Engineering Globin Gene Expression

Hemoglobinopathies, including sickle cell disease and thalassemia, are among the most common inherited genetic diseases worldwide. Due to the relative ease of isolating and genetically modifying hematopoietic stem and progenitor cells, recent gene editing and gene therapy strategies have progressed to clinical trials with promising outcomes; however, challenges remain and necessitate the continued exploration of new gene engineering and cell transplantation protocols. Current gene engineering strategies aim at reactivating the expression of the fetal γ-globin genes in adult erythroid cells. The γ-globin proteins exhibit anti-sickling properties and can functionally replace adult β-globin. Here, we describe and compare the current genetic engineering procedures that may develop into safe and efficient therapies for hemoglobinopathies in the near future. Keywords: hemoglobin, globin, locus control region, gene therapy, gene editing, zinc finger, TALEN, CRISPR/Cas9, hematopoiesi

Allelic Imbalance in Regulation of <i>ANRIL</i> through Chromatin Interaction at 9p21 Endometriosis Risk Locus

<div><p>Genome-wide association studies (GWASs) have discovered numerous single nucleotide polymorphisms (SNPs) associated with human complex disorders. However, functional characterization of the disease-associated SNPs remains a formidable challenge. Here we explored regulatory mechanism of a SNP on chromosome 9p21 associated with endometriosis by leveraging “allele-specific” functional genomic approaches. By re-sequencing 1.29 Mb of 9p21 region and scrutinizing DNase-seq data from the ENCODE project, we prioritized rs17761446 as a candidate functional variant that was in perfect linkage disequilibrium with the original GWAS SNP (rs10965235) and located on DNase I hypersensitive site. Chromosome conformation capture followed by high-throughput sequencing revealed that the protective G allele of rs17761446 exerted stronger chromatin interaction with <i>ANRIL</i> promoter. We demonstrated that the protective allele exhibited preferential binding affinities to TCF7L2 and EP300 by bioinformatics and chromatin immunoprecipitation (ChIP) analyses. ChIP assays for histone H3 lysine 27 acetylation and RNA polymerase II reinforced the enhancer activity of the SNP site. The allele specific expression analysis for eutopic endometrial tissues and endometrial carcinoma cell lines showed that rs17761446 was a <i>cis</i>-regulatory variant where G allele was associated with increased <i>ANRIL</i> expression. Our work illuminates the allelic imbalances in a series of transcriptional regulation from factor binding to gene expression mediated by chromatin interaction underlie the molecular mechanism of 9p21 endometriosis risk locus. Functional genomics on common disease will unlock functional aspect of genotype-phenotype correlations in the post-GWAS stage.</p></div

Prioritization of candidate causal variants via target re-sequencing and DNase-seq data.

<p>A) Distribution of variants exhibiting strong LD (<i>r</i>² > 0.8) with two endometriosis-associated SNPs (rs10965235 and rs1537377). SNPs and indels are coded by diamonds and squares, respectively. The positions of rs10965235 and rs1537377 are shown as vertical lines. The intervals encompassing all the variants showing strong LD with rs10965235 and rs1537377 are highlighted by light green and pink shades, respectively. B) DHSs in endometrial carcinoma cell lines across the two intervals. The transcript structures of <i>ANRIL</i> and the sites of the variants that are in strong LD are depicted. The densities of aligned reads from DNase-seq estimated by F-Seq are plotted. DHSs in which the densities of aligned reads significantly surpassing the threshold are represented by dark blue. Locations in which aligned reads are depleted are depicted by light blue. The positions of the SNPs identified by GWASs (rs10965235 and rs1537377) and candidate causal SNPs (rs17834457 and rs17761446) are highlighted by blue and red arrows, respectively. C) DNase-seq signals at the variant sites showing strong LD. Signals are represented as relative values to the threshold determined by F-Seq. If the relative signal surpasses 1.0, the corresponding variant site coincides with significant DHS.</p

rs17761446 alters binding affinities of TCF7L2 and EP300.

<p>A) Alignments of consensus motifs of TCF/LEF family to sequence surrounding rs17761446. Core motif of HMG class of TFs are highlighted by red. The position of rs17761446 is highlighted by blue. B) ChIP-seq signals for TCF7L2, EP300, H3K27ac, and TBP in HeLa-S3 cells from ENCODE project. Plots are created for 10 kb around rs17761446. Significant peak regions are highlighted by light green. C) ChIP assays for TCF7L2, EP300, H3K27ac, and RNA polymerase II at rs17761446 site in HEC251 cells. <i>MYC</i> promoter and α satellite repeat element were used as positive and negative control regions, respectively. The enrichment of the normalized binding affinity for the candidate SNP site or positive control region was normalized by dividing by that for negative control region. D) Binding strength of the G allele relative to the T allele of rs17761446 in HEC251 cells. Data represents the mean and standard deviation of triplicated ChIP assays. Pol2, RNA polymerase II; TCF7L2 (CS) and TCF7L2 (OG), TCF7L2 antibodies by Cell Signaling Technology and OriGene Technologies, respectively.</p

AS3C-seq for the detection of allele-specific chromatin interaction.

<p>A) The interacting fragments between the candidate causal SNPs (rs17834457 and rs17761446) and the promoter of <i>ANRIL</i> are color-coded by blue and red, respectively. <i>Hin</i>dIII fragments are depicted by gray rectangles. The alternative transcript structures <i>ANRIL</i>, <i>CDKN2A</i>, and <i>CDKN2B</i> are represented. B) Plots of coverage depth for AS3C-seq libraries from two endometrial carcinoma cell lines (HEC251 and HEC265). The fragments between the candidate causal SNPs (rs17834457 and rs17761446) and the promoter of <i>ANRIL</i> are color-coded by blue and red, respectively. The ligation junction (i.e., restriction enzyme recognition sequence of <i>Hin</i>dIII, AAGCTT) is shown by yellow. For heterozygous SNP sites, the proportions of allele-specific paired-end read tag counts supporting the alleles on the risk and protective haplotypes are shown by light blue and dark blue, respectively. It can be seen that a region within the fragment containing <i>ANRIL</i> promoter shows reduced depth of coverage. This seems to be caused by sequence or alignment errors because the region corresponds to a homopolymer stretch with 15 consecutive adenines (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005893#pgen.1005893.s012" target="_blank">S12 Fig</a>), indicating that this does not largely affect our results. C) Proportions of allele-specific paired-end read tag counts supporting the risk and protective haplotypes tagged by T and G alleles of rs17761446, respectively. Results for the two cell lines and for pooled data are shown. D) Simulation study for evaluating mapping bias. Simulation scenarios considering different sequence lengths (50bp, 75bp, and 100bp) and different error rates for each sequenced base (0.01, 0.005, and 0.001). Box plots are based on 10,000 replicates. rs17761446 and rs17834457 are apart from 76bp and in perfect linkage disequilibrium (<i>r</i>² = 1); therefore, we consider the haplotype structure of these two variants in the simulation scenario in which sequence length is 100bp. E) Sensitivity analysis for evaluating mapping bias. In sensitivity analyses, we aligned reads from AS3C-seq to two types of sequences that were different in the base at the SNP site: the “original” and “changed” sequences have “reference” and “alternative” alleles, respectively. Note that the significant result from AS3C-seq exhibited increased proportion of alternative allele.</p

ETV6 Deficiency Unlocks ERG-Dependent Microsatellite Enhancers to Drive Aberrant Gene Activation in B-Lymphoblastic Leukemia.

Distal enhancers play critical roles in sustaining oncogenic gene-expression programs. We identify aberrant enhancer-like activation of GGAA tandem repeats as a characteristic feature of B-cell acute lymphoblastic leukemia (B-ALL) with genetic defects of the ETV6 transcriptional repressor, including ETV6-RUNX1+ and ETV6-null B-ALL. We show that GGAA repeat enhancers are direct activators of previously identified ETV6-RUNX1+/- like B-ALL "signature" genes, including the likely leukemogenic driver EPOR. When restored to ETV6-deficient B-ALL cells, ETV6 directly binds to GGAA repeat enhancers, represses their acetylation, downregulates adjacent genes, and inhibits B-ALL growth. In ETV6-deficient B-ALL cells, we find that the ETS transcription factor ERG directly binds to GGAA microsatellite enhancers and is required for sustained activation of repeat enhancer-activated genes. Together, our findings reveal an epigenetic gatekeeper function of the ETV6 tumor suppressor gene and establish microsatellite enhancers as a key mechanism underlying the unique gene-expression program of ETV6-RUNX1+/- like B-ALL. SIGNIFICANCE: We find a unifying mechanism underlying a leukemia subtype-defining gene-expression signature that relies on repetitive elements with poor conservation between humans and rodents. The ability of ETV6 to antagonize promiscuous, nonphysiologic ERG activity may shed light on other roles of these key regulators in hematolymphoid development and human disease. See related commentary by Mercher, p. 2. This article is highlighted in the In This Issue feature, p. 1.http://deepblue.lib.umich.edu/bitstream/2027.42/175557/2/ETV6_deficiency_Blood_Cancer_Discovery_2023.pdfPublished versionDescription of ETV6_deficiency_Blood_Cancer_Discovery_2023.pdf : Published versio