BET inhibitors enhance embryonic and fetal expression in erythroleukemia cell lines by John Z. Cao, Kristina Bigelow, Amittha Wickrema, and Lucy A. Godley

Received: March 16, 2021. Accepted: August 20, 2021.

Citation: John Z. Cao, Kristina Bigelow, Amittha Wickrema, and Lucy A. Godley. BET inhibitors enhance embryonic and fetal globin expression in erythroleukemia cell lines. Haematologica. 2021 Aug 26. doi: 10.3324/haematol.2021.278791. [Epub ahead of print]

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BET inhibitors enhance embryonic and fetal globin expression

in erythroleukemia cell lines

John Z. Cao1, Kristina Bigelow2, Amittha Wickrema1,2, and Lucy A. Godley1,2*

1 Committee on Cancer Biology, Biological Sciences Division, The University of Chicago, Chicago IL

2 Section of Hematology/Oncology, Department of Medicine, The University of Chicago, Chicago IL

*Corresponding author

Contact information:

5841 S. Maryland Ave.

MC 2115

Chicago, IL 60637

Phone: 773-702-4140

FAX: 773-702-9268

Email: [email protected]

Author Contribution

J.Z.C. designed and performed the experiments, analyzed the data, and wrote the manuscript. K.B. performed additional experiments. L.A.G. conceived of the study and provided insights in experimental design and data interpretation. A.W. provided additional input for experimental design and data interpretation.

Acknowledgement

This work was supported by a grant by the Edwards P. Evans Foundation/EvansMDS to A.W. and

L.A.G. Funding for J.Z.C. was supported by the University of Chicago Biological Sciences Division

Dean’s Office, the University of Chicago Comprehensive Cancer Center Women’s Board, and the

Goldblatt Scholarship. We thank Dr. Alex Ruthenburg (University of Chicago) for providing the K562 cells used in this work and experimental advice; and Dr. Julie-Aurore Losman (Dana-Farber Cancer

Institute) who provided the TF-1 cells.

Declaration Of Interests

L.A.G, A.W., and J.Z.C are owners of the patent PCT/US20/52842 titled “METHODS AND

COMPOSITIONS FOR TREATING AND THALASSEMIA”, filed on

September 25, 2020.

Text word count: 1558

Figures: 3

Supplemental Figures: 3

Reference count: 16 Five encoding the human β- are located in a cluster on

11p15.4 (β-globin locus): 5’-HBE1 (ε)-HBG2 (Gγ)-HBG1 (Aγ)-HBD (δ)-HBB (β)-3’. These genes are expressed in distinct developmental stages, with transitions controlled by a series of transcriptional switches regulated by the interplay between local chromatin structure and erythroid-specific transcription factors (TFs)1, 2. Locally, expression of each β-globin is influenced by the spatial proximity of its promoter to the enhancer-rich (LCR)1, 2. In addition to chromatin looping, a number of TFs, including GATA1, BCL11A, COUP-TF2, NuRD, and MYB, specifically repress the expression of embryonic and fetal ε/γ-globins1, 3, 4. Furthermore, MYB mRNA is targeted for degradation by microRNAs (miRs) miR-15A and miR-16-15.

In mammals, the bromodomain and extra-terminal domain (BET) family consists of histone readers with two acetyl-lysine binding bromodomains (BD1 and BD2), including the ubiquitously expressed BRD2, BRD3, BRD4, and the germ cell-specific BRDT, that are crucial for epigenetic regulation of gene expression through recruitment of the transcription machinery6. Previous studies have established that BET inhibitors (BETi) disrupt adult β-globin expression mediated by GATA1 in mouse G1E cells7 and induce production in UT7 human erythroid cell line8. However, the effects of BETi on the transcription regulation of genes in the β-globin locus have not been evaluated comprehensively. Here, we demonstrate the ability of BETis to reactivate embryonic and fetal ε/γ- globins in erythroleukemia cell lines.

We tested the ability of BETi to induce erythroid differentiation in TF-1 cells, an erythroleukemia cell line that expresses virtually no embryonic ε-globin at baseline (Figure 1A).

Quantitative PCR (qPCR) of β-globins after treatment with JQ1 and/or erythropoietin (EPO) demonstrated that although EPO upregulated all three types of β-globins, JQ1 specifically re- activated HBE1, embryonic ε-globin (Figure 1). Although JQ1 alone did not affect the expression of

HBB, JQ1 antagonized the EPO-induced HBB upregulation, reducing the expression of adult β-globin by 50% (Figure 1C). In contrast, we did not observe expression changes in HBG1/2 with or without

EPO (Figure 1D). Strikingly, JQ1 upregulated the ε-globin by 15-fold alone, and by more than 200- fold when combined with EPO (Figure 1E). Taken together, these expression changes account for the dramatic increase in HBE1 transcripts from <1% to nearly 20% as well as the decrease in HBB transcripts from over 30% to less than 20% of all β-globin transcripts in JQ1 treated cells versus control cells (Figure 1A), indicating that JQ1 is reactivating HBE1 in this cell line. RNA-sequencing

(RNA-seq) on TF-1 cells treated 3 days with DMSO, JQ1, EPO, and EPO+JQ1 confirmed qPCR quantification of β-globin expression (Figure 1B). Moreover, JQ1 treatment also induced the adult α- globin genes HBA1/2, suggesting that BETi treatment is unlikely to lead to an α-thalassemia-like condition (Figure 1B). We performed Western blots for γ- and ε-globins using TF-1 cells treated for 3 days, and observed that HBE1 was increased by the combined treatment compared to EPO alone, as expected given the gene expression changes (Figure 1F).

To test whether this effect was specific to this cell line and/or was a class effect of BETis, we first treated two other erythroleukemia cell lines, K562 and HEL, and one myeloid cell line (HL-60), in a similar fashion. We found that JQ1 induced ε/γ-globin genes only in K562 and HEL cells, without any effect in HL-60 cells (Supplemental Figure 1A-C) and without EPO induction of differentiation.

Two other BETis currently in clinical trials (CPI-0610 and PLX51107) had similar effects to JQ1 in both K562 and TF-1 cells (Supplemental Figure 1D-E), demonstrating that the ability to re-activate

ε/γ-globins is a class effect.

In order to determine the effects of JQ1 on erythroid differentiation, we examined the expression of erythroid- and myeloid-specific genes in TF-1 cells with or without EPO stimulation

(Supplemental Figure 2A). As expected, EPO treatment resulted in erythroid maturation as shown by the upregulation of erythroid-lineage genes and the downregulation of HSPC or myeloid-lineage genes (Supplemental Figure 2A). However, JQ1 only mirrored the effects of EPO in a subset of these genes, including most of those that encode cell surface markers, transporters, and receptors

(Supplemental Figure 2A). JQ1 exhibited opposite effects to those of EPO in many of the genes encoding components of the erythroid cytoskeleton (Supplemental Figure 2A). These observations indicate that JQ1 and EPO have overlapping but distinct effects on the expression of erythroid- lineage genes, and the reactivation of ε/γ-globins by BETis is not purely due to erythroid differentiation and maturation. Transcriptome analyses further revealed the distinct effects of JQ1 and

EPO (Supplemental Table 1 and Supplemental Figure 2). Whereas EPO induces genes involved in erythropoiesis-related pathways (e.g., -biosynthesis and iron ion homeostasis), JQ1 suppresses immune-activation pathways (Supplemental Figure 2B-E). Among the genes regulated by EPO or

JQ1, only a small subset is regulated by both (Supplemental Figure 2F-G).

Since ε/γ-globins are suppressed by a number of TFs, we examined the expression of corresponding genes in our RNA-seq data and further validated the results with qPCR (Figure 2). We found that MYB expression was decreased by JQ1 and EPO, and the combination treatment showed an additive effect (Figure 2A). Since MYB is a target of miR-15A and miR-16-1, we measured the levels of these miRs and found that both were upregulated by either JQ1 and EPO in an additive fashion (Figure 2B-C), showing an inverse correlation compared to MYB expression as expected.

RNA-seq quantification of known miR-15A/16-1 targets indicated that most were downregulated by treatment with JQ1 and/or EPO (Figure 2D), further suggesting that these miRs play a central role in mediating the effects of JQ1. Next, we examined the genes encoding several well-established HBE1 and HBG1/2 inhibitors or HBB activators: IKZF1 (IKAROS), NR2F2 (COUP-TF2), BCL11A, and

GATA1. Each of these genes was downregulated by JQ1 with or without EPO (Figure 2E-G). KLF1 promotes the expression of BCL11A9, and therefore as expected, we observed downregulation of

BCL11A only when we also saw KLF1 repression in the setting of joint JQ1 and EPO treatment

(Figure 2H-I). GATA1 Western blots confirmed a stable level of protein as expected from the qPCR data, whereas Western blotting for BCL11A demonstrated almost no detectable protein after combination treatment (Figure 2J).

To examine how the spatial structure of the locus changes in response to JQ1 treatment, we performed chromatin conformation capture followed by qPCR (3C-qPCR) to quantify the LCR interactions with the β-globin genes under each treatment (Figure 3A). Interestingly, differentiation alone via EPO did not change the interaction frequency in this locus (Figure 3B). In contrast, JQ1 decreased the interaction at multiple loci near or in the fetal and adult γ/β-globin genes (Figure 3B).

Similarly, the LCR interaction in EPO+JQ1 double-treated cells is more similar to that of JQ1-treated cells than that of EPO-treated cells, again showing that JQ1, but not EPO, decreases interactions between the LCR and the γ/β-globin genes (Figure 3C). Notably, JQ1 treatment did not decrease the interaction between the LCR and the HBE1 promoter (Figure 3B,C), suggesting that JQ1 treatment relaxes the looping between the LCR and the γ/β-globin genes, which biases LCR contacts in favor of interactions with the promoter of embryonic HBE1.

Thus, the β-globin gene expression changes under BET inhibition are likely the result of both shifts in local chromatin looping and expression changes of β-globin inhibitors and activators. The shifts in LCR interactions favor HBE1 expression over HBB or HBG1/2. Although the overall interaction between LCR and the β-globin genes decreases under JQ1 treatment, ε/γ-globin expression still increased likely due to reduced inhibition and upregulation of genes involved in erythroid maturation. Together, these factors result in decreased HBB transcription and increased embryonic HBE1 transcription (Figure 1A).

Our data in TF-1 cells show that BETis induces partial erythroid maturation and reactivate the embryonic ε-globin HBE1 even without EPO-mediated erythroid maturation. An important clinical question is whether the HBE1 encoded ε-globin could function as a reasonable substitute for the adult

β-globin chain. Biochemical analysis of the Hb-Gower 2 (α2ε2) shows that its

P50 for oxygen, affinity to 2,3-BPG, Bohr Coefficient, and Hill Coefficient are comparable to those of

HbA10. Hb-Gower 2 also has a comparable tetramer-dimer dissociation constant to that of HbA11. A study in transgenic α/β-thalassemia mice found that human embryonic consist of ζ- globin and ε-globin rescue the lethal phenotype of α/β-thalassemia12. Similarly, a study in sickle cell mice found that the presence of human Hb-Gower 2 (α2ε2) greatly alleviated sickle cell phenotypes, and Hb-Gower 2 inhibits sickle cell hemoglobin (HbS) polymerization13.

Our findings suggest that specific inhibition of bromodomain-containing could provide a reasonable alternative and/or adjuncts to the treatment of β-globinopathies like sickle cell anemia (SCA). SCA patients generally have elevated EPO levels14, and thus treatment with JQ1 alone might be sufficient to induce ε-globin in these patients. Currently, the most common treatment for SCA is administration of hydroxyurea, which elevates production to alleviate symptoms15. In recent years, multiple gene therapy strategies for SCA have emerged, including β-globin gene addition and nuclease-assisted β-globin gene modification/repair16. Although these recent advances in gene therapy offer the potential for cure, they will not be accessible or affordable for all patients, especially those from less affluent countries or health systems. We hope that our work motivates future studies that focus on the effect of BET inhibition on globin expression in other erythroid cell lines and primary cells and how BET inhibition could be harnessed for therapeutic purposes.

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FIGURE LEGENDS

Figure 1. JQ1 specifically induces the ε-globin gene in TF-1 cells.

(A) Percentage of HBB, HBG1/2, and HBE1 transcripts relative to total β-globin transcripts in TF-1 cells after treatment with JQ1 (red), EPO (blue), EPO+JQ1 (green) versus DMSO (black) for 5 days

(N=3). (B) RNA-seq quantification of α- and β-globin genes in TF-1 cells after treatment with JQ1

(red), EPO (blue), EPO+JQ1 (green) versus DMSO (black) for 3 days. (C-E) RT-qPCR quantification of (C) HBB, (D) HBG1/2, and (E) HBE1 expression in TF-1 cells after treatment with JQ1 (red), EPO

(blue), EPO+JQ1 (green) versus DMSO (black) for a total of 5 days (D1-5) (N=3). (F) Western blots of

γ- and ε-globins in TF-1 cells treated for 3 days. The day at which the protein extracts were made is indicated by a black arrow labeled “WB” in panels (D-E). Quantification of the target normalized to loading control is shown to the right of the gel images (N=3). In (A,C-E), *: p<0.05, **: p<0.01, ***: p<0.001 (Student’s t-test). In (B), *: FDR<0.05, **: FDR<0.01, ***: FDR<0.001. FDR: False Discovery

Rate.

Figure 2. JQ1 downregulates known inhibitors of fetal hemoglobin.

(A-C) qPCR quantification of (A) MYB, (B) miR-15A , (C) miR-16-1, in TF-1 cells after treatment with

JQ1 (red), EPO (blue), EPO+JQ1 (green) versus DMSO (control, black) for 3 days. (D) Heatmap representing RNA-seq quantification of expression changes (fold-changes in FPKM) of known miR-

15A and miR-16-1 targets in JQ1 treated TF-1 cells versus control, with or without EPO. Fold-change ranges from -4 (red, suppressed by JQ1) to 4 (green, induced by JQ1). Genes with low expression

(FPKM<1) were excluded or represented with gray box. (E-I) qPCR quantification of (E) IKZF1

(encodes IKAROS), (F) NR2F2 (encodes COUP-TF2), (G) GATA1, (H) BCL11A, and (I) KLF1 in TF-1 cells after treatment with JQ1 (red), EPO (blue), EPO+JQ1 (green) versus DMSO (control, black) for

3 days. (J) Western blots for GATA1 and BCL11A with corresponding loading controls.

Quantifications of signal intensity, normalized to loading controls, are shown as bar graphs to the right of the Western blots. N=3 for panels (B), (C), and (J). N=2 for panel (D). N=4 for all other panels. *: p<0.05, **: p<0.01, ***: p<0.001, n.s.: not statistically significant (Student’s t-test).

Figure 3. JQ1 changes interaction frequencies between the LCR and the β-globin genes.

(A) 3C-qPCR quantification of interaction frequencies between the LCR and segments of the β-globin locus in TF-1 cells after treatment with JQ1 (red), EPO (blue), EPO+JQ1 (green) versus DMSO

(control, black) for 3 days. (N=3). (B) log2 fold-change and statistical significance comparing JQ1 versus DMSO control (red) and EPO versus DMSO control (blue). (C) log2 fold-change and statistical significance comparing EPO+JQ1 versus EPO (green) and EPO+JQ1 versus JQ1 (purple). Track on top indicates chromosomal gene positions and the DNA fragments generated from an EcoRI digestion. Replicates included have EcoRI digestion efficiency >60% at all control sites. LCR: locus control region. HS: DNase hypersensitivity site. The EcoRI-generated fragment containing the bait primer is shown as a yellow bar. *: p<0.05, **: p<0.01, ***: p<0.001 (Student’s t-test). A genomic region containing no EcoRI site (chr7:117,293,465-117,293,547) was used as internal control for the amount of input DNA. All chimeric fragment quantification was normalized to the control region quantification of the respective experimental conditions. Since BET inhibition results in global changes in genomic structure, we were unable to identify a genomic region as a reliable control region for this study.

Supplemental Table 1. Enriched GO terms in induced or suppressed genes. EPO vs. DMSO, Induced (Supplemental Figure 2B, right panel) GO ID Description p.Val FDR GO:0070887 cellular response to chemical stimulus 7.48E-05 7.48E-05 GO:0015669 gas transport 1.45E-04 1.45E-04 GO:0055072 iron ion homeostasis 1.61E-04 1.61E-04 GO:0006779 porphyrin-containing compound biosynthetic process 2.71E-04 2.71E-04 GO:0006778 porphyrin-containing compound metabolic process 4.81E-04 4.81E-04 GO:0033014 tetrapyrrole biosynthetic process 6.34E-04 6.34E-04 GO:0046501 protoporphyrinogen IX metabolic process 7.28E-04 7.28E-04 GO:0006950 response to stress 1.96E-03 1.96E-03 GO:0042744 hydrogen peroxide catabolic process 2.20E-03 2.20E-03 GO:0055076 transition metal ion homeostasis 2.33E-03 2.33E-03 GO:0006879 cellular iron ion homeostasis 5.18E-03 5.18E-03 GO:0015671 oxygen transport 5.76E-03 5.76E-03 GO:0006782 protoporphyrinogen IX biosynthetic process 7.67E-03 7.67E-03 GO:0098754 detoxification 7.92E-03 7.92E-03 GO:0042168 heme metabolic process 9.89E-03 9.89E-03 GO:0006783 heme biosynthetic process 1.33E-02 1.33E-02 GO:0002376 immune system process 2.18E-02 2.18E-02 GO:0006826 iron ion transport 2.28E-02 2.28E-02 GO:0033013 tetrapyrrole metabolic process 2.41E-02 2.41E-02 GO:1990748 cellular detoxification 2.65E-02 2.65E-02 GO:0042743 hydrogen peroxide metabolic process 3.55E-02 3.55E-02 GO:0098869 cellular oxidant detoxification 3.75E-02 3.75E-02 GO:0098656 anion transmembrane transport 3.77E-02 3.77E-02

EPO vs. DMSO, Suppressed (Supplemental Figure 2B, left panel) GO ID Description p.Val FDR GO:0048856 anatomical structure development 3.94E-16 3.94E-16 GO:0048731 system development 7.89E-16 7.89E-16 GO:0007154 cell communication 1.05E-15 1.05E-15 GO:0032502 developmental process 2.13E-15 2.13E-15 GO:0048583 regulation of response to stimulus 2.46E-15 2.46E-15 GO:0009653 anatomical structure morphogenesis 2.97E-15 2.97E-15 GO:0007275 multicellular organism development 7.35E-15 7.35E-15 GO:0023052 signaling 8.35E-15 8.35E-15 GO:0035556 intracellular signal transduction 1.09E-14 1.09E-14 GO:0023051 regulation of signaling 4.42E-14 4.42E-14 GO:0010646 regulation of cell communication 2.12E-13 2.12E-13 GO:0050896 response to stimulus 7.60E-13 7.60E-13 GO:0007165 signal transduction 1.02E-12 1.02E-12 GO:0009966 regulation of signal transduction 1.41E-12 1.41E-12 GO:0040011 locomotion 7.72E-12 7.72E-12 GO:0007399 nervous system development 1.63E-11 1.63E-11 GO:0032501 multicellular organismal process 1.64E-11 1.64E-11 GO:0016477 cell migration 2.67E-11 2.67E-11 GO:0007166 cell surface receptor signaling pathway 2.71E-11 2.71E-11 GO:0050793 regulation of developmental process 8.42E-11 8.42E-11 GO:0022008 neurogenesis 9.06E-11 9.06E-11 GO:0051239 regulation of multicellular organismal process 1.09E-10 1.09E-10 GO:0051240 positive regulation of multicellular organismal process 5.98E-10 5.98E-10 GO:0048699 generation of neurons 1.34E-09 1.34E-09 GO:0051716 cellular response to stimulus 1.39E-09 1.39E-09 GO:0048468 cell development 1.44E-09 1.44E-09 GO:0048870 cell motility 1.52E-09 1.52E-09 GO:0051674 localization of cell 1.52E-09 1.52E-09 GO:0006928 movement of cell or subcellular component 3.65E-09 3.65E-09 GO:0030154 cell differentiation 7.27E-09 7.27E-09

Supplemental Table 1. Enriched GO terms in induced or suppressed genes, continued. GO:0051094 positive regulation of developmental process 9.36E-09 9.36E-09 GO:0030182 neuron differentiation 1.03E-08 1.03E-08 GO:2000026 regulation of multicellular organismal development 1.04E-08 1.04E-08 GO:0048869 cellular developmental process 1.09E-08 1.09E-08 GO:0048585 negative regulation of response to stimulus 1.16E-08 1.16E-08 GO:0009968 negative regulation of signal transduction 1.23E-08 1.23E-08 GO:0001775 cell activation 3.99E-08 3.99E-08 GO:0030155 regulation of cell adhesion 4.67E-08 4.67E-08 GO:0065008 regulation of biological quality 5.18E-08 5.18E-08 GO:0023057 negative regulation of signaling 6.49E-08 6.49E-08 GO:0022610 biological adhesion 1.01E-07 1.01E-07 GO:0000902 cell morphogenesis 1.14E-07 1.14E-07 GO:0010648 negative regulation of cell communication 1.22E-07 1.22E-07 GO:0007155 cell adhesion 1.58E-07 1.58E-07 GO:1902531 regulation of intracellular signal transduction 1.98E-07 1.98E-07 GO:0120036 plasma membrane bounded cell projection organization 1.99E-07 1.99E-07 GO:0030030 cell projection organization 2.33E-07 2.33E-07 GO:0032879 regulation of localization 3.12E-07 3.12E-07 GO:0022603 regulation of anatomical structure morphogenesis 3.41E-07 3.41E-07 GO:0031175 neuron projection development 4.05E-07 4.05E-07

EPO+JQ1 vs. JQ1, Induced (Supplemental Figure 2C, right panel) GO ID Description p.Val FDR GO:0070887 cellular response to chemical stimulus 5.01E-10 5.01E-10 GO:0098754 detoxification 1.24E-08 1.24E-08 GO:0015669 gas transport 6.06E-07 6.06E-07 GO:1990748 cellular detoxification 3.76E-06 3.76E-06 GO:0009636 response to toxic substance 3.85E-06 3.85E-06 GO:0042221 response to chemical 5.44E-06 5.44E-06 GO:0097237 cellular response to toxic substance 9.22E-06 9.22E-06 GO:0042744 hydrogen peroxide catabolic process 1.50E-05 1.50E-05 GO:0010033 response to organic substance 3.34E-05 3.34E-05 GO:0015671 oxygen transport 3.60E-05 3.60E-05 GO:0098869 cellular oxidant detoxification 4.61E-05 4.61E-05 GO:0042168 heme metabolic process 8.86E-05 8.86E-05 GO:0071310 cellular response to organic substance 1.16E-04 1.16E-04 GO:0010035 response to inorganic substance 1.17E-04 1.17E-04 GO:0055076 transition metal ion homeostasis 4.52E-04 4.52E-04 GO:0055072 iron ion homeostasis 4.95E-04 4.95E-04 GO:0006778 porphyrin-containing compound metabolic process 5.76E-04 5.76E-04 GO:0051179 localization 6.06E-04 6.06E-04 GO:0006810 transport 8.35E-04 8.35E-04 GO:0140352 export from cell 1.27E-03 1.27E-03 GO:0051234 establishment of localization 1.78E-03 1.78E-03 GO:0046916 cellular transition metal ion homeostasis 2.08E-03 2.08E-03 GO:0006783 heme biosynthetic process 2.29E-03 2.29E-03 GO:0006879 cellular iron ion homeostasis 3.59E-03 3.59E-03 GO:0046903 secretion 3.65E-03 3.65E-03 GO:0032940 secretion by cell 4.03E-03 4.03E-03 GO:0042743 hydrogen peroxide metabolic process 4.22E-03 4.22E-03 GO:0006779 porphyrin-containing compound biosynthetic process 7.94E-03 7.94E-03 GO:0042127 regulation of cell population proliferation 1.08E-02 1.08E-02 GO:1901700 response to oxygen-containing compound 1.12E-02 1.12E-02 GO:0033014 tetrapyrrole biosynthetic process 1.50E-02 1.50E-02 GO:0033013 tetrapyrrole metabolic process 2.01E-02 2.01E-02 antigen processing and presentation of endogenous peptide GO:0002486 2.05E-02 2.05E-02 antigen via MHC class I via ER pathway, TAP-independent antigen processing and presentation of endogenous peptide GO:0002484 2.05E-02 2.05E-02 antigen via MHC class I via ER pathway GO:0010038 response to metal ion 2.72E-02 2.72E-02 GO:0042592 homeostatic process 2.92E-02 2.92E-02 Supplemental Table 1. Enriched GO terms in induced or suppressed genes, continued.

EPO+JQ1 vs. JQ1, Suppressed (Supplemental Figure 2C, left panel) GO ID Description p.Val FDR GO:0002684 positive regulation of immune system process 1.85E-11 1.85E-11 GO:0002376 immune system process 7.28E-11 7.28E-11 GO:0023052 signaling 2.94E-10 2.94E-10 GO:0007154 cell communication 3.78E-10 3.78E-10 GO:0001775 cell activation 6.75E-10 6.75E-10 GO:0030097 hemopoiesis 1.04E-09 1.04E-09 GO:0002520 immune system development 1.36E-09 1.36E-09 GO:0007165 signal transduction 2.71E-09 2.71E-09 GO:0048534 hematopoietic or lymphoid organ development 3.89E-09 3.89E-09 GO:0045321 leukocyte activation 5.06E-09 5.06E-09 GO:0002696 positive regulation of leukocyte activation 6.35E-09 6.35E-09 GO:0050867 positive regulation of cell activation 1.24E-08 1.24E-08 GO:0035556 intracellular signal transduction 2.31E-08 2.31E-08 GO:0050865 regulation of cell activation 2.76E-08 2.76E-08 GO:0048583 regulation of response to stimulus 3.44E-08 3.44E-08 GO:0002521 leukocyte differentiation 3.72E-08 3.72E-08 GO:0002694 regulation of leukocyte activation 9.95E-08 9.95E-08 GO:0002682 regulation of immune system process 1.67E-07 1.67E-07 GO:0046649 lymphocyte activation 4.71E-07 4.71E-07 GO:0045785 positive regulation of cell adhesion 7.76E-07 7.76E-07 GO:0051239 regulation of multicellular organismal process 7.91E-07 7.91E-07 GO:0007166 cell surface receptor signaling pathway 1.64E-06 1.64E-06 GO:0051716 cellular response to stimulus 1.92E-06 1.92E-06 GO:0040011 locomotion 3.40E-06 3.40E-06 GO:1903039 positive regulation of leukocyte cell-cell adhesion 4.20E-06 4.20E-06 GO:2000026 regulation of multicellular organismal development 1.06E-05 1.06E-05 GO:0030155 regulation of cell adhesion 1.41E-05 1.41E-05 GO:1903131 mononuclear cell differentiation 1.49E-05 1.49E-05 GO:0008283 cell population proliferation 1.49E-05 1.49E-05 GO:0032944 regulation of mononuclear cell proliferation 1.61E-05 1.61E-05 GO:0032943 mononuclear cell proliferation 1.91E-05 1.91E-05 GO:0051251 positive regulation of lymphocyte activation 1.98E-05 1.98E-05 GO:0050896 response to stimulus 2.21E-05 2.21E-05 GO:0016477 cell migration 2.70E-05 2.70E-05 GO:0050776 regulation of immune response 2.72E-05 2.72E-05 GO:1903037 regulation of leukocyte cell-cell adhesion 2.85E-05 2.85E-05 GO:0042110 T cell activation 3.62E-05 3.62E-05 GO:0051240 positive regulation of multicellular organismal process 3.73E-05 3.73E-05 GO:0048584 positive regulation of response to stimulus 4.23E-05 4.23E-05 GO:0050870 positive regulation of T cell activation 4.73E-05 4.73E-05 GO:0048731 system development 4.95E-05 4.95E-05 GO:0032501 multicellular organismal process 5.00E-05 5.00E-05 GO:0070663 regulation of leukocyte proliferation 5.20E-05 5.20E-05 GO:0022409 positive regulation of cell-cell adhesion 5.53E-05 5.53E-05 GO:0006928 movement of cell or subcellular component 6.31E-05 6.31E-05 GO:0007155 cell adhesion 7.91E-05 7.91E-05 GO:0070661 leukocyte proliferation 8.14E-05 8.14E-05 GO:0022610 biological adhesion 9.08E-05 9.08E-05 GO:0046651 lymphocyte proliferation 9.39E-05 9.39E-05 GO:0050670 regulation of lymphocyte proliferation 9.41E-05 9.41E-05

JQ1 vs. DMSO, Induced (Supplemental Figure 2D, right panel) GO ID Description p.Val FDR GO:0071702 organic substance transport 2.55E-02 2.55E-02 GO:0006820 anion transport 4.50E-02 4.50E-02

JQ1 vs. DMSO, Suppressed (Supplemental Figure 2D, left panel) Supplemental Table 1. Enriched GO terms in induced or suppressed genes, continued. GO ID Description p.Val FDR GO:0001775 cell activation 6.50E-21 6.50E-21 GO:0002376 immune system process 2.77E-19 2.77E-19 GO:0006955 immune response 1.99E-17 1.99E-17 GO:0045321 leukocyte activation 4.30E-17 4.30E-17 GO:0002682 regulation of immune system process 2.30E-15 2.30E-15 GO:0009605 response to external stimulus 3.90E-15 3.90E-15 GO:0048583 regulation of response to stimulus 1.14E-14 1.14E-14 GO:0050776 regulation of immune response 2.95E-14 2.95E-14 GO:0050896 response to stimulus 1.19E-13 1.19E-13 GO:0034097 response to cytokine 2.59E-13 2.59E-13 GO:0010033 response to organic substance 7.36E-13 7.36E-13 GO:0051239 regulation of multicellular organismal process 2.46E-12 2.46E-12 GO:0046649 lymphocyte activation 3.41E-12 3.41E-12 GO:0016477 cell migration 3.59E-12 3.59E-12 GO:0002684 positive regulation of immune system process 3.62E-12 3.62E-12 GO:0002252 immune effector process 4.23E-12 4.23E-12 GO:0048584 positive regulation of response to stimulus 6.73E-12 6.73E-12 GO:0030155 regulation of cell adhesion 1.74E-11 1.74E-11 GO:0007166 cell surface receptor signaling pathway 2.78E-11 2.78E-11 GO:0007154 cell communication 7.54E-11 7.54E-11 GO:0045785 positive regulation of cell adhesion 1.34E-10 1.34E-10 GO:0051240 positive regulation of multicellular organismal process 1.53E-10 1.53E-10 GO:1903039 positive regulation of leukocyte cell-cell adhesion 2.01E-10 2.01E-10 GO:0070661 leukocyte proliferation 2.27E-10 2.27E-10 GO:0070663 regulation of leukocyte proliferation 2.80E-10 2.80E-10 GO:0001816 cytokine production 3.24E-10 3.24E-10 GO:0002263 cell activation involved in immune response 3.86E-10 3.86E-10 GO:0007159 leukocyte cell-cell adhesion 5.11E-10 5.11E-10 GO:0040011 locomotion 6.14E-10 6.14E-10 GO:0023052 signaling 6.32E-10 6.32E-10 GO:0002366 leukocyte activation involved in immune response 6.71E-10 6.71E-10 GO:0042110 T cell activation 7.09E-10 7.09E-10 GO:0002274 myeloid leukocyte activation 7.43E-10 7.43E-10 GO:0022409 positive regulation of cell-cell adhesion 1.02E-09 1.02E-09 GO:1903037 regulation of leukocyte cell-cell adhesion 1.38E-09 1.38E-09 GO:0042221 response to chemical 1.65E-09 1.65E-09 GO:0050670 regulation of lymphocyte proliferation 1.88E-09 1.88E-09 GO:0032944 regulation of mononuclear cell proliferation 2.63E-09 2.63E-09 GO:0050900 leukocyte migration 2.64E-09 2.64E-09 GO:0048870 cell motility 3.01E-09 3.01E-09 GO:0051674 localization of cell 3.01E-09 3.01E-09 GO:0098609 cell-cell adhesion 3.03E-09 3.03E-09 GO:0070887 cellular response to chemical stimulus 3.25E-09 3.25E-09 GO:0002443 leukocyte mediated immunity 4.29E-09 4.29E-09 GO:0071345 cellular response to cytokine stimulus 4.61E-09 4.61E-09 GO:0032943 mononuclear cell proliferation 6.72E-09 6.72E-09 GO:0035556 intracellular signal transduction 6.73E-09 6.73E-09 GO:0022610 biological adhesion 7.88E-09 7.88E-09 GO:0022407 regulation of cell-cell adhesion 8.49E-09 8.49E-09 GO:0050865 regulation of cell activation 9.78E-09 9.78E-09

EPO+JQ1 vs. EPO, Induced (Supplemental Figure 2E, right panel) GO ID Description p.Val FDR GO:0006468 protein phosphorylation 2.65E-05 2.65E-05 GO:0120036 plasma membrane bounded cell projection organization 4.75E-05 4.75E-05 GO:0007275 multicellular organism development 9.71E-05 9.71E-05 GO:0030030 cell projection organization 1.14E-04 1.14E-04 GO:0048731 system development 1.41E-04 1.41E-04 GO:0009653 anatomical structure morphogenesis 1.67E-04 1.67E-04 Supplemental Table 1. Enriched GO terms in induced or suppressed genes, continued. GO:0048856 anatomical structure development 1.83E-04 1.83E-04 GO:0032502 developmental process 2.25E-04 2.25E-04 GO:0048522 positive regulation of cellular process 2.79E-04 2.79E-04 GO:0048518 positive regulation of biological process 9.18E-04 9.18E-04 transmembrane receptor protein tyrosine kinase signaling GO:0007169 1.51E-03 1.51E-03 pathway GO:0007154 cell communication 1.91E-03 1.91E-03 GO:0030182 neuron differentiation 2.18E-03 2.18E-03 GO:0007399 nervous system development 2.42E-03 2.42E-03 GO:0051716 cellular response to stimulus 2.50E-03 2.50E-03 GO:0023052 signaling 2.81E-03 2.81E-03 GO:0016310 phosphorylation 3.04E-03 3.04E-03 GO:0022008 neurogenesis 5.34E-03 5.34E-03 GO:0048699 generation of neurons 7.12E-03 7.12E-03 GO:0048468 cell development 7.24E-03 7.24E-03 GO:0007167 enzyme linked receptor protein signaling pathway 8.80E-03 8.80E-03 GO:0048666 neuron development 9.76E-03 9.76E-03 GO:0032989 cellular component morphogenesis 1.01E-02 1.01E-02 GO:0050794 regulation of cellular process 1.12E-02 1.12E-02 GO:0051128 regulation of cellular component organization 1.15E-02 1.15E-02 GO:0000902 cell morphogenesis 1.24E-02 1.24E-02 GO:0050789 regulation of biological process 1.38E-02 1.38E-02 GO:0032990 cell part morphogenesis 1.82E-02 1.82E-02 GO:0048812 neuron projection morphogenesis 1.95E-02 1.95E-02 GO:0031399 regulation of protein modification process 2.34E-02 2.34E-02 GO:0097061 dendritic spine organization 2.59E-02 2.59E-02 GO:0001932 regulation of protein phosphorylation 2.65E-02 2.65E-02 GO:0007165 signal transduction 2.72E-02 2.72E-02 GO:0120039 plasma membrane bounded cell projection morphogenesis 2.87E-02 2.87E-02 GO:0030154 cell differentiation 2.88E-02 2.88E-02 GO:0051338 regulation of transferase activity 2.91E-02 2.91E-02 GO:0065009 regulation of molecular function 3.06E-02 3.06E-02 GO:0048858 cell projection morphogenesis 3.19E-02 3.19E-02 GO:0043549 regulation of kinase activity 3.50E-02 3.50E-02 GO:0031175 neuron projection development 4.29E-02 4.29E-02

EPO+JQ1 vs. EPO, Suppressed (Supplemental Figure 2E left panel) GO ID Description p.Val FDR GO:0002376 immune system process 1.41E-30 1.41E-30 GO:0001775 cell activation 6.71E-27 6.71E-27 GO:0006955 immune response 3.84E-26 3.84E-26 GO:0045321 leukocyte activation 2.12E-25 2.12E-25 GO:0002252 immune effector process 8.48E-19 8.48E-19 GO:0002443 leukocyte mediated immunity 3.95E-18 3.95E-18 GO:0002274 myeloid leukocyte activation 6.14E-18 6.14E-18 GO:0002682 regulation of immune system process 1.25E-17 1.25E-17 GO:0002366 leukocyte activation involved in immune response 6.27E-17 6.27E-17 GO:0002263 cell activation involved in immune response 9.13E-17 9.13E-17 GO:0009605 response to external stimulus 1.04E-16 1.04E-16 GO:0043299 leukocyte degranulation 7.83E-16 7.83E-16 GO:0002444 myeloid leukocyte mediated immunity 1.13E-15 1.13E-15 GO:0050776 regulation of immune response 1.78E-15 1.78E-15 GO:0046649 lymphocyte activation 2.37E-15 2.37E-15 GO:0002275 myeloid cell activation involved in immune response 2.82E-15 2.82E-15 GO:0002684 positive regulation of immune system process 3.57E-15 3.57E-15 GO:0050865 regulation of cell activation 1.44E-14 1.44E-14 GO:0070661 leukocyte proliferation 2.06E-14 2.06E-14 GO:0050896 response to stimulus 2.68E-14 2.68E-14 GO:0051707 response to other organism 3.58E-14 3.58E-14 GO:0043207 response to external biotic stimulus 3.99E-14 3.99E-14 Supplemental Table 1. Enriched GO terms in induced or suppressed genes, continued. GO:0009607 response to biotic stimulus 9.15E-14 9.15E-14 GO:0006887 exocytosis 1.03E-13 1.03E-13 GO:0002694 regulation of leukocyte activation 1.09E-13 1.09E-13 GO:0034097 response to cytokine 1.23E-13 1.23E-13 GO:0045055 regulated exocytosis 2.41E-13 2.41E-13 GO:0042110 T cell activation 2.65E-13 2.65E-13 GO:0022610 biological adhesion 3.00E-13 3.00E-13 GO:0098609 cell-cell adhesion 4.25E-13 4.25E-13 GO:0007155 cell adhesion 5.03E-13 5.03E-13 GO:0046651 lymphocyte proliferation 6.33E-13 6.33E-13 GO:0001816 cytokine production 8.02E-13 8.02E-13 GO:0032943 mononuclear cell proliferation 9.65E-13 9.65E-13 GO:0030155 regulation of cell adhesion 1.16E-12 1.16E-12 GO:0051239 regulation of multicellular organismal process 2.82E-12 2.82E-12 GO:0070663 regulation of leukocyte proliferation 3.30E-12 3.30E-12 GO:0001817 regulation of cytokine production 3.62E-12 3.62E-12 GO:0071345 cellular response to cytokine stimulus 5.89E-12 5.89E-12 GO:0048583 regulation of response to stimulus 7.00E-12 7.00E-12 GO:0006952 defense response 8.12E-12 8.12E-12 GO:0002703 regulation of leukocyte mediated immunity 8.56E-12 8.56E-12 GO:0042119 neutrophil activation 8.70E-12 8.70E-12 GO:0036230 granulocyte activation 1.55E-11 1.55E-11 GO:0050670 regulation of lymphocyte proliferation 1.73E-11 1.73E-11 GO:0098542 defense response to other organism 2.19E-11 2.19E-11 GO:0032944 regulation of mononuclear cell proliferation 2.36E-11 2.36E-11 GO:0140352 export from cell 2.70E-11 2.70E-11 GO:0070887 cellular response to chemical stimulus 3.61E-11 3.61E-11 GO:0042098 T cell proliferation 4.67E-11 4.67E-11

5 DMSO 5 5 A **" JQ1 **" 4 4 4 ***" 3 3 **" *" **" 3 K562 **" **" HBB

2 2 HBE1 2

cells HBG1/2 1 1 1 0 0 0 D1 D2 D3 D4 D5 D1 D2 D3 D4 D5 D1 D2 D3 D4 D5

B 8 DMSO 8 8 JQ1 **" 6 6 6 *" **" HEL 4 4 **" ***" 4 ***" HBB **" ***" HBE1 cells ***" ***" HBG1/2 *" **" 2 ***" ***" 2 2 0 0 0 D1 D2 D3 D4 D5 D1 D2 D3 D4 D5 D1 D2 D3 D4 D5

C 5 DMSO 5 5 4 JQ1 4 4 HL-60 3 3 3 HBB

2 2 HBE1 2

cells HBG1/2 1 1 1 0 0 0 D1 D2 D3 D4 D5 D1 D2 D3 D4 D5 D1 D2 D3 D4 D5

4 DMSO 4 4 D CPI-0610 PLX51107 *" *" *" *" 3 3 **" ***" 3 ***" ***" **" *" **" **" K562 2 2 2 *" HBB

HBE1 **"

cells HBG1/2 **" 1 1 1

0 0 0 D1 D2 D3 D4 D5 D1 D2 D3 D4 D5 D1 D2 D3 D4 D5 DMSO E 4 4 4 CPI-0610 *" PLX51107 *" *" 3 3 3 " *" *" **" TF-1 2 2 2

HBB **" **" **" *" *" HBE1 cells **" *" HBG1/2 1 **" 1 1

0 0 0 D1 D2 D3 D4 D5 D1 D2 D3 D4 D5 D1 D2 D3 D4 D5

Supplemental Figure 1. BET inhibitors induces embryonic and fetal ε/γ-globin genes in erythroid, but not myeloid, cell lines.

Expression of HBB (left), HBG1/2 (middle), HBE1 (right) in cell lines treated with BET inhibitors (red) versus DMSO control (black). (A) K562 cells treated with JQ1 versus DMSO. (B) HEL cells treated with JQ1 versus DMSO. (C) HL-60 cells treated with JQ1 versus DMSO. (D) K562 cells treated with

CPI-0610 or PLX51107 versus DMSO. (E) TF-1 cells treated with CPI-0610 or PLX51107 versus

DMSO. JQ1 (200nM) and PLX51107 (200nM) were applied to the cells at day 0 without additional treatments. CPI-0610 (200nM) was applied to the cells at day 0 and re-applied every subsequent day until day 5. 0.002% DMSO was used in controls. N=3. D, days of treatment. *: p<0.05, **: p<0.01, ***: p<0.001 (Student’s t-test).

A 5 CD34 CD44 SPI1 ITGAMITGA2B GP1BA SPTA1 SPTB EPB42GYPA SLC2A1 TFRC EPORSLC4A1 EPB41 ANK1

DMSO JQ1 EPO

EPO+JQ1 -5

EPO vs. DMSO 4 hydrogen B tetrapyrrole cell signaling peroxide detoxification porphyrin regulation 3 biosynthetic

2 anion iron ion -log10 (q value) (q -log10 1 transmembrane homeostasis transport

0 -8 -4 0 4 8 Suppressed by EPO log2 (Fold Change) Induced by EPO EPO+JQ1 vs. JQ1 C 4 organismal tetrapyrrole cellular positive biosynthetic hematopoietic 3 antigen lymphocyte metabolic activation development response

2 metal ion

-log10 (q value) (q -log10 1 homeostasis

0 -8 -4 0 4 8 Suppressed by EPO log2 (Fold Change) Induced by EPO JQ1 vs. DMSO immune 4 D activation involved 3

organic anion organic 2 substance transport response cell adhesion

regulation value) (q -log10 1

0 -8 -4 0 4 8 Suppressed by JQ1 log2 (Fold Change) Induced by JQ1 EPO+JQ1 vs. EPO chemical 4 E defense response 3 kinase cell phosphorylation projection 2 signaling morphogenesis cell neutrophil proliferation immune adhesion activation value) (q -log10 1

0 -8 -4 0 4 8 log2 (Fold Change) Suppressed by JQ1 Induced by JQ1 GO term clustering Volcano plots GO term clustering (Suppressed genes) (Induced genes)

313 F 300 257

200 198 181 184

Intersectionsize 110 100 87

48

15 19 9 9 4 0 0 0 397 EPO+JQ1 vs. EPO 465 EPO+JQ1 vs. JQ1 501 JQ1 vs. DMSO 631 EPO vs. DMSO 800 600 400 200 000 Set size

504 G 485

400 367 322 298

200

Intersectionsize 106 71 63 38 27 29 35 7 0 0 0 270 EPO+JQ1 vs. JQ1 927 EPO vs. DMSO 998 EPO+JQ1 vs. EPO 1553 JQ1 vs. DMSO 1500 1000 500 0 Set size

Supplemental Figure 2. Effects of EPO or JQ1 on TF-1 cell transcriptome.

(A) Heatmap representing log2 (fold-change) of representative erythroid/myeloid lineage genes. (B-E)

Volcano plots of transcription changes (middle column) and GO term clustering of induced (red, right column) or suppressed (green, left column) genes. Black dots represent genes with log2 (fold- change) less than 1 in either direction, or genes with FDR (q values) greater than 0.05. Genes with

FPKM<1 were excluded from the volcano plots. GO analysis and GO term clustering were performed using g:Profiler and Cytoscape on genes with significant expression changes. Each node represent a

GO term, and edges denote >60% similarity. Size of each node correspond to the number of genes related to the GO term, and lighter fill denotes lower FDR. (B) Genes induced (red) or suppressed

(green) by EPO (comparing EPO versus DMSO). (C) Genes induced (red) or suppressed (green) by

EPO (comparing EPO+JQ1 versus JQ1). (D) Genes induced (red) or suppressed (green) by JQ1

(comparing JQ1 versus DMSO). (E) Genes induced (red) or suppressed (green) by JQ1 (comparing

EPO+JQ1 versus EPO). (F-G) Intersections between (F) induced or (G) suppressed genes as shown in (B-E). Total number of genes in each set is represented by the horizontal bars next to the set labels. Connected dots in the matrix represent common genes in the corresponding sets, with the exact numbers plotted above.