Transcription of the HS2 Enhancer toward a cis-Linked Gene Is Independent of the Orientation, Position, and Distance of the Enhancer Relative to the Gene

Abstract

The locus control region (LCR) of the human ␤-globin gene domain is defined by four erythroid-cell-specific DNase I-hypersensitive (HS) sites, HS1, -2, -3, and -4, located 50 to 70 kb upstream of the ␤-globin gene in a transcriptional direction: 5Ј HS4-HS3-HS2-HS1-//-ε-G ␥-A ␥-␦-␤ 3Ј (17,20,37,50). The LCR has been shown by studies of natural deletions of ␥␦␤-thalassemia (8,11,24) and by gene transfer experiments (14,18,20,51) to be indispensable for erythroid-cell-specific and high-level transcription of cis-linked globin genes and transgenes. The mechanism by which the LCR regulates transcription of the distant embryonic ε-, fetal ␥-, and adult ␤-globin genes at the respective developmental stages is not fully understood.In previous studies examining the mechanism of LCR function, a number of laboratories have investigated the ability of the individual HS sites to activate the transcription of cislinked genes in cell lines and transgenic mice. Among the LCR HS sites, HS2 has been found to possess developmental-stageindependent enhancer function (52). It is capable of stimulating the transcription of embryonic ε-, fetal ␥-, and adult ␤-globin genes in erythroid cells at the corresponding developmental stages (9,28,34,42,45,47) and may therefore constitute a major functional component of the LCR. However, a recent targeted deletion study shows that deletion of the HS2 enhancer from the murine LCR generated only mild effects on the expression of the ␤-like globin genes (15), suggesting that HS2 is not essential for LCR function and that LCR function may be due to the contribution of the other HS sites. Surprisingly, similar targeted deletion of HS3 also did not cause significant changes in the expression of the ␤-like globin genes (22). These findings suggest that LCR function is due not to the contribution of any specific HS site but to a series of interactions among its functional components, including the enhancer elements underlying the HS sites.In the above-described targeted deletion studies, deleting the HS2 or the HS3 sequence from the endogenous murine LCR and inserting in its place an independent transcription unit-a neomycin-resistant gene driven by a strong promoter-has been found to disrupt LCR function and cause severe anemia in and the deaths of homozygous transgenic mice (15,21). In the human ␤-LCR, inserting an independent transcription unit at a location between the HS2 enhancer and the downstream globin genes also disrupted LCR function (23) and caused the distantly downstream ␤-globin gene to be transcriptionally turned off. The mechanism by which the transcription of a foreign gene within the LCR might disrupt LCR function is not clearly understood (21).In an attempt to delineate the functional mechanism of the HS2 enhancer and ultimately of the LCR, we have previously analyzed the transcriptional status of the HS2 enhancer in transfected recombinant chloramphenicol acetyltransferase (CAT) plasmids (53). This earlier study showed that the transfected HS2 enhancer is itself transcribed in eryt...

Analysis of Polygenic Mutants Suggests a Role for Mediator in Regulating Transcriptional Activation Distance in Saccharomyces cerevisiae

“…1997; Kong et al 1997;Carninci et al 2005;Manak et al 2006). Overall, our results demonstrate the successful isolation of mutants with strong long-distance activation phenotypes.…”

“…Previous 59-RACE experiments showed that the HIS3 long transcript initiates proximal to the UAS (Dobi and Winston 2007). Although the level of this long transcript is variable and we do not understand its regulation, we note that transcripts initiating near enhancers have been observed in metazoans (Ashe et al 1997;Kong et al 1997;Carninci et al 2005;Manak et al 2006). Overall, our results demonstrate the successful isolation of mutants with strong long-distance activation phenotypes.…”

Transcription defines the embryonic domains of cis-regulatory activity at the Drosophila bithorax complex

“…The ␤-globin locus in humans also is divided into distinct subdomains, and long, intergenic transcripts are responsible for delineating the active domains by association with chromatin-remodeling activities (38). An active enhancer at the H2 hypersensitive site of the ␤-globin locus control region also was found to be transcribed from transgenes in mice, irrespective of the position and orientation of the enhancer (39). At imprinted loci in mice, a number of cisregulatory elements, including enhancers and the imprinting control regions at H19͞Igf2 and Igf2r, are transcribed (40)(41)(42).…”

Heterogeneous nuclear ribonucleoprotein C1/C2, MeCP1, and SWI/SNF form a chromatin remodeling complex at the β-globin locus control region

“…HS2 acts as a classical enhancer in transient transfection assays and in transgenic mice (6)(7)(8)(9) and has binding sites for several transcription factors (10). A tandem pair of activator protein 1 (AP1)͞NF-E2 sites possesses the core enhancing activity of HS2 (11,12).…”