Linear Distance from the Locus Control Region Determines ε-Globin Transcriptional Activity

Abstract

Enhancer elements modulate promoter activity over vast chromosomal distances, and mechanisms that ensure restrictive interactions between promoters and enhancers are critical for proper control of gene expression. The human ␤-globin locus control region (LCR) activates expression of five genes in erythroid cells, including the proximal embryonic -and the distal adult ␤-globin genes. To test for possible distance sensitivity of the genes to the LCR, we extended the distance between the LCR and genes by 2.3 kbp within the context of a yeast artificial chromosome, followed by the generation of transgenic mice (TgM). In these TgM lines, -globin gene expression decreased by 90%, while the more distantly located ␥-or ␤-globin genes were not affected. Remarkably, introduction of a consensus EKLF binding site into the -globin promoter rendered its expression distance insensitive; when tested in an EKLF-null genetic background, expression of the mutant -globin gene was severely compromised. Thus, the -globin gene differs in its distance sensitivity to the LCR from the other ␤-like globin genes, which is, at least in part, determined by the transcription factor EKLF.Proper temporal and spatial expression of genetic information is tightly regulated by DNA cis elements such as promoters, enhancers, and insulators. Specific combinations of enhancers and promoters often act synergistically to finely tune gene expression. In contrast, inappropriate communication between an enhancer and promoter can disturb their in vivo expression patterns. Enhancers were originally defined by their ability to activate transcription of cis-linked genes over considerable distances in an orientation-independent manner. Increasing evidence suggests that enhancers can physically and functionally interact with promoters over long distances, exceeding several hundreds of kilobase pairs in cis, or even with promoters located on different chromosomes in trans (reviewed in references 5 and 13). This suggests the presence of molecular mechanisms that allow specific enhancer-promoter interactions to take place while protecting promoters from being inappropriately activated by enhancer elements that might be located in neighboring gene loci or even on different chromosomes.The human ␤-globin genes are organized within a 70-kbp span of chromosome 11, with the embryonic ε-globin gene located most 5Ј, followed by the two fetal ␥-globin genes (G␥ and A␥), while the adult ␦-and ␤-globin genes are at the 3Ј end of the locus (Fig. 1A). Expression of all the ␤-like globin genes, in primitive (embryonic) as well as definitive (fetal and adult) erythroid cells, requires the activity of the locus control region (LCR)/enhancer, which consists of five DNase I hypersensitive sites (HSs) and is located 6 or 48 kbp 5Ј to the transcription initiation sites of the ε-and ␤-globin genes, respectively (19, 27).How distal enhancer elements and LCRs activate gene expression has long been a subject of intense debate, and numerous models have been proposed. Among them, two models...

DNase I Hypersensitivity and ϵ-Globin Transcriptional Enhancement Are Separable in Locus Control Region (LCR) HS1 Mutant Human β-Globin YAC Transgenic Mice

“…In this sense, 5ЈHS1 may serve as a local enhancer that preferentially stimulates ⑀-globin, rather than functioning as a part of an LCR holocomplex. We have previously reported that ⑀-globin gene expression in primitive erythroid cells of YAC TgM is sensitive to distance from the LCR enhancer (24). Now, we showed here that efficient ⑀-globin gene transcription is critically dependent on 5ЈHS1 enhancer activity.…”

“…The 5ЈHS1 (formerly termed HS1-3Ј) fragment was excised as a KpnI-XbaI fragment from the pHS1-3Ј/⑀-luc (24) and subcloned into KpnI/NheI-digested p␤-luc and pGV-P2 (TOYO INK) to generate p5ЈHS1/␤-luc and p5ЈHS1/GV-P2, respectively.…”

“…1A) (24). To test for promoter specificity of this enhancer, we linked the 5ЈHS1 fragment to the ⑀-globin, ␤-globin, and SV40 promoters FIGURE 1.…”

The H19 Imprinting Control Region Mediates Preimplantation Imprinted Methylation of Nearby Sequences in Yeast Artificial Chromosome Transgenic Mice

“…In our previous work, we inserted a bacteriophage DNA (HindIIIdigested 2.3-kb) fragment into a human ␤-globin YAC at a position 3= to the locus control region (LCR) and generated YAC-TgM (32). As shown in Fig.…”

EKLF/KLF1, a Tissue-Restricted Integrator of Transcriptional Control, Chromatin Remodeling, and Lineage Determination

“…Together with the effects at the globin locus, these data suggest that the presence of KLF1 is critical for transcription initiation in facilitating the formation of an open chromatin configuration at target loci. These data raise the possibility that in addition to transcription initiation, KLF1 also plays a role in regulating transcription elongation in erythroid cells (67), since the interaction between the upstream LCR and the proximal ␤-globin promoter, whose 3D chromatin structure requires KLF1 (56,58), aids in establishing a postinitiation transcription elongation complex (68,69). KLF1 is also recruited to the ␣-globin promoter and one of its upstream enhancer elements at late stages of differentiation (60,70), possibly aiding in intrachromosomal interactions, preinitiation complex recruitment, and the transition from a poised to an elongating transcriptional state (70).…”