Disruption of the Mechanism of Long Range Activation within the Human α-Globin Complex

Marie-Alice Deville,Roland Ouazana,François Morlé,Agnès Bernet

doi:10.1074/jbc.m312298200

Abstract

The human alpha-globin complex lies at the tip of the short arm of chromosome 16. It comprises three functional globin genes (5'-zeta2-alpha2-alpha1-3'), the expression of which is strictly dependent on a positive regulatory element located 40-kb upstream, HS-40. This DNase I-hypersensitive site is the only known regulatory element displaying strong erythroid-specific enhancer activity within the human alpha-globin complex. How this enhancer activity is shared among different erythroid genes present in the same cluster without affecting the ubiquitous genes present within and around the complex is poorly understood. To address this issue, we used hybrid murine erythroleukemia cells containing a single copy of human chromosome 16 and targeted the insertion of different sequences downstream of HS-40 by recombinase-mediated cassette exchange. We thus demonstrate that (i). HS-40-mediated erythroid-specific activation of the alpha-globin genes is impaired solely by the insertion of a promoter sequence and not a coding sequence, unless it is methylated, and that (ii). the degree of transcriptional repression observed seems to be related directly to the transcriptional rate of the inserted promoter. Taken together, these results emphasize the importance of promoter sequences as the main targets for the activation mechanism of the human alpha-globin genes by HS-40.

Highlights

The human ␣-globin complex lies at the tip of the short arm of chromosome 16
The complex is located in a GC-rich isochore within an early replicating and constitutively DNase I-sensitive chromatin domain in both erythroid and non-erythroid cells, the ␣-globin genes are transcribed exclusively in erythroid cells [2,3,4]. This transcriptional erythroid specificity is controlled by a single positive regulatory element, which corresponds to a DNase I-hypersensitive site located 40 kb upstream of the ␨2 gene and named HS-40 [5, 6]
We have previously demonstrated that the insertion of a new human ␣-globin gene immediately downstream of HS-40 leads to a drastic decrease in HS-40-mediated transcriptional activation of the resident human ␣-globin genes, concomitant with an increase in activation of the inserted erythroid gene [16]

Summary

Introduction

The human ␣-globin complex lies at the tip of the short arm of chromosome 16. It comprises three functional globin genes (5؅-␨2-␣2-␣1–3؅), the expression of which is strictly dependent on a positive regulatory element located 40-kb upstream, HS-40. The data indicate that the level of expression of the human endogenous ␣-globin genes does not significantly differ between the clones bearing the ␣-globin coding sequence and the parental cells harboring no insertion (Fig. 1, C and D, compare lanes 2– 6 with lane 1).

Results

Conclusion