Abstract
MYB plays vital roles in regulating proliferation and differentiation of hematopoietic progenitor cells, dysregulation of MYB has been implicated in the pathogenesis of leukemia. Although the transcription of MYB has been well studied, its detailed underlying regulatory mechanisms still remain elusive. Here, we detected the long-range interaction between the upstream regions, −34k and −88k, and the MYB promoter in K562, U937, and HL-60 cells using circularized chromosome conformation capture (4C) assay, which declined when MYB was downregulated during chemical-induced differentiation. The enrichment of enhancer markers, H3K4me1 and H3K27ac, and enhancer activity at the −34k and −88k regions were confirmed by ChIP-qPCR and luciferase assay respectively. ChIP-qPCR showed the dynamic binding of GATA1, TAL1, and CCAAT/enhancer-binding protein (C/EBPβ) at −34k and −88k during differentiation of K562 cells. Epigenome editing by a CRISPR-Cas9-based method showed that H3K27ac at −34k enhanced TF binding and MYB expression, while DNA methylation inhibited MYB expression. Taken together, our data revealed that enhancer elements at −34k are required for MYB expression, TF binding, and epigenetic modification are closely involved in this process in human myeloid leukemia cells.
Similar content being viewed by others
Introduction
The transcription factor MYB is a key regulator for hematopoiesis1,2. Dysregulation of MYB often associates with various hematological disorders including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoblastic leukemia (ALL)3,4,5. Aberrant expression of MYB has been also reported in malignant solid tumors including colon cancer, breast cancer, adenoid cystic carcinoma, and brain cancer6,7,8,9. Recurrent chromosomal translocation, genomic duplication, C-terminal truncation, and N-terminal truncation contribute to MYB have been reported in human leukemia10,11,12,13.
The expression of MYB is precisely regulated under physiological conditions. Previous studies indicated that MYB transcription is mainly regulated through a transcriptional attenuation site within the first intron14,15. miRNAs including miR-150 and miR-17-92 can target MYB mRNA in a stage-specific manner16,17. PU.1 negatively regulates the c-myb promoter during granulocytic differentiation18. MYB is also an essential downstream target of Hoxa9/Meis1 in hematopoietic cells19.
Recently increasing studies support that distal regulatory elements play vital roles in MYB regulation. Transgene insertion 77 kb upstream of c-myb markedly decreases c-myb expression in mouse20. Multiple distal regions 36, 61, 68, 81, and 109 kb upstream of c-myb are involved in c-myb regulation in erythroid differentiation in mouse21. Retroviral insertions upstream and downstream of c-myb lead to upregulation of c-myb in murine and feline cell lines22,23. And our previous study identified three murine leukemia virus integration regions (located at −25k, −56k, and −70k), which interact with c-myb through DNA looping and facilitate the integrated virus to activate c-myb expression in murine myeloid progenitor M1 cells23,24. We further demonstrated that the regulatory element at the −28k region has an essential role in c-myb regulation during IL-6 induced differentiation in M1 cells25. Distal enhancer elements have been also identified upstream and downstream of MYB in human. The −84k and −71k regions of MYB can regulate MYB and fetal hemoglobin in primary human erythroid progenitors (HEPs)26. An enhancer ~140 kb downstream of MYB was recently identified with improved experimental and computational parameters from single-cell enhancer screens27. Above observations support multiple distal elements regulate MYB in a cell-type-specific manner, however the detailed mechanisms of distal elements in MYB regulation in human leukemia cells remain to be elucidated.
Here, we showed the association of the −34k and −88k regions of MYB and the MYB promoter in human leukemia cells using circularized chromosome conformation capture (4C) assay. And these regions are enriched for enhancer characteristics including hallmark histone modifications, TF binding and enhancer activity in luciferase assay. Further study showed the dynamic DNA looping formation, TF binding, and epigenetic modifications at the −34k region are involved in MYB expression during differentiation of human myeloid leukemia cells.
Results
Long-range interaction between the MYB promoter and its upstream distal regions in human myeloid leukemia cells
To investigate the distal regulatory elements interacting with the MYB promoter, 4C assay was performed in human myeloid leukemia cells. K562, U937, and HL-60 cells all expressed high levels of MYB, while MYB was not detected in HeLa cells (Fig. 1A), the result is consistent with previous studies28,29. Using the MYB promoter as the bait fragment, 4C assay detected strong near-bait interactions on chromosome 6 in all three leukemia cell lines (K562, U937, and HL-60) except for HeLa cells, among which two prominent peaks of interactions were detected around −34k and −88k upstream of the MYB gene (Fig. 1B). The peaks located at the −34k and −88k regions indicate the physical interaction of these regions with the MYB promoter.
We further scanned the potential regulatory elements involved in intra-chromosomal interaction with the MYB promoter, and a Circos diagram was generated to show the 4C-seq contact map for cis interactions on chromosome 6 in all four cell lines (Fig. 1C). A dramatic difference in the abundance of long-range contacts along chromosome 6 was observed between leukemia cells and HeLa cells, much more MYB related intra-chromosomal interactions were detected in leukemia cells than in HeLa cells. Meanwhile, our data also showed the potential regulatory elements interacting with the MYB promoter at the genome-wide level, which showed a high interaction frequency based on the top 100 regions (supplementary Table 2). These observations suggest that multiple distal regions, especially those within the HBS1L-MYB intergenic region, interact with the MYB promoter via long-range DNA interaction.
The −34k and −88k regions of MYB are enriched for enhancer features
H3K4me1 and H3K27ac are two commonly used hallmarks to identify putative genome-wide enhancers30,31 (Fig. 2A). ChIP-seq data from ENCODE (Encyclopedia of DNA Elements) show strong enrichment of H3K4me1 and H3K27ac at −34k, while strong enrichment of only H3K4me1 was detected at −88k. The enrichment of H3K4me1 (Fig. 2B) and H3K27ac (Fig. 2C) at −34k and −88k was further confirmed using ChIP-qPCR. The DNA fragments representing H3K4me1 peaks, named −34ka (1034 bp), −34kb (1055 bp), and −88k (1388 bp), respectively (Fig. 2A), were cloned and inserted upstream of the MYB promoter controlling a firefly luciferase reporter gene (Fig. 2D), a −53k fragment without H3K4me1 enrichment was used as control. The constructs were transfected into HeLa (Fig. 2E) and K562 cells (Fig. 2F), respectively. Compared with the −53k fragment, the −34ka, −34kb, and −88k fragments all showed significantly increased luciferase activity (Fig. 2E, F). The −34kb fragment showed the highest enhancer activity. Taken together, these data indicate that these distal regions contain enhancers for MYB transcription.
Dynamic long-range interaction of the MYB locus during differentiation of human leukemia cells
We treated K562, U937 and HL-60 cells with hemin, 12-O-Tetradecanoylphorbol 13-acetate (TPA) or all-trans-retinoic acid (ATRA) to induce erythroid, monocytic and granulocytic differentiation, respectively. MYB mRNA and protein levels reduced remarkably after treatment in all three cell lines (Fig. 3A–C), which is consistent with previous studies that MYB is highly expressed in immature proliferating haematopoietic cells, and strongly downregulated during terminally differentiation32,33,34. 4C assay was subsequently carried out using the MYB promoter as the bait fragment (Fig. 3D–F). In untreated cells high frequency long-range interaction between the MYB promoter and distal regions was observed, however the frequency of most long-range interactions strikingly diminished upon differentiation, especially at the −34k and −88k regions. Meanwhile, the intra-chromosomal interactions with the MYB promoter significantly altered during differentiation in all tested cell lines (Supplemental Fig. 1A–C). The potential inter-chromosomal interaction with the MYB promoter also showed dramatic changes during differentiation in all tested cell lines (Supplementary Tables 3–5). This finding is consistent with previous study that gene-regulatory chromatin interactions were altered upon ATRA induction in HL-60 cells35. Thus, we concluded that downregulation of MYB upon differentiation is accompanied by a loss of communication between the MYB promoter and above distal enhancers.
Binding of transcription factors at distal enhancers of MYB during differentiation
We further investigated the roles of TFs at above enhancers in MYB regulation. Public ChIP-seq data of histone marks, DNase I hypersensitivity (DNase HS) and TF profiles were generated by the ENCODE project in the HBS1L-MYB region. DNase HS and the enrichment of GATA1, TAL1, C/EBPβ, c-Jun, and PU.1 were observed at the −34ka and/or −34kb regions (Fig. 4A). The enrichment of GATA1, TAL1, and c-Jun were observed at the −88k region (Fig. 4A). Strong enrichment of CTCF and Rad21, which participate in long-range chromatin interactions in the vicinity of 4C interaction sites, was observed mainly near the −34k region (Fig. 4A). The enrichment of GATA1, TAL1, and C/EBPβ was further confirmed by ChIP-qPCR in K562 cells (Supplemental Fig. 2A–C).
We investigated the binding of selected TFs at −34k and −88k regions during differentiation. Western blot showed that GATA1 decreased significantly, while TAL1, C/EBPβ, c-Jun, and PU.1 increased during hemin-induced differentiation in K562 cells (Fig. 4B). After hemin treatment, the binding of GATA1, TAL1, c-Jun decreased at −34kb, PU.1, and C/EBPβ binding increased at −34kb and promoter; while the GATA1 binding decreased and PU.1 binding increased at −88k (Fig. 4C–G).
To further assess the effect of TF binding to these distal regions, we determined the luciferase activity of the −34k enhancer constructs after GATA1 overexpression in 293T cells (Fig. 4H). Unexpectedly, a 3-fold increase of luciferase activity of the −34ka construct was observed after GATA1 overexpression, while only a moderate increase was observed for luciferase activity of the −34kb construct and the construct contains only the MYB promoter, indicating that GATA1 binding is required for enhancer activity of the −34k region. Meanwhile, we overexpressed and knocked down GATA1 in K562 cells via lentiviral transduction. Our results showed that overexpression of GATA1 increased MYB expression (Fig. 4I), while GATA1 knockdown reduced MYB expression (Fig. 4J), indicating GATA1 plays an important role in MYB expression, and corroborating a previous study that GATA1 overexpression leads to failure to repress MYB during erythroid differentiation of K562 cells36. Together, our data showed that TF binding at the −34k enhancer elements play a critical role in MYB expression in K562 cells.
Epigenetic modification of distal enhancers affects MYB expression
Epigenetic modification can affect enhancer activity, probably via regulation of chromatin structure and TF binding37. To test the effect of epigenetic modification on the function of above enhancers, here we applied a dCas9-based epigenome editing method38,39. After co-transfection into K562 cells, a dCas9p300 Core fusion protein with a Flag tag was co-expressed with 2 gRNAs38, which target one specific site, then MYB expression was examined (Fig. 5A). As shown in Fig. 5B, dCas9p300 Core was expressed in K562 cells after transfection. ChIP-qPCR using an antibody against the Flag epitope showed that dCas9p300 Core was recruited to the targeted sites by the gRNAs (Fig. 5C) and upregulated H3K27ac level at these sites specifically (Fig. 5D). And MYB expression was increased by elevated H3K27ac at the promoter, −34k and −88k regions (Fig. 5E), indicating that H3K27ac at these sites upregulates MYB expression.
Meanwhile, the effect of local DNA methylation at these sites on MYB transcription was also investigated using a previously reported dCas9DNMT3A fusion protein40. Figure 5F showed that dCas9DNMT3A was expressed in K562 cells after transfection. ChIP-qPCR using a Flag epitope antibody showed that dCas9DNMT3A was recruited to the targeted sites by the gRNAs (Fig. 5G), and upregulated 5-methylcytosine (5-mC) level at these sites specifically (Fig. 5H). And MYB expression was decreased by elevated 5-mC level at the promoter, −34k and −88k regions (Fig. 5I), indicating that DNA methylation can inhibit the function of these DNA elements.
Epigenetic modification of distal enhancers affects TFs binding and cell differentiation
To further understand the roles of epigenetic modification at above sites in MYB regulation, we examined the effect of H3K27ac on TF binding and cell differentiation in K562 cells. ChIP-qPCR showed that H3K27ac enrichment significantly reduced at the MYB promoter, −34k and −88k enhancer regions during hemin induced differentiation (Fig. 6A). dCas9p300 Core targeting enhanced GATA1 binding at promoter, −34k and −88k (Fig. 6B), while dCas9p300 Core targeting enhanced TAL1 binding at promoter and −34k (Fig. 6C), indicating that H3K27ac helped binding of these two TFs at above DNA elements. Furthermore, dCas9p300 Core targeting at −34k could counter downregulation of MYB during early stages of hemin treatment (Fig. 6D), but failed to efficiently block hemin-induced repression of MYB eventually. As represented in our model (Fig. 6E), these data suggest that epigenetic modification at the MYB distal enhancers could affect TF binding and MYB expression during differentiation in K562 cells.
Discussion
Mounting evidence indicates that the MYB gene is under regulation by distal enhancers. Multiple distal upstream and downstream regulatory elements have been reported from −25k ∼−250k regions of c-myb in mouse, which are involved in c-myb regulation in erythroid differentiation and leukemogenesis20,21,22,23,24,25. In human cells, distal enhancer elements have been identified at regions 84 kb, 71 kb upstream, and 140 kb downstream of MYB26,27. Above studies indicate that the MYB locus is under control of a complex regulatory network involving multiple upstream and downstream regulatory elements. However, how these enhancers coordinate in MYB regulation is unclear so far.
Using 4C assay showed that the −34k, −88k upstream regions interact with the MYB promoter, and binding sites of CTCF and Rad21 were identified near the −34k, −88k regions using public ChIP-seq data. The enhancer activity of the −34k and −88k regions was further confirmed by enrichment of H3K4me1 and H3K27ac and luciferase reporter assay (Figs. 1 and 2). Meanwhile, the binding of TFs (GATA1, TAL1, C/EBPβ, c-Jun, and PU.1) at −34k and −88k during differentiation was confirmed by ChIP-qPCR. Above data showed that the enhancer elements at the −34k, −88k regions interact with the MYB promoter in human K562, U937, and HL-60 leukemia cells. However, in human erythroid progenitors, long-range interactions with the MYB promoter were detected at multiple upstream sites ranging from −63k to −92k, which affect MYB expression26. Above evidence indicates that long-range interactions between distal enhancers and the MYB promoter may perform in a cell-specific manner.
By further analysis of the 4C-assay data, additional long-range interaction with the MYB promoter in K562, U937, and HL-60 cells was detected from more sites, including −107k and +140k regions (Supplementary Table 6), along chromosome 6 at much lower frequency than the −34k and −88k regions. Our results are consistent with a recent report that an enhancer ~140k downstream of MYB can regulate MYB expression in K562 cells via DNA-looping27, supporting that the enhancer at +140k is required for MYB regulation. Meanwhile, many other intrachromosome and interchromosome sites showed interaction with the MYB promoter in our analysis, the potential roles of these interactions are unknown so far.
Distal enhancers contribute to the activation of gene transcription via conformational loops that bring them physically close to gene promoters41. Lineage-specific dynamic and enhancer–promoter contacts cooperate in terminal differentiation37. We observed a loss of long-range interaction frequency from −34k and −88k during differentiation of human leukemia cells, accompanied by MYB downregulation. And a significant decline of long-range interaction from other intrachromosome and interchromosome sites was also observed. The loss of chromatin looping between distal enhancers and the c-myb promoter during differentiation was also reported in MEL cells21. Above data showed the dynamic long-range interaction between the MYB promoter and the −34/−88k regions during hemin induced differentiation in K562 cells.
TFs are required for enhancer function and involved in establishing and stabilizing long-range chromatin interactions42. We first showed that GATA1 binds to the −34k and −88k enhancer regions and upregulates MYB expression (Fig. 4). GATA1 is considered as the “master” transcription factor in erythropoiesis43,44. And it has been reported that GATA1 and TAL1 bind to the −71k and −84k enhancer regions of the MYB locus and positively regulate MYB expression along erythroid cell differentiation26. c-Jun binding at the promoter and −34k regions decreased during differentiation, our results support the idea that c-Jun principally binds to distal enhancers, and promoters and is considered a pioneer factor in modulating chromatin structure of distal enhancers in K562 cells45. PU.1 has been reported to suppress MYB expression through direct binding to the MYB promoter and recruitment corepressors HDAC1 and/or DNMT3a/b46. Here, we found that PU.1 binding at the promoter and −34k regions increased during differentiation, indicating that PU.1 in the MYB enhancer regions can also downregulate MYB expression. Furthermore, C/EBPβ binding at the promoter and −34k regions increased during differentiation, and the expression of C/EBPβ was indeed changed during myeloid differentiation. C/EBPβ acts as a transcription repressor for genes of liver proliferation47. Further studies will be required to determine whether C/EBPβ has a specific function in MYB control during myeloid differentiation.
Recent studies typically suggest that active enhancers display lower 5-mC levels than poised or silent enhancers, along with TF binding as well as the presence of active histone marks H3K4me1 and H3K27ac48,49. Here, we showed that the induction of histone acetylation at the −34k and −88k regions enhanced MYB transcription and TF binding (Figs. 5 and 6). Conversely, DNA methylation at the −34k and −88k regions leads to downregulation of MYB. Our results coincide with that hypermethylation at enhancers is generally associated with reduced chromatin accessibility and decreased TFs binding50. DNA methylation of enhancers can influence cell-type specific gene expression, and regulate relevant genes in acute myeloid leukemia and chronic myeloproliferative neoplasms51. And histone acetylation is required for enhancer function52. Cooperation of TFs with epigenetic modifications of chromatin contributes to the activation of regulatory elements including promoters and enhancers, TFs must gain access to their binding sites, and binding of TFs also modify the chromatin landscape53. Meanwhile, enhanced histone acetylation at the −34k region alone only delayed but failed to block hemin-induced repression of MYB (Fig. 6D), which is consistent with the relatively minor decrease in H3K27ac at this region upon hemin-induced differentiation (Fig. 6A), indicating that H3K27ac at this region is of secondary importance in hemin induced downregulation of MYB.
In the present study, we identified enhancer element at the −34k and −88k regions, which play roles in regulation of MYB in human leukemia cells. Our data will help understanding the mechanisms of regulation/dysregulation of MYB under physiological and pathological conditions in human.
Materials and methods
Cell culture and treatment
K562 (CCL-243, ATCC, Manassas, VA), U937 (CRL-1593.2, ATCC), and HL-60 (CCL-240, ATCC) cells were maintained in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (10099141, Gibco, Auckland, New Zealand). HeLa cell line (CCL-2, ATCC) was cultured in DMEM (10569044, Gibco, USA) supplemented with 10% FBS. Authentication of these cell lines were conducted by short tandem repeat (STR) markers, and no mycoplasma contamination was detected. All cell lines were supplemented with 1% penicillin-streptomycin-glutamine solution (SV30082.01, Hyclone, Utah, USA) and cultured at 37 °C in a humidified atmosphere containing 5% CO2. For treatment, K562, U937, or HL-60 cells were seeded at a density of 1 × 105 cells/ml, then cultured for 72 h with 30 µM Hemin (51280, Sigma-Aldrich, Missouri, USA), 2 µM 12-O-Tetradecanoylphorbol 13-acetate (TPA) (S1819, Beyotime, Shanghai, China) or 0.16 µM all-trans-retinoic acid (ATRA) (R2625, Sigma-Aldrich) to induce erythroid, granulocytic, and monocytic differentiation, respectively.
Antibodies
The following antibodies were used in this study: anti-H3K4me1 (ab8895, Abcam), anti-H3K27ac (ab4729, Abcam), anti-5-methylcytosine (ab10805, Abcam), anti-GATA1 (ab11852, Abcam), anti-TAL1 (C-4) (sc-393287X, Santa Cruz), anti-CEBP/β (ab15050, Abcam), anti-c-Jun (G-4) (sc-74543X, Santa Cruz), anti-PU.1 (B-9) (sc-390659X, Santa Cruz), and anti-Flag (M2) (F1804, Sigma-Aldrich). Anti-mouse IgG HRP-linked antibodies (G-21040, Invitrogen) and anti-rabbit IgG HRP-linked antibodies (G-21234, Invitrogen).
Plasmid construction for expression of dCas9-effector proteins and gRNAs
The constructs, pcDNA-dCas9p300 Core (61357, Addgene)38 pcDNA-dCas9p300 Core (D1399Y) (61358, Addgene)38, pdCas9DNMT3A (71666, Addgene)39, and pdCas9DNMT3A (ANV) (71685, Addgene)39 were from Addgene. gRNAs targeting the MYB promoter and enhancer regions were designed using Feng Zhang lab’s Target Finder software (http://crispr.mit.edu). Best guides, with highest score of the inversed likelihood of off-target binding, were selected, and the gRNA sequences are shown in supplementary Table 1. Expression plasmids for gRNAs were constructed by cloning annealed oligos into pSPgRNA (#47108 Addgene)54 using BbsI (R0539, NEB) and T4 ligase (M0202, NEB). Then these plasmids were transfected into K562 cells using Lipofectamine 3000 (L3000015, Invitrogen) according to the manufacturer’s instructions. Forty-eight hours later, cells were harvested for analysis.
Lentivirus production and cell transduction
For creation of the shGATA1 vectors, oligonucleotides for shGATA1 (sequences are shown in supplementary Table 1) were annealed and ligated into the digested pLKO.1-puro vector at the EcoR I and AgeI sites. For creation of the GATA1 overexpression vector, GATA1 PCR product was cloned into pLVX-IRES-NEO at the EcoRI and XbaI sites. In all, packaging plasmids pCMV-VSVG, pCMV-DR8.91 and the relevant lentiviral transfer vectors in a 3:8:10 mass ratio were cotransfected into 293T cells using TurboFect Transfection Reagent (R0531, ThermoFisher). The media containing lentivirus particles were collected after 48 h. And used to infect K562 cells immediately in the presence of 8 μg/ml hexadimethrine bromide (H9268, Sigma-Aldrich). Cells were collected for analysis after 72 h.
Quantitative real-time PCR analysis
Total RNA was isolated using TRIzol reagent (15596-018, Invitrogen). And 1 μg of total RNA was reverse-transcribed with PrimeScript™ RT reagent Kit with gDNA Eraser (RR047A, TaKaRa, Beijing, China). The levels of specific RNAs were measured using a Light Cycler 480II real-time PCR machine and the iTaq™ Universal SYBR Green Supermix (1725124, Bio-Rad, Hercules, CA, USA) according to the manufacturer instructions. All samples were assayed in triplicate. Data were normalized to a human Gapdh (glyceraldehyde-3-phosphate dehydrogenase) control. Relative quantitation was carried out by the comparative threshold cycle (CT) method. Statistical analysis was performed using the GraphPad Prism 8 software. The primer sequences are listed in supplementary Table 1.
Western blot analysis
Western blot analysis was performed as previously described25. Proteins were isolated from cells and protein concentration was determined by a bicinchoninic acid (BCA) assay kit (P0012, Beyotime). Equal amounts of proteins (30 μg) were electrophoresed in 8% SDS-polyacrylamide gels and transferred to nitrocellulose. The membrane was blocked in PBS containing 5% milk powder and 0.1% Tween 20 and incubated at 4 °C overnight with primary antibody and for 1 h at 25 °C with horseradish peroxidase-conjugated secondary antibody. Antibody binding was visualized using Clarity Western ECL Substrate (1705060, Bio-Rad).
Chromatin immunoprecipitation (ChIP)
ChIP experiments were performed as previously described24. In brief, 1 × 107 cells were fixed in 1% formaldehyde for 10 min at room temperature and sonicated to shear the chromatin. Immunoprecipitation of crosslinked chromatin was performed overnight at 4 °C with antibodies. An equal amount of isotype immunoglobulin G (IgG) was used as background control. Primers for ChIP-qPCR are shown in supplementary Table 1.
Circularized chromosome conformation capture (4 C) assay
4C assay was performed as previously described with minor modification25. In brief, 5 × 106 cells were cross-linked by 1% formaldehyde for 10 min at RT, and 0.125 M glycine was added to prevent further cross-linking. Cells were collected and washed twice with PBS, then suspended in lysis buffer to disrupt membranes and isolate chromatin. HindIII (400 U) was used for the first digestion at 37 °C overnight with shaking followed by diluted ligations. After precipitation, chromatin was further subjected to a second round of digestions with a 4-base cutter DpnII and ligation. Primers for the MYB viewpoint (Forward: 5′-AGTATTAATTTGCCTTGTCC-3′; Reverse: 5′-GCTAATGTTGGATATATTGC-3′) were designed. Inverse polymerase chain reaction (PCR) was carried out to amplify sample libraries. Multiplexed sequencing was performed on the HiSeq2500 platform. 4C-seq data were analyzed visualization via an R package 4C-ker55. Reads were mapped to a reduced genome of unique 29 bp sequences flanking HindIII sites in the hg19 genome.
Dual-luciferase reporter assay
The MYB promoter (chr6:135 501 805–135 502 522, hg19) was amplified, digested with XhoI and BglII and cloned into the pGL4 luciferase reporter vector (Promega). The upstream regions −34ka (chr6:135 467 317-135 468 351, hg19), −34kb (chr6:135 468 624-135 469 679, hg19), −53k (chr6:135 448 094-135 448 922, hg19) and -88k (chr6:135 414 242-135 415 630, hg19) were amplified and cloned into pGL4.10-MYB-promoter mentioned above via KpnI/NheI digestion. Then these reporter vectors were transfected into K562 or HeLa cells using Lipofectamine 3000 (L3000015, Invitrogen) according to the manufacturer’s instructions. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (E1960, Promega) on a FlexStation 3 multi-mode microplate reader. All assays were performed in triplicate and repeated at least three times.
Bioinformatics and statistical analysis
The ChIP-seq datasets were obtained from the ENCODE project were visualized with the WashU Epigenome Browser (https://epigenomegateway.wustl.edu/)56. 4C-seq data were analyzed via the R package 4C-ker (https://github.com/rr1859/R.4Cker)55. Statistical significance (P < 0.05) for RT-qPCR, ChIP-qPCR, and luciferase reporter assay experiments was assessed by Student’s two-tailed t-test. Data were obtained from at least three independent experiments and are expressed as the means ± standard deviation (SD).
Data availability
The data used in this study has been deposited in NCBI’s Gene Expression Omnibus repository and are accessible through GEO accession number GSE140321.
References
Ramsay, R. G. & Gonda, T. J. MYB function in normal and cancer cells. Nat. Rev. Cancer 8, 523–534 (2008).
Greig, K. T., Carotta, S. & Nutt, S. L. Critical roles for c-Myb in hematopoietic progenitor cells. Semin. Immunol. 20, 247–256 (2008).
Nguyen, N. et al. Myb expression is critical for myeloid leukemia development induced by Setbp1 activation. Oncotarget 7, 86300–86312 (2016).
Negi, V. et al. Hoxa9 and Hoxa10 induce CML myeloid blast crisis development through activation of Myb expression. Oncotarget 8, 98853–98864 (2017).
Nakano, K., Uchimaru, K., Utsunomiya, A., Yamaguchi, K. & Watanabe, T. Dysregulation of c-Myb pathway by aberrant expression of proto-oncogene MYB provides the basis for malignancy in adult T-cell leukemia/lymphoma cells. Clin. Cancer Res. 22, 5915–5928 (2016).
Qu, X. et al. c-Myb promotes growth and metastasis of colorectal cancer through c-fos-induced epithelial-mesenchymal transition. Cancer Sci. 110, 3183–3196 (2019).
Li, Y. et al. c-Myb enhances breast cancer invasion and metastasis through the Wnt/beta-Catenin/Axin2 pathway. Cancer Res. 76, 3364–3375 (2016).
Drier, Y. et al. An oncogenic MYB feedback loop drives alternate cell fates in adenoid cystic carcinoma. Nat. Genet. 48, 265–272 (2016).
Zhang, J. et al. Whole-genome sequencing identifies genetic alterations in pediatric low-grade gliomas. Nat. Genet. 45, 602–612 (2013).
Clappier, E. et al. The C-MYB locus is involved in chromosomal translocation and genomic duplications in human T-cell acute leukemia (T-ALL), the translocation defining a new T-ALL subtype in very young children. Blood 110, 1251–1261 (2007).
Lahortiga, I. et al. Duplication of the MYB oncogene in T cell acute lymphoblastic leukemia. Nat. Genet. 39, 593–595 (2007).
Tomita, A. et al. Truncated c-Myb expression in the human leukemia cell line TK-6. Leukemia 12, 1422–1429 (1998).
Frerich, C. A. et al. N-terminal truncated Myb with new transcriptional activity produced through use of an alternative MYB promoter in salivary gland adenoid cystic carcinoma. Cancers 12, 45 (2019).
Hugo, H. et al. Mutations in the MYB intron I regulatory sequence increase transcription in colon cancers. Genes Chromosomes Cancer 45, 1143–1154 (2006).
Drabsch, Y. et al. Mechanism of and requirement for estrogen-regulated MYB expression in estrogen-receptor-positive breast cancer cells. Proc. Natl Acad. Sci. USA 104, 13762–13767 (2007).
Xiao, C. et al. MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb. Cell 131, 146–159 (2007).
Spagnuolo, M. et al. Transcriptional activation of the miR-17-92 cluster is involved in the growth-promoting effects of MYB in human Ph-positive leukemia cells. Haematologica 104, 82–92 (2019).
Bellon, T., Perrotti, D. & Calabretta, B. Granulocytic differentiation of normal hematopoietic precursor cells induced by transcription factor PU.1 correlates with negative regulation of the c-myb promoter. Blood 90, 1828–1839 (1997).
Hess, J. L. et al. c-Myb is an essential downstream target for homeobox-mediated transformation of hematopoietic cells. Blood 108, 297–304 (2006).
Mukai, H. Y. et al. Transgene insertion in proximity to the c-myb gene disrupts erythroid-megakaryocytic lineage bifurcation. Mol. Cell Biol. 26, 7953–7965 (2006).
Stadhouders, R. et al. Dynamic long-range chromatin interactions control Myb proto-oncogene transcription during erythroid development. EMBO J. 31, 986–999 (2012).
Hanlon, L. et al. Long-range effects of retroviral insertion on c-myb: overexpression may be obscured by silencing during tumor growth in vitro. J. Virol. 77, 1059–1068 (2003).
Haviernik, P. et al. Linkage on chromosome 10 of several murine retroviral integration loci associated with leukaemia. J. Gen. Virol. 83, 819–827 (2002).
Zhang, J., Markus, J., Bies, J., Paul, T. & Wolff, L. Three murine leukemia virus integration regions within 100 kilobases upstream of c-myb are proximal to the 5’ regulatory region of the gene through DNA looping. J. Virol. 86, 10524–10532 (2012).
Zhang, J. et al. Distal regulation of c-myb expression during IL-6-induced differentiation in murine myeloid progenitor M1 cells. Cell Death Dis. 7, e2364 (2016).
Stadhouders, R. et al. HBS1L-MYB intergenic variants modulate fetal hemoglobin via long-range MYB enhancers. J. Clin. Invest. 124, 1699–1710 (2014).
Xie, S., Armendariz, D., Zhou, P., Duan, J. & Hon, G. C. Global analysis of enhancer targets reveals convergent enhancer-driven regulatory modules. Cell Rep. 29, 2570–2578 (2019).
Srutova, K., Curik, N., Burda, P., Savvulidi, F., Silvestri, G. & Trotta, R. et al. BCR-ABL1 mediated miR-150 downregulation through MYC contributed to myeloid differentiation block and drug resistance in chronic myeloid leukemia. Haematologica 103, 2016–2025 (2018).
Ye, P., Zhao, L., McGirr, C. & Gonda, T. J. MYB down-regulation enhances sensitivity of U937 myeloid leukemia cells to the histone deacetylase inhibitor LBH589 in vitro and in vivo. Cancer Lett. 343, 98–106 (2014).
Buecker, C. & Wysocka, J. Enhancers as information integration hubs in development: lessons from genomics. Trends Genet. 28, 276–284 (2012).
Wang, A. et al. Epigenetic priming of enhancers predicts developmental competence of hESC-derived endodermal lineage intermediates. Cell Stem Cell 16, 386–399 (2015).
Emambokus, N. et al. Progression through key stages of haemopoiesis is dependent on distinct threshold levels of c-Myb. EMBO J. 22, 4478–4488 (2003).
Fuglerud, B. M. et al. A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function. Nucleic Acids Res. 45, 7681–7696 (2017).
Zhao, L., Ye, P. & Gonda, T. J. The MYB proto-oncogene suppresses monocytic differentiation of acute myeloid leukemia cells via transcriptional activation of its target gene GFI1. Oncogene 33, 4442–4449 (2014).
Li, Y. et al. Alterations of specific chromatin conformation affect ATRA-induced leukemia cell differentiation. Cell Death Dis. 9, 200 (2018).
Halsey, C. et al. The GATA1s isoform is normally down-regulated during terminal haematopoietic differentiation and over-expression leads to failure to repress MYB, CCND2 and SKI during erythroid differentiation of K562 cells. J. Hematol. Oncol. 5, 45 (2012).
Rubin, A. J. et al. Lineage-specific dynamic and pre-established enhancer-promoter contacts cooperate in terminal differentiation. Nat. Genet. 49, 1522–1528 (2017).
Hilton, I. B. et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–U225 (2015).
Vojta, A. et al. Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Nucleic Acids Res. 44, 5615–5628 (2016).
Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247 (2016).
Heintzman, N. D. et al. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459, 108–112 (2009).
van den Heuvel, A., Stadhouders, R., Andrieu-Soler, C., Grosveld, F. & Soler, E. Long-range gene regulation and novel therapeutic applications. Blood 125, 1521–1525 (2015).
de Thonel, A. et al. HSP27 controls GATA-1 protein level during erythroid cell differentiation. Blood 116, 85–96 (2010).
Gutierrez, L., Caballero, N., Fernandez-Calleja, L., Karkoulia, E. & Strouboulis, J. Regulation of GATA1 levels in erythropoiesis. IUBMB Life 72, 89–105 (2020).
Bejjani, F., Evanno, E., Zibara, K., Piechaczyk, M. & Jariel-Encontre, I. The AP-1 transcriptional complex: local switch or remote command? Biochim Biophys. Acta 11-23, 2019 (1872).
Shinichiro, T. in Myeloid Leukemia—Basic Mechanisms of Leukemogenesis 1 edn (eds. Koschmieder, S. & Krug, U.) 239–262 (IntechOpen, 2011).
Jin, J. et al. Cooperation of C/EBP family proteins and chromatin remodeling proteins is essential for termination of liver regeneration. Hepatology 61, 315–325 (2015).
Angeloni, A. & Bogdanovic, O. Enhancer DNA methylation: implications for gene regulation. Essays Biochem. 63, 707–715 (2019).
Shlyueva, D., Stampfel, G. & Stark, A. Transcriptional enhancers: from properties to genome-wide predictions. Nat. Rev. Genet. 15, 272–286 (2014).
Luo, C., Hajkova, P. & Ecker, J. R. Dynamic DNA methylation: In the right place at the right time. Science 361, 1336–1340 (2018).
Ordonez, R., Martinez-Calle, N., Agirre, X. & Prosper, F. DNA methylation of enhancer elements in myeloid neoplasms: think outside the promoters? Cancers 11, 1424 (2019).
Pradeepa, M. M. Causal role of histone acetylations in enhancer function. Transcription 8, 40–47 (2017).
Brettingham-Moore, K. H., Taberlay, P. C. & Holloway, A. F. Interplay between transcription factors and the epigenome: insight from the role of RUNX1 in leukemia. Front. Immunol. 6, 499 (2015).
Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).
Raviram, R. et al. 4C-ker: a method to reproducibly identify genome-wide interactions captured by 4C-Seq experiments. PLoS Comput. Biol. 12, e1004780 (2016).
Li, D., Hsu, S., Purushotham, D., Sears, R. L. & Wang, T. WashU epigenome browser update 2019. Nucleic Acids Res. 47, W158–W165 (2019).
Funding
This study was funded by the National Natural Science Foundation of China (81770165 to B.H., and 31372516 to J.Z.). The work was also supported by the First Class Discipline Program for Fishery from the Shanghai municipal government.
Author information
Authors and Affiliations
Contributions
B.H. and J.Z. conceived the study and designed the experiments. B.H., J.Z., M.L., and P.J. performed development of methodology and writing, review and revision of the paper. M.L., P.J., K.C., Z.Z., S.L., X.L., L.Z., and Y.W. provided acquisition, analysis and interpretation of data, and statistical analysis. J.C., T.J., and X.W. provided technical and material support. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests.
Ethics approval and consent to participate
No ethical approval was required for this study.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Edited by M. Diederich
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Li, M., Jiang, P., Cheng, K. et al. Regulation of MYB by distal enhancer elements in human myeloid leukemia. Cell Death Dis 12, 223 (2021). https://doi.org/10.1038/s41419-021-03515-z
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41419-021-03515-z
- Springer Nature Limited
This article is cited by
-
Enhancer engagement sustains oncogenic transformation and progression of B-cell precursor acute lymphoblastic leukemia
Journal of Experimental & Clinical Cancer Research (2024)
-
BRD9 determines the cell fate of hematopoietic stem cells by regulating chromatin state
Nature Communications (2023)
-
Genetic variation in CSF2 (5q31.1) is associated with longitudinal susceptibility to pediatric malaria, severe malarial anemia, and all-cause mortality in a high-burden malaria and HIV region of Kenya
Tropical Medicine and Health (2022)