Chromatin looping as a target for altering erythroid gene expression

Abstract

The β-hemoglobinopathies are the most common monogenic disorders in humans, with symptoms arising after birth when the fetal γ-globin genes are silenced and the adult β-globin gene is activated. There is a growing appreciation that genome organization and the folding of chromosomes are key determinants of gene transcription. Underlying this function is the activity of transcriptional enhancers that increase the transcription of target genes over long linear distances. To accomplish this, enhancers engage in close physical contact with target promoters through chromosome folding or looping that is orchestrated by protein complexes that bind to both sites and stabilize their interaction. We find that enhancer activity can be redirected with concomitant changes in gene transcription. Both targeting the β-globin locus control region (LCR) to the γ-globin gene in adult erythroid cells by tethering and epigenetic unmasking of a silenced γ-globin gene lead to increased frequency of LCR/γ-globin contacts and reduced LCR/β-globin contacts. The outcome of these manipulations is robust, pancellular γ-globin transcription activation with a concomitant reduction in β-globin transcription. These examples show that chromosome looping may be considered a therapeutic target for gene activation in β-thalassemia and sickle cell disease.

Keywords: chromatin looping; enhancer; fetal hemoglobin; globin switching; thalassemia.

Figure 1

Role of the LDB1 complex in regulation of expression of the β-globin genes. (A) Schematic diagram of the human β-globin gene locus. Rectangles represent genes, arrows represent DNase I–hypersensitive sites composing the LCR. (B) Domain organization of the LDB1 protein. (C) The LDB1 complex occupies chromatin by virtue of GATA1 and TAL1 binding to compound DNA motifs. These proteins are bridged by LMO2, which in turn binds to LDB1 through the LID.

Figure 2

Transcription-independent mechanism of enhancer–promoter interaction. Enhancer–promoter proximity is established by interaction between enhancer and promoter, each occupied by transcription factors. The looped locus then migrates to Pol II factories in the nuclear interior to achieve a high level of gene expression.

Figure 3

Mechanisms of manipulating long-range enhancer–promoter interaction to reactivate silenced fetal γ-globin gene in adult erythroid cells. (A) Model of the forced-looping approach. Artificial targeting of the LDB1 dimerization domain to the promoter of silenced γ-globin genes stimulates de novo loop formation with the LCR and reactivation of γ-globin gene expression. (B) UNC0638 treatment is most effective in raising HbF levels when carried out in the early stage of erythroid differentiation of progenitor cells. (C) Model illustrating that UNC0638 inhibition of G9a methyltransferase activity eliminates repressed chromatin from the silenced γ-globin gene promoters and stimulates LDB1 complex occupancy, de novo LCR looping, and reactivation of the expression of γ-globin genes.