The Leukemic Protein Core Binding Factor Beta (CBFbeta)-Smooth-Muscle Myosin Heavy Chain Sequesters CBFalpha2 into Cytoskeletal Filaments and Aggregates

The fusion gene CBFB-MYH11 is generated by the chromosome 16 inversion associated with acute myeloid leukemias. This gene encodes a chimeric protein involving the core binding factor β (CBFβ) and the smooth-muscle myosin heavy chain (SMMHC). Mouse model studies suggest that this chimeric protein CBFβ-SMMHC dominantly suppresses the function of CBF, a heterodimeric transcription factor composed of DNA binding subunits (CBFα1 to 3) and a non-DNA binding subunit (CBFβ). This dominant suppression results in the blockage of hematopoiesis in mice and presumably contributes to leukemogenesis. We used transient-transfection assays, in combination with immunofluorescence and green fluorescent protein-tagged proteins, to monitor subcellular localization of CBFβ-SMMHC, CBFβ, and CBFα2 (also known as AML1 or PEBP2αB). When expressed individually, CBFα2 was located in the nuclei of transfected cells, whereas CBFβ was distributed throughout the cell. On the other hand, CBFβ-SMMHC formed filament-like structures that colocalized with actin filaments. Upon cotransfection, CBFα2 was able to drive localization of CBFβ into the nucleus in a dose-dependent manner. In contrast, CBFα2 colocalized with CBFβ-SMMHC along the filaments instead of localizing to the nucleus. Deletion of the CBFα-interacting domain within CBFβ-SMMHC abolished this CBFα2 sequestration, whereas truncation of the C-terminal-end SMMHC domain led to nuclear localization of CBFβ-SMMHC when coexpressed with CBFα2. CBFα2 sequestration by CBFβ-SMMHC was further confirmed in vivo in a knock-in mouse model. These observations suggest that CBFβ-SMMHC plays a dominant negative role by sequestering CBFα2 into cytoskeletal filaments and aggregates, thereby disrupting CBFα2-mediated regulation of gene expression. The pericentric inversion of chromosome 16 [inv(16)(p13q22)] is a cytogenetic abnormality consistently associated with acute myeloid leukemia (AML) subtype M4Eo (2, 21), a variant of subtype M4 with abnormal eosinophils in the bone marrow and sometimes in the peripheral blood. The inversion results in the reciprocal fusions of two genes: theMYH11 gene (16p13), which encodes the smooth-muscle myosin heavy chain (SMMHC), and the CBFB gene (16q22), which encodes the β subunit of the core binding factor (CBFβ) (24). The chimeric gene CBFB-MYH11 fuses most of the 5′ coding region of CBFB in frame with the 3′ portion of MYH11, resulting in the production of the chimeric protein CBFβ-SMMHC. The reciprocal fusion,MYH11-CBFB, is not believed to be important since its expression is below detectable levels in leukemic cells and it is deleted in some patients with an unbalanced inversion (24, 25,29). CBFβ is the heterodimeric partner of CBFα proteins, and together they constitute the core binding factors (CBF). CBF was initially identified as a transcriptional regulator of Moloney murine leukemia virus (50, 51) and polyomavirus (4, 18, 36, 37,43) in mice, and it was subsequently shown to be an important transcriptional activator of genes involved in mammalian hematopoiesis and bone development (5, 12, 14, 15, 20, 32, 40, 46). Monomeric CBFα proteins bind DNA, albeit weakly (3, 50). Although CBFβ does not make any detectable direct contact with DNA (50), it enhances the DNA binding affinity of the CBFα proteins (4, 36). While CBFβ is expressed from a single gene in the human and mouse, there are three CBFα genes, all of which encode the so-called runt domain (3, 22,54), which is required for both DNA binding and interaction with CBFβ. One of the three genes, CBFA2, also known as AML1 or PEBP2αB, is located on human chromosome 21 and is involved in several different leukemias as a result of translocations (16, 31, 34, 35, 41). Chromosomal inversions and translocations involving either CBFB orCBFA2 are the most frequent cytogenetic abnormalities in human AMLs (27). Gene-targeting experiments in mice have demonstrated that the CBFα2 and CBFβ subunits are likely to function together as a complex in vivo. Homozygous disruption of either Cbfa2 (39,48) or Cbfb (42, 49) in mice produces an identical phenotype: both Cbfa2 −/− andCbfb −/− embryos demonstrate a failure of definitive hematopoiesis in the liver, and in both cases the embryos die at around day 12.5 due to extensive hemorrhages. The inv(16) chimeric gene CBFB-MYH11 has been shown to exert a dominant negative effect in vivo by a mouse knock-in experiment (8). CBFB-MYH11was introduced into the mouse genome to replace one copy of theCbfb gene. The expression of this chimeric gene was controlled by the endogenous Cbfb promoter, thus simulating the condition in leukemic patients. CBFβ-SMMHC was found to dominantly suppress the function of the CBFα2:CBFβ heterodimer, since mouse embryos heterozygous for the knock-inCbfb-MYH11 gene (CbfbCBFB-MYH11/+ ) displayed a phenotype similar to that of Cbfb−/− andCbfa2−/− embryos, i.e., failure of definitive hematopoiesis and midgestation lethality. In vitro, the chimeric protein was shown to retain its ability to interact with CBFα proteins and participate in the formation of protein-DNA complexes (23). Although presence of the chimeric protein reduces CBF DNA-binding activity in cultured Ba/F3 lymphoid and 32D c13 myeloid cells (6), it is not clear how this reduction was achieved. Unlike wild-type CBFβ, CBFβ-SMMHC can potentially form dimers and multimers via the rod-like domain of the myosin chain (23, 25). Two possible mechanisms could explain the dominant negative effect of the chimeric CBFβ-SMMHC protein. One is that CBFβ-SMMHC, via heterodimerization with CBFα2, can assemble into a ternary complex at the core sites within promoters of target genes and interfere with the regulation of gene expression. The second possibility is that CBFβ-SMMHC, with its capacity to form multimers, can sequester CBFα2 into nonfunctional complexes, thus preventing it from regulating transcription of target genes. Previous studies by our group demonstrated that CBFβ-SMMHC can form rod-like nuclear structures as well as cytoplasmic stress fibers in NIH 3T3 cells stably transfected with a CBFB-MYH11cDNA construct (53). However, the effect on CBFα2 subcellular localization by CBFβ-SMMHC has not been fully examined. In this study, we used transient-transfection assays in combination with immunofluorescence and green fluorescent protein (GFP) tags to demonstrate that CBFβ-SMMHC does, in fact, sequester CBFα2 in abnormal locations. We also demonstrated that the sequestration requires the abilities of CBFβ-SMMHC to interact with CBFα2 and to multimerize. This observed sequestration can at least partially explain the dominant negative effect of the CBFβ-SMMHC protein on CBF function in leukemogenesis

Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination

Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63–linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)–induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress

Diffusion-based Time Series Data Imputation for Microsoft 365

Reliability is extremely important for large-scale cloud systems like Microsoft 365. Cloud failures such as disk failure, node failure, etc. threaten service reliability, resulting in online service interruptions and economic loss. Existing works focus on predicting cloud failures and proactively taking action before failures happen. However, they suffer from poor data quality like data missing in model training and prediction, which limits the performance. In this paper, we focus on enhancing data quality through data imputation by the proposed Diffusion+, a sample-efficient diffusion model, to impute the missing data efficiently based on the observed data. Our experiments and application practice show that our model contributes to improving the performance of the downstream failure prediction task

The effect of volume change and stack pressure on solid‐state battery cathodes

Solid-state lithium batteries may provide increased energy density and improved safety compared with Li-ion technology. However, in a solid-state composite cathode, mechanical degradation due to repeated cathode volume changes during cycling may occur, which may be partially mitigated by applying a significant, but often impractical, uniaxial stack pressure. Herein, we compare the behavior of composite electrodes based on Li4Ti5O12 (LTO) (negligible volume change) and Nb2O5 (+4% expansion) cycled at different stack pressures. The initial LTO capacity and retention are not affected by pressure but for Nb2O5, they are significantly lower when a stack pressure of <2 MPa is applied, due to inter-particle cracking and solid-solid contact loss because of cyclic volume changes. This work confirms the importance of cathode mechanical stability and the stack pressures for long-term cyclability for solid-state batteries. This suggests that low volume-change cathode materials or a proper buffer layer are required for solid-state batteries, especially at low stack pressures

The Greenland Telescope: Antenna Retrofit Status and Future Plans

Since the ALMA North America Prototype Antenna was awarded to the Smithsonian Astrophysical Observatory (SAO), SAO and the Academia Sinica Institute of Astronomy & Astrophysics (ASIAA) are working jointly to relocate the antenna to Greenland. This paper shows the status of the antenna retrofit and the work carried out after the recommissioning and subsequent disassembly of the antenna at the VLA has taken place. The next coming months will see the start of the antenna reassembly at Thule Air Base. These activities are expected to last until the fall of 2017 when commissioning should take place. In parallel, design, fabrication and testing of the last components are taking place in Taiwan

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation