Background
MLL leukemia, which originates from a rearrangement of the mixed-lineage leukemia (
MLL) gene by 11q23 translocation, is one of the most aggressive subtypes of acute leukemia with approximate 70% of infant leukemia patients and 7–10% of adult cases [
1,
2]. Strong self-renewal capacity of this aggressive disease is generally deemed to be an important factor that results in limited treatment options and poor survival rates [
3‐
7]. Recently, the
MLL gene can recombine with more than 60 partners to acquire abnormal gain-of-function effects on aberrant epigenetic modification and proto-oncogene activation, as a result triggering dysregulated increased expression of the clustered homeobox A (
HOXA) genes, which results in limitless self-renewal capacity necessary for leukemia initiation and propagation [
3]. Hematopoietic malignancy caused by
MLL translocation, especially
MLL-AF9, has been reported to have an apex self-renewal to generate more distinct self-renewing progenitor-like leukemia cells that are resistant to chemotherapy and are responsible for relapse [
6,
8‐
10]. Persistence of
MLL leukemia clones after treatment including available cell cycle-based or signaling protein-targeted therapies is the critical factor for unsatisfactory treatment outcome [
11,
12]. Thus, understanding the mechanism of how
MLL leukemia cells to promote self-renewal and block differentiation could provide novel therapeutic strategies.
Ectopic and higher order H3K79 methylation, which triggers dysregulated activation of MLL fusion protein targets such as the
HOXA genes, is believed to play an essential role in leukemogenesis and propagation of primary
MLL leukemias [
13,
14]. DOT1L is the only known histone methyltransferase that catalyzes histone 3 lysine 79 monomethylation (H3K79me1), dimethylation (H3K79me2), and trimethylation (H3K79me3), and higher degrees of H3K79 methylation have been indicated to be associated with elevated gene expression [
15,
16]. Recent studies have demonstrated that the functions of DOT1L are regulated on multiple levels by the recruitment or blocking effector molecules, and misdirected localization and/or enhanced methyltransferase activity of DOT1L could lead to higher order H3K79 methylation and activation of MLL fusion protein target genes [
14]. Thus, the identification and understanding of specific regulators of DOT1L in
MLL leukemia could provide a precise target to block
MLL leukemia cell stemness. However, little is known about the potential regulatory molecules that mediate the chromatin-modifying activity of DOT1L.
lncRNAs have been demonstrated to be key regulators in cellular metabolism processes, such as stemness maintenance [
17‐
20], through diverse functions, including affecting chromatin epigenetic modification, protein complex constitution, and protein translation or degradation [
18,
19,
21‐
23]. Remarkably, recent evidence indicates that lncRNAs could direct the development of hematopoiesis and leukemia. For instance, mouse lncRNA Spehd influences HSPC fate by participating in the oxidative phosphorylation pathway [
24]. H19 lncRNA is pivotal for the development of embryonic hematopoietic stem cells [
17]. LncRNA DANCR is upregulated in leukemia stem cells (LSCs), and its knockdown results in the decreased self-renewal of LSCs [
25]. However, whether lncRNAs could specifically play regulatory roles in the balance between self-renewal and differentiation in
MLL leukemia or serve as effector molecules of DOT1L to affect the abnormal activation of
HOXA genes has not been reported yet.
We previously carried out a transcriptome microarray analysis of patient samples in the context of either
MLL rearrangements or wild-type
MLL (
MLL-wt) and identified a distinct set of lncRNAs associated with
MLL leukemia progression [
26]. Specifically, we found that lncRNA LAMP5-AS1 showed the most significant difference between the two groups and prominently high expression in
MLL leukemia patients. In this study, we reported that LAMP5-AS1 is necessary for cell self-renewal in
MLL leukemia in view of its capacity to enhance the methyltransferase activity of DOT1L, which facilitates H3K79me2 and H3K79me3 modifications on the locus of the
HOXA genes to upregulate their expression. LAMP5-AS1 knockdown remarkably inhibits the self-renewal capacity and promotes differentiation of
MLL leukemia cells both in vivo and in vitro. We demonstrated that LAMP5-AS1 is crucial for the regulation of self-renewal in
MLL leukemia and may be a valuable therapeutic target in this subtype of leukemia.
Methods
Leukemia patient samples
The clinical leukemia samples were obtained at the time of diagnosis or relapse and with informed consent from the first Affiliated Hospital of Sun Yat-sen University. Sample collection was approved by the Hospital’s Protection of Human Subjects Committee. The detail clinicopathological characteristics of the patients were summarized in Additional file
1: Table S1 and 2. The leukemia samples were stored in liquid nitrogen until used.
Cell culture
Human
MLL leukemia cells MOLM13, THP1, MV4-11, RS4-11, and HEK293T cells were purchased from American Type Culture Collection (ATCC, USA). MOLM13, THP1, and RS4-11 were cultured in RPMI-1640 medium (HyClone, USA); MV4-11 cells were cultured in IMDM (HyClone), and HEK 293T (ATCC) were cultured in DMEM (Gibco) supplemented with 10% FBS (HyClone) at 37 °C in a 5% CO
2 atmosphere. The primary cells were from the patients with
MLL leukemia and cultured in IMDM (HyClone) supplemented with 20% FBS [
27,
28]. Primary CD34+ blasts cells sorted from
MLL leukemia patients were either cultured in IMDM medium supplemented with 20% FBS and 10 ng/mL of SCF, TPO, Flt-3 L, IL-3, and IL-6 [
29].
Xenotransplantation experiments
Five-week-old NOD-SCID mice were maintained under specific pathogen-free conditions in the Laboratory Animal Center of Sun Yat-sen University. All experiments on animals were performed according to the institutional ethical guidelines for animal experiments. MOLM13 cells transduced with control or knockdown lentivirus (GFP+ cell populations) were tail vein injected into the mice (5 × 106 cells in 150 μL PBS per mouse). Three weeks after inoculation, xenografted mice were sacrificed for analysis. Human cell engraftment (GFP+ cell populations) in the bone marrow, peripheral blood, liver, and spleen was evaluated by flow cytometry or hematoxylin and eosin (H&E) staining performed as described. The remaining mice were performed the survival assay.
RNA electrophoretic mobility shift assays (EMSA)
The biotin-labeled RNA probes of variant LAMP5-AS1 truncated mutants were generated using the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher, USA) according to the manufacturer’ s guidelines. Recombinant DOT1L (residues 1-416aa) protein was mixed with variant RNA probes in the EMSA binding buffer (10 mM HEPES, pH 7.3, 20 mM KCl, 1 mM MgCl2, 1 mM DTT, 5% glycerol, 100 μg/mL transfer RNA). The reaction systems were incubated at room temperature for 30 min. Then, the complexes were separated using non-denaturing polyacrylamide gel electrophoresis. Biotinylated RNA was measured in the blots with a chemiluminescent EMSA Kit (Beyotime, China).
Histone methyltransferase assay
Histone methyltransferase assay was performed as previously described [
30]. Oligonucleosomes purified from HeLa cells were purchased from Millipore. Briefly, recombinant DOT1L (residues 1-416aa) were incubated with oligonucleosomes in the reaction buffer (50 mM NaCl, 50 mM Tris-HCl, pH 8.5, 5 mM MgCl
2, 1 mM DTT, and 10 μM SAM) at 30 °C for 1 h. Additional LAMP5-AS1 with different concentration was added to the reactions. The reaction systems were supplemented with 1× SDS loading buffer and were boiled at 100 °C for 10 min. The proteins were separated in a 12% SDS-PAGE gel, and H3K79me2 and H3K79me3 were assessed by immunoblotting. H3 was served as a control.
Culture and transduction of primary leukemia cells
Cells were thawed in a 37 °C water bath, washed once with Iscove’s modified Dulbecco medium (IMDM) containing 20% fetal bovine serum (FBS). Then, the cells were culture in IMDM supplemented with 20% FBS. CD34+ leukemia stem/progenitor cells were purified using human CD34 MicroBead Kit (Miltenyi Biotec, Auburn, CA) and fluorescence-activated cell sorting (FACS). The transient transfections of primary leukemia cells were performed using the Neon Transfection System with 10 μL reactions according to the manufacturer’s guidelines (Invitrogen, USA).
Plasmid construction
The wild-type DOT1L-CDS-full length and DOT1L truncated mutants (1-416aa, 417-822aa, 1-822aa, and 823-1537aa) were PCR-amplified from THP1 cDNA using the primers shown in Additional file
1: Table S3. Then, the PCR product was purified and cloned into pCDH-CMV-MCS-EF1-Puro eukaryotic expression vector (pCDH-DOT1L) which has be reconstructed with a FLAG-tag fusion at the N terminal and then derived a FLAG-DOT1L and truncated mutants fusion protein once they expressed. Similarly, the DOT1L (1-416aa) truncated mutants (1-416aa, 1-390aa, 1-407aa, 360-416aa, 1-416aa deletion 390-407) were also PCR-amplified from THP1 cDNA using the primers shown in Additional file
1: Table S3, and then were purified and cloned into pcDNA3.1 eukaryotic expression vector which has been reconstructed with a HA-tag fusion at the N terminal and then derived a HA-DOT1L truncated mutant fusion protein once they expressed.
For purification of 1-416aa DOT1L in vitro, corresponding cDNA fragment of the 1-416aa DOT1L was amplified by PCR and then the cDNA fragment was cloned into a prokaryotic expression vector pET-N-GST-Thrombin-C-His that contains an N-terminal fusion of a GST tag.
For stable LAMP5-AS1 knockdown vectors, the DNA oligos encoding shRNAs targeting LAMP5-AS1 (sh-LAMP5-AS1-1 and sh-LAMP5-AS1-2) were synthesized and cloned into the pGreenPuro™ eukaryotic expression vector, with sh-NC as the negative control (Additional file
1: Table S4).
Lentiviral preparation and infection
Lentivirus carrying shRNAs was made in the 60-mm culture dish by transfecting packaging cell HEK293T with Lentivector Expression Systems (System Biosciences, Germany) consisting of pPACKH1-GAG, pPACKH1-REV, and pVSV-G. Virus was harvested 48 and 72 h after transfection. Lentivirus Precipitation Solution (System Biosciences, Germany) was used to precipitate the virus.
For stable expression assays, 3 × 105 MOLM13, THP1, and MV4-11 were prepared for each infection system. The cells were centrifuged and resuspended in 300 μL virus suspension, followed by incubation at 37 °C and 5% CO2 for 24 h. Then, the cells were centrifuged, washed, and resuspended in fresh medium containing 1 μg/ml puromycin and 1% penicillin-streptomycin. To confirm target knockdown, cells were collected for qRT-PCR analysis.
Transfection of cell lines
SiRNAs were transfected into MOLM13, THP1, MV4-11, and RS4-11 cells at a final concentration of 50 nM with Neon™ Transfection System 10 μL Kit using the Neon Transfection System (Invitrogen, USA). HEK293T cells were transfected using the Lipofectamine 2000/3000 (Invitrogen, USA). Cells were collected 48 or 72 h after transfection. The siRNA sequences were listed in Additional file
1: Table S4.
RNA isolation and quantitative real-time PCR (RT-PCR)
Total RNA was extracted from bone marrow and cell samples using an Invitrogen™ TRIzol™ Kit (Thermo Fisher, USA) according to the manufacturer’s instructions. All RNA samples were stored at − 80 °C before reverse transcription and quantitative RT-PCR. RNA was reverse-transcribed into cDNA with the PrimeScript® RT reagent Kit with gDNA Eraser (Takara, Japan). Quantitative RT-PCR for lncRNA and mRNA was performed using the SYBR Premix ExTaq real-time PCR Kit (Takara, Japan) according to the manufacturer’s instructions. All of the data were normalized to GAPDH expression as a control. The expression level for each lncRNA and mRNA was determined using the 2
−△△Ct method. All primers were confirmed by sequencing the PCR product fragments, as shown in Additional file
1: Table S3.
Cell nucleus/cytoplasm fraction isolation
Cell nuclear and cytoplasmic fractions were isolated from cell samples using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher, USA) according to the manufacturer’s instructions.
Total protein was extracted from cells using RIPA lysis buffer (Beyotime Biotechnology, China) with 1× complete ULTRA (Roche, USA); protein was extracted from bone marrow samples using an Invitrogen™ TRIzol™ Kit (Thermo Fisher, USA) with the Thermo Scientific™ Halt™ Protease Inhibitor Cocktail (Thermo Fisher, USA) according to the manufacturer’s instructions.
A total of 10,000 MLL leukemia patient cells or CD34+ MLL leukemia cells were used per CFC assay and were added to 3 mL of methylcellulose media (R&D, USA). The vial was vigorously vortexed to thoroughly mix cells with the media. The media was allowed to stand until air bubbles disappeared. Next, we added 1.1 mL of the final cell mixture to each 35-mm culture dish. Two sample dishes and an uncovered dish containing 3–4 mL of sterile water were then placed into a 100-mm culture dish with a cover. Cells were incubated at 37 °C and 5% CO2, and the total colonies were counted after 7 days post plating.
Colony assay for MLL leukemia cell lines MOLM13 and THP1 was performed by plating 5000 cells on methylcellulose media (R&D, USA). Cells were incubated for 14 days respectively at 37 °C and 5% CO2.
Cell differentiation and morphological assay
For flow cytometric analysis, primary MLL leukemia patient cells, CD34+ MLL leukemia cells, and MOLM13, THP1, and RS4-11 with different treatments were harvested and washed with cooling PBS and were stained with the specific antibodies of differentiation markers (anti-CD14 and anti-CD11b for the differentiation of MLL myeloid leukemia cells, anti-CD19 for the differentiation of MLL lymphoid leukemia cells, BD Biosciences) with 0.5% FBS. The cells were incubated at 37 °C in the dark for 30 min and then were analyzed on a BD FACSCalibur analyzer (BD Biosciences, USA).
For morphological analysis, 50,000 cells of MOLM13 or THP1 were collected at 1000 rpm for 5 min. Slides were let dry and stained with Wright-Giemsa Stain Solution (Sangon biotech, China).
Limiting dilution assay
MOLM13 cells transduced with control or LAMP5-AS1 knockdown lentivirus were tail vein injected into 5-week-old NOD-SCID mice with four (10,0000, 50,000, 10,000, 5000) different doses of cells for each group of 4 mice. The number of recipient mice developed leukemia within 4 weeks after inoculation, and a recipient mouse was considered positive if GFP+ cells constituted more than 1.0% of all nucleated cells in the blood. Limiting dilution analysis (ELDA) online software (
http://bioinf.wehi.edu.au/software/elda/) [
31] was used to estimate the frequency of leukemia stem/progenitor-like cells in MOLM13 upon different treatments in vivo.
5′ RACE and 3′ RACE
Total RNA from THP1 cells was extracted using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer’ s guidelines. The 5′- and 3′-ends of cDNA were acquired using a 5′-FULL RACE Kit with TAP (Takara, Japan) and 3′-FULL RACE Core Set with PrimeScript RTase (Takara, Japan) respectively according to the manufacturer’s instructions. PCR products were obtained and then cloned into pEASY-T3 (TransGen Biotech, China) for further sequencing. All of the primers for RACE experiments are listed in Additional file
1: Table S3.
RNA pull-down assay
We performed pull-down assays with biotinylated LAMP5-AS1 using a Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher, USA) according to the manufacturer’s instructions. First, the LAMP5-AS1 and LAMP5-AS1 antisense sequences (primers are listed in Additional file
1: Table S3) were reverse-transcribed in vitro by PCR with T7 RNA polymerase using the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher, USA) with biotin-labeled UTP mix, and then the RNAs were purified using the Thermo GeneJET RNA Purification Kit (Thermo Fisher, USA). After folding, biotin-labeled RNAs were mixed with THP1 cell extract (containing 2 mg total protein) in 400 μL RIP buffer and incubated at RT (room temperature) for 1 h. Next, 50 μL of washed streptavidin magnetic beads was added to each reaction and further incubated at RT for another hour. Beads were washed briefly with RIP buffer six times and then boiled in SDS loading buffer. Finally, the enriched proteins were resolved via SDS-PAGE and silver stained followed by mass spectrometry (MS) identification (FitGene Biotechnology, China) and western blotting.
For LAMP5-AS1 truncated mutant pull-down assays, we performed the tRSA RNA pull-down system based on previous publications with modifications. LAMP5-AS1 full-length or truncated mutant sequences were first cloned into pcDNA3.1 plasmid containing the 5′ terminal tRSA tag. The plasmids were used as templates to in vitro transcribe RNA products using the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher, USA). Then, the RNA products were purified using the GeneJET RNA Purification Kit (Thermo Fisher, USA). The following procedures were based on the Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher, USA).
RNA fluorescence in situ hybridization (FISH) and immunofluorescence microscopy
To detect the subcellular location of LAMP5-AS1 RNA, we carried out FISH in THP1 and MV4-11 cells using the Ribo
TM Fluorescent In Situ Hybridization Kit (RiboBio, China). Cells were washed briefly with PBS and then fixed in 4% formaldehyde for 15 min at room temperature. Cells were permeabilized in PBS containing 0.5% Triton X-100 on ice for 5 min and then blocked in the preliminary hybridization solution for 30 min at room temperature after three washes with PBS for 10 min each. Hybridization was carried out using the LAMP5-AS1 FISH Probe Mix (RiboBio, China) in a humidified chamber at 37 °C for 12–16 h. Cells were rinsed with SSC buffer in accordance with the order 4×, 2×, and 1×. For co-localization studies, after RNA FISH, cells were fixed again for 5 min in 2% formaldehyde and subjected to immunofluorescence with anti-DOT1L primary antibody and fluorescent secondary antibody were sequentially (antibodies were listed in Additional file
1: Table S5). The nuclei were counterstained with DAPI. Cells were observed on a Zeiss7 DUO NLO confocal laser microscope (Carl Zeiss, Germany).
Purification of DOT1L (1-416aa)
The construction of GST-1-416aa DOT1L expression vector was described above in Plasmid Construction. Recombinant DOT1L (residues 1-416aa) with a termination codon in pET-N-GST-Thrombin-C-His was transformed into E. coli expression strain BL21 [Transetta(DE3) chemically competent cell (Transgen biotech, CD801)] for expression of GST-DOT1L (1-416aa). Five milliliters of Luria-Bertani (LB) culture supplemented with kanamycin was inoculated with a single colony at 37 °C. After overnight growth, the culture was diluted 100-fold into 300 mL LB supplemented with kanamycin. Protein expression was induced in the presence of 0.4 mM IPTG at 16 °C overnight. Then, the cell pellets were collected by centrifugation at 5000 rpm, 4 °C for 10 min, and were suspended in lysis buffer (500 mM NaCl, 50 mM Tris pH 8.0, 5% Glycerol, 0.5 mM (DTT), and 1x Protease Inhibitor (cOmplete EDTA free, Roche)). The cells were lysed on ice for 30 min, followed by sonication on ice with 4 s on/6 s off for 25 min. After centrifugation, the supernatant was applied to a ProteinIso® GST Resin column (TransGen Biotech, China) and the column was washed 3 times using lysis buffer. GST-DOT1L (1-416aa) protein was digested with Thrombin (sigma, USA) in digestion buffer (75 mM NaCl, 20 mM Tris pH 8.0, 5% Glycerol, 1 mM DTT) overnight at 4 °C to cleave off GST-tag. Finally, the tag-free DOT1L (residue 1-416aa) was pooled and the buffer exchanged into storage buffer (200 mM NaCl, 30 mM Tris pH 8.0,1 mM Tris(2-carboxyethyl)phosphine (TCEP), 20% glycerol) using an Amicon Ultra spin concentrator (Millipore, USA). The pure concentrated protein at 25 μM was then stored at − 80 °C until use.
Chromatin immunoprecipitation (ChIP) and ChIP-seq
ChIP analyses were performed on chromatin extracts from THP1 and MOM13 cells using a Magna ChIP™ G - Chromatin Immunoprecipitation Kit (17-611) (Merck Millipore, Germany) with di- and tri-Histone H3 (Lys79) according to the manufacturer’s standard protocol. In this assay, samples incubated with Rabbit IgG served as the negative control. The fold enrichment of H3K79me2/3 was quantified by quantitative RT-PCR and calculated relative to the input chromatin. The primers used for ChIP-qPCR analysis were listed in Additional file
1: Table S3. Eluted DNA fragments were also subjected to sequencing using the next-generation Illumina sequencing. The ChIP-seq data are uploading to the gene expression omnibus (GEO) database and the number is GSE150483.
Immunoblotting and RNA immunoprecipitation (RIP)
Proteins extracted from primary patient cells or cell lines were resolved by 7.5%, 10%, or 12% Bis-Tris polyacrylamide gels and were transferred to polyvinylidene fluoride membranes. Membranes were blocked in 5% BSA for 1 h and probed with the appropriate antibody overnight at 4 °C and then were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Membranes were visualized with an enhanced chemiluminescence detection system. Detail information of antibody was mentioned in Additional file
1: Table S5.
For RIP assays, FLAG- or HA-tagged fusion proteins were used. In the RIP experiment with FLAG-DOT1L (N- terminal) or HA-DOT1L (N- terminal) truncated mutants in 293T cells, anti-FLAG (Sigma, USA) or anti-HA (Sigma, USA) was used along with an EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (17-701) (Merck Millipore, Germany) according to the manufacturer’s instructions. All proteins for RIP were lysed with cell lysis buffer supplemented with Thermo Scientific™ Halt™ Protease Inhibitor Cocktail (Thermo Fisher, USA). Finally, all samples were suspended in 5× loading buffer and then denatured for 5 min at 100 °C, separated via SDS-PAGE, transferred to PVDF membranes, and blotted.
Statistical analysis
Pearson’s correlation coefficient was used to determine the correlation between lncRNA and MLL fusion protein levels. Mann-Whitney test was used to analyze the LAMP5-AS1 levels between patients with or without MLL fusion proteins. Fisher’s exact test was used to determine the significance of differentially expressed lncRNA and mRNA levels between the two groups. Data are expressed as the mean ± SEM of three independent experiments. One-way ANOVA was performed to compare multiple groups, and the LSD t test was used to analyze multiple comparisons. The probability of leukemia-free survival at 5 years was the study end-point. Leukemia-free survival was calculated from the date of complete remission (CR) until either relapse or death in remission. Leukemia-free survival was analyzed using the Kaplan-Meier method with a log-rank test. Two-tailed tests were used for univariate comparisons. For univariate and multivariate analysis of prognostic factors, a Cox proportional hazard regression model was used. p < 0.05 was considered statistically significant
Discussion
MLL leukemia is one of the most aggressive acute leukemia subtypes and has an apex self-renewal capacity [
6,
8]. It remains a challenge to directly inhibit rearranged
MLL itself in view of its multiple fusion partners [
1,
48,
49]. Nevertheless, the dependency of the
MLL fusion-driven gene expression program on the DOT1L pathway provides potential therapeutic opportunities for
MLL leukemia [
46,
48,
50,
51]. Therefore, further study of the DOT1L biology in
MLL leukemias could offer alternative strategies to inhibit the acquired self-renewal in
MLL leukemia stem and progenitor cells by targeting DOT1L pathways. In this study, we found that the lncRNA LAMP5-AS1, which is specifically expressed in
MLL leukemias, serves as an effector molecule to upregulate DOT1L methyltransferase activity by binding to its active center. We demonstrated that LAMP5-AS1 knockdown triggered a decreased H3K79 methylation state on the locus of the
HOXA genes and
MEIS1, which remarkably inhibited
MLL leukemia cell self-renewal and promoted differentiation (Fig.
6e)
. We are the first to demonstrate that lncRNAs can play a crucial role in the progression of
MLL leukemias and can directly influence the enzyme activity of H3 methyltransferase as effector molecules like coenzymes. We showed that LAMP5-AS1 is crucial for self-renewal in
MLL leukemia cells and may be a valuable therapeutic target for
MLL leukemia treatment.
LncRNAs have been illustrated to play a pivotal role in the progression of cell fate and cancer development, including hematopoiesis and leukemogenesis [
17,
21,
24,
52]. They exert their functions via cotranscriptional regulation, gene expression modulation, scaffolding of protein complexes, and pairing with other RNAs [
22,
53]. Notably, recent studies showed that lncRNAs modulated gene expression as epigenetic histone modifiers [
54,
55]. For instance, lncRNA HOTTIP interacts with WDR5 to recruit the MLL H3K4 methylase complex to facilitate H3K4me3 [
56]. LncRNA XIST recruits polycomb repressive complex 2 to induce H3K27me3 and silence the X chromosome [
57]. Nevertheless, in most studies on lncRNA functions in the regulation of epigenetic histone modification, lncRNAs are described as simple scaffolds to regulate the localization of activating or repressive chromatin modification machinery [
54,
55]. However, little is known about whether lncRNAs could directly regulate the activity of modification machines that would influence modification of the whole chromatin. In this study, we revealed that LAMP5-AS1 enhanced the methyltransferase activity of DOT1L by directly binding to its activity site. LAMP5-AS1 knockdown significantly reduced the global H3K79me2/me3 levels in
MLL leukemia cells, resulting in significantly decreased expression of the stem gene
HOXA cluster and
MEIS1 and decreased cell self-renewal capacity
. We demonstrate a novel function of lncRNAs to modulate global epigenetic histone modification by directly regulating enzymatic activity as effector molecules like coenzymes, rather than as simple scaffolds. The results highlight the extensive functions of lncRNAs on the regulation of epigenetic histone modification.
Recently, the positive feedback mechanisms in which posttranscriptional regulation is connected to transcriptional programs highlight a novel way to sustain the program of leukemic stem and progenitor cells [
58]. Interestingly, we showed that LAMP5-AS1 interacted with the methyltransferase active center of the mature DOT1L protein and could enhance its enzyme activity at the posttranslational level. LAMP5-AS1 knockdown significantly decreased global H3K79me2/me3 levels, which sharply inhibited the expression of the stem genes in the
HOXA cluster and
MEIS1 at the transcriptional level. These results suggest that the LAMP5-AS1-DOT1L axis can partially integrate the posttranslational regulation into the transcription program to sustain self-renewal capacity in
MLL leukemia.
DOT1L, a H3K79 methyltransferase without the SET domain that is found in most histone lysine methyltransferases, requires a more complicated complex to perform selective H3K79 methylation at different levels [
15]. Importantly, LAMP5-AS1 has been reported to be specifically highly expressed in
MLL leukemia cells, while it has an extra low expression level in
MLL-wt leukemia and general blood cells [
26]. Thus, we speculated that the MLL fusion protein purposely enhanced the methyltransferase activity of DOT1L through upregulation of LAMP5-AS1 to obtain a high self-renewal capacity. A recent study also showed that a cofactor of DOT1L AF10, which is highly expressed in mouse LSK cells, can facilitate the methyltransferase activity of DOT1L to obtain higher order H3K79 methylation [
14]. Recently, AF10 has been verified to bind to CC0 (amino acids 483 to 502) and a longer CC1 helix (amino acids 510 to 549) of DOT1L in
MLL-AF10 leukemia [
59]; both domains are near to the K-rich region that LAMP5-AS1 interacted with, suggesting that LAMP5-AS1 and AF10 might cooperate to enhance the methyltransferase activity of DOT1L in
MLL leukemia. Taken together, these results explain the specific oncogenicity of the ubiquitously expressed DOT1L from another perspective. Although there is a new controversy about whether DOT1L is necessary for
MLL LSC maintenance [
60], the essential role of DOT1L in sustaining
MLL leukemia cell self-renewal ability is undisputed [
14,
61]. Due to the complicated pathogenesis of
MLL leukemia and the potential limitation on direct DOT1L-targeted strategies, optional approaches with more precise targeted therapies, including lncRNA and other drugs highly specific to LSC, are therefore required, particularly as combination regimens to treat different clinical responses.
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