Long-range interactions between enhancers and promoters play key roles in regulating gene expression profiles during development. The locus control region (LCR) is a powerful enhancer that activates -globin genes by forming developmentally restricted long-distance interactions with the embryonic, fetal, and adult globin gene promoters. These contacts are mediated in part by a looping factor termed Ldb1. This nuclear factor occupies both the LCR and globin promoters, and is thought to favor looping interactions by self- association. Recently, our laboratory has shown that tethering Ldb1 to the fetal globin promoter via artificial zinc finger (ZF) proteins triggers promoter contacts with the LCR, and re-activation of the fetal genes with up to 85% of total globin synthesis in cultured adult human erythroid cells. This is of particular interest as high fetal globin levels mitigate th clinical severity of hemoglobinopathies such as Sickle Cell Disease (SCD). While the levels achieved by this approach would be therapeutic for SCD patients, it is still unclear if this stratey can be applied in a clinical setting. Towards this goal, we will test this approach in transgenic mice that specifically express human sickle hemoglobin. These mice contain naturally configured human transgenes encoding the fetal and adult sickle -globin. We will transduce hematopoietic stem cells from these mice with either ZF-Ldb1 fusion constructs or empty vector and transplant the transduced cells into lethally irradiated recipients. We will compare complete blood counts, red blood cell parameters, organ pathologies, and survival rates of the ZF- Ldb1 treated mice to empty vector treated mice. Furthermore, while the forced chromatin looping approach appears to be a robust strategy to control globin genes, it is unclear if this strategy may be more broadly applicable. ChIP-seq studies have shown that Ldb1 occupies a substantial number of sites throughout the genome. Knockdown experiments have shown that Ldb1 is required for the proper activation of a wide range of erythroid genes. However, the specific long-range interactions Ldb1 may mediate genome-wide remain unclear. We will characterize long-range interactions in an unbiased manner for a select group of Ldb1-bound distal regulatory elements at high-resolution with the Capture-C method, which is well established in our laboratory. To gain insight into looping interactions mediated by Ldb1 genome-wide, we will use Chromatin Interaction Analysis using Paired-End Tag sequencing (ChIA-PET), in collaboration with Dr. Ali Mortazavi (UC Irvine). Together, the proposed experiments are designed to elucidate basic mechanisms of chromatin looping, to explore the therapeutic potential of Ldb1-mediated looping for the treatment of SCD, and broaden the scope of the approach to other genes and diseases.
Enhancers play key roles in gene regulation. Our laboratory has shown that tethering the looping factor Ldb1 to globin promoters triggers chromatin looping and activates transcription. In particular, this approach can robustly reactivate fetal globin in adult erythroid cells. We aim to determine if this strategy can ameliorate sickle cell phenotypes in a humanized mouse model of the disease. We also aim to globally characterize gene targets of Ldb1-bound enhancers in erythroid cells to broaden the scope of the forced looping approach.
| Grevet, Jeremy D; Lan, Xianjiang; Hamagami, Nicole et al. (2018) Domain-focused CRISPR screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells. Science 361:285-290 |
