Deficiency of the Fanconi anemia E2 ubiqitin conjugase UBE2T only partially abrogates Alu-mediated recombination in a new model of homology dependent recombination.

The primary function of the UBE2T ubiquitin conjugase is in the monoubiquitination of the FANCI-FANCD2 heterodimer, a central step in the Fanconi anemia (FA) pathway. Genetic inactivation of UBE2T is responsible for the phenotypes of FANCT patients; however, a FANCT patient carrying a maternal duplication and a paternal deletion in the UBE2T loci displayed normal peripheral blood counts and UBE2T protein levels in B-lymphoblast cell lines. To test whether reversion by recombination between UBE2T AluYa5 elements could have occurred in the patient's hematopoietic stem cells despite the defects in homologous recombination (HR) in FA cells, we constructed HeLa cell lines containing the UBE2T AluYa5 elements and neighboring intervening sequences flanked by fluorescent reporter genes. Introduction of a DNA double strand break in the model UBE2T locus in vivo promoted single strand annealing (SSA) between proximal Alu elements and deletion of the intervening color marker gene, recapitulating the reversion of the UBE2T duplication in the FA patient. To test whether UBE2T null cells retain HR activity, the UBE2T genes were knocked out in HeLa cells and U2OS cells. CRISPR/Cas9-mediated genetic knockout of UBE2T only partially reduced HR, demonstrating that UBE2T-independent pathways can compensate for the recombination defect in UBE2T/FANCT null cells.

IL-2 Inducible Kinase ITK is Critical for HIV-1 Infection of Jurkat T-cells.

Successful replication of Human immunodeficiency virus (HIV)-1 depends on the expression of various cellular host factors, such as the interleukin-2 inducible T-cell kinase (ITK), a member of the protein family of TEC-tyrosine kinases. ITK is selectively expressed in T-cells and coordinates signaling pathways downstream of the T-cell receptor and chemokine receptors, including PLC-1 activation, Ca2+-release, transcription factor mobilization, and actin rearrangements. The exact role of ITK during HIV-1 infection is still unknown. We analyzed the function of ITK during HIV-1 replication and showed that attachment, fusion of virions with the cell membrane and entry into Jurkat T-cells was inhibited when ITK was knocked down. In contrast, reverse transcription and provirus expression were not affected by ITK deficiency. Inhibited ITK expression did not affect the CXCR4 receptor on the cell surface, whereas CD4 and LFA-1 integrin levels were slightly enhanced in ITK knockdown cells and heparan sulfate (HS) expression was completely abolished in ITK depleted T-cells. However, neither HS expression nor other attachment factors could explain the impaired HIV-1 binding to ITK-deficient cells, which suggests that a more complex cellular process is influenced by ITK or that not yet discovered molecules contribute to restriction of HIV-1 binding and entry.

HSP90 affects the stability of BMAL1 and circadian gene expression.

The mammalian circadian clock comprises a system of interconnected transcriptional and translational feedback loops. Proper oscillator function requires the precisely timed synthesis and degradation of core clock proteins. Heat shock protein 90 (HSP90), an adenosine triphosphate (ATP)-dependent molecular chaperone, has important functions in many cellular regulatory pathways by controlling the activity and stability of its various client proteins. Despite accumulating evidence for interplay between the heat shock response and the circadian system, the role of HSP90 in the mammalian core clock is not known. The results of this study suggest that inhibition of the ATP-dependent chaperone activity of HSP90 impairs circadian rhythmicity of cultured mouse fibroblasts whereby amplitude and phase of the oscillations are predominantly affected. Inhibition of HSP90 shortened the half-life of BMAL1, which resulted in reduced cellular protein levels and blunted expression of rhythmic BMAL1-CLOCK target genes. Furthermore, the HSP90 isoforms HSP90AA1 and HSP90AB1, and not HSP90B1-GRP94 or TRAP1, are responsible for maintaining proper cellular levels of BMAL1 protein. In summary, these findings provide evidence for a model in which cytoplasmic HSP90 is required for transcriptional activation processes by the positive arm of the mammalian circadian clock.

IL-2-inducible T-cell kinase deficiency: clinical presentation and therapeutic approach.

Mutations in the IL-2-inducible T-cell kinase gene have recently been shown to cause an autosomal recessive fatal Epstein Barr virus (EBV) associated lymphoproliferation. We report 3 cases from a single family who presented with EBV-positive B-cell proliferation diagnosed as Hodgkin's lymphoma. Single nucleotide polymorphism array-based genome-wide linkage analysis revealed IL-2-inducible T-cell kinase as a candidate gene for this disorder. All 3 patients harbored the same novel homozygous nonsense mutation C1764G which causes a premature stop-codon in the kinase domain. All cases were initially treated with chemotherapy. One patient remains in durable remission, the second patient subsequently developed severe hemophagocytic lymphohistiocytosis with multi-organ failure and died, and the third patient underwent a successful allogeneic bone marrow transplantation. IL-2-inducible T-cell kinase deficiency underlies a new primary immune deficiency which may account for part of the spectrum of Epstein Barr virus related lymphoproliferative disorders which can be successfully corrected by bone marrow transplantation.

C-terminal regions of topoisomerase IIalpha and IIbeta determine isoform-specific functioning of the enzymes in vivo.

Topoisomerase II removes supercoils and catenanes generated during DNA metabolic processes such as transcription and replication. Vertebrate cells express two genetically distinct isoforms (alpha and beta) with similar structures and biochemical activities but different biological roles. Topoisomerase IIalpha is essential for cell proliferation, whereas topoisomerase IIbeta is required only for aspects of nerve growth and brain development. To identify the structural features responsible for these differences, we exchanged the divergent C-terminal regions (CTRs) of the two human isoforms (alpha 1173-1531 and beta 1186-1621) and tested the resulting hybrids for complementation of a conditional topoisomerase IIalpha knockout in human cells. Proliferation was fully supported by all enzymes bearing the alpha CTR. The alpha CTR also promoted chromosome binding of both enzyme cores, and was by itself chromosome-bound, suggesting a role in enzyme targeting during mitosis. In contrast, enzymes bearing the beta CTR supported proliferation only rarely and when expressed at unusually high levels. A similar analysis of the divergent N-terminal regions (alpha 1-27 and beta 1-43) revealed no role in isoform-specific functions. Our results show that it is the CTRs of human topoisomerase II that determine their isoform-specific functions in proliferating cells. They also indicate persistence of some functional redundancy between the two isoforms.