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Comparative Analysis of Three-Dimensional Chromosomal Architecture Identifies a Novel Fetal Hemoglobin Regulatory Element

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Comparative Analysis of Three-Dimensional Chromosomal Architecture Identifies a Novel Fetal Hemoglobin Regulatory Element Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Comparative analysis of three-dimensional chromosomal architecture identifies a novel fetal hemoglobin regulatory element Peng Huang,1 Cheryl A. Keller,2 Belinda Giardine,2 Jeremy D. Grevet,1,3 James O.J. Davies,4 Jim R. Hughes,4 Ryo Kurita,5 Yukio Nakamura,6 Ross C. Hardison,2 and Gerd A. Blobel1,3 1Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA; 2Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA; 3Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA; 4Medical Research Council (MRC) Molecular Hematology Unit, Weatherall Institute of Molecular Medicine, Oxford University, Oxford OX3 9DS, United Kingdom; 5Research and Development Department, Central Blood Institute, Blood Service Headquarters, Japanese Red Cross Society, Koto-ku, Tokyo 135-8521, Japan; 6Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan Chromatin structure is tightly intertwined with transcription regulation. Here we compared the chromosomal ar- chitectures of fetal and adult human erythroblasts and found that, globally, chromatin structures and compartments A/B are highly similar at both developmental stages. At a finer scale, we detected distinct folding patterns at the developmentally controlled β-globin locus. Specifically, new fetal stage-specific contacts were uncovered between a region separating the fetal (γ) and adult (δ and β) globin genes (encompassing the HBBP1 and BGLT3 noncoding genes) and two distal chromosomal sites (HS5 and 3′HS1) that flank the locus. In contrast, in adult cells, the HBBP1– BGLT3 region contacts the embryonic ε-globin gene, physically separating the fetal globin genes from the enhancer (locus control region [LCR]). Deletion of the HBBP1 region in adult cells alters contact landscapes in ways more closely resembling those of fetal cells, including increased LCR–γ-globin contacts. These changes are accompanied by strong increases in γ-globin transcription. Notably, the effects of HBBP1 removal on chromatin architecture and gene expression closely mimic those of deleting the fetal globin repressor BCL11A, implicating BCL11A in the function of the HBBP1 region. Our results uncover a new critical regulatory region as a potential target for thera- peutic genome editing for hemoglobinopathies and highlight the power of chromosome conformation analysis in discovering new cis control elements. [Keywords: chromatin structure; transcription; globin switching; fetal hemoglobin] Supplemental material is available for this article. Received June 17, 2017; revised version accepted August 21, 2017. Chromosomal folding patterns differ between tissues, re- Among the genes that undergo well-known develop- flecting distinct gene expression patterns. How chromatin mental stage-specific changes in transcription are the β- architecture is modified and interconnects with gene ex- type globin genes. The human β-globin locus comprises pression as cells of a given lineage traverse developmental five coding genes that are specifically expressed in ery- stages has not been studied in great depth. Erythropoiesis throid cells; namely, the embryonic-specific ε (HBE1), fe- offers an ideal model system to examine the molecular tal-restricted Gγ and Aγ (HBG2 and HBG1), and adult mechanisms of gene regulation within the same cell line- expressed δ and β (HBD and HBB) globin genes. Expression age during development. Erythroid precursors from fetal of all of these genes requires a strong upstream enhancer or adult stages have been characterized in much detail, known as the locus control region (LCR), which encom- and it was discovered that in spite of their phenotypical passes five DNaseI-hypersensitive sites (HSs). The LCR and functional similarities, they differ in their gene ex- is in physical proximity to the relevant β-type globin pression, enhancer utilization, DNA methylation, and gene promoters in a developmental stage-specific manner chromatin signatures (Xu et al. 2012; Lessard et al. 2015; (Palstra et al. 2003). The switch from fetal to adult globin Huang et al. 2016). © 2017 Huang et al. This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publi- cation date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After Corresponding author: [email protected] six months, it is available under a Creative Commons License (At- Article published online ahead of print. Article and publication date are tribution-NonCommercial 4.0 International), as described at http://creati- online at http://www.genesdev.org/cgi/doi/10.1101/gad.303461.117. vecommons.org/licenses/by-nc/4.0/. 1704 GENES & DEVELOPMENT 31:1704–1713 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/17; www.genesdev.org Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Chromosome architecture in human erythroid cells gene expression, which normally occurs around the time chromatin folding patterns between these stages. We of birth, is of particular importance, as its impairment show that, globally, chromatin structures of human eryth- (e.g., due to mutations) or reversal (e.g., via therapeutic in- roblast are largely preserved during development. Nota- tervention) benefits patients with sickle cell disease (SCD) bly, at the β-globin locus, the HBBP1 region forms fetal and some forms of β-thalassemia (Smith and Orkin 2016). stage-specific contacts with 3′HS1 and HS5, two The intergenic region between the Aγ- and δ-globin DNaseI-sensitive regions bound by the architectural nu- genes contains a pseudogene (HBBP1) and a noncoding clear factor CTCF that flank the locus (Fig. 1A). In con- gene (BGLT3) (Fig. 1A; Kiefer et al. 2011). Genome-wide trast, the HBBP1 region forms adult stage-specific association studies (GWASs) identified two single-nucleo- contacts with the ε-globin region. CRISPR/Cas9-mediated tide polymorphisms (SNPs), rs10128556 and rs2071348, deletion of the HBBP1 region in an adult-type human ery- both of which reside within the second intron of HBBP1 throid cell line strongly reactivates γ-globin transcription, and are associated with elevated fetal hemoglobin levels suggesting that HBBP1 chromatin contacts contribute (Galarneau et al. 2010; Nuinoon et al. 2010), suggesting to the developmental control of β-globin expression. the presence of developmental regulatory elements in BCL11A is a well-characterized transcriptional repressor this region. of γ-globin transcription (Sankaran et al. 2008). Deletion The major trans-acting regulatory factors of erythroid of BCL11A or the HBBP1 region triggers similar chroma- development and gene expression have been identified, tin conformational changes at the β-globin locus, impli- and it is well-established that changes in chromatin struc- cating BCL11A in the function of the HBBP1 region. ture are tightly coordinated with the control of gene ex- Finally, our study illustrates that fine-scale studies of pression. However, little is known about the dynamics chromosomal folding patterns enable identification of of chromosomal architectural features in erythroblasts critical regulatory elements. at different developmental stages. Here we carried out comparative Hi-C (chromosome conformation capture [3C] followed by high-throughput sequencing) and Cap- Results ture-C (a multiplexed derivative of 3C) experiments in pri- Gene expression profiles of primary fetal and adult mary human fetal- and adult-type erythroblasts. We human erythroblasts describe global and local similarities and distinctions in To study human erythroblasts at different developmental stages, we used a three-phase in vitro culture system to expand and differentiate fetal and adult CD34+ hematopoi- A 3’HS1 βδ HBBP1BGLT3Aγ Gγ ε HS1 HS2 HS3 HS4HS5 etic stem/progenitor cells (Fig. 1B). Cell morphology and LCR surface marker phenotyping confirmed that fetal and Capture-C adult erythroid cells were at similar maturation stages B Human RT-PCR Hi-C RNA-seq fetal liver (Supplemental Fig. S1A,B,E), allowing comparisons be- tween these two populations. As expected, RT-qPCR re- SCF, IL3, EPO SCF Heparin CD34+ hydrocortisone EPO EPO, insulin sults revealed that fetal and adult erythroblasts expressed Day 8 Day 11 Day 14 Human adult predominantly γ-globin and β-globin genes, respectively peripheral blood (Fig. 1C,D). Hence, the in vitro culture system does not γ-globin δ+β-globin RNA-seq (day11) perturb normal developmental stage specificity of gene C 1500 1500 Fetal Fetal E fetal vs. adult expression. Adult Adult LIN28B HBD 1000 1000 We determined transcription profiles of fetal and adult IGF2BP1 150 erythroid cells each at two stages of differentiation by 500 500 To spike-in RNA sequencing (RNA-seq). Spike-in controls were added 0 0 100 D8 D11 D14 D8 D11 D14 C6ORF223 for normalization purposes, since global changes in gene BCL11A Fetal Adult 50 HBG1 expression during erythroid differentiation can distort D 100 100 measurements of RNA abundance (Stonestrom et al. -log10(adjusted P-value) HBG2 0 HBB 60 60 2015). The results revealed highly similar transcription -8 -4 0 4 Fold change adult/fetal (log2) patterns between fetal and adult stages when analyzed 20 20 Percentage at matched time points of maturation (Supplemental Fetal highly
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