RUNX1 Binding Sites Research Articles

Inherited thrombocytopenia associated with a variant in the FLI1 binding site in the 5' UTR of ANKRD26.

Variants in the 5' UTR of ANKRD26 are a common cause of inherited thrombocytopenia (ANKRD26-RT), and are associated with sustained ANKRD26 expression, which inhibits megakaryocyte maturation and proplatelet formation. ANKRD26 expression is controlled by the binding of a RUNX1/FLI1 complex to the 5' UTR. To date, all reported ANKRD26-RD associated variants have been within the RUNX1 binding site and a 22 base pair flanking region. Here, we report a novel variant in the 5' UTR of ANKRD26, c.-107C>T. This variant is in the FLI1 binding site, and is predicted to disrupt FLI1 binding due to loss of a hydrogen bond with FLI1. Differentiated PBMCs from affected family members showed impaired megakaryocyte maturation and proplatelet formation and sustained expression of ANKRD26, and platelets from affected family members had higher ANKRD26 expression than control platelets. The variant increased activity of the ANKRD26 promotor in a reporter assay. We also provide evidence that the previously reported c.-140C>G ANKRD26 5' UTR variant is benign and not associated with thrombocytopenia. Identification of the c.-107C>T variant extends the range of the regulatory region in the 5' UTR of ANKRD26 that is associated with ANKRD26-RT.

RUNX1 regulates promoter activity in the absence of cognate DNA binding motifs.

Runt-related transcription factor 1 (RUNX1) plays an important role in normal haematopoietic cell development and function, and its function is frequently disrupted in leukaemia. RUNX1 is widely recognised as a sequence-specific DNA binding factor that recognises the motif 5'-TG(T/C)GGT-3' in promoter and enhancer regions of its target genes. Moreover, RUNX1 fusion proteins, such as RUNX1-ETO formed by the t(8;21) translocation, retain the ability to recognise and bind to this sequence to elicit atypical gene regulatory effects on bona fide RUNX1 targets. However, our analysis of publicly available RUNX1 chromatin immunoprecipitation sequencing (ChIP-Seq) data has provided evidence challenging this dogma, revealing that this motif-specific model of RUNX1 recruitment and function is incomplete. Our analyses revealed that the majority of RUNX1 genomic localisation occurs outside of promoters, that 20% of RUNX1 binding sites lack consensus RUNX motifs, and that binding in the absence of a cognate binding site is more common in promoter regions compared to distal sites. Reporter assays demonstrate that RUNX1 can drive promoter activity in the absence of a recognised DNA binding motif, in contrast to RUNX1-ETO. RUNX1-ETO supresses activity when it is recruited to promoters containing a sequence specific motif, while interestingly, it binds but does not repress promoters devoid of a RUNX1 recognition site. These data suggest that RUNX1 regulation of target genes occurs through multiple mechanisms depending on genomic location, the type of regulatory element and mode of recruitment.

Novel regulatory variant in ABO intronic RUNX1 binding site inducing A3 phenotype.

Mixed-field agglutination in ABO phenotyping (A3, B3) has been linked to genetically different blood cell populations such as in chimerism, or to rare variants in either ABO exon 7 or regulatory regions. Clarification of such cases is challenging and would greatly benefit from sequencing technologies that allow resolving full-gene haplotypes at high resolution. We used long-read sequencing by Oxford Nanopore Technologies to sequence the entire ABO gene, amplified in two overlapping long-range PCR fragments, in a blood donor presented with A3B phenotype. Confirmation analyses were carried out by Sanger sequencing and included samples from other family members. Our data revealed a novel heterozygous g.10924C>A variant on the ABO*A allele located in the transcription factor binding site for RUNX1 in intron 1 (+5.8 kb site). Inheritance was shown by the results of the donor's mother, who shared the novel variant and the anti-A specific mixed-field agglutination. We discovered a regulatory variant in the 8-bp RUNX1 motif of ABO, which extends current knowledge of three other variants affecting the same motif and also leading to A3 or B3 phenotypes. Overall, long-range PCR combined with nanopore sequencing proved powerful and showed great potential as an emerging strategy for resolving cases with cryptic ABO phenotypes.

Canonical BAF complex regulates the oncogenic program in human T-cell acute lymphoblastic leukemia

AbstractAcute leukemia cells require bone marrow microenvironments, known as niches, which provide leukemic cells with niche factors that are essential for leukemic cell survival and/or proliferation. However, it remains unclear how the dynamics of the leukemic cell–niche interaction are regulated. Using a genome-wide CRISPR screen, we discovered that canonical BRG1/BRM-associated factor (cBAF), a variant of the switch/sucrose nonfermenting chromatin remodeling complex, regulates the migratory response of human T-cell acute lymphoblastic leukemia (T-ALL) cells to a niche factor CXCL12. Mechanistically, cBAF maintains chromatin accessibility and allows RUNX1 to bind to CXCR4 enhancer regions. cBAF inhibition evicts RUNX1 from the genome, resulting in CXCR4 downregulation and impaired migration activity. In addition, cBAF maintains chromatin accessibility preferentially at RUNX1 binding sites, ensuring RUNX1 binding at these sites, and is required for expression of RUNX1-regulated genes, such as CDK6; therefore, cBAF inhibition negatively impacts cell proliferation and profoundly induces apoptosis. This anticancer effect was also confirmed using T-ALL xenograft models, suggesting cBAF as a promising therapeutic target. Thus, we provide novel evidence that cBAF regulates the RUNX1-driven leukemic program and governs migration activity toward CXCL12 and cell-autonomous growth in human T-ALL.

RUNX1 Isoforms Regulate RUNX1 and Target-Genes Differentially in Platelets/Megakaryocytes: Association with Clinical Cardiovascular Events

RUNX1 is an essential transcription factor for hematopoiesis. Germline RUNX1 haplodeficiency is associated with thrombocytopenia, platelet dysfunction and predisposition to myeloid malignancies. Three major RUNX1 isoforms (A, B and C) are recognized and share a DNA binding RUNT domain; RUNX1A lacks the transactivation domain. Two distinct promoters regulate RUNX1 expression: distal P1 regulates RUNX1C; proximal P2 regulates RUNX1B and RUNX1A. Little is known regarding the differential effects of isoforms in RUNX1 autoregulation, target-gene regulation and association with clinical events. We performed studies in megakaryocytic HEL cells, HeLa cells (which do not express RUNX1) and in platelets of healthy subjects (n=74) and provide evidence for isoform-specific differential autoregulation and target-gene regulation by RUNX1. We show an association between expression of specific RUNX1-regulated genes in whole blood with cardiovascular events in patients. There are 5 RUNX1 consensus binding sites in P1 promoter and 1 in P2 promoter within ~1000 bp from ATG. Wild type RUNX1 P1 and P2 promoter regions were individually cloned into PGL4 luciferase promoter vector. ChIP studies using PMA-treated megakaryocytic HEL cells showed RUNX1 binding to chromatin regions encompassing the RUNX1 binding sites in both promoters. Mutations of 2 RUNX1 binding sites in intron region of P1 promoter reduced promoter activity; mutations of first 2 RUNX1 binding sites at the exon region of P1 promoter increased activity. Mutation of single RUNX1 binding site in P2 promoter increased promoter activity. Thus, RUNX1 binds to P1 and P2 promoters to regulate activities. To examine the RUNX1 autoregulation by individual isoforms, we co-transfected each isoform with P1 or P2 promoter vector in HeLa cells, which do not express endogenous RUNX1. In response to RUNX1B over-expression, both P1 and P2 promoters showed a dose-dependent decrease in promoter activity. RUNX1C over-expression increased P1 and P2 promoter activities. Thus, RUNX1B and RUNX1C regulate P1 and P2 promoters differentially. In HEL cells, which have endogenous RUNX1, RUNX1B overexpression decreased RUNX1C and RUNX1A mRNA/protein expression by ~50%. RUNX1C overexpression increased RUNX1B and RUNX1A mRNA/protein expression. Thus, RUNX1B decreases RUNX1C and RUNX1A expression; RUNX1C increases RUNX1B and RUNX1A. We studied the regulation by RUNX1 isoforms of target genes PCTP (phosphatidylcholine transfer protein), MYL9 (myosin light chain), PDE5A (phosphodiesterase 5A) and F13A (factor XIIIA) . In HeLa cells. RUNX1B overexpression increased PCTP, MYL9 and PDE5A (protein and mRNA); RUNX1C overexpression reduced PCTP and MYL9. In HEL cells RUNX1B increased PCTP, MYL9, PDE5A and F13A expression; RUNX1C reduced PCTP and MYL9 expression. To obtain evidence in vivo, we studied the relationships between RUNX1 isoforms and their relationships to RUNX1-target genes by RNAseq in leukocyte-poor platelets (74 healthy donors; ages 30-75 yrs). RUNX1B correlated negatively with RUNX1C (R= -0.413 and p=1.5e-06) and RUNX1A (R= -0.493 and p=0.00e+oo); RUNX1C correlated positively with RUNX1A (R=0.45, p<0.0001). RUNX1B correlated positively with PCTP, F131A, PDE5A and RAB1B, and negatively with MYL9 (Figure 1); no significant relationships were observed with RUNX1C or RUNX1A. To understand the relationships to cardiovascular events (MI or death), we explored whole blood gene expression using microarrays in 587 patients (2 cohorts, 190 and 397 patients) presenting to cardiac catherization for chest pain and followed for 3.8 years. In our previous studies RUNX1B transcripts were higher and RUNX1C lower in those with MI or death. We now found that higher expressions of F13A1(Figure 2) and RAB31, which have been shown by us to be RUNX1-targets, were associated with cardiovascular events. Conclusions: RUNX1 isoforms autoregulate RUNX1 expression differentially and regulate target genes in a differential manner, a pattern consistent with in vivo platelet gene expression. The relative expression of RUNX1B and RUNX1C isoforms and of their target genes associate with clinical cardiovascular events. RUNX1-isoform specific effects need to be considered in clinical scenarios where RUNX1 is involved and in its pharmacologic modulation.

Control of focal adhesion kinase activation by RUNX1-regulated miRNAs in high-risk AML.

We recently described a 16-gene expression signature for improved risk stratification of acute myeloid leukemia (AML) patients called the AML Prognostic Score (APS). A subset of APS-high-risk AML patients showed increased levels of focal adhesion kinase (FAK), encoded by the Protein Tyrosine Kinase 2 (PTK2) gene, which was correlated with RUNX1 mutations. RUNX1 mutant cells are more sensitive to PTK2 inhibitors. As we were not able to detect RUNX1-binding sites in the PTK2 promoter, we hypothesized that RUNX1 might regulate micro(mi)RNAs that repress PTK2, such that loss-of-function RUNX1 mutations would result in reduced miRNA expression and derepression of PTK2. Examination of paired RNA-seq and miRNA-seq data from 301 AML cases revealed two miRNAs that positively correlated with RUNX1 expression, contained RUNX1-binding sites in their promoters and were predicted to target PTK2. We show that the hsa-let7a-2-3p and hsa-miR-135a-5p promoters are regulated by RUNX1, and that PTK2 is a direct target of both miRNAs. Even in the absence of RUNX1 mutations, hsa-let7a-2-3p and hsa-miR-135a-5p regulate PTK2 expression, and reduced expression of these two miRNAs sensitizes AML cells to PTK2 inhibition. These data explain how RUNX1 regulates PTK2, and identify potential miRNA biomarkers for targeting AML with PTK2 inhibitors.

DLX5 promotes Col10a1 expression and chondrocyte hypertrophy and is involved in osteoarthritis progression

Osteoarthritis (OA) has been considered non-reversible as articular cartilage wears down with limited repair capacity. Enhanced chondrocyte hypertrophy and increased type X collagen gene (COL10A1) expression have been associated with OA. Therefore, regulators controlling collagen X expression and chondrocyte hypertrophy may play a role in OA intervention. Here, we investigated how Distal-less homeobox 5 (DLX5), the distal-less homeobox family member, controls murine Col10a1 gene expression and chondrocyte hypertrophy in chondrogenic cell models and its role in a murine OA model. Through qRT-PCR and Western blot analyses, we detected significantly increased levels of COL10A1 and DLX5 in hypertrophic MCT and ATDC5 cells compared to their proliferative stage. Forced expression of Dlx5 further increases, while knockdown of Dlx5 decreases COL10A1 expression in hypertrophic MCT cells. We have performed dual-luciferase reporter and ChIP assays and demonstrated that DLX5 promotes reporter activity through direct interaction with Col10a1 cis-enhancer. We established a murine OA model and detected markedly increased COL10A1 and DLX5 in the articular cartilage and subchondral bone of the OA mice compared with the controls. Notably, forced overexpression of DLX5 in hypertrophic MCT cells up-regulates RUNX2, and adjacent DLX5 and RUNX2 binding sites have previously been found within the Col10a1 cis-enhancer. Together, our data suggest that DLX5 may cooperate with RUNX2 to control cell-specific Col10a1 expression and chondrocyte hypertrophy and is involved in OA pathogenesis.

Regulome analysis in B-acute lymphoblastic leukemia exposes Core Binding Factor addiction as a therapeutic vulnerability

The ETV6-RUNX1 onco-fusion arises in utero, initiating a clinically silent pre-leukemic state associated with the development of pediatric B-acute lymphoblastic leukemia (B-ALL). We characterize the ETV6-RUNX1 regulome by integrating chromatin immunoprecipitation- and RNA-sequencing and show that ETV6-RUNX1 functions primarily through competition for RUNX1 binding sites and transcriptional repression. In pre-leukemia, this results in ETV6-RUNX1 antagonization of cell cycle regulation by RUNX1 as evidenced by mass cytometry analysis of B-lineage cells derived from ETV6-RUNX1 knock-in human pluripotent stem cells. In frank leukemia, knockdown of RUNX1 or its co-factor CBFβ results in cell death suggesting sustained requirement for RUNX1 activity which is recapitulated by chemical perturbation using an allosteric CBFβ-inhibitor. Strikingly, we show that RUNX1 addiction extends to other genetic subtypes of pediatric B-ALL and also adult disease. Importantly, inhibition of RUNX1 activity spares normal hematopoiesis. Our results suggest that chemical intervention in the RUNX1 program may provide a therapeutic opportunity in ALL.

Differential RUNX1-Isoform Specific Autoregulation of RUNX1 and Regulation of Target Genes (PCTP, MYL9) in Megakaryocytic Cells

RUNX1 is an essential transcription factor for hematopoiesis. Human germline RUNX1 haplodeficiency is associated with impaired megakaryocyte differentiation, thrombocytopenia, platelet dysfunction and predisposition to myeloid malignancies. The two major RUNX1 isoforms RUNX1C and RUNX1B differ at N-terminus by 14AA, and are regulated by two distinct promoters P1 and P2, respectively. RUNX1B and RUNX1C have distinct roles and tissue expression. Little is known regarding the differential effects of RUNX1 isoforms in RUNX1 autoregulation, and on their regulation of target genes. There are 5 RUNX1 consensus binding sites located in P1 promoter and one RUNX1 site in P2 promoter. To study RUNX1 promoters, both WT RUNX1 P1 and P2 promoter regions were individually cloned into PGL4 luciferase promoter vector. ChIP studies using PMA-treated megakaryocytic HEL cells showed RUNX1 binding to chromatin regions encompassing the RUNX1 binding sites in both promoters. Mutations of 2 RUNX1 binding sites in intron region of P1 promoter reduced promoter activity; mutations of first 2 RUNX1 binding sites in at the exon region of P1 promoter increased activity. Mutation of single RUNX1 binding site in P2 promoter increased promoter activity. Thus, RUNX1 binds to P1 and P2 promoters to regulate their activities. To examine the RUNX1 autoregulation by individual isoforms, we co-transfected each isoform with P1 or P2 promoter vector in HeLa cells, which express negligible endogenous RUNX1. In response to RUNX1B, P1 promoter showed a dose-dependent decrease in promoter activity. RUNX1C expression, increased P1 promoter activity in a dose-dependent manner. With constant RUNX1C expression, increase in RUNX1B co-expression led to a dose-dependent decrease in P1 promoter activity. In HeLa cells RUNX1B decreased P2 promoter activity in a dose-dependent manner. With constant RUNX1B expression, an increase in RUNX1C increased RUNX1 P2 promoter activity. Thus, RUNX1B and RUNX1C regulate both P1 and P2 promoters in a distinct manner. In studies in megakaryocytic HEL cells, which express endogenous RUNX1, RUNX1B overexpression decreased RUNX1C expression by 50%. RUNX1C overexpression increased by 2-fold RUNX1B protein. In studies using RUNX1C-specific antibody, and expression plasmids for hemagglutinin-tagged-RUNX1B and c-Myc-tagged RUNX1C, RUNX1B overexpression decreased RUNX1C; RUNX1C overexpression increased RUNX1B. These studies indicate that RUNX1 isoforms autoregulate in a differential manner at the protein level. We studied the effects of RUNX1 isoforms on two established downstream RUNX1 target genes PCTP (phosphatidylcholine transfer protein) and MYL9(myosin light chain) in HeLa cells. RUNX1B overexpression increased both PCTP and MYL9 protein on immunoblotting, while RUNX1C overexpression reduced both proteins. This suggests RUNX1B and RUNX1C have differential effects in regulating downstream genes. These findings are in line with our studies in whole blood from a large cohort of patients with CAD showing a positive correlation between PCTP (Circulation 2017136(10):927-939) with RUNX1B transcript levels and a negative relationship with those for RUNX1C. Conclusions: RUNX1 isoforms autoregulate RUNX1 expression differentially in an isoform-specific manner. Moreover, they regulate target genes (MYL9 and PCTP) in a differential manner. These RUNX1 isoform-specific effects may be important in understanding the consequences of germline RUNX1 haplodeficiency and in modulating RUNX1 levels for therapeutic purposes.

MYCN Regulates Cell Survival Via EIF4G1 in Inv(16) Acute Myeloid Leukemia

Transcription factors are frequently deregulated in leukemia and are pivotal to its pathogenesis. The core-binding factor (CBF) transcription factor is a heterodimer of CBFβ and RUNX proteins, is critical to hematopoiesis, and dysregulated in AML. The fusion oncogene CBFβ-SMMHC expressed in AML with the chromosome inversion inv(16)(p13q22) acts as a driver oncogene in hematopoietic stem cells by sequestering RUNX1 by protein-protein interaction to induce AML. The CBFβ-SMMHC inhibitor, AI-10-49, specifically disrupts its binding to RUNX1 resulting in genome-wide RUNX1 binding, apoptosis induction, and enhanced survival in inv(16) mouse model [Illendula* and Pulikkan* et al, Science 2015]. While previous studies have demonstrated that AI-10-49 induced cell death is partly mediated by repression of MYC expression in inv(16) AML cells [Pulikkan et al, Cell 2018], the function of other MYC family members, in inv(16) cell survival, and overall in AML, remains poorly understood. By conducting Real Time RT-PCR in CD34+ bone marrow cells from human AML samples, we identified MYCN upregulation in inv(16) AML. Analysis of RNA-seq data from inv(16) AML cells treated with AI-10-49 revealed that MYCN expression is downregulated (7-fold). MYCN silencing via CRISPR/Cas9 induced apoptosis in inv(16) AML cells. Furthermore, MYCN genetic targeting by CRISPR/Cas9-RNP approach in human primary inv(16) AML cells transplanted in humanized NSGS xenograft mouse model displayed significant delay in leukemia development, underlining the role of MYCN in inv(16) AML maintenance. ChIP-seq analysis conducted in inv(16) AML cells treated with AI-10-49 demonstrated increased RUNX1 occupancy at MYCN distal genomic element located at +26 Kb (named 'MYCN-e26') downstream from MYCN TSS without any RUNX1 binding at MYCN TSS. ChIP-seq analysis for histone marks such as H3K27ac and H3K4Me1, and chromatin accessibility by ATAC-seq suggested MYCN-e26 acts as a transcriptionally active enhancer element. Analysis of DNase I hypersensitive sites (DHSs) representing accessible chromatin regions in primary AML samples revealed that MYCN-e26 is a bona fide AML enhancer. Finally, deletion of a genomic element in MYCN-e26 surrounding the RUNX1 binding site (200 bp) by CRISPR/Cas9 genome editing downregulated MYCN transcript levels and inhibited inv(16) AML cell survival. Taken together, MYCN-e26 is instrumental in MYCN transcription and cell survival in inv(16) AML cells as a RUNX1 target. To identify the mechanism(s) by which MYCN regulates inv(16) AML cell survival, we performed CUT&Tag sequencing for MYCN and H3K27ac histone mark in inv(16) AML cells. Gene ontology analysis for MYCN binding peaks in transcriptionally active regions revealed mRNA metabolism and stability as the major pathways orchestrated by MYCN in inv(16) AML cells. By comparing the mRNA metabolism/ stability signature genes with transcripts significantly deregulated in human primary inv(16) AML samples, we identified eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) as one of the major genes regulated by MYCN in inv(16) AML cell survival. Our CUT&Tag sequencing analysis demonstrated MYCN binds to EIF4G1 promoter and enhances its transcription. MYCN silencing by CRISPR/Cas9 approach in inv(16) AML cells showed significant downregulation of EIF4G1, suggesting MYCN is required for EIF4G1 expression in inv(16) AML cells. EIF4G1 genetic silencing via CRISPR/Cas9 approach induced apoptosis in inv(16) AML cells in vitro, and significantly delayed leukemia development in NSGS xenograft mouse model, suggesting EIF4G1 is required for the survival of inv(16) leukemia-initiating cells. Our studies demonstrate that CBFβ-SMMHC modulates MYCN levels by inhibition of RUNX1 binding to MYCN distal enhancer in inv(16) AML cells. In addition, our studies bring novel function of MYCN in inv(16) leukemic cell survival by regulating EIF4G1. Future therapeutic approaches targeting MYCN/EIF4G1 axis in combination with AI-10-49 warrant better clinical outcome for the treatment of inv(16) AML.

Cyclophilin A Promotes Osteoblast Differentiation by Regulating Runx2.

Cyclophilin A (CypA) is a ubiquitously expressed and highly conserved protein with peptidyl-prolyl cis-trans isomerase activity that is involved in various biological activities by regulating protein folding and trafficking. Although CypA has been reported to positively regulate osteoblast differentiation, the mechanistic details remain largely unknown. In this study, we aimed to elucidate the mechanism of CypA-mediated regulation of osteoblast differentiation. Overexpression of CypA promoted osteoblast differentiation in bone morphogenic protein 4 (BMP4)-treated C2C12 cells, while knockdown of CypA inhibited osteoblast differentiation in BMP4-treated C2C12. CypA and Runx2 were shown to interact based on immunoprecipitation experiments and CypA increased Runx2 transcriptional activity in a dose-dependent manner. Our results indicate that this may be because CypA can increase the DNA binding affinity of Runx2 to Runx2 binding sites such as osteoblast-specific cis-acting element 2. Furthermore, to identify factors upstream of CypA in the regulation of osteoblast differentiation, various kinase inhibitors known to affect osteoblast differentiation were applied during osteogenesis. Akt inhibition resulted in the most significant suppression of osteogenesis in BMP4-induced C2C12 cells overexpressing CypA. Taken together, our results show that CypA positively regulates osteoblast differentiation by increasing the DNA binding affinity of Runx2, and Akt signaling is upstream of CypA.

Metformin and lipopolysaccharide regulate transcription of NFATc2 gene via the transcription factor RUNX2

To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene. The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn Ⅰ and Hind Ⅲ. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription. We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively. pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3- NFATc2-promoter in a RUNX2-dependent manner.

Stimulation of the Runx2 P1 promoter by collagen-derived dipeptide prolyl-hydroxyproline bound to Foxg1 and Foxo1 in osteoblasts.

Collagen-derived dipeptide prolyl-hydroxyproline (Pro-Hyp) directly binds to the forkhead box g1 (Foxg1) protein and causes it to undergo structural alteration. Pro-Hyp also promotes the production of a regulator of osteoblast differentiation, Runt-related transcription factor 2 (Runx2), through Foxg1, inducing osteoblast differentiation. In addition, Pro-Hyp disrupts the interaction between Foxg1 and Runx2, and Foxg1 appears to interact with Runx2 in the absence of Pro-Hyp. To elucidate the mechanism of Pro-Hyp that promotes osteoblast differentiation, we investigated whether Pro-Hyp regulates the Runx2 P1 promoter together with Foxg1. The present study revealed that Pro-Hyp is taken up by osteoblastic cells via the solute carrier family 15 member (Slc15a) 4. In the presence of Pro-Hyp, Runx2 is translocated from the nucleus to the cytoplasm and Foxg1 is translocated from the cytoplasm to the nucleus. We also found that Pro-Hyp promoted the interaction between Forkhead box o1 (Foxo1) and Runx2 and the dissociation of Foxg1 from Runx2. Moreover, we identified the Pro-Hyp response element in the Runx2 distal P1 promoter at nt −375 to −316, including the Runx2 binding sites and Fox core sequence. In the presence of Pro-Hyp, Runx2 is dissociated from the Pro-Hyp response element in the Runx2 distal P1 promoter. Subsequently, Foxg1 and Foxo1 activated the Runx2 promoter by binding to the Pro-Hyp response element. In summary, we delineated the mechanism by which Pro-Hyp stimulates the bone-related Runx2 distal P1 promoter activity in osteoblastic cells through Foxg1, Foxo1, and Runx2.

Epigenetic silencing of UBXN8 contributes to leukemogenesis in t(8;21) acute myeloid leukemia

The formation of the RUNX1-RUNX1T1 fusion protein, resulting from the t(8;21) translocation, is considered to be one of the initiating events of t(8;21) acute myeloid leukemia (AML). However, the mechanisms of the oncogenic mechanism of RUNX1-RUNX1T1 remain unclear. In this study, we found that RUNX1-RUNX1T1 triggers the heterochromatic silencing of UBXN8 by recognizing the RUNX1-binding sites and recruiting chromatin-remodeling enzymes to the UBXN8 promoter region. Decitabine, a specific inhibitor of DNA methylation, upregulated the expression of UBXN8 in RUNX1-RUNX1T1+ AML cell lines. Overexpression of UBXN8 inhibited the proliferation and colony-forming ability of and promoted cell cycle arrest in t(8;21) AML cell lines. Enhancing UBXN8 levels can significantly inhibit tumor proliferation and promote the differentiation of RUNX1-RUNX1T1+ cells in vivo. In conclusion, our results indicated that epigenetic silencing of UBXN8 via methylation of its promoter region mediated by the RUNX1-RUNX1T1 fusion protein contributes to the leukemogenesis of t(8;21) AML and that UBXN8 targeting may be a potential therapeutic strategy for t(8;21) AML.

The Polymorphism at PLCB4 Promoter (rs6086746) Changes the Binding Affinity of RUNX2 and Affects Osteoporosis Susceptibility: An Analysis of Bioinformatics-Based Case-Control Study and Functional Validation.

PurposeGenome-wide association studies have identified numerous genetic variants that are associated with osteoporosis risk; however, most of them are present in the non-coding regions of the genome and the functional mechanisms are unknown. In this study, we aimed to investigate the potential variation in runt domain transcription factor 2 (RUNX2), which is an osteoblast-specific transcription factor that normally stimulates bone formation and osteoblast differentiation, regarding variants within RUNX2 binding sites and risk of osteoporosis in postmenopausal osteoporosis (PMOP).MethodsWe performed bioinformatics-based prediction by combining whole genome sequencing and chromatin immunoprecipitation sequencing to screen functional SNPs in the RUNX2 binding site using data from the database of Taiwan Biobank; Case-control studies with 651 postmenopausal women comprising 107 osteoporosis patients, 290 osteopenia patients, and 254 controls at Tri-Service General Hospital between 2015 and 2019 were included. The subjects were examined for bone mass density and classified into normal and those with osteopenia or osteoporosis by T-scoring with dual-energy X-ray absorptiometry. Furthermore, mRNA expression and luciferase reporter assay were used to provide additional evidence regarding the associations identified in the association analyses. Chi-square tests and logistic regression were mainly used for statistical assessment.ResultsThrough candidate gene approaches, 3 SNPs in the RUNX2 binding site were selected. A novel SNP rs6086746 in the PLCB4 promoter was identified to be associated with osteoporosis in Chinese populations. Patients with AA allele had higher risk of osteoporosis than those with GG+AG (adjusted OR = 6.89; 95% confidence intervals: 2.23–21.31, p = 0.001). Moreover, the AA genotype exhibited lower bone mass density (p < 0.05). Regarding mRNA expression, there were large differences in the correlation between PLCB4 and different RUNX2 alleles (Cohen’s q = 0.91). Functionally, the rs6086746 A allele reduces the RUNX2 binding affinity, thus enhancing the suppression of PLCB4 expression (p < 0.05).ConclusionsOur results provide further evidence to support the important role of the SNP rs6086746 in the etiology of osteopenia/osteoporosis, thereby enhancing the current understanding of the susceptibility to osteoporosis. We further studied the mechanism underlying osteoporosis regulation by PLCB4.

PPARγ-Induced Global H3K27 Acetylation Maintains Osteo/Cementogenic Abilities of Periodontal Ligament Fibroblasts.

The periodontal ligament is a soft connective tissue embedded between the alveolar bone and cementum, the surface hard tissue of teeth. Periodontal ligament fibroblasts (PDLF) actively express osteo/cementogenic genes, which contribute to periodontal tissue homeostasis. However, the key factors maintaining the osteo/cementogenic abilities of PDLF remain unclear. We herein demonstrated that PPARγ was expressed by in vivo periodontal ligament tissue and its distribution pattern correlated with alkaline phosphate enzyme activity. The knockdown of PPARγ markedly reduced the osteo/cementogenic abilities of PDLF in vitro, whereas PPARγ agonists exerted the opposite effects. PPARγ was required to maintain the acetylation status of H3K9 and H3K27, active chromatin markers, and the supplementation of acetyl-CoA, a donor of histone acetylation, restored PPARγ knockdown-induced decreases in the osteo/cementogenic abilities of PDLF. An RNA-seq/ChIP-seq combined analysis identified four osteogenic transcripts, RUNX2, SULF2, RCAN2, and RGMA, in the PPARγ-dependent active chromatin region marked by H3K27ac. Furthermore, RUNX2-binding sites were selectively enriched in the PPARγ-dependent active chromatin region. Collectively, these results identified PPARγ as the key transcriptional factor maintaining the osteo/cementogenic abilities of PDLF and revealed that global H3K27ac modifications play a role in the comprehensive osteo/cementogenic transcriptional alterations mediated by PPARγ.

Genome-Wide Analysis of the Regulatory Landscape in Different Pre-B-ALL Subtypes

BackgroundAcute pre-B leukemia results from a failure in cell differentiation. In normal development, cells commit to their lineage and differentiated phenotype state by the activation of transcription factors (TFs), or TF modules, in a series of decision points at which a choice is made between alternative lineage fates. Previously, we showed that the ETV6-RUNX1 gene translocation that occurs in 25% of pediatric leukemia patients silences regulatory regions containing RUNX binding sites (enhancers) at target gene loci implicated in lymphoid lineage differentiation (Teppo et al. 2016). However, the genome-wide changes in enhancer activities at binding sites of other TFs functioning in B-cell differentiation remain poorly understood. Moreover, the genome-wide identification of enhancers altered during leukemogenesis is incomplete.Material & MethodsTo measure transcripts generated both at enhancer and gene regions, global run-on sequencing (GRO-seq) technique was used to measure nascent RNAs (Heinäniemi et al. 2016) from human pre-B-ALL patient samples (N=35) and several cell lines representing different subtypes. For validation, additional samples were generated from cell line models with overexpression or silencing of leukemic TFs. To this collection of 151 nascent transcriptomes, additional 92 GRO-seq samples representing different cell lineages (Liivrand et al 2017) were used to compare the leukemic cells to primary cells and other cancers. Enhancer RNA (eRNA) levels were compared at TF binding motif centered enhancer regions. TF ChIP-seq data from leukemic cells and HSC were acquired from the NCBI Gene Expression Omnibus for validation of this approach.ResultsTo gain insight into the derailed regulatory landscape in leukemic cells, we identified eRNA regions both from normal and leukemic samples and performed statistical analysis distinguishing regulatory regions with differential activity between pre-B-ALL-subtypes or compared to normal CD34+ bone marrow precursors. Next, association of the significantly altered enhancers with gene transcripts was utilized to build regulatory maps for differentially expressed gene loci. For this purpose, we developed a transcript identification and quantification pipeline suitable for detecting altered expression at different coding and non-coding RNA loci from nascent transcriptomes (Liivrand et al 2017; unpublished). To globally evaluate altered TF activities across pre-B-ALL subtypes underlying the differences observed, we present here an unbiased approach based on eRNA quantification at TF motif-centered enhancers. The analysis of eRNA levels at binding sites of TFs with established role in leukemia was used as a benchmark: we could confirm low eRNA transcription at RUNX1 binding sites in the ETV6-RUNX1 subtype, elevated eRNA levels at HOXA9 and MEIS1 binding sites in the MLL rearranged subtype, while TCF3, BCL6 and PBX1 bound sites were most active in TCF3-PBX1 samples.ConclusionsOur results provide the first genome-wide transcriptional regulatory landscape of different pre-B-ALL subtypes. We identified novel subtype-specific active enhancers and annotated regulatory non-coding regions that can be utilized to evaluate the functional impact of genetic alterations outside gene regions. DisclosuresNo relevant conflicts of interest to declare.

The Impacts of Transcriptional Regulatory Mechanism and Protein Interaction Relationship between RUNX1 and KLF4 on the Biological Functions of t (8;21) Leukemia Cells

Our previous study has reported that sodium phenylbutyrate (PB), one of the HDAC inhibitors, could induce t (8;21) leukemia cells to undergo differentiation and apoptosis. However, genes that promote differentiation and apoptosis in this process are not fully identified. Identification of these genes and elucidation of their interaction relationships are essential to develop novel therapy strategies for t (8;21) leukemia.In this study, we found that the expressions of RUNX1, KLF4 and P57 at both mRNA and protein level were significantly up-regulated in Kasumi-1 cells after PB treatment. Bioinformatics analysis further demonstrated that there might be some transcriptional regulatory mechanism and protein interaction relationship among these three genes.For the study of transcriptional regulatory mechanism, a KLF4 promoter/enhancer reporter plasmid containing RUNX1 binding sites and a P57 promoter/enhancer reporter plasmid containing KLF4 binding sites were constructed. Luciferase assay showed that RUNX1 exhibited strong transcriptional activation effect on KLF4 promoter/enhancer reporter plasmid in a dose-dependent manner, while RUNX1-ETO showed slight effect. KLF4 exhibited dose-dependent transcriptional activation effect on P57 promoter /enhancer reporter plasmid. These transcriptional regulatory relationships were further confirmed by Western blot assay. Transient expression of exogenous RUNX1 up-regulated the expression of KLF4 and P57 in 293T cells and Kasumi-1 cells, while RUNX1-ETO had no effect.In the study of protein interaction relationship, co-immunoprecipitation (CO-IP) and immunofluorescence confocal imaging showed that RUNX1 interacted with KLF4 and co-localized with it in nucleus, and Runt homology domain (RHD) was responsible for this interaction. RUNX1-ETO competed with RUNX1 for binding to KLF4 in a dose-dependent manner through RHD and the ETO domains. PB has no effect on the interactions between RUNX1/RUNX1-ETO with KLF4. To further investigate the effects of RUNX1 and RUNX1-ETO on KLF4-dependent transactivation of target genes, a luciferase reporting plasmid containing KLF4 binding sites was constructed. Luciferase assay was then performed and showed that RUNX1 could coactivate KLF4 target genes while RUNX1-ETO had little effect.Finally,RUNX1, KLF4 and P57 were overexpressed in Kasumi-1 cells by a pCDH lentivirus system, and their biological functions were investigated by MTS assay and flow cytometry. Overexpression of either of the three genes could inhibit cell proliferation, block cell cycle and induce apoptosis of t (8;21) leukemia cells. Moreover, in vivo experiment demonstrated that overexpression of KLF4 and P57 in mouse bone marrow leukemic cells could reduce the incidence of leukemia development in recipient mice.In conclusion, we reported “RUNX1-KLF4-P57” as a novel signaling pathway in t (8;21) leukemia cells in this study. RUNX1 transactivated KLF4 and then P57 in leukemia cells to inhibit cell proliferation, block cell cycle and induce cell apoptosis, while oncoprotein RUNX1-ETO had little effect. Moreover, RUNX1 interacted with KLF4 and coactivated its target genes, which could be blocked by the competed binding of RUNX1-ETO with KLF4. Our study revealed the regulatory relationship between RUNX1 and KLF4 and the impacts of RUNX1-ETO on this relationship, which helps to elucidate the mechanism of t (8;21) leukemogenensis. DisclosuresNo relevant conflicts of interest to declare.

Cross Talk between Ikaros and RUNX1 in Myeloid Leukemia

Background: RUNX1 is critically important for normal hematopoiesis within both the lymphoid and myeloid lineages. Mutations or translocations involving RUNX1 have been identified in a variety of myeloid and lymphoid lineage hematopoietic malignancies. RUNX1 interacts with chromatin remodeling complexes and modulates transcriptional regulation of target genes via epigenetic mechanisms.IKAROS is a transcription factor that plays an essential role in normal lymphopoiesis, and mutations of IKZF1, the gene encoding IKAROS, are frequently seen in acute lymphoblastic leukemia (ALL). Despite reports of the presence of IKZF1 mutations in acute myeloid leukemia (AML), there remains a paucity of information regarding a possible role for disrupted IKAROS function in AML.Emerging evidence indicates that the transcriptional programs of these two transcription factors converge on critical pathways in hematopoiesis, such as the Notch1 signaling pathway. We are testing the hypothesis that IKAROS not only contributes to a gene regulatory program in AML, but specifically that the IKAROS and RUNX1 regulatory networks intersect in the pathogenesis of myeloid malignancies.Methods: Chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) was utilized to identify the genome-wide binding sites of IKAROS and RUNX1 in the human myeloid sarcoma U937 cell-line. Independent ChIP-seq replicates were generated for these transcription factors, as well as replicates for a panel of activating and repressive histone modifications. The genes bound by IKAROS and RUNX1 as well as the chromatin patterns of these binding sites were analyzed with respect to their pathways were analyzed to understand the mutual and shared roles of these transcription factors.Results: We identified that greater than 30% of RUNX1 binding sites in this myeloid sarcoma cell line are also bound by IKAROS. These sites of co-occupancy are enriched at distal sites marked by the enhancer histone mark H3K27ac. Of the high-confidence IKAROS-RUNX1 shared binding sites, RUNX1 binding motifs predominate; while ETS, IRF4 and CEBPA motifs are also present. A Genome Regions Enrichment of Annotations (GREAT) analysis of the high confidence IKAROS-RUNX1 shared binding sites reveals enrichment for several biological associations, including leukocyte activation, migration, and chemotaxis. Importantly, genes associated with the regulation of chromatin organization and Notch and NFKB signaling cascades are bound by these factors.We have additionally found that IKAROS and RUNX1 are bound to the RUNX1 gene promoter. IKAROS is also independently bound to the RUNX1 gene at an upstream enhancer, also marked by H3K27ac.Conclusions: The role of IKAROS as a tumor suppressor in lymphoid leukemias is well established. Here, we begin to examine a role for IKAROS in myeloid leukemogenesis by defining the IKAROS cistrome in a myeloid sarcoma cell line. We have additionally identified cross talk between IKAROS and the important pan-hematopoietic transcription factor RUNX1 in myeloid malignancy by showing bidirectional transcriptional regulation between these two tumor suppressors, and defining an overlapping set of transcriptionally regulated genes. Future studies, including RNA-Seq experiments in the presence and absence of IKAROS and RUNX1 knockdown, will define the transcriptome, explore the functional consequences of co-occupancy at these identified binding sites, and functionally characterize the relationship between IKAROS and RUNX1 in the context of acute myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Identification of therapeutic targets of the hijacked super-enhancer complex in EVI1-rearranged leukemia

Deregulation of the EVI1 proto-oncogene by the GATA2 distal hematopoietic enhancer (G2DHE) is a key event in high-risk acute myeloid leukemia carrying 3q21q26 aberrations (3q-AML). Upon chromosomal rearrangement, G2DHE acquires characteristics of a super-enhancer and causes overexpression of EVI1 at 3q26.2. However, the transcription factor (TF) complex of G2DHE remains poorly characterized. The aim of this study was to unravel key components of G2DHE-bound TFs involved in the deregulation of EVI1. We have identified several CEBPA and RUNX1 binding sites to be enriched and critical for G2DHE function in 3q-AML cells. Using ChIP-SICAP (ChIP followed by selective isolation of chromatin-associated proteins), a panel of chromatin interactors of RUNX1 and CEBPA were detected in 3q-AML, including PARP1 and IKZF1. PARP1 inhibition (PARPi) caused a reduction of EVI1 expression and a decrease in EVI1–G2DHE interaction frequency, highlighting the involvement of PARP1 in oncogenic super-enhancer formation. Furthermore, 3q-AML cells were highly sensitive to PARPi and displayed morphological changes with higher rates of differentiation and apoptosis as well as depletion of CD34 + cells. In summary, integrative analysis of the 3q-AML super-enhancer complex identified CEBPA and RUNX1 associated proteins and nominated PARP1 as a potential new therapeutic target in EVI1 + 3q-AML.