A dominant chromatin-opening activity in 5′ hypersensitive site 3 of the human beta-globin locus control region.

Abstract

Single‐copy human beta‐globin transgenes are very susceptible to suppression by position effects of surrounding closed chromatin. However, these position effects are overcome by a 20 kbp DNA fragment containing the locus control region (LCR). Here we show that the 6.5 kbp microlocus LCR cassette reproducibly directs full expression from independent single‐copy beta‐globin transgenes. By testing individual DNase I‐hypersensitive sites (HS) present in the microlocus cassette, we demonstrate that the 1.5 kbp 5′HS2 enhancer fragment does not direct beta‐globin expression from single‐copy transgenes. In contrast, the 1.9 kbp 5′HS3 fragment directs beta‐globin expression in five independent single‐copy transgenic mouse lines. Moreover, the 5′HS3 core element and beta‐globin proximal promoter sequences are DNase I hypersensitive in fetal liver nuclei of these expressing transgenic lines. Taken together, these results demonstrate that LCR activity is the culmination of at least two separable functions including: (i) a novel activity located in 5′HS3 that dominantly opens and remodels chromatin structure; and (ii) a recessive enhancer activity residing in 5′HS2. We postulate that the different elements of the LCR form a ‘holocomplex’ that interacts with the individual globin genes.

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

“…Our observation that single copies of the integrated HS2 sequence possess enhancer function is in agreement with similar findings in K562 cells reported by other investigators (31,54). However, it has been reported recently that single copies of the HS2 sequence integrated into the fetal liver erythroid cells of transgenic mice did not exhibit enhancer activity (13). This apparently discordant observation may be due to the relative inactivity of the HS2 enhancer in fetal liver erythroid cells, since the HS2 site, as compared with the HS3 site, exists in a relatively inaccessible chromatin structure in fetal liver erythroid cells (17), whereas it exists in a more accessible chromatin structure in K562 cells (50,56).…”

“…Because of the concerns that the HS2 enhancer may be active only when it is integrated in multiple tandem copies (13) and that the subsequently detected HS2 transcripts are therefore not RNA transcribed from the HS2 in each individual plasmid but are nonspecific readthrough transcripts of the tandemly integrated upstream plasmids, we also transfected a number of the plasmids by a modified calcium phosphate method in an attempt to integrate the plasmids in single copies into K562 cells (see Materials and Methods). For purpose of comparison, a number of plasmids were also transfected by the electroporation method.…”

“…The possibility of the existence of a common set of initiation sites suggests that specific DNA motifs in HS2 may be capable of initiating the transcription of enhancer RNAs. Indeed, the HS2 sequence has been shown to contain DNA motifs that bind to many transcription factors such as NF-E2 and Ap-1 (33,35,46,48,49), GATA (22,39,49), YY1 (5,12), and USF (4,5,12,40,49). NF-E2, AP-1, and GATA have been suggested to be capable of recruiting and assembling the transcriptional machinery (16,53), and USF and YY1 have been reported to be capable of stimulating transcriptional initiation from their respective binding sites (25,26,41,44).…”

Function and Factor Interactions of a Locus Control Region Element in the Mouse T Cell Receptor-α/Dad1 Gene Locus

“…In the ␤:6 mice, the values of expression level per copy varied over a range of 11-fold in the thymus, 4-fold in spleen, 5-fold in heart, and 6-fold in liver. These values are well within the range described for the activity of a "partial-LCR" (27). In contrast, in the ␤:⌬6 mice, the range of variation in expression level per copy was over 110-fold in thymus, 190-fold in spleen, 56-fold in heart, and 150-fold in liver.…”

“…Nevertheless, their effect on transgene expression in the chromatin of the whole animal is now well documented. Such elements include HS3 of the ␤-globin LCR (27), HSS3 of the CD2 LCR (44), and the facilitator elements of the adenosine deaminase LCR (38). We have also recently described such an element in the TCR␣ LCR.…”