Summary Chromatin loops juxtapose distal enhancers with active promoters but their molecular architecture and relationship with transcription remain unclear. In erythroid cells, the locus control region (LCR) and β-globin promoter form a chromatin loop that requires transcription factor GATA1 and the associated molecule Ldb1. We employed artificial zinc fingers (ZF) to tether Ldb1 to the β-globin promoter in GATA1 null erythroblasts in which the β-globin locus is relaxed and inactive. Remarkably, targeting Ldb1 or only its self-association domain to the β-globin promoter substantially activated β-globin transcription in the absence of GATA1. Promoter-tethered Ldb1 interacted with endogenous Ldb1 complexes at the LCR to form a chromatin loop, causing recruitment and phosphorylation of RNA polymerase II. ZF-Ldb1 proteins were inactive at alleles lacking the LCR, demonstrating that their activities depend on long-range interactions. Our findings establish Ldb1 as critical effector of GATA1-mediated loop formation and indicate that chromatin looping causally underlies gene regulation.

The ␤-globin locus control region (LCR) is necessary for high-level ␤-globin gene transcription and differentiation-dependent relocation of the ␤-globin locus from the nuclear periphery to the central nucleoplasm and to foci of hyperphosphorylated Pol II "transcription factories" (TFys). To determine the contribution of individual LCR DNaseI hypersensitive sites (HSs) to transcription and nuclear location, in the present study, we compared ␤-globin gene activity and location in erythroid cells derived from mice with deletions of individual HSs, deletions of 2 HSs, and deletion of the whole LCR and found all of the HSs had a similar spectrum of activities, albeit to different degrees. Each HS acts as an independent module to activate expression in an additive manner, and this is correlated with relocation away from the nuclear periphery. In contrast, HSs have redundant activities with respect to association with TFys and the probability that an allele is actively transcribed, as measured by primary RNA transcript FISH. The limiting effect on RNA levels occurs after ␤-globin genes associate with TFys, at which time HSs contribute to the amount of RNA arising from each burst of transcription by stimulating transcriptional elongation. IntroductionAnalysis of naturally occurring and targeted deletions of the endogenous ␤-globin locus control region (LCR) and of transgenic mice has revealed that the LCR is required for high-level ␤-globin transcription in erythroid cells. [1][2][3] The LCR DNase1 hypersensitive sites (HSs) contain similar, albeit distinct sequence motifs, and vary in their activities in vitro and in transgenic mouse assays. [2][3][4][5][6][7][8][9][10] Therefore, each HS may play a unique role in LCR function or may contribute a similar spectrum of activities. Transgenic mice bearing individual LCR HSs, combinations of HSs, or an intact LCR linked to the human ␤-globin locus have led to models in which the LCR HSs interact to form a holocomplex in which the individual HSs 1-4 act synergistically to ensure a permissive chromatin environment and activate expression, whereas 5ЈHSs 5 and 6 act as a chromatin barrier. 5,[11][12][13][14][15][16][17][18][19] In addition, it has been suggested that 5ЈHS2 has a dominant or unique activity compared with other LCR HSs. In contrast to these transgenic studies, our analysis of the transcriptional phenotypes of mice in which each LCR HS was deleted individually from the endogenous locus suggested that each site might contribute additively to LCR-mediated gene activation. [20][21][22] The LCR has also been implicated in the localization of the ␤-globin locus during erythroid differentiation. During differentiation, the ␤-globin locus relocates away from the nuclear periphery and increasingly associates with foci of hyperphosphorylated Pol II "transcription factories" (TFys), where high-level transcription is activated. 23 Deletion of the HSs that comprise the endogenous mouse ␤-globin LCR results in both decreased relocation of the locus to the nucleoplasm and asso...

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...