Pharmacologic control of chromatin looping

S ickle cell disease (SCD) and b-thalassemia are widespread genetic disorders that result from inherited mutations in the adultb-globin gene. An important aspect of SCD and b-thalassemia is that diseasecausing mutations affect the adult b-globin gene but leave intact its fetal counterparts Ggand Ag-globin, a point that explains why SCD and b-thalassemia patients first experience major symptoms in late infancy when the fetal g-globin genes become developmentally extinguished. Furthermore, rare mutations that lead to persistence of fetal g-globin expression in adults significantly ameliorate SCD and b-thalassemia symptoms, highlighting the clinical benefits of elevated levels of HbF. Therefore, a major research objective is the development of methods to reactivate fetal g-globin in adult erythroid cells. The b-like globin genes reside in a single cluster where they are arranged in the order of their expression during development. Highlevel expression of these genes is mediated by the locus control region (LCR), a distal array of multiple enhancers that act in an additive manner to increase the rate of transcriptional elongation. During development, when the embryonic, fetal, and adult b-globin genes undergo sequential phases of expression followed by gene silencing, the LCR alters its spatial positioning within the nucleus to remain in close proximity to the promoter of the developmentally appropriate, active b-like globin gene through a 3-dimensional looping of chromatin. Although the mechanism through which looping is established is not entirely clear, the authors have previously identified the Lim-domain binding 1 (LDB1) protein as a key factor that mediates loop formation. Furthermore, it has been shown that in adult erythroid cells, tethering the dimerization domain of LDB1 to the fetal g-globin gene promoters via an artificial zinc-finger protein brings the LCR in close proximity to the fetal genes and stimulates their expression. Although this shows that forced looping through an artificial transcription factor allows reactivation of HbF in adult erythroid cells (see figure), such an approach requires genetic manipulation of erythroblasts, which may complicate its application in a clinical setting. Here, Krivega et al describe a novel pharmacologic approach to modulate b-globin gene expression where they use a small molecule inhibitor of the histone H3 lysine 9 (H3K9) methyltransferase enzymes G9a and G9a-like protein (GLP) to reactivate HbF production in adult erythroid cells. Interestingly, the authors show that this reactivation is associated with spatial reconfiguration of the locus whereby the LCR alters its nuclear positioning to gain proximity to the fetal g-globin genes (see figure). This finding is important because it provides proofof-principle that structural reconfiguration of the b-globin locus can be achieved through pharmacologic modification of its chromatin state. In addition, the study provides new insights into the mechanism of long-distance enhancer-gene communication by showing that the chromatin-modifying enzyme G9a, previously shown to spread across theb-globin locus, contributes to the regulation of chromatin loop formation. This finding offers the first clue that chromatin spreading and looping may be functionally linked. G9a and its paralog GLP are methyltransferases that can monoand dimethylate H3K9. Furthermore, G9a and GLP possess ankyrin repeat domains, which allow them to bind to their own substrate, albeit with different specificities (ie, H3K9me1 for GLP and H3K9me2 for G9a). It has been previously shown that G9a is recruited to the b-globin LCR by the transcription factor NF-E2, and spreads across the b-globin locus. Furthermore, knocking down G9a through RNA interference in murine erythroid cells, or inhibiting its enzymatic activity in human hematopoietic progenitors, leads to reactivation of the embryonic/fetal b-like globin genes,