Distinct Ldb1/NLI complexes orchestrate γ-globin repression and reactivation through ETO2 in human adult erythroid cells

Abstract

The Ldb1/GATA-1/TAL1/LMO2 complex mediates long-range interaction between the ␤-globin locus control region (LCR) and gene in adult mouse erythroid cells, but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. We investigated NLI (Ldb1 homolog) complex occupancy and chromatin conformation of the ␤-globin locus in human erythroid cells. In addition to the LCR, we found robust NLI complex occupancy at a site downstream of the A ␥-globin gene within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing ␤-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members, together with corepressor ETO2 and by ␥-globin repressor BCL11A. The LCR and ␤-globin gene establish proximity in these cells. In contrast, when ␥-globin transcription is reactivated in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and ␥-globin are transcribed. In these cells, proximity between the BGL3/␥-globin region and the LCR is established. We conclude that alternative NLI complexes mediate ␥-globin transcription or silencing through long-range LCR interactions involving an intergenic site of noncoding RNA transcription and that ETO2 is critical to this process. (Blood. 2011; 118(23):6200-6208) IntroductionThe human ␤-globin locus consists of 5 genes expressed sequentially during development: embryonic ⑀, fetal G ␥ and A ␥, and adult ␦ and ␤. An enhancer, the locus control region (LCR), lies far upstream of the genes and is necessary for the high level of transcription achieved by these genes in erythroid cells. 1 Developmental regulation of globin gene expression is incompletely understood. In particular, repression of fetal (␥) globin expression in adult cells is of great interest because an increase in fetal hemoglobin (HbF) in response to drugs or in patients with hereditary persistence of HbF ameliorates the severity of clinical symptoms associated with sickle cell disease and ␤-thalassemia. Therefore, reactivation of the fetal ␥-globin genes in adult cells is a therapeutic target. Recent genetic and biochemical studies have implicated the zinc finger transcription factor BCL11A as a direct negative regulator of HbF expression [2][3][4] ; however, the mechanism of action of BCL11A is largely unknown.Achieving a high level of globin gene expression is dependent on a physical interaction between the LCR and globin genes. 5,6 Proximity between the LCR and ␤-globin gene in mouse erythroid cells requires GATA-1 and EKLF, as well as the widely expressed protein NLI/Ldb1 (hereafter NLI). 7-9 NLI stabilizes a complex including DNA-binding proteins GATA-1 and SCL/TAL1 (hereafter TAL1) and the bridging protein LMO2 on both the ␤-globin promoter and LCR, and mediates loop formation between them. 9 Although EKLF shares many sites genome-wide with GATA-1 or TAL1, sites shared by all 3 proteins appear to be rare in mouse erythroid cells, leaving it unclear whether EKLF and t...

Chromatin Loop Formation in the β-Globin Locus and Its Role in Globin Gene Transcription

“…In addition, occupancy of GATA-1 and SCL/TAL1 at the LCR HSs and the β-globin gene promoter was decreased by reduction of NLI/Ldb1 (Song et al, 2010). Interestingly, in the human β-globin locus, the NLI/Ldb1 complex occupies a position 3′ of the γ-globin genes that is the site of an erythroid specific non-coding transcript (Kiefer et al, 2011). This region also comes into proximity with the LCR when the γ-globin genes are active, suggesting that not all LCR loops are directly to globin genes.…”

“…The widely expressed nuclear protein NLI/Ldb1 forms a complex with erythroid specific activators GATA-1 and SCL/TAL1 (Wadman et al, 1997) that binds to the LCR HSs of the human and mouse β-globin loci (Brand et al, 2004;Kiefer et al, 2011;Song et al, 2007). Reduction of NLI/Ldb1 by RNAi in MEL cells inhibits transcriptional activation of the βmaj globin gene with loss of spatial proximity between LCR HS2 and βmaj (Song et al, 2007).…”

Inhibition of G9a methyltransferase stimulates fetal hemoglobin production by facilitating LCR/γ-globin looping

“…8 Relative crosslinking between the anchor fragment and fragments of interest were analyzed by SYBR Green real-time qPCR using published primers. 21 The primer used for detecting interaction with g-globin genes does not distinguish between A g-and G g-globin genes, and the value presented in Figure 5 is the average signal from both. Interaction between 2 fragments within the a-tubulin gene was used as the internal normalization control for 3C.…”

“…21 Primers used for g-globin genes do not distinguish between A g-and G g-globin genes and the values presented in Figures 2-4 are the average signal from both. The data for H3K9me2, H3K27me2, and H3K36me3 were normalized to the H3 signal.…”

“…Given the role of the LDB1 complex in facilitating long-range interactions in the b-globin locus 8,21,27 and its redistribution after G9a inactivation, looping between an anchor LCR fragment and the b-globin genes was examined using the 3C assay. UNC0638 treatment established an interaction between the LCR and the g-globin genes that is absent in control cells ( Figure 5).…”