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myeloid cells. EMP-derived erythroid progenitors later on will generate the first “definitive” erythroid cells, which synthesize mainly adult βmaj and βmin globins and little βH1, prior to the emergence of the “adult” aorta-gonad- mesonephros-derived hematopoietic stem cells.7,8 In mice transgenic for the human β-locus, EryP synthesize e- and γ-globin chains, whereas the EMP erythroid progeny mainly expresses γ- and some β-globin; hematopoietic stem cell-derived definitive cells express human β- and very little, if any, γ-globin.8
In this work, we focused on the chicken ovalbumin upstream promoter-transcription factor II (Coup-TFII or NR2F2/ARP1). Coup-TFII is an orphan nuclear receptor essential for many biological processes, as demonstrated by early embryonic death of Coup-TFII knockout mice.9 Conditional knockout models further demonstrated the requirement of COUP-TFII in different developmental programs, such as angiogenesis, organogenesis, cell fate determination and metabolic homeostasis.10 Within the erythroid lineage, Coup-TFII is expressed in the early embryo and its levels decline around embryonic day E12.5 11,12
Coup-TFII was originally identified as part of the NF-E3 complex, proposed to act as a repressor of e- and γ-globin genes on the basis of in vitro DNA binding studies.11,13,14 Subsequently, transfection experiments showed that the binding of Coup-TFII to both e- and γ-globin promoters interferes with that of NF-Y and possibly of other tran- scription factors and/or cofactors,15-17 resulting in either cooperation or competition.15
Here we show that Coup-TFII is co-expressed with embryonic globins in cells of yolk sac origin. Significantly, Coup-TFII overexpression induces γ-globin (and βH1) in different adult erythroid cellular systems. At the molecu- lar level, overexpression or knockout of Coup-TFII in β- K562 cells18 (to our knowledge the only erythroid model system expressing Coup-TFII), alters the γ/β expression ratio and the frequencies of the interactions within the β- locus in opposite directions, further demonstrating that Coup-TFII favors the expression of embryonic/fetal glo- bins.
Methods
Cell cultures
Coup-TFII activates γ-globin blotting and flow cytometry (Figure 3 and Online Supplementary
Figures S2, 3 and 5).
Coup-TFII CRISPR/Cas9 knockout
The 39900 single guide RNA from the GeCKOv2 library (https://www.addgene.org/crispr/libraries/geckov2) was cloned into the LentiCrisprV2 plasmid (Addgene #52961), as described in the Online Supplementary Methods. After selection, single knockout (KO) cells were isolated to obtain single clones.
Lentiviral production and transduction experiments
HEK-293T cells were transfected with the Coup-TFII or empty vector plus the packaging plasmids psPAX2 and pMD-VSVG (www.lentiweb.com). Seventy-two hours after transfection, recom- binant particles were collected, concentrated and resuspended in phosphate-buffered saline.
RNA isolation and polymerase chain reaction
The procedures used for RNA isolation and RTqPCR are detailed in the Online Supplementary Methods.
Western blotting
Protein extracts were prepared according to standard protocols21 and subjected to sodium dodecylsulfate polyacrylamide gel elec- trophoresis and blotting.
Immunofluorescence
Cells were fixed in 4% paraformaldehyde, washed, permeabi- lized and incubated overnight with the appropriate antibodies. DAPI was added 20 min before the final wash in phosphate- buffered saline. Cells were analyzed using an ArrayScan VTI high- content screening reader (Thermo-Fisher), as described by Durlak et al.,18 or by flow cytometry.
β-K562 cells were cultured in RPMI with 10% fetal bovine serum.18 Ex vivo cultures of mouse E13.5 fetal liver were estab- lished from human β-locus transgenic mice (a gift from Prof. Frank Grosveld). After expansion of erythroblasts, cells were infected, with a multiplicity of infection of 100, and analyzed 72 h after transduction.19 Human burst-forming unit erythroid cultures from peripheral blood were obtained essentially as described by Migliaccio et al.,20 by using a two-phase protocol (details in the Online Supplementary Methods). Cells were monitored by flow cytometry for the expression of CD71, Ter119/CD235a, CD117 and DNGFR. Experiments on mouse and human samples were approved by the Italian Ministry of Health.
β-K562 overexpressing Coup-TFII were fixed with 1% formaldehyde for 10 min at room temperature. Chromatin was sonicated to a size of about 500 bp. DNA immunoprecipitation was obtained by incubation with the appropriate antibodies and subsequent isolation with protein A-agarose beads. DNA was sequenced on an Illumina platform. The sequencing data were uploaded to the Galaxy web platform.22 Reads were mapped to the human genome (GRCh37/hg19) using the Burrows-Wheeler aligner23 and peaks were detected using the HOMER tool package.24 Peaks in different experiments were called as the same if the minimal overlap of intervals was 1 bp. To identify peaks bound in one experiment but not in another, intervals should not overlap.25,26 Motif discovery was conducted using Multiple Expression motifs for Motif Elicitation (MEME)27 with default parameters.
Coup-TFII overexpression
Coup-TFII murine cDNA (a gift from Dr. Michèle Studer) was cloned into a bicistronic IRES-eGFP or IRES-DNGFR CSI vector (a gift from Prof. Tariq Enver) (Online Supplementary Figure S1). Exogenous expression was monitored by quantitative real-time polymerase chain reaction (RTqPCR) analysis and/or western
Chromosome conformation capture
Chromosome conformation capture (3C) experiments28,29 are described in detail in the Online Supplementary Methods.
Flow cytometry
Transduced cells were washed in phosphate-buffered saline, fixed in 4% paraformaldehyde, stained with appropriate antibod- ies, acquired on FACSCalibur (Becton-Dickinson) or a CytoflexS (Beckman-Coulter) and analyzed with Summit V4.3 software or CytExpert, respectively.
Primers, antibodies and reagents
The primers, antibodies and reagents used in this study are list- ed in Online Supplementary Tables S1 and S2.
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Chromatin immunoprecipitation, sequencing and analysis
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