Paediatric patients with acute leukaemia and KMT2A (MLL) rearrangement show a distinctive expression pattern of histone deacetylases.

Histone deacetylase inhibitors (HDACi) had emerged as promising drugs in leukaemia, but their toxicity due to lack of specificity limited their use. Therefore, there is a need to elucidate the role of HDACs in specific settings. The study of HDAC expression in childhood leukaemia could help to choose more specific HDACi for selected candidates in a personalized approach. We analysed HDAC1-11, SIRT1, SIRT7, MEF2C and MEF2D mRNA expression in 211 paediatric patients diagnosed with acute leukaemia. There was a global overexpression of HDACs, while specific HDACs correlated with clinical and biological features, and some even predicted outcome. Thus, some HDAC and MEF2C profiles probably reflected the lineage and the maturation of the blasts and some profiles identified specific oncogenic pathways active in the leukaemic cells. Specifically, we identified a distinctive signature for patients with KMT2A (MLL) rearrangement, with high HDAC9 and MEF2D expression, regardless of age, KMT2A partner and lineage. Moreover, we observed an adverse prognostic value of HDAC9 overexpression, regardless of KMT2A rearrangement. Our results provide useful knowledge on the complex picture of HDAC expression in childhood leukaemia and support the directed use of specific HDACi to selected paediatric patients with acute leukaemia.

Thrombin-induced, TNFR-dependent miR-181c downregulation promotes MLL1 and NF-κB target gene expression in human microglia.

BACKGROUND: Controlling thrombin-driven microglial activation may serve as a therapeutic target for intracerebral hemorrhage (ICH). Here, we investigated microRNA (miRNA)-based regulation of thrombin-driven microglial activation using an in vitro thrombin toxicity model applied to primary human microglia. METHODS: A miRNA array identified 22 differential miRNA candidates. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) identified miR-181c as the most significantly downregulated miRNA. TargetScan analysis identified mixed lineage leukemia-1 (MLL1) as a putative gene target for miR-181c. qRT-PCR was applied to assess tumor necrosis factor-alpha (TNF-α), miR-181c, and MLL1 levels following thrombin or proteinase-activated receptor-4-specific activating peptide (PAR4AP) exposure. Anti-TNF-α antibodies and tumor necrosis factor receptor (TNFR) silencing were employed to test TNF-α/TNFR dependence. A dual-luciferase reporter system and miR-181c mimic transfection assessed whether mir-181c directly binds to and negatively regulates MLL1. Nuclear factor kappa-B (NF-κB)-dependent luciferase reporter assays and NF-κB target gene expression were assessed in wild-type (MLL1+) and MLL1-silenced cells. RESULTS: Thrombin or PAR4AP-induced miR-181c downregulation (p < 0.05) and MLL1 upregulation (p < 0.05) that were dependent upon TNF-α/TNFR. miR-181c decreased wild-type MLL1 3'-UTR luciferase reporter activity (p < 0.05), and a miR-181c mimic suppressed MLL1 expression (p < 0.05). Thrombin treatment increased, while miR-181c reduced, NF-κB activity and NF-κB target gene expression in both wild-type (MLL1+) and MLL1-silenced cells (p < 0.05). CONCLUSIONS: Thrombin-induced, TNF-α/TNFR-dependent miR-181c downregulation promotes MLL1 expression, increases NF-κB activity, and upregulates NF-κB target gene expression. As miR-181c opposes thrombin's stimulation of pro-inflammatory NF-κB activity, miR-181c mimic therapy may show promise in controlling thrombin-driven microglial activation following ICH.

KMT2A promotes melanoma cell growth by targeting hTERT signaling pathway.

Melanoma is an aggressive cutaneous malignancy, illuminating the exact mechanisms and finding novel therapeutic targets are urgently needed. In this study, we identified KMT2A as a potential target, which promoted the growth of human melanoma cells. KMT2A knockdown significantly inhibited cell viability and cell migration and induced apoptosis, whereas KMT2A overexpression effectively promoted cell proliferation in various melanoma cell lines. Further study showed that KMT2A regulated melanoma cell growth by targeting the hTERT-dependent signal pathway. Knockdown of KMT2A markedly inhibited the promoter activity and expression of hTERT, and hTERT overexpression rescued the viability inhibition caused by KMT2A knockdown. Moreover, KMT2A knockdown suppressed tumorsphere formation and the expression of cancer stem cell markers, which was also reversed by hTERT overexpression. In addition, the results from a xenograft mouse model confirmed that KMT2A promoted melanoma growth via hTERT signaling. Finally, analyses of clinical samples demonstrated that the expression of KMT2A and hTERT were positively correlated in melanoma tumor tissues, and KMT2A high expression predicted poor prognosis in melanoma patients. Collectively, our results indicate that KMT2A promotes melanoma growth by activating the hTERT signaling, suggesting that the KMT2A/hTERT signaling pathway may be a potential therapeutic target for melanoma.

KMT2A and KMT2B Mediate Memory Function by Affecting Distinct Genomic Regions.

Kmt2a and Kmt2b are H3K4 methyltransferases of the Set1/Trithorax class. We have recently shown the importance of Kmt2b for learning and memory. Here, we report that Kmt2a is also important in memory formation. We compare the decrease in H3K4 methylation and de-regulation of gene expression in hippocampal neurons of mice with knockdown of either Kmt2a or Kmt2b. Kmt2a and Kmt2b control largely distinct genomic regions and different molecular pathways linked to neuronal plasticity. Finally, we show that the decrease in H3K4 methylation resulting from Kmt2a knockdown partially recapitulates the pattern previously reported in CK-p25 mice, a model for neurodegeneration and memory impairment. Our findings point to the distinct functions of even closely related histone-modifying enzymes and provide essential insight for the development of more efficient and specific epigenetic therapies against brain diseases.

Prognostic Significance of Mixed-Lineage Leukemia (MLL) Gene Detected by Real-Time Fluorescence Quantitative PCR Assay in Acute Myeloid Leukemia.

BACKGROUND The overall prognosis of acute myeloid leukemia (AML) patients with mixed-lineage leukemia (MLL) gene-positivity is unfavorable. In this study, we evaluated the expression levels of the MLL gene in AML patients. MATERIAL AND METHODS We enrolled 68 MLL gene-positive patients out of 433 newly diagnosed AML patients, and 216 bone marrow samples were collected. Real-time fluorescence quantitative PCR (RQ-PCR) was used to precisely detect the expression levels of the MLL gene. RESULTS We divided 41 patients into 2 groups according to the variation of MRD (minimal residual disease) level of the MLL gene. Group 1 (n=22) had a rapid reduction of MRD level to ≤10^-4 in all samples collected in the first 3 chemotherapy cycles, while group 2 (n=19) had MRD levels constantly >10^-4 in all samples collected in the first 3 chemotherapy cycles. Group 1 had a significantly better overall survival (p=0.001) and event-free survival (p=0.001) compared to group 2. Moreover, the patients with >10^-4 MRD level before the start of HSCT (hematopoietic stem cell transplantation) had worse prognosis and higher risk of relapse compared to patients with ≤10^-4 before the start of HSCT. CONCLUSIONS We found that a rapid reduction of MRD level to ≤10^-4 appears to be a prerequisite for better overall survival and event-free survival during the treatment of AML. The MRD levels detected by RQ-PCR were basically in line with the clinical outcome and may be of great importance in guiding early allogeneic HSCT (allo-HSCT) treatment.

SON and Its Alternatively Spliced Isoforms Control MLL Complex-Mediated H3K4me3 and Transcription of Leukemia-Associated Genes.

Dysregulation of MLL complex-mediated histone methylation plays a pivotal role in gene expression associated with diseases, but little is known about cellular factors modulating MLL complex activity. Here, we report that SON, previously known as an RNA splicing factor, controls MLL complex-mediated transcriptional initiation. SON binds to DNA near transcription start sites, interacts with menin, and inhibits MLL complex assembly, resulting in decreased H3K4me3 and transcriptional repression. Importantly, alternatively spliced short isoforms of SON are markedly upregulated in acute myeloid leukemia. The short isoforms compete with full-length SON for chromatin occupancy but lack the menin-binding ability, thereby antagonizing full-length SON function in transcriptional repression while not impairing full-length SON-mediated RNA splicing. Furthermore, overexpression of a short isoform of SON enhances replating potential of hematopoietic progenitors. Our findings define SON as a fine-tuner of the MLL-menin interaction and reveal short SON overexpression as a marker indicating aberrant transcriptional initiation in leukemia.

[Biological microchip for establishing the structure of fusion transcripts involving MLL in children with acute leukemia].

MLL is involved in fusion genes with more than 100 partner genes, approximately 80 of which have been characterized at the molecular level. MLL fusion genes are often found in infants (60-80% of acute lymphoblastic leukemia (ALL) cases and 40-50% of acute myeloblastic leukemia (AML) cases) and are appreciably rarer (8-10%) in children older than 1 year of age. MLL rearrangements are important markers in diagnosis and treatment choice. To identify the partner gene is of primary importance for prognosis and minimal residual disease monitoring. The structure of the fusion gene, including localization of the MLL breakpoints, is also informative. A method was developed to examine the fusion transcripts in order to identify the partner gene among the six most common ones and to establish the exon structure of the rearranged MLL. The method includes a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify and to fluorescently label a fusion transcript fragment and subsequent hybridization of the product on a biological microchip with immobilized oligonucleotides complementary to exons of MLL and its partner genes AFF1, MLLT1, MLLT3, MLLT4, MLLT10, and ELL. Hybridization results were verified by sequencing the RT-PCR products and, in some cases, performing long-distance inverse PCR (LDI-PCR). The study involved 38 bone marrow samples from ALL patients (including 33 children younger than 1 year of age) and 15 samples from AML patients (including 10 from children younger than 1 year of age). The main partner genes were AFF1 (49%), MLLT1 (27%), MLLT3 (12%), and MLLT10 (12%) in ALL and MLLT3 (80%), MLLT10 (10%), and MLLT4 (10%) in AML. Fusion gene transcripts most commonly included MLL exon 11 (58% of ALL cases and 50% of AML cases), suggesting a breakpoint in MLL intron 11.

Targeting DOT1L and HOX gene expression in MLL-rearranged leukemia and beyond.

Leukemias harboring mixed-lineage leukemia gene (MLL1) abnormalities are associated with poor clinical outcomes, and new therapeutic approaches are desperately needed. Rearrangement of the MLL1 gene generates chimeric proteins that fuse the NH3 terminus of MLL1 to the COOH terminus of its translocation partners. These MLL1 fusion oncoproteins drive the expression of homeobox genes such as HOXA cluster genes and myeloid ecotropic viral integration site 1 homolog (MEIS1), which are known to induce leukemic transformation of hematopoietic progenitors. Genomewide histone methylation studies have revealed that the abnormal expression of MLL1 fusion target genes is associated with high levels of H3K79 methylation at these gene loci. The only known enzyme that catalyzes methylation of H3K79 is disruptor of telomeric-silencing 1-like (DOT1L). Loss-of-function mouse models, as well as small molecular inhibitors of DOT1L, illustrate that leukemias driven by MLL1 translocations are dependent on DOT1L enzymatic activity for proliferation and for the maintenance of HOXA gene expression. Furthermore, DOT1L also appears to be important for HOXA gene expression in other settings including leukemias with select genetic abnormalities. These discoveries have established a foundation for disease-specific therapies that target chromatin modifications in highly malignant leukemias harboring specific genetic abnormalities. This review focuses on the molecular mechanisms underlying MLL1 translocation-driven leukemogenesis and the latest progress on DOT1L-targeted epigenetic therapies for MLL1-rearranged and other leukemias.

Epigenetic regulation of IL-12-dependent T cell proliferation.

It is well established that the cytokine IL-12 and the transcription factor STAT4, an essential part of the IL-12 signaling pathway, are critical components of the Th1 differentiation process in T cells. In response to pathogenic stimuli, this process causes T cells to proliferate rapidly and secrete high amounts of the cytokine IFN-γ, leading to the Th1 proinflammatory phenotype. However, there are still unknown components of this differentiation pathway. We here demonstrated that the expression of the histone methyltransferase Mll1 is driven by IL-12 signaling through STAT4 in humans and mice and is critical for the proper differentiation of a naïve T cell to a Th1 cell. Once MLL1 is up-regulated by IL-12, it regulates the proliferation of Th1 cells. As evidence of this, we show that Th1 cells from Mll1(+/-) mice are unable to proliferate rapidly in a Th1 environment in vitro and in vivo. Additionally, upon restimulation with cognate antigen Mll1(+/-), T cells do not convert to a Th1 phenotype, as characterized by IFN-γ output. Furthermore, we observed a reduction in IFN-γ production and proliferation in human peripheral blood stimulated with tetanus toxoid by use of a specific inhibitor of the MLL1/menin complex. Together, our results demonstrate that the MLL1 gene plays a previously unrecognized but essential role in Th1 cell biology and furthermore, describes a novel pathway through which Mll1 expression is regulated.

Global transcriptional disturbances underlie Cornelia de Lange syndrome and related phenotypes.

Cornelia de Lange syndrome (CdLS) is a genetically heterogeneous disorder that presents with extensive phenotypic variability, including facial dysmorphism, developmental delay/intellectual disability (DD/ID), abnormal extremities, and hirsutism. About 65% of patients harbor mutations in genes that encode subunits or regulators of the cohesin complex, including NIPBL, SMC1A, SMC3, RAD21, and HDAC8. Wiedemann-Steiner syndrome (WDSTS), which shares CdLS phenotypic features, is caused by mutations in lysine-specific methyltransferase 2A (KMT2A). Here, we performed whole-exome sequencing (WES) of 2 male siblings clinically diagnosed with WDSTS; this revealed a hemizygous, missense mutation in SMC1A that was predicted to be deleterious. Extensive clinical evaluation and WES of 32 Turkish patients clinically diagnosed with CdLS revealed the presence of a de novo heterozygous nonsense KMT2A mutation in 1 patient without characteristic WDSTS features. We also identified de novo heterozygous mutations in SMC3 or SMC1A that affected RNA splicing in 2 independent patients with combined CdLS and WDSTS features. Furthermore, in families from 2 separate world populations segregating an autosomal-recessive disorder with CdLS-like features, we identified homozygous mutations in TAF6, which encodes a core transcriptional regulatory pathway component. Together, our data, along with recent transcriptome studies, suggest that CdLS and related phenotypes may be "transcriptomopathies" rather than cohesinopathies.

Monitoring mixed lineage leukemia expression may help identify patients with mixed lineage leukemia--rearranged acute leukemia who are at high risk of relapse after allogeneic hematopoietic stem cell transplantation.

To evaluate the prognostic value of the expression of the mixed lineage leukemia (MLL) gene for predicting the relapse of patients with MLL-rearranged acute leukemia (AL) after allogeneic hematopoietic stem cell transplantation (allo-HSCT), the levels of MLL transcripts in bone marrow (BM) specimens were monitored serially by real-time quantitative polymerase chain reaction (RQ-PCR) at predetermined time points in 40 patients with MLL-rearranged AL who were treated with allo-HSCT. These patients were followed for a median of 24.5 months (range, 8 to 60 months). A total of 236 BM samples were collected and analyzed. Of these, 230 were monitored concurrently for minimal residual disease (MRD) by flow cytometry (FCM) for leukemia-associated aberrant immune phenotypes and by RQ-PCR for the expression of the Wilms tumor (WT1) gene. The 3-year cumulative incidence of relapse in patients who experienced MLL-positive patients (MLL > .0000%) (n = 9) after HSCT was 93.5% (95% confidence interval [CI], 87% to 100%) compared with 12.5% (95% CI, 5.6% to 19.4%) for MLL-negative patients (n = 31) (P < .001). For these 2 patient groups, the 3-year overall survival (OS) was 12.5% (95% CI, .8% to 24.2%) and 77.8% (95% CI, 68.4% to 87.2%) (P < .001), respectively, and the 3-year leukemia-free survival (LFS) was 0% and 72.2% (95% CI, 61.1% to 83.3%), respectively (P < .001). MLL positivity was associated with a higher rate of relapse (hazard ratio [HR], 18.643; 95% CI, 3.449 to 57.025; P = .001), lower LFS (HR, 7.267; 95% CI, 2.038 to 25.916; P = .002), and lower OS (HR, 8.259; 95% CI, 2.109 to 32.336; P = .002), as determined by Cox multivariate analysis. The expression of the MLL gene had a higher specificity and sensitivity than WT1 or MRD monitored by FCM for predicting the relapse of the patients with MLL + AL. Our results suggest that monitoring the expression of the MLL gene may help to identify patients with MLL + AL who are at high risk of relapse after allo-HSCT and may provide a guide for suitable intervention.

FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia.

Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are aberrations associated with leukemia which indicate unsatisfactory prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined. We have analyzed the expression of MDR-1, multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) messenger RNA (mRNA) in relation to the mutational status of FLT3-ITD and MLL-PTD in 185 acute myeloid leukemia (AML) adult patients. The real-time quantitative polymerase chain reaction method was performed to assess the expression of the MDR-1, MRP-1, BCRP, and LRP mRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. Significantly higher expressions of MDR-1 mRNA were found in patients who did not harbor FLT3-ITD (0.20 vs. 0.05; p = 0.0001) and MRP-1 mRNA in patients with this mutation (0.96 vs. 0.70; p = 0.002) and of BCRP mRNA in patients with MLL-PTD (0.61 vs. 0.38; p = 0.03). In univariate analysis, the high expression of MDR-1 mRNA (≥0.1317) negatively influenced the outcome of induction therapy (p = 0.05), whereas the high expression of BCRP mRNA (≥1.1487) was associated with a high relapse rate (RR) (p = 0.013). We found that the high expression of MDR-1 (≥0.1317), MRP-1 (≥0.8409), and BCRP mRNA (≥1.1487) significantly influenced disease-free survival (DFS; p = 0.059, 0.032, and 0.009, respectively) and overall survival (0.048, 0.014, and 0.059, respectively). Moreover, a high expression of BCRP mRNA (≥1.1487) proved to be an independent prognostic factor for RR (p = 0.01) and DFS (p = 0.002) in multivariate analysis. The significant correlation between the expression of MDR-1, MRP-1, and BCRP mRNA and FLT3-ITD or MLL-PTD in AML patients requires further investigation.

The leukemia-associated Mll-Ell oncoprotein induces fibroblast growth factor 2 (Fgf2)-dependent cytokine hypersensitivity in myeloid progenitor cells.

The subset of acute myeloid leukemias (AML) with chromosomal translocations involving the MLL gene have a poor prognosis (referred to as 11q23-AML). The MLL fusion proteins that are expressed in 11q23-AML facilitate transcription of a set of HOX genes, including HOXA9 and HOXA10. Because Hox proteins are transcription factors, this suggests the possibility that Hox target genes mediate the adverse effects of MLL fusion proteins in leukemia. Identifying such Hox target genes might provide insights to the pathogenesis and treatment of 11q23-AML. In the current study we found that Mll-Ell (an MLL fusion protein) induced transcriptional activation of the FGF2 gene in a HoxA9- and HoxA10-dependent manner. FGF2 encodes fibroblast growth factor 2 (also referred to as basic fibroblast growth factor). Fgf2 influences proliferation and survival of hematopoietic stem cells and myeloid progenitor cells, and increased Fgf2-expression has been described in AMLs. We determined that expression of Mll-Ell in myeloid progenitor cells resulted in autocrine production of Fgf2 and Fgf2-dependent cytokine hypersensitivity. Therefore, our results implicated increased Fgf2 expression in progenitor proliferation and expansion in 11q23-AML. Because small molecule inhibitors of Fgf-receptors are in human clinical trials, this suggested a potential therapeutic approach to this treatment refractory leukemia.

Reversible growth factor dependency and autonomy during murine myelomonocytic leukemia induced by oncogenes.

When murine fetal liver cells were transduced with either of the human acute myeloid leukemia fusion oncogenes MLL-ENL or MLL-AF9 and then transplanted to irradiated recipient mice, myelomonocyte leukemias rapidly developed from the transplanted cells. Analysis of initial events following transduction showed that both oncogenes immediately induced a wide range of enhanced proliferative states, the most extreme of which could generate continuous lines of cells. Maturation defects accompanied the enhanced proliferative states. At all times, the transformed cells exhibited complete dependency on hematopoietic growth factors, particularly GM-CSF and IL-3. Myelomonocytic leukemic cells from primary or transplanted mice formed colonies in semisolid agar. The large majority were dependent on hematopoietic growth factors, but a low frequency of autonomous colonies was also detected. Unexpectedly, reculture of autonomous leukemic colonies generated large numbers of growth factor-dependent clonogenic progeny. Similarly, transplanted clonal autonomous leukemic cells produced leukemias containing a majority of factor-dependent cells. Conversely, recultures of factor-dependent colonies in vitro always produced small numbers of autonomous colonies among the dependent progeny. The reversible relationship between factor dependency and autonomy is surprising because autonomy would have been presumed to represent the final, irreversible, leukemic state.

Ataxia telangiectasia mutated-dependent regulation of topoisomerase II alpha expression and sensitivity to topoisomerase II inhibitor.

Topoisomerase II alpha (TOP2A) has a crucial role in proper chromosome condensation and segregation. Here we report the interaction of TOP2A with ataxia telangiectasia mutated (ATM) and its phosphorylation in an ATM-dependent manner after DNA damage. In vitro kinase assay and site-directed mutagenesis studies revealed that serine 1512 is the target of phosphorylation through ATM. Serine 1512 to Alanine mutation of TOP2A showed increased stability of the protein, retaining TOP2A activity at least with regard to cell survival activity. Ataxia telangiectasia-derived cell lines showed high levels of TOP2A that were associated with hypersensitivity to the TOP2 inhibitor etoposide. These findings suggest that ATM-dependent TOP2A modification is required for proper regulation of TOP2 stability and subsequently of the sensitivity to TOP2 inhibitor. In a lymphoblastoid cell line derived from a patient who developed MLL rearrangement, positive infant leukemia, defective ATM expression, and increased TOP2A expression were shown. It was intriguing that hypersensitivity to TOP2 inhibitor and susceptibility to MLL gene rearrangement were shown by low-dose etoposide exposure in this cell line. Thus, our findings have clinically important implications for the pathogenesis of infantile acute leukemia as well as treatment-associated secondary leukemia following exposure to TOP2 inhibitors.

Photoperiod influences growth and mll (mixed-lineage leukaemia) expression in Atlantic cod.

Photoperiod is associated to phenotypic plasticity of somatic growth in several teleost species. However, the molecular mechanisms underlying this phenomenon are currently unknown but it is likely that epigenetic regulation by methyltransferases is involved. The MLL (mixed-lineage leukaemia) family comprises histone methyltransferases that play a critical role in regulating gene expression during early development in mammals. So far, these genes have received scant attention in teleost fish. In the present study, the mean weight of Atlantic cod juveniles reared under continuous illumination was found to be 13% greater than those kept under natural photoperiod conditions for 120 days. We newly determined cDNA sequences of five mll (mll1, mll2, mll3a, mll4b and mll5) and two setd1 (setd1a and setd1ba) paralogues from Atlantic cod. Phylogenetic analysis revealed that the cod genes clustered within the appropriate mll clade and comparative mapping of mll paralogues showed that these genes lie within a region of conserved synteny among teleosts. All mll and setd1 genes were highly expressed in gonads and fast muscle of adult cod, albeit at different levels, and they were differentially regulated with photoperiod in muscle of juvenile fish. Following only one day of exposure to constant light, mll1, mll4b and setd1a were up to 57% lower in these fish compared to the natural photoperiod group. In addition, mRNA expression of myogenic regulatory factors (myog and myf-5) and pax7 in fast muscle was also affected by different photoperiod conditions. Notably, myog was significantly elevated in the continuous illumination group throughout the time course of the experiment. The absence of a day/night cycle is associated with a generalised decrease in mll expression concomitant with an increase in myog transcript levels in fast muscle of Atlantic cod, which may be involved in the observed epigenetic regulation of growth by photoperiod in this species.

The histone methyltransferase Wbp7 controls macrophage function through GPI glycolipid anchor synthesis.

Histone methyltransferases catalyze site-specific deposition of methyl groups, enabling recruitment of transcriptional regulators. In mammals, trimethylation of lysine 4 in histone H3, a modification localized at the transcription start sites of active genes, is catalyzed by six enzymes (SET1a and SET1b, MLL1-MLL4) whose specific functions are largely unknown. By using a genomic approach, we found that in macrophages, MLL4 (also known as Wbp7) was required for the expression of Pigp, an essential component of the GPI-GlcNAc transferase, the enzyme catalyzing the first step of glycosylphosphatidylinositol (GPI) anchor synthesis. Impaired Pigp expression in Wbp7(-/-) macrophages abolished GPI anchor-dependent loading of proteins on the cell membrane. Consistently, loss of GPI-anchored CD14, the coreceptor for lipopolysaccharide (LPS) and other bacterial molecules, markedly attenuated LPS-triggered intracellular signals and gene expression changes. These data link a histone-modifying enzyme to a biosynthetic pathway and indicate a specialized biological role for Wbp7 in macrophage function and antimicrobial response.

miR-196b directly targets both HOXA9/MEIS1 oncogenes and FAS tumour suppressor in MLL-rearranged leukaemia.

HOXA9 and MEIS1 have essential oncogenic roles in mixed lineage leukaemia (MLL)-rearranged leukaemia. Here we show that they are direct targets of miRNA-196b, a microRNA (miRNA) located adjacent to and co-expressed with HOXA9, in MLL-rearranged leukaemic cells. Forced expression of miR-196b significantly delays MLL-fusion-mediated leukemogenesis in primary bone marrow transplantation through suppressing Hoxa9/Meis1 expression. However, ectopic expression of miR-196b results in more aggressive leukaemic phenotypes and causes much faster leukemogenesis in secondary transplantation than MLL fusion alone, likely through the further repression of Fas expression, a proapoptotic gene downregulated in MLL-rearranged leukaemia. Overexpression of FAS significantly inhibits leukemogenesis and reverses miR-196b-mediated phenotypes. Targeting Hoxa9/Meis1 and Fas by miR-196b is probably also important for normal haematopoiesis. Thus, our results uncover a previously unappreciated miRNA-regulation mechanism by which a single miRNA may target both oncogenes and tumour suppressors, simultaneously, or, sequentially, in tumourigenesis and normal development per cell differentiation, indicating that miRNA regulation is much more complex than previously thought.

Effect of ICSI on gene expression and development of mouse preimplantation embryos.

BACKGROUND: In vitro culture (IVC) and IVF of preimplantation mouse embryos are associated with changes in gene expression. It is however not known whether ICSI has additional effects on the transcriptome of mouse blastocysts. METHODS: We compared gene expression and development of mouse blastocysts produced by ICSI and cultured in Whitten's medium (ICSI(WM)) or KSOM medium with amino acids (ICSI(KSOMaa)) with control blastocysts flushed out of the uterus on post coital Day 3.5 (in vivo). In addition, we compared gene expression in embryos generated by IVF or ICSI using WM. Global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. RESULTS: Blastocysts from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared with embryos generated in vivo. Approximately 1000 genes are differentially expressed between ICSI blastocyst and in vivo blastocysts; proliferation, apoptosis and morphogenetic pathways are the most common pathways altered after IVC. Unexpectedly, expression of only 41 genes was significantly different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOM(aa)). CONCLUSIONS: Our results suggest that fertilization by ICSI may play a more important role in shaping the transcriptome of the developing mouse embryo than the culture media used.