difference in sensitivity of the human locus to LCR mutations at different developmental stages.…”

Control of globin gene expression during development and erythroid differentiation

“…For example, HS5 (in humans) or HS4 (in chicken) function as insulators [48,49]. HS3 functions as an activator of ε and γ globin gene expression [50]. The LCR confers lineage-specific expression on the globin genes; it acts as the major enhancer of the β locus; it insulates the locus from surrounding inactive chromatin.…”

“…Deletion of the core element of HS3 produces a distinct phenotype characterized by absence of ε gene expression in the yolk sac and absence of γ gene expression in the fetal liver stage of erythropoiesis (Fig. 6A), indicating that the HS3 core is required for γ gene activation [50]. The HS3 core is studded with transcriptional motifs, six of which, the GT motifs bind SP1/KLF type transcriptional factors.…”

Deletion of the core region of 5′ HS2 of the mouse β-globin locus control region reveals a distinct effect in comparison with human β-globin transgenes

“…This model is supported by transfection studies of constructs with different HS combinations 13,19,20 and by deletion studies of human LCR HS core regions in human ␤-globin yeast artificial chromosome (YAC) transgenic mice. 18,[21][22][23] In YAC transgenic mice, deletion of individual human HS cores (HS2, HS3, or HS4) results in severe reduction of all ␤-like globin gene expression and failure to form the remaining HSs, 21,24,25 suggesting that LCR HSs are mutually interdependent for their formation. Findings that complicate a simple interpretation of these studies are that larger deletions of these same human 5Ј HSs, including the core plus the flanking sequences, result in a more moderate reduction of ␤-like globin gene expression 26 comparable to that of full site deletions within the mouse locus.…”