Dicer-Dependent Turnover of Intergenic Transcripts from the Human β-Globin Gene Cluster

Abstract

The widespread occurrence of intergenic transcription in eukaryotes is increasingly evident. Intergenic transcription in the ␤-globin gene cluster has been described in murine and human cells, and models for a role in gene and chromatin activation have been proposed. In this study, we analyze intergenic transcription and the chromatin state throughout the human ␤-globin gene cluster and find that the data are not consistent with such activation-linked models. Thus, intergenic transcript levels correlate with neither chromatin activation nor globin gene expression. Instead, we find that intergenic transcripts of the ␤-globin gene cluster are specifically upregulated in Dicer-deficient cells. This is accompanied by a shift towards more activated chromatin as indicated by changes in histone tail modifications. Our results strongly implicate RNA interference (RNAi)-related mechanisms in regulating intergenic transcription in the human ␤-globin gene cluster and further suggest that RNAi-dependent chromatin silencing in vertebrates is not restricted to the centromeres.The human ␤-globin gene cluster spans over 70 kb on chromosome 11 consisting of five ␤-globin-like genes. It is regulated by an upstream locus control region (LCR) which confers high-level and tissue-specific expression on linked globin genes in transgenic mice (18). Although the LCR autonomously opens chromatin, presumably by recruiting chromatin remodeling complexes through transcription factor association, a region upstream of the LCR has been inferred to modulate ␤-globin locus chromatin as well (21,36). This region consists of a number of repetitive elements, one of which is an endogenous retroviral (ERV) solitary long terminal repeat (LTR) (ERV solo LTR). This element has been identified as a major promoter of intergenic transcription in both erythroid cells and nonerythroid HeLa cells following treatment with the histone deacetyltransferase inhibitor trichostatin A (TSA) (35).The presence of intergenic transcription in the human ␤-globin gene cluster has given rise to various models trying to explain their existence (reviewed in reference 43). All of these models essentially positively link intergenic transcription to globin gene activity. The notion that intergenic transcription follows the same 5Ј-3Ј polarity as the structural globin genes (subsequently referred to as "sense" orientation) (2) triggered speculation that intergenic transcription may be a means of delivering polymerases to the globin gene promoters during gene activation and development (30). A variation of this tracking model explains the spatially and temporally coordinated activation of globin genes during development by arguing that intergenic transcription would be necessary for opening chromatin at the appropriate developmental stages, thus facilitating globin gene expression (16). Intergenic transcription should therefore be active at all erythropoietic stages in the LCR, consistent with its role as a constitutive globin enhancer, while it should be coupled to resident globi...

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“…Thus, despite the collection of the reports below, one should still approach the nuclear aspect cautiously. One of the earlier implications for nuclear localization of Dicer was the reported Dicer-dependent turnover of intergenic transcripts from the human beta-globin gene cluster (Haussecker and Proudfoot, 2005). However, this study showed mostly correlation of abundance of nuclear transcripts.…”

“…A number of works directly or indirectly implies nuclear localization of Dicer (Haussecker and Proudfoot, 2005;Sinkkonen et al, 2010;Gullerova and Proudfoot, 2012;Ohrt et al, 2012;Doyle et al, 2013;Drake et al, 2014;Gagnon et al, 2014a;White et al, 2014;Neve et al, 2016), which contrasts with a recent in vivo study on mouse Dicer where tagging of an endogenous Dicer gene with an antibody epitope yielded exclusively cytoplasmic localization in all analyzed tissues with an extremely sensitive detection limit for nuclear Dicer (Much et al, 2016). Thus, despite the collection of the reports below, one should still approach the nuclear aspect cautiously.…”

Eukaryotic regulatory RNAs: an answer to the ‘genome complexity’ conundrum

“…Similarly, studies by Haussecker and Proudfoot (2005) did not observe a positive correlation between intergenic transcript abundance and chromatin activation and/or globin gene expression. Instead, this study suggested that intergenic transcription at the ␤-globin locus mediates the formation of silent chromatin in the absence of erythrocyte-specific transcription factors (Haussecker and Proudfoot 2005).…”

Identification of distal KIR promoters and transcripts

“…Alu repeats within the human b-globin gene cluster have been shown to play a role in the silencing of fetal g-globin during adult erythropoiesis, and a recent report has implicated Dicer-mediated silencing as a mechanism associated with the loss of fetal hemoglobin expression during development. 26 The possible role of distal KIR transcripts in generating doublestranded RNA in conjunction with a recently discovered antisense KIR transcript is currently under investigation. (SK Anderson, unpublished data).…”