Runt-related transcription factor 1 (RUNX1) is an essential regulator of normal hematopoiesis. Its dysfunction, caused by either fusions or mutations, is frequently reported in acute myeloid leukemia (AML). However, RUNX1 mutations have been largely under-explored compared with RUNX1 fusions mainly due to their elusive genetic characteristics. Here, based on 1741 patients with AML, we report a unique expression pattern associated with RUNX1 mutations in AML. This expression pattern was coordinated by target repression and promoter hypermethylation. We first reanalyzed a joint AML cohort that consisted of three public cohorts and found that RUNX1 mutations were mainly distributed in the Runt domain and almost mutually exclusive with NPM1 mutations. Then, based on RNA-seq data from The Cancer Genome Atlas AML cohort, we developed a 300-gene signature that significantly distinguished the patients with RUNX1 mutations from those with other AML subtypes. Furthermore, we explored the mechanisms underlying this signature from the transcriptional and epigenetic levels. Using chromatin immunoprecipitation sequencing data, we found that RUNX1 target genes tended to be repressed in patients with RUNX1 mutations. Through the integration of DNA methylation array data, we illustrated that hypermethylation on the promoter regions of RUNX1-regulated genes also contributed to dysregulation in RUNX1-mutated AML. This study revealed the distinct gene expression pattern of RUNX1 mutations and the underlying mechanisms in AML development.
Core Binding Factor Alpha 2 Subunit/metabolism* ; DNA Methylation ; Gene Expression ; Humans ; Leukemia, Myeloid, Acute/genetics* ; Mutation ; Promoter Regions, Genetic

OBJECTIVE: To investigate the effects of Bax inhibitor 1 (BI- 1) and optic atrophy protein 1 (OPA1) on vascular calcification (VC). METHODS: Mouse models of VC were established in ApoE-deficient (ApoE^-/-) diabetic mice by high-fat diet feeding for 12 weeks followed by intraperitoneal injections with N^ε-carboxymethyl-lysine for 16 weeks. ApoE^-/- mice (control group), ApoE^-/- diabetic mice (VC group), ApoE^-/- diabetic mice with BI-1 overexpression (VC + BI-1^TG group), and ApoE^-/- diabetic mice with BI-1 overexpression and OPA1 knockout (VC+BI-1^TG+OPA1^-/- group) were obtained for examination of the degree of aortic calcification using von Kossa staining. The changes in calcium content in the aorta were analyzed using ELISA. The expressions of Runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP-2) were detected using immunohistochemistry, and the expression of cleaved caspase-3 was determined using Western blotting. Cultured mouse aortic smooth muscle cells were treated with 10 mmol/L β-glycerophosphate for 14 days to induce calcification, and the changes in BI-1 and OPA1 protein expressions were examined using Western blotting and cell apoptosis was detected using TUNEL staining. RESULTS: ApoE^-/- mice with VC showed significantly decreased expressions of BI-1 and OPA1 proteins in the aorta (P=0.0044) with obviously increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P= 0.0041). Overexpression of BI-1 significantly promoted OPA1 protein expression and reduced calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P=0.0006). OPA1 knockdown significantly increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 in the aorta (P=0.0007). CONCLUSION BI-1 inhibits VC possibly by promoting the expression of OPA1, reducing calcium deposition and inhibiting osteogenic differentiation and apoptosis of the vascular smooth muscle cells.
Animals ; Apolipoproteins E/metabolism* ; Calcium/metabolism* ; Caspase 3/metabolism* ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Diabetes Mellitus, Experimental/pathology* ; GTP Phosphohydrolases/metabolism* ; Membrane Proteins/metabolism* ; Mice ; Mice, Knockout ; Muscle, Smooth, Vascular/pathology* ; Myocytes, Smooth Muscle/pathology* ; Optic Atrophy, Autosomal Dominant/pathology* ; Osteogenesis ; Vascular Calcification/pathology* ; bcl-2-Associated X Protein/metabolism*

As a dioxygenase, Ten-Eleven Translocation 2 (TET2) catalyzes subsequent steps of 5-methylcytosine (5mC) oxidation. TET2 plays a critical role in the self-renewal, proliferation, and differentiation of hematopoietic stem cells, but its impact on mature hematopoietic cells is not well-characterized. Here we show that Tet2 plays an essential role in osteoclastogenesis. Deletion of Tet2 impairs the differentiation of osteoclast precursor cells (macrophages) and their maturation into bone-resorbing osteoclasts in vitro. Furthermore, Tet2 mice exhibit mild osteopetrosis, accompanied by decreased number of osteoclasts in vivo. Tet2 loss in macrophages results in the altered expression of a set of genes implicated in osteoclast differentiation, such as Cebpa, Mafb, and Nfkbiz. Tet2 deletion also leads to a genome-wide alteration in the level of 5-hydroxymethylcytosine (5hmC) and altered expression of a specific subset of macrophage genes associated with osteoclast differentiation. Furthermore, Tet2 interacts with Runx1 and negatively modulates its transcriptional activity. Our studies demonstrate a novel molecular mechanism controlling osteoclast differentiation and function by Tet2, that is, through interactions with Runx1 and the maintenance of genomic 5hmC. Targeting Tet2 and its pathway could be a potential therapeutic strategy for the prevention and treatment of abnormal bone mass caused by the deregulation of osteoclast activities.
5-Methylcytosine ; analogs & derivatives ; chemistry ; metabolism ; Animals ; Cell Differentiation ; Cells, Cultured ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; DNA-Binding Proteins ; physiology ; Genome ; Genomics ; Mice ; Mice, Knockout ; Osteoclasts ; cytology ; metabolism ; Proto-Oncogene Proteins ; physiology

No abstract available.
Bone Marrow/pathology ; Chromosomes, Human, Pair 12 ; Chromosomes, Human, Pair 9 ; Core Binding Factor Alpha 2 Subunit/*genetics ; DNA/metabolism ; Gene Rearrangement ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis/*genetics ; Male ; Middle Aged ; Oncogene Proteins, Fusion/*genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Translocation, Genetic

BACKGROUNDThe acute myeloid leukemia 1 (AML1)-eight-twenty-one (ETO) fusion protein generated by the t(8;21)(q22;q22) translocation is considered to display a crucial role in leukemogenesis in AML. By focusing on the anti-leukemia effects of eyes absent 4 (EYA4) gene on AML cells, we investigated the biologic and molecular mechanism associated with AML1-ETO expressed in t(8;21) AML.

METHODSQualitative polymerase chain reaction (PCR), quantitative reverse transcription PCR (RT-PCR), and Western blotting analysis were used to observe the mRNA and protein expression levels of EYA4 in cell lines. Different plasmids (including mutant plasmids) of dual luciferase reporter vector were built to study the binding status of AML1-ETO to the promoter region of EYA4. Chromatin immunoprecipitation assay was used to study the epigenetic silencing mechanism of EYA4. Bisulfite sequencing was applied to detect the methylation status in EYA4 promoter region. The influence of EYA4 gene in the cell proliferation, apoptosis, and cell clone-forming ability was detected by the technique of Cell Counting Kit-8, flow cytometry, and clonogenic assay.

RESULTSEYA4 gene was hypermethylated in AML1-ETO+ patients and its expression was down-regulated by 6-fold in Kasumi-1 and SKNO-1 cells, compared to HL-60 and SKNO-1-siA/E cells, respectively. We demonstrated that AML1-ETO triggered the epigenetic silencing of EYA4 gene by binding at AML1-binding sites and recruiting histone deacetylase 1 and DNA methyltransferases. Enhanced EYA4 expression levels inhibited cellular proliferation and suppressed cell colony formation in AML1-ETO+ cell lines. We also found EYA4 transfection increased apoptosis of Kasumi-1 and SKNO-1 cells by 1.6-fold and 1.4-fold compared to negative control, respectively.

CONCLUSIONSOur study identified EYA4 gene as targets for AML1-ETO and indicated it as a novel tumor suppressor gene. In addition, we provided evidence that EYA4 gene might be a novel therapeutic target and a potential candidate for treating AML1-ETO+ t (8;21) AML.

Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; genetics ; physiology ; Chromatin Immunoprecipitation ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; DNA Methylation ; genetics ; Epigenesis, Genetic ; genetics ; Gene Silencing ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; pathology ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; RUNX1 Translocation Partner 1 Protein ; Radioimmunoprecipitation Assay ; Trans-Activators ; genetics ; metabolism

OBJECTIVETo explore the effect of total flavonoids of Herba Epimedium (FHE) on BMP-2/RunX2/OSX signaling pathway in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).

METHODSPassage 3 BMSCs were randomly divided into the control group, the experimental group, and the inhibitor group. BMSCs in the control group were cultured in 0.2% dimethyl sulfoxide + Osteogenuxic Supplement (OS) fluid + DMEM/F12 culture media. BMSCs in the experimental group were intervened by 20 microg/mL FHE. BMSCs in the inhibitor group were intervened by 20 microg/mL FHE and 1 microg/mL NOGGIN recombinant protein. At day 9 alkaline phosphatase (ALP) activity was measured. Calcium nodules were stained by alizarin red staining and the density was observed. The transcription expression of osteogenic differentiation-related proteins (type I collagen, osteocalcin, and osteopontin) and related factors of BMP-2/RunX2/OSX signaling pathway was assayed by RT-PCR.

RESULTSCompared with the control group, ALP activities were enhanced and the density of calcium nodules significantly increased; type I collagen, osteocalcin, and osteopontin expression levels were increased in the experimental group. The expression of osteogenesis-related transcription factor was also increased in the experimental group. Noggin recombinant protein inhibited FHE promoting BMSCs osteogenesis in the inhibitor group. Compared with the experimental group, ALP activity decreased (P < 0.05), the density of calcium nodules was lowered, expression levels of type I collagen, osteocalcin, osteopontin significantly decreased (P < 0.05) in the inhibitor group.

CONCLUSION20 microg/mL FHE promoted osteogenic differentiation process of BMSCs by BMP-2/RunX2/OSX signaling pathway.

Bone Morphogenetic Protein 2 ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Collagen Type I ; metabolism ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Epimedium ; chemistry ; Flavonoids ; pharmacology ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Osteocalcin ; metabolism ; Osteogenesis ; drug effects ; Osteopontin ; metabolism ; Signal Transduction ; Sp7 Transcription Factor ; Transcription Factors ; metabolism

OBJECTIVETo observe the effect of Bushen Huoxue Recipe (BHR) on inhibiting vascular calcification (VC) in chronic renal failure (CRF) rats by regulating BMP-2/Runx2/Osterix signal pathway, and to explore its possible mechanism.

METHODSThirty SD rats were randomly divided into the normal group, the model group, and the BHR group, 10 in each group. Rats in the model group and the BHR group were administered with 250 mg/kg adenine suspension by gastroagavage and fed with 1.8% high phosphorus forage, once per day in the first 4 weeks, and then gastric administration of adenine suspension was changed to once per two days in the following 5-8 weeks. Rats in the BHR group were administered with BHR at the daily dose of 55 g/kg by gastrogavage in the first 8 weeks, once per day. Equal volume of normal saline was given to rats in the normal group by gastrogavage for 8 weeks. Histological changes in renal tissue and aorta VC were observed by HE staining and alizarin red staining respectively. Levels of calcium (Ca), phosphorus (P), serum creatinine (Cr), blood urea nitrogen (BUN), and intact parathyroid hormone (iPTH) in serum were detected. Protein expression levels of bone morphogenetic protein (BMP-2), Runt related transcription factor (Runx2) , and Osterix were detected by Western blot.

RESULTSHE staining showed that compared with the normal group, disordered glomerular structure, tubular ectasia and dropsy, intracavitary inflammatory cell infiltration, dark brown crystal deposition in kidney tubules, renal interstitial fibrosis, and decreased number of renal blood vessels in the model group. Compared with the model group, normal glomerular numbers increased more, reduced degree of tubular ectasia, decreased number of inflammatory cells, and reduced adenine crystal deposition in the BHR group. Alizarin red staining showed that compared with the normal group, calcified nodes could be found in the model group, with extensive deposition of red particle in aorta. Compared with the model group, calcified nodes were reduced in the BHR group. Compared with normal group, serum levels of P, SCr, BUN, and iPTH significantly increased, serum Ca level significantly decreased, protein expressions of BMP-2, Runx2, Osterix also increased in the model group (P < 0.05, P < 0.01). Compared with the model group, serum levels of P, SCr, BUN, and iPTH levels significantly decreased, serum Ca level significantly increased, protein expressions of BMP-2, Runx2, Osterix also decreased in the BHD group (P < 0.05, P < 0.01).

CONCLUSIONBHD could improve renal function, Ca-P metabolism, and renal histological changes in CHF rats, down-regulate the expression level of BMP-2/Runx2/Osterix signal pathway in vascular calcification of CRF, which might be one of the mechanisms for inhibiting VC in CHF.

Animals ; Blood Urea Nitrogen ; Bone Morphogenetic Protein 2 ; metabolism ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Kidney ; pathology ; Kidney Failure, Chronic ; drug therapy ; metabolism ; Kidney Function Tests ; Kidney Tubules ; pathology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Transcription Factors ; metabolism ; Vascular Calcification ; drug therapy

Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; Humans ; Leukemia, B-Cell ; genetics

BACKGROUND: Intrachromosomal amplification of chromosome 21 (iAMP21) is known to be associated with poor prognosis in B-cell ALL (B-ALL). To determine the frequency and clinical characteristics of iAMP21 in Korean B-ALL patients, we performed FISH and multiplex ligation-dependent probe amplification (MLPA) analyses. METHODS: A total of 102 childhood B-ALL patients were screened with ETV6-RUNX1 FISH probes (Abbott Molecular, USA). The presence of an iAMP21 was confirmed by using MLPA P327 iAMP21-ERG probemix (MRC Holland, The Netherlands). RESULTS: iAMP21 was detected in one of the screened B-ALL patients (1/102 patients, 1.0%) who presented the ALL immunophenotype and complex karyotype at initial diagnosis. The patient relapsed twice after bone marrow transplantation. MLPA showed 12.5-Mb and 4.28-Mb regions of amplification and deletion, respectively. CONCLUSIONS: The frequency of iAMP21 is considerable in Korean pediatric patients. Our report suggests that iAMP21 in childhood B-ALL has very unfavorable impact on patient's prognosis. Additional methods such as MLPA analysis is essential to rule out patients with equivocal interphase FISH results.
Adolescent ; Asian Continental Ancestry Group/*genetics ; B-Lymphocytes/*metabolism ; Child ; Child, Preschool ; *Chromosomes, Human, Pair 21 ; Core Binding Factor Alpha 2 Subunit/genetics ; DNA Probes/metabolism ; Female ; Humans ; Immunophenotyping ; In Situ Hybridization, Fluorescence ; Infant ; Infant, Newborn ; Male ; Multiplex Polymerase Chain Reaction ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/*diagnosis/genetics ; Proto-Oncogene Proteins c-ets/genetics ; Repressor Proteins/genetics ; Republic of Korea ; Translocation, Genetic ; Young Adult

No abstract available.
Adult ; Amino Acid Sequence ; Bone Marrow/metabolism/pathology ; Chromosomes, Human, Pair 21 ; Chromosomes, Human, Pair 8 ; Core Binding Factor Alpha 2 Subunit/*genetics ; Exons ; Female ; Humans ; INDEL Mutation ; Leukemia, Myeloid, Acute/*genetics/pathology ; Multiplex Polymerase Chain Reaction ; Proto-Oncogene Proteins/*genetics ; Proto-Oncogene Proteins c-kit/*genetics ; Transcription Factors/*genetics ; *Translocation, Genetic