Jianhua Ling ^a, Boris Baibakov, Wenhu Pi ^a, Beverly M. Emerson ^c, and Dorothy Tuan ^{a, @},

^a Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA
^b Imaging Core Laboratory, Medical College of Georgia, Augusta, GA 30912, USA
^c Salk Institute for Biological Studies, La Jolla, CA 92138, USA

^@ dtuanlo@mail.mcg.edu

The HS2 enhancer in the b-globin locus control region (LCR) regulates transcription of the globin genes 10-50kb away. Earlier studies show that a transcription mechanism initiated by the HS2 enhancer through the intervening DNA in the direction of the cis-linked promoter and gene mediates long-range enhancer function. Here, we further analyzed the enhancer-initiated RNAs and their mode of transcription from the HS2 enhancer in the endogenous genome of erythroid K562 cells, in plasmids integrated into K562 cells and in purified DNA used as template in in vitro transcription reactions. We found that the HS2 enhancer was able to initiate transcription autonomously in the absence of a cis-linked globin promoter. The enhancer-initiated, intergenic RNAs were different from the mRNA synthesized at the promoter in several aspects. The enhancer RNAs were synthesized not from a defined site but from multiple sites both within and as far as 1kb downstream of the enhancer. The enhancer RNAs did not appear to contain a normal cap structure at the 5' ends. They were polyadenylated at multiple sites within 3kb downstream of their initiation sites and were therefore shorter than 3kb in lengths. The enhancer RNAs remained in discrete spots within the nucleus and were not processed into mRNA or translated into proteins. These particular features of enhancer-initiated transcription indicate that the transcriptional complex assembled by the enhancer was different from the basal transcription complex assembled at the promoter. The results suggest that in synthesizing non-coding, intergenic RNAs, the enhancer-assembled transcription complex could track through the intervening DNA to reach the basal promoter complex and activate efficient mRNA synthesis from the promoter.

Introduction:

The locus control region (LCR) of the human b-globin gene domain, defined by four erythroid specific DNase I hypersensitive sites HS1, HS2, HS3 and HS4, and a ubiquitous HS5 site [1-3], regulates transcription of the far downstream embryonic e, fetal Gg and Ag, and the adult d and b-globin genes during erythroid cell differentiation and development. Among the LCR HS sites, the HS2 site located 11 kb and 55 kb 5' of the e and b-globin genes, respectively (see Figure 1(a)) possesses strong enhancer activity [4], and can regulate transcription of the globin genes over a long distance [5, 6]. Whether the HS2 enhancer and the LCR act over the long distance by a looping or a tracking mechanism is not fully understood [7-11].

Figure 1. Promoter-independent transcription of the HS2 enhancer detected by RT-PCR.

Figure 1. Promoter-independent transcription of the HS2 enhancer detected by RT-PCR.

(a) Map of the human globin gene locus. Vertical arrows: the DNase I hypersensitive sites HS1-5 defining the b-globin LCR: filled boxes, the five b-like globin genes. Angled arrow, direction of globin mRNA synthesis. In the enlarged HS2 site, H, S and Bg: HindIII, StuI and BglII sites. Numbers, sizes (in bases) of the HS2 enhancers and of the downstream DNA present in the CAT plasmids (see (b)).

(b) CAT plasmid maps. ep, the 200 bp e-globin promoter; Bg/B and S/S, BglII/BamHI and StuI/StuI junction sites between HS2 and downstream DNAs. Sense and anti-sense arrows: the HS2 RNAs and cDNAs synthesized from the HS2 RNAs by random hexamer primers, (N)6, in reverse transcription (RT). Horizontal lines 1 and 2, PCR products generated by primer pairs 1 and 2, represented by the filled triangles. Numbers below the lines, sizes (in bp) of the PCR products.

(c) HS2 enhancer transcripts analyzed by semi-quantatitive RT-PCRs. In vivo and in vitro: RT-PCR bands generated by RNAs transcribed respectively from integrated plasmids and RNAs transcribed from plasmid DNA in in vitro transcription reactions. Lanes 1 and 2, RT-PCR bands generated by primer pairs 1 and 2. Lane 0, control RT-PCR of in vitro transcribed RNA sample amplified with primer pair 2 with no reverse transcriptase in the RT step. M, 100 bp ladder of size markers. Numbers in the margins, sizes (in bp) of the size marker and the RT-PCR bands.

In attempts to define the functional mechanism of the HS2 enhancer and ultimately of the LCR, we, as well as others, have analyzed the transcriptional status of the HS2 enhancer [12-14]. The HS2 enhancer is transcribed in erythroid cells but not in non-erythroid cells. In the endogenous genome of erythroid cells and in integrated plasmids, the HS2 enhancer initiates transcription predominantly in the direction toward the downstream globin or reporter gene. Furthermore, a transcriptional terminator inserted in the intervening DNA between the HS2 enhancer and a distant, cis-linked promoter reduced HS2 enhancer activity drastically [15]. These findings provide strong support for the pivotal importance of the enhancer-initiated transcription in mediating HS2 enhancer function over a long distance. Consistent with the involvement of a transcription mechanism in long-range HS2 enhancer function, replacement of the HS2 enhancer in the genome of mice with an independent transcription unit, a neomycin-resistant gene driven by a strong promoter, to disrupt the transcription traffic at the HS2 enhancer, suppresses globin gene transcription and causes severe anemia and death of the homozygous transgenic mice [16].

However, in these transgenic mice, transcriptional suppression of the globin genes may be due to competition between the inserted foreign promoter and the further downstream globin promoters for interaction with the LCR enhancers through a looping mechanism. Similarly, HS2 transcription in the integrated plasmids and the endogenous genome may result from direct loop formation between the HS2 enhancer and the cis-linked globin promoter, which then initiates transcription in the HS2 enhancer. Hence, HS2 enhancer transcription could be initiated not by the enhancer but by the cis-linked promoter.

In this study, we used reverse transcription (RT)-PCR, RNase protection assay (RPA), 5' and 3' rapid amplification of cDNA end (RACE) and RNA fluorescent in situ hybridization (FISH) visualized with two-photon laser scanning microscopy to analyze the RNAs transcribed from the HS2 enhancer in the endogenous genome of K562 cells and in plasmids integrated into K562 cells and in purified plasmid DNA used as template for in vitro transcription reactions either in the presence or in the absence of a cis-linked globin promoter. The results indicate that the HS2 enhancer was able to initiate transcription autonomously in the absence of a cis-linked promoter. Furthermore, the enhancer/LCR-initiated, intergenic RNAs were polyadenylated and relatively short (< 3 kb); they could be spliced and appeared to be synthesized by RNA polymerase II (pol II). However, the enhancer RNAs remained in discrete foci in the nucleus and were not translated into protein products. The results suggest that the HS2 enhancer assembled a pol II transcription complex, which could mediate long-range enhancer function by synthesizing non-coding, intergenic RNAs through the intervening DNA to reach the distant promoter and activate efficient mRNA synthesis of the cis-linked gene.

...

Discussion:

In this study, we characterized the HS2 enhancer RNAs and their mode of transcription by RT-PCR, RPA, 5' and 3' RACE and RNA FISH. The results indicate that the HS2 enhancer, in the endogenous genome of erythroid K562 cells, in CAT plasmids integrated into K562 cells, and in purified DNA used as templates for in vitro transcription reactions, was able to initiate RNA synthesis from multiple sites within HS2. In the integrated plasmids and in in vitro transcription reactions, the HS2 enhancer was able to initiate RNA synthesis in the absence of a cis-linked globin promoter. This result indicates that the enhancer-initiated transcription was an autonomous process and did not require the enhancer to loop and interact with the promoter in order for the promoter to initiate RNA synthesis from within the enhancer. Our finding is consistent with the results of an earlier study, which showed that the human HS2 enhancer integrated into the genome of transgenic mice in the absence of a cis-linked promoter and gene could form an erythroid-specific DNase I hypersensitive site autonomously, and was thus able to organize an open chromatin domain in erythroid cells autonomously [21]. In addition, the HS2 enhancer activated RNA synthesis from distant sites in the downstream DNA, which in the absence of the HS2 enhancer was unable to initiate transcription. These results indicate that the HS2 enhancer-assembled transcription complex was able to track and transcribe RNAs from the enhancer through the intervening DNA to reach the distant promoter and activate efficient mRNA synthesis from the cis-linked gene.

Further analyses of the enhancer-initiated RNAs showed that they were polyadenylated and many of the polyadenylated RNAs were transcribed through internal poly(A) signals. In particular, a strong polyadenylation signal was located in the middle of the 1.2 kb genomic DNA immediately downstream of the HS2 enhancer. The presence of poly(A) signals in the 1.2 kb DNA and in the DNA further downstream of HS2 raised the question of whether termination of enhancer-initiated RNAs at downstream poly(A) sites diminished HS2 enhancer activity. Earlier enhancer assays, however, showed that the HS2-1.2-ep-CAT plasmid containing the 1.2 kb DNA and thus the poly(A) signal interposed between HS2 enhancer and the e-globin promoter exhibited strong enhancer activity at a level similar to that of the HS2-ep-CAT plasmid containing the enhancer linked directly to the promoter [13, 15]. This finding was not surprising, since the HS2 enhancer was able to initiate transcription from multiple sites further downstream of the poly(A) signal in the 1.2 kb DNA. In addition, precursors for polyadenylated RNAs are known to be transcribed past the poly(A) signals for up to thousands of bases before they are truncated by nucleases and polyadenylated [22]. Hence, the enhancer transcription machinery could have proceeded by thousands of bases and reached its target before the polyadenylation was initiated.

In the integrated plasmids containing inserted splice signals, the enhancer RNAs were spliced correctly. However, the enhancer RNAs transcribed from the endogenous K562 genome were not spliced due, apparently, to the absence of appropriate splice signals in this region of the b-LCR.

The detection of enhancer RNAs that could be polyadenylated and spliced indicates that pol II is involved in HS2 enhancer-initiated transcription, since pol II through its unique C-terminal domain (CTD) has been reported to participate in both polyadenylation and splicing of the RNAs it transcribes [18[. Indeed, a-amanitin at a low concentration that specifically inhibited pol II inhibited HS2 enhancer transcription from a number of initiation sites in HS2 in in vitro transcription reactions. These results are in agreement with earlier reports that the HS2 enhancer, in the endogenous genome of K562 cells and in a 140 kb YAC DNA spanning the entire b-globin gene locus, is transcribed by pol II [14, 23]. Moreover, recent chromatin immunoprecipitation (ChIP) studies show that the HS2 enhancer in both human and murine erythroid cells is associated in vivo with pol II [24 -26]. Our results cannot exclude the possibility that pol I and pol III may be involved in HS2 transcription. However, the HS2 enhancer RNAs are preferentially transcribed in erythroid cells (Figure 7(a)) [12-14], whereas pol I and pol III transcripts are ubiquitous in tissue distribution. These observations suggest that pol II is the primary polymerase involved in transcribing the HS2 enhancer in erythroid cells.

The enhancer-assembled pol II transcription complex appeared to be different in several aspects from the pol II complex assembled at the promoter. First, as opposed to pol II transcription at the e-globin promoter, which initiates mRNA synthesis at the cap site in the promoter [17, 27], the pol II enhancer complex initiated transcription from multiple sites both within the enhancer and in downstream DNA as far as 1 kb away. Second, unlike the capped 5' end of globin mRNA, the 5' ends of the HS2 enhancer RNAs frequently contained from one to five extra, non-cap bases not present in the HS2 DNA template, suggesting a different mechanism of 5' end processing by the pol II enhancer complex. Third, the enhancer RNAs were relatively short, as they were polyadenylated within 3 kb downstream of their initiation sites. Indeed, HS2 and LCR transcripts of longer than 3 kb were not detected by further analyses with Northern blots and long-range RT-PCR (J.L.D.T., unpublished results). In contrast, the chicken a-globin gene locus has been observed to be transcribed from the putative LCR through the intergenic DNA and the globin genes to produce giant, nuclear RNAs of longer than 20 kb [28-29]. Fourth, the polyadenylated enhancer RNAs were not processed into mRNA or translated into protein products. Fifth, similar to the HS3 and LCR transcripts visualized in RNA FISH in murine erythroid cells[14, 30], the HS2 enhancer RNAs in human erythroid K562 cells were confined within two or three discrete nuclear foci corresponding probably to the location of the transcription factories of the globin gene locus [19]. These findings suggest that the non-coding, intergenic RNAs were transcribed by a pol II transcription complex that is different from the basal transcription machinery assembled at the promoter.

Our findings are consistent with transcriptional studies in yeast, which show that the predominant function of enhancers is to recruit and deliver the pol II machinery to the cis-linked promoters [31-32], since the promoters, packaged in nucleosomes, are unable to bind TBP stably and recruit the pol II transcription machinery [33]. In erythroid K562 cells expressing the embryonic globin gene program, the entire 10 kb of the intergenic DNA between the HS2 enhancer and the embryonic e-globin promoter is transcribed by pol II in a relay fashion to produce not one long contiguous RNA of 10 kb but many shorter, overlapping, polyadenylated, non-coding RNAs in the sense direction co-linear with globin mRNA synthesis (this study; and D.T. et al., unpublished results) [14, 17, and 25]. In support of the functional significance of the transcription mechanism of this HS2-assembled pol II machinery in mediating long-range enhancer function between the HS2 enhancer and the e-globin gene, inserting a transcriptional terminator or an insulator [34] between HS2 and the e-globin promoter to block the enhancer-assembled pol II machinery from tracking to the promoter also blocked HS2 enhancer function [15, 26]. In the endogenous genome of human erythroid cells expressing the fetal or adult globin gene program, the functional significance of the tracking and transcription mechanism of the HS2/LCR-assembled transcription machinery in transmitting long-range enhancer/LCR function through the embryonic e-globin gene to the further downstream fetal g-globin or adult b-globin gene remains to be investigated.
...

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