Debunking the "junk": Unraveling the role of lncRNA–miRNA–mRNA networks in fetal hemoglobin regulation

Fetal hemoglobin (HbF) induction is considered to be a promising therapeutic strategy to ameliorate the clinical severity of β-hemoglobin disorders, and has gained a significant amount of attention in recent times. Despite the enormous efforts towards the pharmacological intervention of HbF reactivation, progress has been stymied due to limited understanding of γ-globin gene regulation. In this study, we intended to investigate the implications of lncRNA-associated competing endogenous RNA (ceRNA) interactions in HbF regulation. Probe repurposing strategies for extraction of lncRNA signatures and subsequent in silico analysis on publicly available datasets (GSE13284, GSE71935 and GSE7874) enabled us to identify 46 differentially expressed lncRNAs (DElncRNAs). Further, an optimum set of 11 lncRNAs that could distinguish between high HbF and normal conditions were predicted from these DElncRNAs using supervised machine learning and a stepwise selection model. The candidate lncRNAs were then linked with differentially expressed miRNAs and mRNAs to identify lncRNA-miRNA-mRNA ceRNA networks. The network revealed that 2 lncRNAs (UCA1 and ZEB1-AS1) and 4 miRNAs (hsa-miR-19b-3p,hsa-miR-3646,hsa-miR-937 and hsa-miR-548j) sequentially mediate cross-talk among different signaling pathways which provide novel insights into the lncRNA-mediated regulatory mechanisms, and thus lay the foundation of future studies to identify lncRNA-mediated therapeutic targets for HbF reactivation.

Exploring epigenetic and microRNA approaches for γ-globin gene regulation

Therapeutic interventions aimed at inducing fetal hemoglobin and reducing the concentration of sickle hemoglobin is an effective approach to ameliorating acute and chronic complications of sickle cell disease, exemplified by the long-term use of hydroxyurea. However, there remains an unmet need for the development of additional safe and effective drugs for single agent or combination therapy for individuals with β-hemoglobinopathies. Regulation of the γ-globin to β-globin switch is achieved by chromatin remodeling at the HBB locus on chromosome 11 and interactions of major DNA binding proteins, such as KLF1 and BCL11A in the proximal promoters of the globin genes. Experimental evidence also supports a role of epigenetic modifications including DNA methylation, histone acetylation/methylation, and microRNA expression in γ-globin gene silencing during development. In this review, we will critically evaluate the role of epigenetic mechanisms in γ-globin gene regulation and discuss data generated in tissue culture, pre-clinical animal models, and clinical trials to support drug development to date. The question remains whether modulation of epigenetic pathways will produce sufficient efficacy and specificity for fetal hemoglobin induction and to what extent targeting these pathways form the basis of prospects for clinical therapy.

NRF2 mediates γ-globin gene regulation through epigenetic modifications in a β-YAC transgenic mouse model

NRF2 is the master regulator for the cellular oxidative stress response and regulates γ-globin gene expression in human erythroid progenitors and sickle cell disease mice. To explore NRF2 function, we established a human β-globin locus yeast artificial chromosome transgenic/NRF2 knockout (β-YAC/NRF2−/−) mouse model. NRF2 loss reduced γ-globin gene expression during erythropoiesis and abolished the ability of dimethyl fumarate, an NRF2 activator, to enhance γ-globin transcription. We observed decreased H3K4Me1 and H3K4Me3 chromatin marks and association of TATA-binding protein and RNA polymerase II at the β-locus control region (LCR) and γ-globin gene promoters in β-YAC/NRF2−/− mice. As a result, long-range chromatin interaction between the LCR DNase I hypersensitive sites and γ-globin gene was decreased, while interaction with the β-globin was not affected. Further, NRF2 loss silenced the expression of DNA methylcytosine dioxygenases TET1, TET2, and TET3 and inhibited γ-globin gene DNA hydroxymethylation. Subsequently, protein-protein interaction between NRF2 and TET3 was demonstrated. These data support the ability of NRF2 to mediate γ-globin gene regulation through epigenetic DNA and histone modifications. Impact statement Sickle cell disease is an inherited hemoglobin disorder that affects over 100,000 people in the United States causing high morbidity and early mortality. Although new treatments were recently approved by the FDA, only one drug Hydroxyurea induces fetal hemoglobin expression to inhibit sickle hemoglobin polymerization in red blood cells. Our laboratory previously demonstrated the ability of the NRF2 activator, dimethyl fumarate to induce fetal hemoglobin in the sickle cell mouse model. In this study, we investigated molecular mechanisms of γ-globin gene activation by NRF2. We observed the ability of NRF2 to modulate chromatin structure in the human β-like globin gene locus of β-YAC transgenic mice during development. Furthermore, an NRF2/TET3 interaction regulates γ-globin gene DNA methylation. These findings provide potential new molecular targets for small molecule drug developed for treating sickle cell disease.

The HRI-regulated transcription factor ATF4 activates BCL11A transcription to silence fetal hemoglobin expression

Reactivation of fetal hemoglobin remains a critical goal in the treatment of patients with sickle cell disease and β-thalassemia. Previously, we discovered that silencing of the fetal γ-globin gene requires the erythroid-specific eIF2α kinase heme-regulated inhibitor (HRI), suggesting that HRI might present a pharmacologic target for raising fetal hemoglobin levels. Here, via a CRISPR-Cas9–guided loss-of-function screen in human erythroblasts, we identify transcription factor ATF4, a known HRI-regulated protein, as a novel γ-globin regulator. ATF4 directly stimulates transcription of BCL11A, a repressor of γ-globin transcription, by binding to its enhancer and fostering enhancer-promoter contacts. Notably, HRI-deficient mice display normal Bcl11a levels, suggesting species-selective regulation, which we explain here by demonstrating that the analogous ATF4 motif at the murine Bcl11a enhancer is largely dispensable. Our studies uncover a linear signaling pathway from HRI to ATF4 to BCL11A to γ-globin and illustrate potential limits of murine models of globin gene regulation.

NRF2 Mediates Epigenetic Changes in DNA and Chromatin Structure to Regulate γ-Globin Gene Expression in a Human βYAC Transgenic Mouse Model

NRF2 is the master regulator for the cellular anti-oxidative stress response and previously shown to activate γ-globin gene expression in human erythroid progenitor cells. The goal of this study was to expand on these findings by exploring the in vivo function of NRF2 using the human β-globin locus YAC transgenic (βYAC) mouse carrying the entire 248kb human β-globin locus (HBB). We observed that NRF2 activation by chronic dimethyl fumarate treatment of βYAC mice, induced human γ-globin gene expression, but had no effect on the adult β-globin gene. Subsequently in a novel βYAC/NRF2 knockout mouse model established in our laboratory, we demonstrated that NRF2 loss increased mouse erythroid CD71 levels while reducing human γ-globin gene expression during erythropoiesis in mouse embryonic E13.5 and E18.5 day fetal livers and peripheral blood. Furthermore, the ability of dimethyl fumarate to induce γ-globin gene expression was abolished after NRF2 loss. By western blot analysis of nuclear protein, we confirmed that part of the mechanism of globin gene regulation by NRF2 loss involves a decline of global histone H3 lysine 4 trimethylation levels, without changing histone acetylation. Interestingly, NRF2 loss decreased the protein levels of the DNA methylcytosine dioxygenases including TET1, TET2 and TET3. Analysis of DNA methylation/hydroxymethylation levels by DNA dot-blot assay of mouse E13.5 fetal livers isolated from βYAC/NRF2 knockout mice, showed inhibition of genome wide DNA hydroxymethylation, while DNA methylation was not affected. In addition, DNA immunoprecipitation confirmed decreased hydroxymethylation in the HBB locus control region (LCR) enhancer and γ-globin gene region. These data suggest an essential role of NRF2 in modifying chromatin structure and assembling transcription complexes to regulate γ-globin gene expression. ChIP assay to assess in vivo DNA-protein interactions showed decreased associations of histone H3 lysine 4 monomethylation and trimethylation, TATA-binding protein and RNA polymerase II to the LCR and γ-globin promoter after NRF2 loss. Final studies were conducted to evaluate long-range chromatin interactions between the LCR and individual globin genes by chromosome conformation capture assay. We observed decreased interactions between the LCR and γ-globin gene promoter region after NRF2 loss while interactions in the adult β-globin was not affected suggesting NRF2 preferentially mediates γ-globin gene regulation. In conclusion, our data suggest that NRF2 alters γ-globin expression through epigenetic DNA/histone modifications in addition to direct DNA binding. Therefore, activation of NRF2 expression using small chemical compounds is an innovative strategy to induce γ-globin gene transcription for the treatment of β-hemoglobinopathies. This work was supported by funding from the National Heart, Lung, and Blood Institute to XZ through the Hemoglobinopathy Translational Research Skills Core component of U01 grant HL117684 and R01 grant HL069234 to BSP. Disclosures No relevant conflicts of interest to declare.

In Situ Capture of the Molecular Composition of Erythroid Transcriptional Enhancers

Elucidating mechanisms that regulate gene transcription during erythropoiesis is critical for identifying fundamental principles of cellular differentiation, and for developing new therapies for blood disorders. Transcriptional enhancers determine cell identity by directing spatiotemporal gene expression, yet the molecular processes controlling enhancer activation and deactivation during lineage differentiation remain largely unknown. Recently, we and others have identified human erythroid cell-specific enhancers by mapping histone modifications, transcription factor binding and transcriptomic changes in primary fetal and adult hematopoietic stem/progenitor cells and lineage-committed erythroid progenitors. While these studies establish a comprehensive catalog of erythroid transcriptional enhancers, the molecular composition and in vivo function of the vast majority of these enhancers remain unknown. This is a major impediment for understanding the coordinated control of gene transcription in normal and diseased erythroid cells.Given that enhancers are frequently targeted by disease-associated genetic variations, there is a critical need to correct this gap in knowledge. Identifying the complete composition of a specific enhancer within its native chromatin can provide unprecedented insight into the molecular mechanisms regulating its activity. To facilitate the molecular characterization of enhancers within their native chromatin environment, we have developed a new method, CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements), to isolate enhancer-associated molecular interactions by repurposing the CRISPR/Cas9 system. By using the endonuclease-deficient Cas9 (dCas9) and enhancer-targeting guide RNA (sgRNA), the enhancer-associated protein, DNA and RNA complexes are isolated by affinity purification and identified by high-throughput sequencing and proteomics, respectively. Using these methods, we isolated chromatin-regulating protein complexes associated with several critical cis -regulatory elements within the human β-globin cluster such as the locus control region (LCR) in human erythroid cells, and identified many known factors such as GATA1, TAL1, NFE2, LDB1 and the SWI/SNF chromatin remodeling complexes involved in globin gene regulation. More importantly, we also identified several new factors, such as the nuclear pore proteins NUP98 and NUP153, which have not previously been implicated in globin gene regulation. Subsequent studies of these factors established their roles in the developmental regulation of enhancer activities for globin gene transcription. In situ capture of individual constituents of the enhancer cluster controlling human β-globin genes also establishes evidence for composition-based hierarchical organization of enhancer structure. Furthermore, unbiased analysis of cis -element-mediated long-range chromatin interactions at disease-associated non-coding elements and developmentally controlled super-enhancers reveals spatial features causally regulate gene transcription. Therefore, the dCas9-mediated affinity purification has the potential to advance the characterization of chromatin-templated hierarchical events by providing a method to examine the complete molecular composition of transcriptional enhancers and how composition changes in cellular differentiation. Our studies not only help elucidate the underlying principles regulating lineage-defining enhancer elements, but also establish new approaches for in situ molecular dissection of disease-associated cis -regulatory elements in globin gene transcription and erythropoiesis. Disclosures No relevant conflicts of interest to declare.