Globin Gene Locus Research Articles

Mechanisms of Pol II Recruitment to the Human Beta-Globin Locus Control Region Super-Enhancer

The human ꞵ-globin gene locus consists of five genes that encode subunits of hemoglobin. The locus control region (LCR) regulates these genes, acting as a super-enhancer to promote high-level of β-globin expression in erythroid cells. The LCR, like other super-enhancers, is known to recruit transcription complexes and generate non-coding enhancer RNA (eRNA). Past research has suggested that super-enhancers form phase-separated transcription initiation domains that concentrate RNA polymerase II (Pol II) and transfer Pol II to target gene promoters during transient looping interactions. The aim of this project is to examine the role of TFIIH, involved transcription initiation, in enhancer RNA production and Pol II transfer in the globin gene locus. The LCR was immobilized on magnetic beads and incubated with erythroid nuclear protein extracts. Proteins not bound were washed away and bound proteins were subjected to western blotting experiments. The data show that TFIIH is bound at the LCR and thus may be involved in eRNA production. Cut & Run experiments were performed to examine the localization of TFIIH in the globin gene locus in intact cells. In the future, the laboratory plans to deplete TFIIH and examine production of eRNA and Pol II recruitment at the LCR.

Gene Therapy for β-Hemoglobinopathies: From Discovery to Clinical Trials.

Investigations to understand the function and control of the globin genes have led to some of the most exciting molecular discoveries and biomedical breakthroughs of the 20th and 21st centuries. Extensive characterization of the globin gene locus, accompanied by pioneering work on the utilization of viruses as human gene delivery tools in human hematopoietic stem and progenitor cells (HPSCs), has led to transformative and successful therapies via autologous hematopoietic stem-cell transplant with gene therapy (HSCT-GT). Due to the advanced understanding of the β-globin gene cluster, the first diseases considered for autologous HSCT-GT were two prevalent β-hemoglobinopathies: sickle cell disease and β-thalassemia, both affecting functional β-globin chains and leading to substantial morbidity. Both conditions are suitable for allogeneic HSCT; however, this therapy comes with serious risks and is most effective using an HLA-matched family donor (which is not available for most patients) to obtain optimal therapeutic and safe benefits. Transplants from unrelated or haplo-identical donors carry higher risks, although they are progressively improving. Conversely, HSCT-GT utilizes the patient's own HSPCs, broadening access to more patients. Several gene therapy clinical trials have been reported to have achieved significant disease improvement, and more are underway. Based on the safety and the therapeutic success of autologous HSCT-GT, the U.S. Food and Drug Administration (FDA) in 2022 approved an HSCT-GT for β-thalassemia (Zynteglo™). This review illuminates the β-globin gene research journey, adversities faced, and achievements reached; it highlights important molecular and genetic findings of the β-globin locus, describes the predominant globin vectors, and concludes by describing promising results from clinical trials for both sickle cell disease and β-thalassemia.

A Common Alpha Globin Gene Deletion is Associated with Decreased Arterial Alpha Globin Gene Expression and Altered Vasoreactivity among Black Americans

Alpha globin binds to endothelial nitric oxide (NO) synthase (eNOS) and limits the diffusion of NO from endothelial cells into vascular smooth muscle. Previously, we demonstrated that pharmacologic disruption of alpha globin‐eNOS binding enhanced NOS‐dependent vasodilation in human arteries. Here, we explore the hypothesis that individuals with alpha globin gene deletions will have reduced alpha globin gene expression in resistance arteries and altered arterial vasoreactivity.To test this hypothesis, we screened healthy Black Americans (clinical trial # 02692872) for a common 3.7 kb gene deletion in the alpha globin gene locus (HBA) that reduces the number of functional gene copies. We excluded people with risk factors for cardiovascular disease and created a nested case control study that included 15 people with four copies, 8 people with three copies, and 2 people with two copies of HBA. Each individual underwent an incisional subcutaneous adipose biopsy (clinical trial # 03937817), from which we isolated resistance arteries less than 200 μm in diameter. Resistance arteries were cannulated and perfused free of blood before undergoing multiphoton imaging, gene expression analysis, or pressure myography.Multiphoton imaging of adipose resistance arteries revealed alpha globin to co‐localize with eNOS near the myoendothelial junction between endothelial and smooth muscle cells. Arteries from individuals with three functional copies of HBA had lower expression of HBA compared to arteries from individuals with four functional copies of HBA(1715 copies/ng cDNA vs 1176 copies/ng cDNA, p = 0.03). The concentration of phenylephrine required to constrict the human adipose arteries to 50% of baseline was 8 times greater for arteries from subjects with three copies of HBA compared to arteries from subjects with four copies of HBA (log10[EC50]: ‐4.78 (95% CI: ‐4.29 to ‐5.22) vs ‐5.68 ± (95%CI: ‐5.33 to ‐5.98) p = 0.003). Arteries from individuals with three copies of HBA also had reduced maximal constriction to phenylephrine compared to subjects with four copies of HBA(p < 0.01).These results offer compelling new genetic evidence from humans supporting the role of alpha globin as a regulator of NO signaling in resistance arteries. We demonstrate that HBA deletions reduce arterial expression of HBA and change arterial vasoreactivity. This clinical investigation defines a novel vascular phenotype for individuals with alpha thalassemia and provides insight into a vascular mechanism by which alpha globin gene deletions protect against severe malaria, chronic kidney disease, and vascular complications of sickle cell disease.

Super-enhancer mediated regulation of adult β-globin gene expression: the role of eRNA and Integrator.

Super-enhancers (SEs) mediate high transcription levels of target genes. Previous studies have shown that SEs recruit transcription complexes and generate enhancer RNAs (eRNAs). We characterized transcription at the human and murine β-globin locus control region (LCR) SE. We found that the human LCR is capable of recruiting transcription complexes independently from linked globin genes in transgenic mice. Furthermore, LCR hypersensitive site 2 (HS2) initiates the formation of bidirectional transcripts in transgenic mice and in the endogenous β-globin gene locus in murine erythroleukemia (MEL) cells. HS2 3′eRNA is relatively unstable and remains in close proximity to the globin gene locus. Reducing the abundance of HS2 3′eRNA leads to a reduction in β-globin gene transcription and compromises RNA polymerase II (Pol II) recruitment at the promoter. The Integrator complex has been shown to terminate eRNA transcription. We demonstrate that Integrator interacts downstream of LCR HS2. Inducible ablation of Integrator function in MEL or differentiating primary human CD34+ cells causes a decrease in expression of the adult β-globin gene and accumulation of Pol II and eRNA at the LCR. The data suggest that transcription complexes are assembled at the LCR and transferred to the globin genes by mechanisms that involve Integrator mediated release of Pol II and eRNA from the LCR.

Mechanisms mediating suppression of globin gene transcription in Danio rerio nonerythroid cells

We studied the repression of adult and embryo-larval genes of the major globin gene locus in D. rerio fibroblasts. The results obtained suggest that at least some of the globin genes are repressed by Polycomb, similarly to human α-globin genes. Furthermore, within two α/β globin gene pairs, repression of α-type and β-type genes appears to be mediated by different mechanisms, as increasing the level of histone acetylation can activate transcription of only β-type genes.

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

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.

Simultaneous detection of chromosomal aneuploidy and a monogenic disease by next-generation sequencing with linkage analysis

Introduction Mutations giving rise to single gene disorders (SGD) are currently directly detected in PGD-M using PCR and Sanger sequencing. However, amplification failures, allelic drop-out (ADO), mosaicism and contamination limits PGT precision and diagnostic efficiency. The current approaches for PGT-M generally uses standard panels pre-designed for each disease. The present study applied whole genome amplification (WGA) of biopsied trophectoderm cells and concurrent next-generation sequencing (NGS)-based single nucleotide polymorphism (SNP) haplotyping on an Ion Torrent Personal Genome Machine (PGM) with the aim of customized testing for each family. Material and Methods Exome sequencing data of a consanguineous family applying for ART (mother, father and child), who give their informed consent, was used to establish the haplotype of the thalassemia gene locus-based on informative SNPs by using the VcfTools and R software. A total of 88 loci containing 116 SNPs located 2 Mb upstream and 2 Mb downstream of the beta globin gene locus (HBB) were selected for NGS-based SNP haplotyping. These loci were then submitted to a primer design website ( www.ampliseq.com ; Thermo Fisher Scientific). The WGA products of three embryos generated during the IVF cycle of the couple and which were diagnosed as aneuploid and genomic DNA of the child were used for the Amplicon Library Preparation (using Ion AmpliSeq™ Library kits) for PGT-M and in parallel for PGT-A. To validate the SNP-based haplotype, a STR markers’-based haplotype was also established. Results The SNP-based haplotypes of the tested embryos and of the child were successfully constructed by NGS with linkage analysis, using GeneHunter v2.15 software. Out of the three embryos tested with family customized PGT-M panel, the first embryo was found to be haploidentical with the child for the HBB locus. The second embryo shared only the paternal allele, whereas the third embryo was carrying the other parental alleles when compared to the child's alleles. These haplotypes were also verified by classical STR marker-based haplotyping. Read depth of amplicons ranged between 70 and 6800. The parallel PGS diagnoses of the three embryos were identical with the original diagnoses of the respective embryos. The variations of SNP call rates and read depths of the trophectoderm samples were correlated with library concentrations. Conclusions In conclusion, this approach allows for a successful simultaneous detection of chromosomal aneuploidy and a monogenic disease by NGS with linkage analysis. Especially in but not limited to consanguineous families, establishing family-specific gene panels for PGT-M maximizes allelic information with a minimum number of SNPs without the need for direct mutation testing thus lowering the cost of testing per embryo.

ERV-9 LTR Retrotransposon Modulates Globin Gene Switching

In the human beta-globin gene locus, an LTR retrotransposon derived from ERV-9 human endogenous retrovirus is located near the locus control region (LCR) far upstream of the globin genes. In transgenic mice carrying the 100 kb human globin gene locus, deleting the ERV-9 LTR by cre-loxP mediated in situ recombination inactivates transcription of beta-globin gene by ~50% and activates that of gamma-globin gene by up to 5 fold, to at least 20% the level of beta-globin mRNA in both fetal and adult erythroid cells. Chromosome-conformation-capture (3C) shows that the ERV-9 LTR preferentially loops with beta-globin gene, even in fetal erythroid cells where gamma-globin gene is predominantly transcribed. Unique in the locus, the ERV-9 LTR contains high density of CCAAT motifs that strongly bind transcription factor (TF) NF-Y critical for assembling the LTR enhancer complex but no CCNCNCCC motifs that bind master erythroid TF KLF1, while the beta-globin promoter contains two tandem CCNCNCCC motifs that bind KLF1 but a CCAAT motif that only weakly bind NF-Y. However, both the LTR and beta-globin promoter associate in vivo with high levels of NF-Y and KLF1 in ChIP assays. Protein immunoprecipitation shows that neither NF-Y nor KLF1 associates with transcription co-regulator Ldb1, which has been reported to mediate chromosome looping of the LCR with the globin genes. Thus, the Ldb1/LMO2/GATA -1, -2 complex potentially assembled by the GATA motifs distributed throughout the locus does not appear to mediate the preferential looping of ERV-9 LTR with beta-globin promoter. Through interaction of NF-Y and KLF1 mediated by the to be identified co-regulator, the ERV-9 LTR interacts with and activates beta-globin gene, and suppresses gamma-globin gene by a competitive mechanism. Deletion of the ERV-9 LTR removes the competitive advantage of beta-globin gene, thus re-activating gamma-globin gene. Our findings suggest that deleting the ERV-9 LTR from the globin locus by genome-editing could provide a potential new therapy for beta-hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

Simultaneous detection of chromosomal aneuploidy and a monogenic disease by next-generation sequencing with linkage analysis

The current approaches for PGT-M generally uses standard panels pre-designed for each disease. The present study applied whole genome amplification (WGA) of biopsied trophectoderm cells and concurrent next-generation sequencing (NGS)-based single nucleotide polymorphism (SNP) haplotyping on an Ion Torrent Personal Genome Machine (PGM) with the aim of customized testing for each family. Exome sequencing data of a consanguineous family applying for ART (mother, father and child), who gave their informed consent, was used to establish the haplotype of the thalassemia gene locus-based on informative SNPs by using the VcfTools and R software. A total of 88 loci containing 116 SNPs located 2 Mb upstream and 2 Mb downstream of the beta globin gene locus (HBB) were selected for NGS-based SNP haplotyping. These loci were then submitted to a primer design website (www.ampliseq.com; Thermo Fisher Scientific). The WGA products of three embryos generated during the IVF cycle of the couple and which were diagnosed as aneuploid were used for the Amplicon Library Preparation in addition to the genomic DNA of the child (using Ion AmpliSeq™ Library kits) for PGT-M and in parallel for PGT-A. To validate the SNP-based haplotype, a STR markers’-based haplotype was also established. The SNP-based haplotypes of the tested embryos and of the child were successfully constructed by NGS with linkage analysis, using GeneHunter v2.15 software. Out of the three embryos tested with family customized PGT-M panel, the first embryo was found to be haploidentical with the child for the HBB locus. The second embryo shared only the paternal allele, whereas the third embryo was carrying the other parental alleles when compared to the child’s alleles. These haplotypes were also verified by classical STR marker-based haplotyping. Read depth of amplicons ranged between 70 and 6800. The parallel PGS diagnoses of the three embryos were identical with the original diagnoses of the respective embryos. The variations of SNP call rates and read depths of the trophectoderm samples were correlated with library concentrations. In conclusion, this approach allows for a successful simultaneous detection of chromosomal aneuploidy and a monogenic disease by NGS with linkage analysis. Especially in but not limited to consanguineous families, establishing family-specific gene panels for PGT-M maximizes allelic information with a minimum number of SNPs without the need for direct mutation testing thus lowering the cost of testing per embryo.

Recent progress in understanding and manipulating haemoglobin switching for the haemoglobinopathies

The major β-haemoglobinopathies, sickle cell disease and β-thalassaemia, represent the most common monogenic disorders worldwide and a steadily increasing global disease burden. Allogeneic haematopoietic stem cell transplantation, the only curative therapy, is only applied to a small minority of patients. Common clinical management strategies act mainly downstream of the root causes of disease. The observation that elevated fetal haemoglobin expression ameliorates these disorders has motivated longstanding investigations into the mechanisms of haemoglobin switching. Landmark studies over the last decade have led to the identification of two potent transcriptional repressors of γ-globin, BCL11A and ZBTB7A. These regulators act with additional trans-acting epigenetic repressive complexes, lineage-defining factors and developmental programs to silence fetal haemoglobin by working on cis-acting sequences at the globin gene loci. Rapidly advancing genetic technology is enabling researchers to probe deeply the interplay between the molecular players required for γ-globin (HBG1/HBG2) silencing. Gene therapies may enable permanent cures with autologous modified haematopoietic stem cells that generate persistent fetal haemoglobin expression. Ultimately rational small molecule pharmacotherapies to reactivate HbF could extend benefits widely to patients.

Elevational divergence in the great tit complex revealed by major hemoglobin genes.

Gene flow and demographic history can play important roles in the adaptive genetic differentiation of species, which is rarely understood in the high-altitude adaptive evolution of birds. To elucidate genetic divergence of populations in the great tit complex (Parus major, P. minor and P. cinereus) at different elevations, we compared the genetic structure and gene flow in hemoglobin genes with neutral loci. Our results revealed the elevationally divergent structure of αA-globin gene, distinctive from that of the βA-globin gene and neutral loci. We further investigated gene flow patterns among the populations in the central-northern (> 1,000 m a.s.l.), south-eastern (< 1,000 m a.s.l.) regions and the Southwest Mountains (> 2,000 m a.s.l.) in China. The high-altitude (> 1,000 m a.s.l.) diverged αA-globin genetic structure coincided with higher αA-globin gene flow between highland populations, in contrast to restricted neutral gene flow concordant with the phylogeny. The higher αA-globin gene flow suggests the possibility of adaptive evolution during population divergence, contrary to the lower αA-globin gene flow homogenized by neutral loci during population expansion. In concordance with patterns of historical gene flow, genotypic and allelic profiles provide distinctive patterns of fixation in different high-altitude populations. The fixation of alleles at contrasting elevations may primarily due to highland standing variants αA49Asn/72Asn/108Ala originating from the south-western population. Our findings demonstrate a pattern of genetic divergence with gene flow in major hemoglobin genes depending on population demographic history.

Evolution of the Genome 3D Organization: Comparison of Fused and Segregated Globin Gene Clusters.

The genomes are folded in a complex three-dimensional (3D) structure. Some features of this organization are common for all eukaryotes, but little is known about its evolution. Here, we have studied the 3D organization and regulation of zebrafish globin gene domain and compared its organization and regulation with those of other vertebrate species. In birds and mammals, the α- and β-globin genes are segregated into separate clusters located on different chromosomes and organized into chromatin domains of different types, whereas in cold-blooded vertebrates, including Danio rerio, α- and β-globin genes are organized into common clusters. The major globin gene locus of Danio rerio is of particular interest as it is located in a genomic area that is syntenic in vertebrates and is controlled by a conserved enhancer. We have found that the major globin gene locus of Danio rerio is structurally and functionally segregated into two spatially distinct subloci harboring either adult or embryo-larval globin genes. These subloci demonstrate different organization at the level of chromatin domains and different modes of spatial organization, which appears to be due to selective interaction of the upstream enhancer with the sublocus harboring globin genes of the adult type. These data are discussed in terms of evolution of linear and 3D organization of gene clusters in vertebrates.

Elevated Pre-Treatment Fetal Hemoglobin Predicts Better Outcome in MDS/AML Patients Receiving 5-Aza-2'-Deoxycytidine (DAC)

Introduction: Aberrant DNA methylation occurs frequently in hematologic malignancies and is associated with altered gene expression. Consequently, DNA hypomethylating agents (HMAs), e.g. 5-aza-2'-deoxycytidine (DAC), have been tested for in vivo demethylation and have become accepted as first-line treatment of older MDS and AML patients (pts). While HMAs are already routinely used for the treatment of MDS and AML, only very few outcome predictors have been established thus far. Expression of the β-like globin gene locus is tightly regulated by methylation, is HMA-sensitive in vitro, and fetal hemoglobin (HbF) expression is under study as a potential biomarker for response of MDS pts to 5-azacytidine. Here, we present the first study of serial HbF measurements in MDS and AML pts receiving DAC in order to investigate a potential clinical application of HbF as a predictor of outcome to this treatment.Methods: 16 MDS and 36 AML pts enrolled on two clinical trials, the 06011 EORTC-GMDSSG phase III trial of higher-risk MDS, and the 00331 phase II trial of older, non-fit AML pts (Lübbert M et al., Haematologica 2012), were treated with DAC. MDS pts received nine 4-hour infusions of 15 mg/m2 given over 72 hours, repeated every 6 weeks for a minimum of four courses. AML pts were treated with nine 3-hour infusions of 15 mg/m2 given over 72 hours, repeated every 6 weeks for four courses followed by maintenance treatment with DAC at 20 mg/m2 when responding (defined by CR, PR or an antileukemic effect). HbF levels were measured in peripheral blood by HPLC before treatment and sequentially, i. e. after the end of each treatment course (every 6 weeks). HbF levels >1% of total hemoglobin were considered elevated.Results: Baseline HbF was elevated (>1.0%) in 7/16 MDS and 12/36 AML pts. Clinical baseline characteristics were uniformly distributed between pts with normal and elevated HbF levels. HbF induction was observed in 81% of MDS pts after a median of 2 (range 2-6) courses of DAC and in 54% of AML pts after a median of 3 (2-11) courses. Four AML pts receiving standard cytarabine-based induction chemotherapy and four pts with pancreatic cancer receiving gemcitabine-based treatment did not show HbF induction to >1%. When analyzing clinical survival endpoints, elevated baseline HbF was associated with longer median overall survival (OS) for MDS: 26.6 vs. 8.6 months (HR 8.56, 95% CI 1.74-42.49, p=0.008). Similarly, median PFS and AMLFS was prolonged in "HbF high" MDS pts with 15.4 compared to 5.9 months (p=0.016) and 26.3 compared to 8.8 months (p=0.03), respectively. A statistically non-significant trend towards higher HbF baseline values (median 1.5%, range 0.3% to 3.9%) in MDS pts achieving a CR, PR or hematological improvement compared to non-responders (median baseline 0.4%, range 0.1% - 1.9%) was noted. Likewise, OS for AML pts with elevated pre-treatment HbF was prolonged with 10.0 vs. 2.9 months OS (HR 3.01, 95% CI 1.26-7.22, p=0.014). HbF baseline values were comparable in the group that did achieve any response (CR, PR, antileukemic effect) vs. the non-responding group. In a multivariate analysis including LDH and age, the predictive value of HbF was retained. Time-dependent Cox models revealed that the predictive value of treatment-induced HbF induction was markedly inferior to that of baseline HbF.Conclusion: We provide first evidence for in vivo induction of HbF by DAC in MDS and AML pts. Notably, HbF values elevated already prior to treatment harbor highly significant predictive value for survival benefit upon DAC whereas HbF induction is inferior to a stronger effect of pre-treatment HbF. Our findings warrant incorporation of HbF as potential predictive biomarker in larger prospective studies. DisclosuresClaus:Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria, Other: Travel Funding. Becker:BMS: Honoraria; Novartis: Honoraria. Lübbert:Ratiopharm: Other: Study drug valproic acid; Janssen-Cilag: Other: Travel Funding, Research Funding; Celgene: Other: Travel Funding.

Characterization of the enhancer element of the Danio rerio minor globin gene locus.

In Danio rerio, the alpha- and beta-globin genes are present in two clusters: a major cluster located on chromosome 3 and a minor cluster located on chromosome 12. In contrast to the segregated alpha- and beta-globin gene domains of warm-blooded animals, in Danio rerio, each cluster contains both alpha- and beta-globin genes. Expression of globin genes present in the major cluster is controlled by an erythroid-specific enhancer similar to the major regulatory element of mammalian and avian alpha-globin gene domains. The enhancer controlling expression of the globin genes present in the minor locus has not been identified yet. Based on the distribution of epigenetic marks, we have selected two genomic regions that might harbor an enhancer of the minor locus. Using transient transfection of constructs with a reporter gene, we have demonstrated that a ~500-bp DNA fragment located ~1.7Kb upstream of the αe4 gene possesses an erythroid-specific enhancer active with respect to promoters present in both the major and the minor globin gene loci of Danio rerio. The identified enhancer element harbors clustered binding sites for GATA-1, NF-E2, and EKLF similar to the enhancer of the major globin locus on chromosome 3. Both enhancers appear to have emerged as a result of independent evolution of a duplicated regulatory element present in an ancestral single alpha-/beta-globin locus that existed before teleost-specific genome duplication.

Non-Coding RNAs Transcribed from ERV-9 LTR Retrotransposons Regulate Erythropoiesis

Long noncoding RNAs (lncRNAs) regulate diverse cellular processes in development, differentiation and malignancy. In human cells, over 80% of the lncRNAs contain retrotransposon sequences transcribed from Alu, L1 and LTR retrotransposons, which comprise ~40% of the human genome. The functional significance of the retrotransposon lncRNAs is largely unknown. The human genome contains ~4000 copies of the ERV-9 LTR retrotransposon, which exhibits strong enhancer activity and initiates synthesis of ERV-9 lncRNAs in erythroid progenitor cells. Recently, we discovered that depletion of the ERV-9 lncRNAs in human erythroid progenitor cells cultured ex vivo from peripheral blood CD34+ cells inhibited ex vivo erythropoiesis. Whole genome RNA sequencing (RNA-seq) found that depletion of ERV-9 lncRNAs significantly suppressed transcription of 608 genes including ~50 key erythroid genes. We hypothesize that the ERV-9 lncRNAs together with the ERV-9 LTR act in cis to regulate transcription of these key erythroid genes and other genes to set up a transcriptional network that promotes erythropoiesis.In the human b-globin gene locus, we showed previously that the ERV-9 LTR retrotransposon performs a beneficial biological function: The ERV-9 LTR enhancer binds NF-Y and GATA-1 and -2 to assemble an LTR-pol II transcription complex, which transcribes long, noncoding RNAs (lncRNAs) from the LTR R-U5 regions through the downstream locus control region (LCR) and intergenic DNAs to reach and activate transcription of b-globin gene 70 kb away. In this tracking and transcription (T&T) mechanism of long-range LTR enhancer function, the ERV-9 lncRNAs could be merely by-products of the tracking and transcribing process of the LTR complex without any functional significance. However, we found recently that depletion of the ERV-9 lncRNAs suppressed transcription of the entire human globin gene locus, diminished occupancies of NF-Y, GATA-1 and -2 and pol II at the ERV-9 LTR and reduced the looping frequency of the ERV-9 LTR with the globin gene locus in erythroid progenitor cells. Thus, the ERV-9 lncRNAs acted in cis to facilitate assembly of the LTR-pol II complex and modulate long-range LTR enhancer function in transcriptional activation of b-globin gene. Genome-wide, whether the ERV-9 lncRNAs transcribed from other gene loci perform similar biological function is under investigation. DisclosuresNo relevant conflicts of interest to declare.

A Feedback Loop Consisting of MicroRNA 23a/27a and the β-Like Globin Suppressors KLF3 and SP1 Regulates Globin Gene Expression

The developmental stage-specific expression of the human β-like globin genes has been studied for decades, and many transcriptional factors as well as other important cis elements have been identified. However, little is known about the microRNAs that potentially regulate β-like globin gene expression directly or indirectly during erythropoiesis. In this study, we show that microRNA 23a (miR-23a) and miR-27a promote β-like globin gene expression in K562 cells and primary erythroid cells through targeting of the transcription factors KLF3 and SP1. Intriguingly, miR-23a and miR-27a further enhance the transcription of β-like globin genes through repression of KLF3 and SP1 binding to the β-like globin gene locus during erythroid differentiation. Moreover, KLF3 can bind to the promoter of the miR-23a∼27a∼24-2 cluster and suppress this microRNA cluster expression. Hence, a positive feedback loop comprised of KLF3 and miR-23a promotes the expression of β-like globin genes and the miR-23a∼27a∼24-2 cluster during erythropoiesis.

Cell‐cycle specific association of transcription factors and RNA polymerase ii with the human β‐globin gene locus

The human β-globin genes are regulated by a locus control region (LCR) and are expressed at extremely high levels in erythroid cells. How transcriptional fidelity of highly expressed genes is regulated and maintained during the cell cycle is not completely understood. Here, we analyzed the association of transcription factor USF, the co-activator CBP, topoisomerase I (Topo I), basal transcription factor TFIIB, and RNA polymerase II (Pol II) with the β-globin gene locus at specific cell-cycle stages. The data demonstrate that while association of Pol II with globin locus associated chromatin decreased in mitotically arrested cells, it remained bound at lower levels at the γ-globin gene promoter. During early S-phase, association of CBP, USF, and Pol II with the globin gene locus decreased. The re-association of CBP and USF2 with the LCR preceded re-association of Pol II, suggesting that these proteins together mediate recruitment of Pol II to the β-globin gene locus during S-phase. Finally, we analyzed the association of Topo I with the globin gene locus during late S-phase. In general, Topo I association correlated with the binding of Pol II. Inhibition of Topo I activity reduced Pol II binding at the LCR and intergenic regions but not at the γ-globin gene promoter. The data demonstrate dynamic associations of transcription factors with the globin gene locus during the cell cycle and support previous results showing that specific components of transcription complexes remain associated with highly transcribed genes during mitosis.

Role of ZBP-89 in human globin gene regulation and erythroid differentiation

AbstractThe molecular mechanisms underlying erythroid-specific gene regulation remain incompletely understood. Closely spaced binding sites for GATA, NF-E2/maf, and CACCC interacting transcription factors play functionally important roles in globin and other erythroid-specific gene expression. We and others recently identified the CACCC-binding transcription factor ZBP-89 as a novel GATA-1 and NF-E2/mafK interacting partner. Here, we examined the role of ZBP-89 in human globin gene regulation and erythroid maturation using a primary CD34+ cell ex vivo differentiation system. We show that ZBP-89 protein levels rise dramatically during human erythroid differentiation and that ZBP-89 occupies key cis-regulatory elements within the globin and other erythroid gene loci. ZBP-89 binding correlates strongly with RNA Pol II occupancy, active histone marks, and high-level gene expression. ZBP-89 physically associates with the histone acetyltransferases p300 and Gcn5/Trrap, and occupies common sites with Gcn5 within the human globin loci. Lentiviral short hairpin RNAs knockdown of ZBP-89 results in reduced Gcn5 occupancy, decreased acetylated histone 3 levels, lower globin and erythroid-specific gene expression, and impaired erythroid maturation. Addition of the histone deacetylase inhibitor valproic acid partially reverses the reduced globin gene expression. These findings reveal an activating role for ZBP-89 in human globin gene regulation and erythroid differentiation.

MicroRNA-96 Inhibits Fetal Hemoglobin Expression During Erythropoiesis

AbstractAbstract 3220 Background:Fetal hemoglobin, HbF (α2γ2), is the main hemoglobin synthesized until birth; it subsequently declines and switches to adult hemoglobin, HbA (α2β2). Certain mutations within the globin gene locus can cause persistent HbF synthesis after birth, ameliorating symptoms in hemoglobinopathies. Analysis from e.g. Hb Corfu and δβ-thalassemia indicates that γ-globin gene expression is post-transcriptionally regulated (Efremov DG., Br J Haematol, Oct 1994; Chakalova L., Blood, 2005). The post-transcriptional regulation of HbF might involve regulatory non-coding RNAs. MicroRNAs (miRNAs) are a small non-coding family of 21 nucleotide RNAs that regulate gene expression post-transcriptionally by targeting mRNAs. After transcription and maturation the miRNA is incorporated into the miRNA-induced silencing complex (miRISC). The miRISC, which contains argonaute proteins (AGO), binds and silences the target mRNAs. Previous studies have reported a role of miRNAs in the developmental switch from HbF to HbA synthesis (Noh SJ, J Transl Med, 2009; Bianchi N., BMB Rep, 2009), however so far no direct interaction between miRNAs and the γ-globin mRNA has been reported. Moreover a function of miRISC in mature red blood cells (RBC) has not been shown yet. Therefore, we aimed (I) to characterize the miRISC composition in RBCs, (II) to identify miRNAs that are directly involved in the regulation of HbF and (III) to study their function during erythropoiesis. Methods:To characterize miRISC in RBCs, we performed co-immunoprecipitations of the miRISC using antibodies directed against human AGO. The immunoprecipitated complexes were analyzed by Western-blot, mass spectrometry and qPCR. To identify miRNAs potentially involved in the regulation of HbF miRNA expression patterns in RBCs from adult and umbilical cord blood were analyzed by qPCR. To confirm the role of selected miRNAs, γ-globin protein levels were measured by ELISA in HEL cell cultures transfected with miRNA precursors or inhibitors as well as in primary erythroid cultures overexpressing miRNAs after lentiviral transduction. The direct interaction between miRNAs and the γ-globin mRNA was analyzed by luciferase reporter assay. Results:AGO1, 2, 3 and 4 and Importin 8 were detected in RBCs both from adult and cord blood. In adult RBC samples (n=3), expressing <1% HbF of total Hb, γ-globin mRNA was bound to AGO2 but not to AGO1 containing miRISC. However in cord blood (n=3), containing 90 % HbF and 30 times more γ-globin mRNA molecules than adult RBCs, the amount of γ-globin mRNA bound to AGO2 containing miRISC was 200 times less (p<0.05). With these indications, we started to analyze miRNA patterns in adult and cord blood RBCs. miR-96, miR-146a, miR-330-3p, let-7a and miR-888 were significantly downregulated in cord blood RBCs compared to adult RBCs. However, luciferase reporter assays demonstrated that only miR-96 was able to bind to the predicted target site within the γ-globin mRNA. Transfection of HEL cells with miR-96 inhibitor showed a twofold induction (p<0.01; n=3) of the γ-globin protein level compared to control cells transfected with scramble RNA. Consistently, transfection with miR-96 precursor in HEL cells showed a 15% reduction (p<0.05, n=3) of the γ-globin protein level. To study the miR-96 mediated inhibition of HbF during erythropoiesis, cord blood derived primary erythroid cultures were transduced with miR-96 lentiviruses. A 40% decrease (p<0.05, n=3) of γ-globin protein expression was observed in erythroid cells overexpressing miR-96 compared to cells transduced with the negative control virus. This γ-globin reduction was observed on 10 and 14 days old cultures, which contained 80% and 65% CD235+ CD71+ erythroblasts, respectively. Conclusions:This study identifies miR-96 as direct inhibitor of the HbF expression during erythropoiesis. Specifically we observed that in cells with low HbF content, γ-globin mRNAs are bound to miRISC and are therefore directly inhibited by miRNAs. We demonstrated that miR-96 is binding to a seedless target site within the γ-globin open reading frame. Finally we showed that miR-96 overexpression reduces the expression of HbF in erythroleukemia cells and during late stages of cord blood derived erythropoiesis. Disclosures:No relevant conflicts of interest to declare.

The PcG Protein L3MBTL1 Transcriptionally Represses Human Embryonic and Fetal Globin Genes: a Novel Prospect for HbF Activation

AbstractAbstract 1014L3MBTL1 is the human homolog of the Drosophila Polycomb Group tumor suppressor gene, lethal(3)malignant brain tumor. We demonstrated that human L3MBTL1 functions as a transcriptional repressor and after crystallizing the MBT repeat domain determined that L3MBTL1 compacts chromatin by binding mono- and di-methylated lysine residues in histones H1 (H1K26) and H4 (H4K20).Despite the known role of L3MBTL1 in affecting chromatin structure, the function of L3MBTL1 in human hematopoiesis has remained largely unknown. We recently demonstrated that L3MBTL1 enforces cell fate decision toward the erythroid lineage and that knockdown of L3MBTL1 accelerates the erythroid differentiation of human hematopoietic stem/progenitor cells, suggesting that its deletion contributes to the pathogenesis of 20q- erythroid malignancies. Consistently with its role in erythropoiesis, here we reveal that L3MBTL1 is a novel transcriptional repressor of fetal globin genes and it may work in concert with BCL11A and EKLF to control globin gene expression.By utilizing RNA interference to reduce L3MBTL1 expression, we have found that knockdown of L3MBTL1 in human cord blood hematopoietic stem/progenitor cells consistently upregulates the expression of the epsilon, gamma, and zeta globin genes, but not the beta globin gene. Similar effects were seen following knockdown of L3MBTL1 in the human erythroleukemia cell line K562, and knockdown of L3MBTL1 in human embryonic stem cells (ESCs) led to the inappropriate expression of fetal and embryonic globin genes (which increases more than 50-fold after the L3MBTL1-KD). These data suggest a role for L3MBTL1 in regulating the globin switch.To investigate the mechanism by which L3MBTL1 silences embryonic and fetal globin gene expression, we used chromatin immunoprecipitation (ChIP) assays to show that L3MBTL1 directly associates with the human β-globin locus. L3MBTL1 occupies several discrete regions within the human β-globin cluster and colocalizes with H4K20me within the Locus Control Region (LCR), a primary attachment site for chromatin modifiers. As confirmation, we found that treatment of K562 cells with hemin, which broadly increases H3K9 acetylation over the β-globin locus and activates the transcription of globin genes, leads to decreases in expression of the repressive H4K20me2 methylmark and L3MBTL1 to the beta-globin cluster.Given the recent identification of the repressor of gamma globin gene expression, BCL11A, we investigated a potential relationship between L3MBTL1 and BCL11A. We found that knockdown of L3MBTL1 led to downregulation of BCL11A mRNA. Accordingly, we have also found that overexpression of L3MBTL1 is associated with an upregulation of BCL11A mRNA, suggesting that L3MBTL1 and BCL11A may function cooperatively to silence globin gene expression. Knockdown of L3MBTL1 also upregulated EKLF mRNA levels which could relate to the decreased BCL11A expression.In summary our data demonstrate that knock-down of L3MBTL1 upregulates embryonic and fetal globin genes in cell contexts where they are usually silenced, indicating the functional importance of this Polycomb protein for repressing the globin gene locus. The clearance of L3MBTL1 and its associated histone mark (H4K20me2) during treatments that induce potent transcriptional activation of globin genes suggest that repression induced by L3MBTL1 is dynamic and may be involved in the fetal-to-adult globin switch. L3MBTL1 therefore emerges as a novel transcriptional repressor of fetal globin genes whose expression may be coordinated with that of BCL11A and EKLF. Understanding the role of L3MBTL1 and the H4K20 methylmark in globin gene switching offers the prospect of the targeted activation of HbF in erythroid cells of patients with hemoglobin disorders. Disclosures:No relevant conflicts of interest to declare.