Transcription of the hypersensitive site HS2 enhancer in erythroid cells.

Abstract

In the human genome, the erythroidspecfic hypersensitive site HS2 enhancer regulates the trsiption of the downstream 1-like globin genes 10-50 kilobses away. The mechanism of HS2 eancr fuction Is not known. The present study employs RNA protection assays to analyze the transcriptional status of the HS2 enancer in trneced recombinant chloramphenicol acetltrnsferase (CAT) pasmids. In erythroid K562 cells in which the HS2 enhancer is active, the HS2 sequence directs the synthesis of long enhancer transcripts that are initiated apparently from within the enhancer and elongated through the intervening DNA into the cis-linked CAT gene. In nonerythroid HL-60 cells in which the HS2 enhancer is inactive, long enhancer tra rpt are not detectable. Splitting the HS2 encer between two tandem Apl sites abolishes the synthesis of a group of log enhancer transcripts and results in loss of enhancer function and transcriptional silencing ofthe cis-linked CAT gene. In directing the synthesis ofRNA through the intervening DNA and the gene by a tracking and transcription mechan , the HS2 enhaner may (i) open up the chromatin structure of a gene domain and (it) deliver enhancer binding proteins to the promoter sequence where they may stimulate the transcription of the gene at the cap site.In seeking to identify the cis regulatory elements of the human (-like globin genes ( this laboratory (1) and others (2, 3) have mapped four erythroid-specific and developmentally stable DNase I-hypersensitive sites HS1, -2, -3, and -4 in the locus control region (LCR) between 50 and 70 kilobases (kb) upstream of the (3-globin gene. HS2 at -11 kb 5' of the embryonic E-and thus at -54 kb 5' of the ,B-globin gene has been shown to possess an erythroidspecific and developmentally stable enhancer function (4-6). It is capable of stimulating the transcription of embryonic E-, fetal -, and adult (B-globin genes in erythroid cells (7-10). In y6f3thalassemia, the deletion of HS2 and more upstream DNA is associated with transcriptional silencing of the far downstream f3-globin gene (11-13) and possibly of the whole (-like globin gene domain.How the HS2 enhancer cooperates with distant globin promoter sequences to stimulate the E-, y-, and (-globin genes at the respective developmental stage is not clear. Looping (14) and tracking (15) mechanisms have been proposed to explain how a distant enhancer may communicate with the promoter of a cis-linked gene (14,16,17). In the present study, the detection of HS2 enhancer transcripts in erythroid cells suggests that the tandem Apl sites and other sequence motifs in the enhancer may provide entry sites for transcription factors associated with the transcriptional machinery that track the DNA and synthesize long strands of RNA through the intervening DNA into the cis-linked gene.The possible biological significance of such long transcripts in enhancer function will be discussed. MATERIALS AND METHODSConstruction ofRecombinant Constructs. HS2-eP-CAT and EP-CAT have been described (5). 5'-HS2-eP-CAT was made by ...

Intergenic transcription and transinduction of the human β-globin locus

“…Support for this comes from the observations that the LCR microlocus (a fragment containing the four LCR HSs linked together) and HS2 are transcribed (Collis et al 1990;Tuan et al 1992). The reason why only a proportion of erythroid cells transcribe the intergenic regions as shown in Figure 4 is unknown, although this could reflect a stage dependence of transcription.…”

“…It is also possible that the role of the LCR and intergenic transcripts is to deliver proteins and/or Pol II to the globin gene promoters, and such a tracking mechanism has been proposed previously (Tuan et al 1992).…”

“…As HS2 and the microlocus, which can substitute for the entire LCR, are transcribed (Collis et al 1990;Tuan et al 1992), it is possible that LCR transcription is important for its function. Indeed, transcription has been correlated to LCR activity for the keratin 18 gene.…”

“…Also, it is possible that the LCR is transcribed as microlocus and HS2 transcription have been detected in constructs integrated into erythroid cells (Collis et al 1990;Tuan et al 1992). We have therefore investigated transcription upstream of the ⑀ gene using NRO probes with ∼500-bp inserts from the LCR, as shown in Figure 2A.…”

“…The ␤-globin LCR contains five erythroid-specific, developmentally stable hypersensitive sites, HS1-HS5, situated upstream of the ⑀-globin gene (Grosveld et al 1987;Zafarana et al 1996). Transcription has been detected both in a microlocus fragment containing HS1-HS4 linked together and in HS2 as part of a chimeric construct, raising the possibility that the ␤-globin LCR is transcribed (Collis et al 1990;Tuan et al 1992). In addition, the existence of large transcripts from the avian and murine ␤-globin clusters, up to seven times longer than the globin mRNAs, was described over two decades ago (Imaizumi et al 1973;Bastos and Aviv 1977).…”