Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4.

Abstract

Proper tissue-and developmental stage-specific transcriptional control over the five genes of the human ~-globin locus is elicited in part by the locus control region (LCR), but the molecular mechanisms that dictate this determined pattern of gene expression during human development are still controversial. By use of homologous recombination in yeast to generate mutations in the LCR within a yeast artificial chromosome (YAC) bearing the entire human 13-globin gene locus, followed by injection of each of the mutated YACs into murine ova, we addressed the function of LCR hypersensitive site (HS) elements 3 and 4 in human 13-globin gene switching. The experiments revealed a number of unexpected properties that are directly attributable to LCR function. First, deletion of either HS3 or HS4 core elements from an otherwise intact YAC results in catastrophic disruption of globin gene expression at all erythroid developmental stages, despite the presence of all other HS elements in the YAC transgenes. If HS3 is used to replace HS4, gene expression is normal at all developmental stages. Conversely, insertion of the HS4 element in place of HS3 results in significant expression changes at every developmental stage, indicating that individual LCR HS elements play distinct roles in stage-specific [~-type globin gene activation. Although the HS4 duplication leads to alteration in the levels of ¢-and ~/-globin mRNAs during embryonic erythropoiesis, total [3-type globin mRNA synthesis is balanced, thereby leading to the conclusion that all of the human [3-locus genes are competitively regulated. In summary, the human [3-globin HS elements appear to form a single, synergistic functional entity called the LCR, and HS3 and HS4 appear to be individually indispensable to the integrity of this macromolecular complex.

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.…”

“…7). One model assumes that the LCR forms a large protein-DNA complex that physically interacts with the promoter of the gene it is activating, with the intervening DNA looped out (looping) (8,9). An alternative is that activators bound to the LCR either proceed down the chromatin fiber until they reach and bind to a promoter (tracking) or form a chain of proteins that reaches and activates the promoter (linking).…”

Dynamic GATA Factor Interplay at a Multicomponent Regulatory Region of the GATA-2 Locus

“…Deletion of individual HSs of the endogenous ␤-globin locus control region yielded incremental decreases in ␤-globin expression, consistent with a mechanism involving additivity (46,47). In contrast, deletion of HSs from the intact ␤-globin locus within a yeast artificial chromosome (48,49) and transfection experiments (50,51) reveal synergism between the HSs.…”

Neutralizing the function of a β-globin–associated cis -regulatory DNA element using an artificial zinc finger DNA-binding domain

“…The plasmid pMSCV-neo containing the −90 β-ZF-DBD was linearized with Acl I (NEB), and purified from an agarose gel using the Qiagen gel extraction kit, resuspended in injection buffer at a concentration of 2 ng/μL, and injected into FvB oocytes as described previously (40). After transfer to pseudopregnant recipients, embryos were taken at day 13.5 dpc and imaged with a stereomicroscope (Leica MZ16-FA, Houston, TX) and processed with Q-capture software (Q-imaging, BC, Canada).…”