The DNMT3A R882H mutation is frequently observed in acute myeloid leukemia (AML). It is located in the subunit and DNA binding interface of DNMT3A and has been reported to cause a reduction in activity and dominant negative effects. We investigated the mechanistic consequences of the R882H mutation on DNMT3A showing a roughly 40% reduction in overall DNA methylation activity. Biochemical assays demonstrated that R882H does not change DNA binding affinity, protein stability or subnuclear distribution of DNMT3A. Strikingly, DNA methylation experiments revealed pronounced changes in the flanking sequence preference of the DNMT3A-R882H mutant. Based on these results, different DNA substrates with selected flanking sequences were designed to be favored or disfavored by R882H. Kinetic analyses showed that the R882H favored substrate was methylated by R882H with 45% increased rate when compared with wildtype DNMT3A, while methylation of the disfavored substrate was reduced 7-fold. Our data expand the model of the potential carcinogenic effect of the R882H mutation by showing CpG site specific activity changes. This result suggests that R882 is involved in the indirect readout of flanking sequence preferences of DNMT3A and it may explain the particular enrichment of the R882H mutation in cancer patients by revealing mutation specific effects.

Somatic DNMT3A mutations at R882 are frequently observed in AML patients including the very abundant R882H, but also R882C, R882P and R882S. Using deep enzymology, we show here that DNMT3A-R882H has more than 70-fold altered flanking sequence preferences when compared with wildtype DNMT3A. The R882H flanking sequence preferences mainly differ on the 3′ side of the CpG site, where they resemble DNMT3B, while 5′ flanking sequence preferences resemble wildtype DNMT3A, indicating that R882H behaves like a DNMT3A/DNMT3B chimera. Investigation of the activity and flanking sequence preferences of other mutations of R882 revealed that they cause similar effects. Bioinformatic analyses of genomic methylation patterns focusing on flanking sequence effects after expression of wildtype DNMT3A and R882H in human cells revealed that genomic methylation patterns reflect the details of the altered flanking sequence preferences of R882H. Concordantly, R882H specific hypermethylation in AML patients was strongly correlated with the R882H flanking sequence preferences. R882H specific DNA hypermethylation events in AML patients were accompanied by R882H specific mis-regulation of several genes with strong cancer connection, which are potential downstream targets of R882H. In conclusion, our data provide novel and detailed mechanistic understanding of the pathogenic mechanism of the DNMT3A R882H somatic cancer mutation.

Terminally mature megakaryocytes undergo dramatic cellular reorganization to produce hundreds of virtually identical platelets. A hallmark feature of this process is the generation of an elaborate system of branched protrusions called proplatelets. We recently identified RanBP10 as a tubulin-binding protein that is concentrated along polymerized microtubules in mature megakaryocytes. RanBP10 depletion in vitro caused the disturbance of polymerized filaments.Here we study the function of RanBP10 in vivo by generating deficient mice using a gene-trap approach. Mutant mice show normal platelet counts, and fetal liverderived megakaryocytes reveal only slightly reduced proplatelet formation. However, ultrastructural analysis unveiled a significantly increased geometric axis ratio for resting platelets, and many platelets exhibited disorders in microtubule filament numbers and localization. Mutant mice showed a markedly prolonged bleeding time. Granule release, a process that depends on internal contraction of the microtubule marginal coil, also was reduced. Flow cytometry analysis revealed reduced expression of CD62P and CD63 after PAR4-peptide stimulation. These data suggest that RanBP10 plays an essential role in hemostasis and in maintaining microtubule dynamics with respect to both platelet shape and function. (Blood. 2009;114:5532-5540) IntroductionPlatelets develop from megakaryocytes (MKs), large polyploid cells localized in the bone marrow. Mature MKs rearrange their entire cytoplasm into pseudopod protrusions (designated proplatelets), a process that is driven by and dependent on microtubules (MTs). 1,2 MT filaments emanate from the cell cortex into the proplatelet tips, where they coil into a loop and later build the platelet marginal band. In wild-type platelets, this MT loop is normally coiled 8 to 12 times. 3 Ultrastructural data previously suggested that the platelet marginal bundle contains a single coiled filament, [3][4][5][6] and isolation of MT coils by platelet extraction affirmed this idea. However, the study by Patel-Hett et al 7 on living platelets revealed up to 7 free plus-ends, suggesting that several shorter MT filaments associate dynamically with one longer filament, forming a bipolar array.Each MT filament is composed of 13 protofilament heteropolymers of ␣-and ␤-tubulins. Mammalian genomes encode 6 ␣-tubulin and 5 ␤-tubulin genes. ␤1-tubulin (also referred to as class VI) is the most diverse isotype 8 and is found exclusively in cells that harbor a marginal band: MKs, platelets, and erythroblasts. 9 More than 90% of proplatelet MT filaments are composed of this ␤-tubulin isotype, 10 whose expression depends on the MK-specific transcription factor NF-E2. Genetically engineered mice that lack a functional Tubb1 gene are thrombocytopenic, and Tubb1 Ϫ/Ϫ platelets lack lentiform shape; the marginal band consists of only 2 to 3 coils, resulting in platelet spherocytosis. 11,12 Although ubiquitously expressed tubulin isotypes ␤2 and ␤5 are up-regulated, the equilibrium is shifted toward monomer...

The DNA methyltransferase DNMT3A R882H mutation is observed in 25% of all AML patients. DNMT3A is active as tetramer and the R882H mutation is located in one of the subunit/subunit interfaces. Previous work has reported that formation of mixed wildtype/R882H complexes leads to a strong loss of catalytic activity observed in in vitro DNA methylation assays (Russler-Germain et al., 2014, Cancer Cell 25:442–454). To investigate this effect further, we have prepared mixed wildtype/R882H DNMT3A complexes by incubation of individually purified subunits of the DNMT3A catalytic domain and full-length DNMT3A2. In addition, we have used a double affinity tag approach and specifically purified mixed catalytic domain complexes formed after co-expression of R882H and wildtype subunits in E. coli cells. Afterwards, we determined the catalytic activity of the mixed complexes and compared it to that of purified complexes only consisting of one subunit type. In both settings, the expected catalytic activities of mixed R882H/wildtype complexes were observed demonstrating an absence of a dominant negative effect of the R882H mutation in purified DNMT3A enzymes. This result suggests that heterocomplex formation of DNMT3A and R882H is unlikely to cause dominant negative effects in human cells as well. The limitations of this conclusion and its implications are discussed.

The crucial function of blood platelets in hemostasis is to prevent blood loss by stable thrombus formation. This process is driven by orchestrated mechanisms including several signal transduction cascades and morphologic transformations. The cytoplasmic microtubule modulator RanBP10 is a Ran and ␤1-tubulin binding protein that is essential for platelet granule release and mice lacking RanBP10 harbor a severe bleeding phenotype. In this study, we demonstrate that RanBP10-nullizygous platelets show normal adhesion on collagen and von Willebrand factor under flow conditions. However, using a ferric chloride-induced arterial thrombosis model, the formation of stable thrombi was significantly impaired, preventing vessel occlusion or leading to recanalization and thromboembolization. Deltagranule secretion was normal in mutant mice, whereas platelet shape change in aggregometry was attenuated. Lack of RanBP10 leads to increased ␤1-tubulin protein, which drives ␣-monomers into polymerized microtubules. In mutant platelets agonists failed to contract the peripheral marginal band or centralize granules. Pretreatment of wild-type platelets with taxol caused microtubule stabilization and phenocopied the attenuated shape change in response to collagen, suggesting that RanBP10 inhibits premature microtubule polymerization of ␤1-tubulin and plays a pivotal role in thrombus stabilization. (Blood. 2012;120(17): 3594-3602) IntroductionMammals share a unique mechanism to ensure that injured blood vessels are sealed and loss of blood is minimized. Although specialized cells for this purpose are even found in invertebrates, the generation of anuclear cell fragments, designated platelets, are only present in mammals where they bud off cytoplasmic protrusions emanating from the megakaryocyte periphery. 1 These precursor cells form proplatelets and release nascent platelets across the endothelial barrier into the blood stream. 2 Endothelial cell lesions trigger platelets to make direct cellular contact with the subcellular matrix, which leads to platelet adhesion at the site of injury. Circulating platelets bind to an adhered layer of platelets, resulting in aggregation and thrombus formation, which is restricted to the site of injury and may ultimately occlude the vessel. 3 This process is tightly regulated as false activation leads to thrombosis and infarction, one of the major causes of death in the Western countries. Platelet activation is a multistep process. A hallmark of activation is the release of bioactive small molecules and proteins stored in 3 defined sets of granules present within the platelet cytoplasm: release of ␣-granules leads to surface expression of CD62P; CD63 is a marker for lysosomal granules; and dense granules harbor nucleotides such as ATP, ADP, amines, and bivalent cations. Although the granule markers are wellcharacterized, the molecular and cell biologic mechanisms underlying the process of granule release are less well understood. [4][5][6] Platelets harbor a cortical ring of bundled microtubules. In r...