Date Published: August 14, 2019
Publisher: Public Library of Science
Author(s): Emmanuel Debrand, Lyubomira Chakalova, Joanne Miles, Yan-Feng Dai, Beatriz Goyenechea, Sandra Dye, Cameron S. Osborne, Alice Horton, Susanna Harju-Baker, Ryan C. Pink, Daniel Caley, David R. F. Carter, Kenneth R. Peterson, Peter Fraser, Andrew C. Wilber.
Transcriptome analyses show a surprisingly large proportion of the mammalian genome is transcribed; much more than can be accounted for by genes and introns alone. Most of this transcription is non-coding in nature and arises from intergenic regions, often overlapping known protein-coding genes in sense or antisense orientation. The functional relevance of this widespread transcription is unknown. Here we characterize a promoter responsible for initiation of an intergenic transcript located approximately 3.3 kb and 10.7 kb upstream of the adult-specific human β-globin genes. Mutational analyses in β-YAC transgenic mice show that alteration of intergenic promoter activity results in ablation of H3K4 di- and tri-methylation and H3 hyperacetylation extending over a 30 kb region immediately downstream of the initiation site, containing the adult δ- and β-globin genes. This results in dramatically decreased expression of the adult genes through position effect variegation in which the vast majority of definitive erythroid cells harbor inactive adult globin genes. In contrast, expression of the neighboring ε- and γ-globin genes is completely normal in embryonic erythroid cells, indicating a developmentally specific variegation of the adult domain. Our results demonstrate a role for intergenic non-coding RNA transcription in the propagation of histone modifications over chromatin domains and epigenetic control of β-like globin gene transcription during development.
A staggering proportion of the mammalian genome is transcribed. More than half of the transcribed genomic regions in mammalian cells are intergenic regions [1–3] producing vast amounts of non-coding RNAs . Such intergenic transcription patterns can be complex, with multiple overlapping sense, and antisense transcripts detected for a single locus . During the process of mammalian development, proper temporal and spatial expression of genetic information must be achieved. The multigene β-globin locus has been intensively studied as a model for these events. In humans, it spans over 70 kb on chromosome 11 and its five genes (HBE, HBG1, HBG2, HBD and HBB) are arranged in the order of their developmental expression in erythroid cells. The locus includes a locus control region (LCR) , located 6–22 kb upstream of the embryonic HBE gene, which acts as a long-range regulatory element that physically interacts with the active globin genes during transcription [7, 8].
Intergenic transcription is a widespread phenomenon. Aside from the handful of individual gene loci in which intergenic transcripts have been recognized and studied, it is now apparent from various transcriptome studies that most of the sequence of a mammalian genome is transcribed at one time or another, and that the vast majority of these transcribed regions are non-coding . Many of these non-coding transcripts overlap known coding transcript regions and can be sense or antisense. In many cases, the non-coding transcripts correlate with activity of nearby genes and appear to be regulated in a similar fashion. In other cases, especially where antisense transcripts are involved it has been proposed that these transcripts may play a repressive role. Here we present the characterization of the δβ intergenic promoter, which initiates sense intergenic transcription toward the downstream HBD and HBB genes. We show that deletion of a 300 bp sequence that contains the δβ promoter results in dramatic alterations to the normal active histone modification profile over a 30 kb downstream region in YAC transgenic mice. As a result, expression of the adult-specific HBD and HBB genes is severely compromised in erythroid tissues. Our data show that intergenic promoter activity is knocked down by insertion of loxP sites flanking the δβ promoter, with a corresponding knock down of adult gene expression. It is not clear why the inserted loxP sites cause this knockdown of promoter activity. One possibility is that the upstream loxP site disrupts a critical cis element needed for full intergenic promoter activity. A search for potential transcription factor binding sites indicates that the upstream loxP sequence disrupts a potential E-box binding site and could potentially bind TAL1 and/or E47. Transgenic line FX14, which has only one loxP site in the downstream position, does not suffer from a decrease in intergenic promoter activity, further implicating the upstream site. However, deletion of the region containing this putative binding site results in a doubling of the number of transfected cells that express the EGFP reporter gene in stably transfected K562 cells (compare BstXI and Apo in Fig 1B). This is seemingly at odds with the transgenic data but is important to point out that K562 cells express embryonic and fetal globins, HBE and HBG, and do not appear to initiate sense transcripts from the δβ promoter . In combination, these data suggest that this sequence element may be of importance in regulating δβ intergenic promoter activity by repressing transcription in primitive erythroid cells and promoting it in definitive cells.