Does Transcription of Remote α-Globin Regulatory Elements Affect Their Function?.

Abstract 4060

Poster Board III-995

Expression of many tissue and developmental stage specific genes is controlled by remote regulatory elements. The mammalian a-globin gene cluster includes four previously characterised remote regulatory elements (MCS-R1 to R4) upstream of the a-globin genes. Naturally occurring deletions and experiments in transgenic mice, have shown that one or more of these elements are necessary for fully regulated expression of the a-like globin genes. At present it is unclear exactly how these elements interact with the a-globin genes to enhance their expression, although ultimately we know that they physically interact with the promoters forming a chromatin loop. Of interest, three of the MCS-R sequences are located within introns of a widely expressed gene C16orf35, which lies adjacent to the a-globin locus and we now know that such complex, intermingled arrangements are common in the mammalian genome. We have asked two questions: can the MCS-R elements be transcribed while activating the globin genes, and does transcription of the MCS-R elements play any role in their activation?

We have analysed nascent transcription of C16orf35 and a-globin, and shown that both genes are simultaneously transcribed in a high proportion of erythroid cells. Thus it appears that the MCS-R elements can interact with the globin genes while being transcribed. We next asked whether transcription of the C16orf35 gene (through the upstream regulatory elements) is necessary for their activation. The promoter region and a putative erythroid-specific promoter of the C16orf35 gene were deleted (singly and in combination) by homologous recombination in mice. Provisional analysis has shown no evidence of a-thalassaemia in these mice, suggesting that activation of the MCS-R elements occurs independently of their transcription. In addition to their importance in globin gene regulation, these findings have general implications for the relationship between structure and function in the mammalian genome and the mechanisms by which long-range elements interact with their cognate promoters.