•microRNAs (miRNAs) are one of the molecular participants involved in regulating the translational process during oocyte and early embryo development.•Cellular proteins and the mRNAs that encode them ...are key factors in oocyte and sperm development.•MiRNAs have been identified as a group of testosterone-dependent disruptors in SCs that play an essential role in androgen-mediated processes.
Cellular proteins and the mRNAs that encode them are key factors in oocyte and sperm development, and the mechanisms that regulate their translation and degradation play an important role during early embryogenesis. There is abundant evidence that expression of microRNAs (miRNAs) is crucial for embryo development and are highly involved in regulating translation during oocyte and early embryo development. MiRNAs are a group of short (18–24 nucleotides) non-coding RNA molecules that regulate post-transcriptional gene silencing. The miRNAs are secreted outside the cell by embryos during preimplantation embryo development. Understanding regulatory mechanisms involving miRNAs during gametogenesis and embryogenesis will provide insights into molecular pathways active during gamete formation and early embryo development. This review summarizes recent findings regarding multiple roles of miRNAs in molecular signaling, plus their transport during gametogenesis and embryo preimplantation.
The oncogenic cluster miR‐17‐92 encodes seven related microRNAs that regulate cell proliferation, apoptosis and development. Expression of miR‐17‐92 cluster is decreased upon cell differentiation. ...Here, we report a novel mechanism of the regulation of miR‐17‐92 cluster. Using transgenic PU.1−/− myeloid progenitors we show that upon macrophage differentiation, the transcription factor PU.1 induces the secondary determinant Egr2 which, in turn, directly represses miR‐17‐92 expression by recruiting histone demethylase Jarid1b leading to histone H3 lysine K4 demethylation within the CpG island at the miR‐17‐92 promoter. Conversely, Egr2 itself is targeted by miR‐17‐92, indicating existence of mutual regulatory relationship between miR‐17‐92 and Egr2. Furthermore, restoring EGR2 levels in primary acute myeloid leukaemia blasts expressing elevated levels of miR‐17‐92 and low levels of PU.1 and EGR2 leads to downregulation of miR‐17‐92 and restored expression of its targets p21CIP1 and BIM. We propose that upon macrophage differentiation PU.1 represses the miR‐17‐92 cluster promoter by an Egr‐2/Jarid1b‐mediated H3K4 demethylation mechanism whose deregulation may contribute to leukaemic states.
This study unravels an epigenetic mechanism for the regulation of the oncogenic miRNA cluster 17‐92, involving the master hematopoietic transcription factor PU.1/Egr‐2 and Jarid1b.
Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In this study, we present evidence that MYB (v-myb myeloblastosis viral oncogene ...homolog) is overexpressed in a subset of B-CLL patients. MYB physically associates with the promoter of miR-155 host gene (MIR155HG, also known as BIC, B-cell integration cluster) and stimulates its transcription. This coincides with the hypermethylated histone H3K4 residue and spread hyperacetylation of H3K9 at MIR155HG promoter. Our data provide evidence of oncogenic activities of MYB in B-CLL that include its stimulatory role in MIR155HG transcription.
Introduction: Somatic mutation detection in myelodysplastic syndrome (MDS) is very important in deciphering clonal pathogenesis of every patient and if determined correctly will become useful tool in ...followup studies such as testing individual susceptibility to epigenetic therapy with azacitidine (AZA). While some patients respond to AZA by restoring hematologic parameters, others progress to AML. Recent identification of quite heterogeneous sets of mutated genes (Bejar R et al. 2013) suggested that: patients with specific mutation pattern/s may respond to epigenetic therapy differently.
Aim: We herein set to determine mutation profiles of MDS cohort indicated to and treated by AZA and utilized TrueSight DNA amplicon NGS sequencing approach containing 54 genes all previously associated with MDS or AML.
Patients: We analyzed immunomagnetically CD3-depleted bone marrows of two MDS patients - AZA responders. First patient (male, 68y), was diagnosed with RAEB2, IPSS int-2, transfusion dependent (4 TU/Mo), intermediate cytogenetics (tri21). Following 4 cycles of AZA (75 mg/m2 s.c., 5+2) the patient responded by partial remission, and AZA was discontinued after 17 cycles. Twelve months after discontinuation he progressed and AZA was readministered for additional 3 cycles and the patient achieved again partial remission. Analyzed are samples after 11 (P394) and 20 (P1380) cycles of Vidaza. Second patient (female, 64y), was diagnosed with RAEB2, IPSS high, transfusion dependent (2 TU/Mo), favorable cytogenetics (46XX). Following 4 cycles of AZA (75 mg/m2 s.c., 5+2) she responded by hematology improvement and later by partial remission. Analyzed is a sample after 4 (P1510) cycles of Vidaza. As negative controls we used two normal donor bone marrows from 41y male and 32y female. As a positive control we also used: 1 MDS/AML cell line MOLM-13 with previously identified mutations of CBL and FLT3 (DSMZ; ACC 554).
Methods and approach: Samples were sequenced on Illumina MiSeq sequencer. The mapping was performed using Burrows-Wheeler Aligner algorithm. Illumina Somatic variant caller was used to identify mutations. Then we applied following filters on the data: sequencing coverage should be higher than 1000 per mutation (~80% data left), mutation should be heterozygous (~95% data left), mutation frequency should be higher than 10% (~10% data left), Illumina Somatic variant caller should flag the mutation as "PASS" (~50% data left), mutation should not be synonymous (~75% data left) and mutation should be exonic (~40% data left). These filters were also applied to find mutations in the two control samples. Those mutations which were identified also in the control samples were removed from the analysis of patient samples (~50% data left).
Results: the MDS/AML cell line MOLM-13 contained mutations (SNVs or InDels) in ABL1 (SNV/frequency=46.5%), ASXL1 (SNV/49.8%), CEBPA (In/47.9%), HRAS (SNV/54.5%), TET2 (SNV/49.7%), and as expected also in the genes encoding CBL (delta-exon8/52.4%) and FLT3 (ITD/50.6%). Patient’ sample P1510 contained mutations in CBL (SNV/67.9%), CUX1 (SNV/51.8%), IKZF1 (2 different SNVs/41.9 and 50.7%), KDM6A (SNV/51.6%), SF3B1 (SNV/38.9%), and SMC3 (SNV/33.1%). Patient samples P394 and P1380 contained mutations in the ASXL1 (SNV/ 35.5% and 32.6% respectively), CUX1 (2x SNVs, first SNV/46.1->64.5%, second 48.4->57.9%), and IKZF1 (SNV, 50.4->44.5%) in similar frequencies in the sample before and after 2.5 years (including 9 cycles of AZA) suggesting limited genetic heterogeneity in this AZA-responding patient. Consequently, to gain more insight into how AZA modulates mutation pattern in MDS, we now analyze a set of fourty nine additional patients before and following at least 4 cycles on AZA treatment.
Conclusions: Our data support use of immunomagnetic CD3-depletion of bone marrow and addition of normal control samples in the sequencing of MDS patient samples and support this approach for testing genetic heterogeneity during MDS disease course upon AZA treatment.
Stopka:GAČR P305/12/1033 and UNCE 204021: Research Funding; Celgene: Research Funding; PersMed ltd.: Equity Ownership. Vargova:GAČR P305/12/1033 and P305/11/1745: Research Funding; UNCE 204021: Research Funding; PRVOUK P24/LF1/3: Research Funding. Kulvait:PersMed ltd.: Equity Ownership. Jonasova:PRVOUK P24/LF1/1: Research Funding; Celgene: Research Funding.
Abstract 3848
5-azacitidine (AZA) represents very promising albeit not fully efficient therapy for int-2 and high risk MDS patients. Molecules that interfere with AZA therapy are not known. In ...significant proportion of MDS patients, PU.1 gene is methylated at −17-kb-located upstream regulatory element (URE) where several key transcription factors regulate PU.1 expression. PU.1 represents major factor that controls normal myeloid differentiation. Methylated URE in MDS progenitors can be efficiently demethylated by AZA leading to restoration of cell differentiation capacity (Curik et al 2012). PU.1 gene contains several binding sites for transcription factor CTCF. CTCF represents very important modulator of gene expression, whose binding to DNA can be prevented by DNA methylation. We herein asked if CTCF regulates PU.1 and if so, whether its association with PU.1 gene coincides with DNA methylation status of MDS blasts.
Human high risk MDS patient CD34+ progenitors and MDS-derived erytroleukaemia OCI-M2 and murine erythroleukaemia cell (MEL) lines were studied by RT-PCR, immunoblotting, and chromatin immunoprecipitation (ChIP) assays. Manipulation of gene expression was done by transfection of cDNA or siRNA.
We herein show that CTCF binding sites at PU.1 gene similarly to URE are severely methylated in CD34+ progenitors from high risk MDS patients and MDS-derived erytroleukaemia cell line, and as expected, AZA induced their rapid demethylation. Methylated CTCF binding sites are not occupied by CTCF. However upon AZA-mediated demethylation, CTCF is recruited to the binding sites at PU.1 gene as determined by ChIP. Our other data provided evidence that CTCF interacts with the ISWI ATPse SNF2H (SMARCA5). Indeed, the recruitment of CTCF at PU.1 gene in MDS/AML cells was coincident with recruitment of its interacting partner SMARCA5. In addition, SMARCA5 facilitates CTCF binding to the DNA as demonstrated at ICR locus (near H19 and Igf2 genes) upon siRNA-mediated downregulation of SMARCA5. To understand role of CTCF-SMARCA5 recruitment to the PU.1 gene and its effects on PU.1 expression we upregulated CTCF expression by transfecting an expression plasmid encoding CTCF cDNA and observed that upon increasing CTCF levels the PU.1 protein level was downregulated. Conversely, downregulation of SMARCA5 by siRNA caused upregulation of PU.1 levels. These data indicated that PU.1 is negatively regulated by CTCF and SMARCA5. Furthermore, inhibitory effects of CTCF and SMARCA5 on PU.1 expression were also demonstrated in presence of AZA in MDS cells following DNA demethylation of PU.1 gene.
Our results indicate that CTCF and SMARCA5 are cooperating inhibitory factors to downregulate PU.1 and that AZA-mediated demethylation facilitates the CTCF-SMARCA5 binding to PU.1 gene in MDS patients. CTCF and SMARCA5 are novel factors that interfere with positive prodifferentiation effects of AZA. (Grant support: P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/3, SVV-2012–264507, P301/12/P380, GAUK 251070 45410 and 251135 82210).
No relevant conflicts of interest to declare.
Abstract 769
Downregulation of tumour suppressor transcription factor PU.1 in haematopoietic stem and progenitor cells represents primary underlying mechanism for the development of acute myeloid ...leukaemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. Human AML often display differences in aggressiveness that are associated with mutations of a well known tumour suppressor p53. We produced murine model carrying mutations of p53 and URE that develops highly aggressive AML and focused on molecular mechanisms that are responsible for AML aggressiveness.
PU.1ure/ure (Rosenbauer F, et al. 2004) and p53−/− (Jacks T, et al. 1994) mice were used. Conditional deletion of the URE leads to downregulation of PU.1 and is marked by clonal accumulation of myeloid c-Kit+Mac-1low Gr-1low blast cells within bone marrow, spleen, and peripheral blood mirrored by lower numbers of lymphoid and erythroid cells. AML development in PU.1ure/ure mice involves a preleukaemic phase (at 2–3 months) marked by proliferation of myeloid c-Kit+Gr-1+ cells and splenomegaly. Interestingly, p53−/−mice do not develop AML, instead loss of p53 predisposes mice to solid tumours, mostly lymphomas, by 6 months of age.
Deletion of TP53 in the PU.1ure/ure mice (PU.1ure/ure p53−/−) results in more aggressive AML with significantly shortened overall survival, prominent hepatosplenomegaly and cachexia (wasting syndrome). Mild differences in cell surface phenotype of bone marrow derived cells were observed between PU.1ure/ure and PU.1ure/ure p53−/− mice by flow cytometry (these included: blasts expansion and lymphopenia). Next, the PU.1 expression was determined in all genotypes at progenitor and stem cell levels. PU.1 mRNA level in more aggressive PU.1ure/ure p53−/− murine AML is decreased in the entire c-Kit+tumour cell population compared to AML in PU.1ure/ure mice including haematopoietic stem and progenitor cells (HSPCs). Correspondingly to RNA level, in the PU.1ure/ure progenitors the PU.1 protein was decreased compared to p53−/− progenitors and is yet further reduced in the PU.1ure/ure p53−/− c-Kit+ Mac1+progenitors. p53−/− progenitors express similar level of PU.1 as wild type progenitors indicating that despite p53 can bind DNA as a transcription factor, it does not regulate PU.1 level directly. In addition to URE deletion we searched for other mechanisms that control PU.1 levels and found that PU.1-inhibiting microRNA miR-155 gene display altered chromatin structure and expression of both pri-miR-155 as well as its spliced mature form in the AML of PU.1ure/ure and (to higher extent in) PU.1ure/ure p53−/− murine progenitors. Upregulation of miR-155 coincides with upregulation of the Mir155hg activators: Myc and Myb. Finally, upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukaemic cell growth.
In summary, PU.1 and p53 double mutant mice develop aggressive AML with dysplastic features. Defective control of PU.1 levels in PU.1ure/ure and PU.1ure/ure p53−/−AML involves miR-155. Lastly, restored PU.1 level and cell differentiation capacity are achieved by inhibiting either Myb or miR-155 in the PU.1ure/ure p53−/− progenitors. (Grant support: P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/3, SVV-2012-264507, P301/12/P380. MK was sponsored by GAUK 251070 45410, 251135 82210)
No relevant conflicts of interest to declare.
Abstract 4624
Mantle cell lymphoma (MCL) represents B-cell lymphoma derived from the mantle zone that surrounds normal germinal center follicles. Pathophysiology of this hardly curable disease ...involves t(11,14)(q13,q32) translocation which leads to upregulation of Cyclin D1(CCND1). Recently, microRNAs were demonstrated to significantly modify MCL pathogenesis (Jian-Jun Zhao et al. 2010) and therapy responsiveness (Jiang et al. 2010). In order to broaden our knowledge of regulatory pathways in MCL we searched for differentially expressed microRNAs and their differentially expressed mRNA targets between MCL samples and control samples. Samples consist of magnetically separated B cells derived from peripheral blood. We used 1) Microarray mRNA hybridization Affymetrix Human Genome U133 Plus 2.0 Array, N(MCL)=5, N(control)=5 and 2) microRNA profiling TaqMan® Array Human MicroRNA Card A v2.0 technology, N(MCL)=5, N(control)=5 followed by statistical analysis limma (Smyth 2005). Differentially regulated targets of the deregulated microRNAs were selected from the databases involving both predicted (Betel et al. 2007) or confirmed (Hsu et al. 2010) target mRNAs. Among the most significant upregulated microRNAs (exceeding 10 fold) are miR-9, miR-124 and miR-183. We have found that upregulated miR-9 has confirmed downregulated target gene PRDM1 (PR domain zinc finger protein 1) which plays a role in B cell maturation (Turner et al. 1994) and may act as a tumor suppressor (Pasqualucci et al. 2006). Upregulation of miR-9 and downregulation of its targets was recently demonstrated in Burkitt lymphoma (Onnis A et al. 2010) and Hodgkin lymphoma (Nie K et al. 2008). Two additional upregulated microRNAs, miR-124 and miR-183, yet not associated with lymphomas, have downregulated target gene Integrin beta-1 (CD29) which regulates survival (Fukumori et al. 2003). Among most significant downregulated microRNAs is miR-101 (FC=-7). Downregulated microRNA miR-101 has known oncogene N-Myc (MYCN) as upregulated confirmed target and DNA (cytosine-5)-methyltransferase 3A (DNMT3A) as upregulated target. Overexpression of a well known hematopoietic oncogene MYCN and epigenetic repressor DNMT3A may represent additional pathogenesis-related factors in MCL. Our data support importance of the candidate mechanisms involving microRNAs and their target programs in pathogenesis of MCL. They are currently extended on a larger patient cohort by analyzing expression and functional significance of the candidate microRNAs. (Grants: NT10310-3/2009, MPO FR-TI2/509, NPVII 2B06077, MSM 0021620806, LC 06044, SVV-2011-262507).
No relevant conflicts of interest to declare.
Abstract 124
The myelodysplastic syndrome (MDS) represents a heterogeneous disorder characterized by ineffective hematopoiesis and evolution to acute myelogenous leukemia that is strikingly ...refractory to current therapeutic approaches. Novel epigenetic drugs including DNA-methyltransferase inhibitor 5-Azacitidine (5-AZA, Vidaza) are currently considered to improve clinical response in patients with MDS. MDS is characterized by abnormal differentiation and blocked maturation responsive to 5-AZA, therefore we studied major regulator of hematopoietic differentiation, transcription factor PU.1 as a candidate target of the epigenetic therapy. Transcription factor PU.1 represents very important myelo-lymphoid regulator of differentiation. PU.1 expression is regulated by Upstream Regulatory Element (URE) and its deletion in mouse caused downregulation of PU.1 leading to acute leukemia (Rosenbauer 2004). Our laboratory recently demonstrated that PU.1 in murine acute leukemic cells binds and promotes derepression of CCAAT/enhancer binding protein (C/EBP) alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) (Burda 2009) that encode two key hematopoietic transcription factors involved in myeloid differentiation. Furthermore, transcriptional regulation through PU.1 binding sites of Cebpa and Cbfb loci involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3 lysine K9. Others reported that Cebpa expression is augmented by G-CSF (Dahl 2003). To determine if 5-AZA regulates PU.1 and its targets we determined their expression and chromatin structure following the 5-AZA treatment in MDS patient-derived blasts and in cell lines derived from MDS (MOLM-13, OCI-M2, SKM-1) and AML (K562). Our data provide evidence that in the chosen cell lines and in so far limited number of patients-derived cells (N=4) the gene expression of PU.1 and its direct targets Cebpa and Cbfb is stimulated by 5-AZA and this effect is further enhanced by G-CSF. Furthermore, marks of activated chromatin structure including histone H3K9 hyperacetylation and H3K4 hypermethylation are increased at the URE of the PU.1 gene again documenting its transcriptional activation. Conversely, levels of H3K9 methylation at URE are significantly reduced upon 5-AZA treatment documenting 5-AZA stimulates loss of repressive chromatin structure near PU.1 gene. These observations are currently compared with responsiveness of the patients to 5-AZA in vivo and expanded to larger set of patients. Our data collectively supports importance of the chromatin structure upstream of PU.1 gene and of its direct targets Cebpa and Cbfb in patients with MDS that may add to better understanding of effectiveness of epigenetic therapy in MDS. (Grants # IGA 10310-3, MSMT 2B06077, SVV-2010-254260507, MPO FR-TI2/509, GAUK 251135 82210).
No relevant conflicts of interest to declare.
Abstract 390
The miR-17-92 cluster (Oncomir1) encodes seven related microRNAs associated with cell proliferation, apoptosis and development and is overexpressed in number of malignancies including ...myeloid leukemias. The miR-17-92 cluster is highly expressed in myeloid progenitors, while it becomes downregulated upon the onset of macrophage differentiation. Conversely, sustained expression of miR-17-92 is associated with a differentiation blockade (Fontana 2007). Here we report a novel mechanism of the regulation of miR-17-92 cluster within differentiating myeloid progenitors. During macrophage differentiation, the myeloid transcription factor PU.1 transcriptionally induces the secondary determinant Early growth factor 2 (Egr2). Subsequently, Egr2 binds to the miR-17-92 cluster promoter and recruits histone demethylase Jarid1b resulting in histone H3 lysine K4 (H3K4) demethylation within the CpG island upstream miR-17-92 cluster leading to the repression of miR-17-92 expression. Reporter assays using deletion constructs of the miR-17-92 promoter region revealed that Egr2 is required for targeting the Jarid1b onto critical minimal region within CpG island of upstream miR-17-92 locus and its decreased H3K4 methylation. In addition, functional assays identified the 3'UTR of Egr2 as the target of multiple miRNAs of the miR-17-92 cluster, indicating existence of a mutual regulation between miR-17-92 cluster and Egr2, putatively involved in macrophage differentiation characterized by a bistable state, where Egr2 negatively regulates miR-17-92 cluster in differentiating cells and, in turn, miR-17-92 cluster negatively regulates Egr2 in highly proliferating progenitor cells to achieve homeostatic regulation. Ectopic expression of miR-17-92 cluster within myeloid progenitors blocked macrophage differentiation indicating that leukemogenesis may involve miR-17-92 mediated differentiation blockade. To determine if the newly identified negative regulatory mechanism of miR-17-92 is involved in leukemogenesis, we tested peripheral blood mononuclear cells isolated from acute myeloid leukemia (AML) patients (N=27). 14 of 27 AML patients exhibited significantly downregulated transcriptional factors PU.1 and Egr2 and high levels of the miR-17-92 cluster expression. Ectopic expression of Egr2 within AML blast cells (N=2) led to decreased levels of miR-17-92 and restored the expression of p21 and BIM, two established miR-17-92 targets downregulated in AML. We conclude that PU.1 represses miR-17-92 cluster during macrophage differentiation by Egr2 mediated recruitment of the Jarid1b demethylase and that dysregulation in the miR-17-92 repression mechanism may contribute to the pathogenesis of AML. (Grants # IGA 10310-3, MSMT 2B06077, 0021620806, LC06044, SVV-2010-254260507).
No relevant conflicts of interest to declare.
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