Mantu Bhaumik

Two Critical Hits for Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations that always involve the RARalpha gene, which variably fuses to one of several distinct loci, including PML or PLZF (X genes). Due to the reciprocity of the translocation, X-RARalpha and RARalpha-X fusion proteins coexist in APL blasts. PLZF-RARalpha transgenic mice (TM) develop leukemia that lacks the differentiation block at the promyelocytic stage that characterizes APL. We generated TM expressing RARalpha-PLZF and PLZF-RARalpha in their promyelocytes. RARalpha-PLZF TM do not develop leukemia. However, PLZF-RARalpha/RARalpha-PLZF double TM develop leukemia with classic APL features. We demonstrate that RARalpha-PLZF can interfere with PLZF transcriptional repression and that this is critical for APL pathogenesis, since leukemias in PLZF(-/-)/PLZF-RARalpha mutants and in PLZF-RARalpha/RARalpha-PLZF TM are indistinguishable. Thus, both products of a cancer-associated translocation are crucial in determining the distinctive features of the disease.

Myeloid leukemia with promyelocytic features in transgenic mice expressing hCG-NuMA-RARα

Acute promyelocytic leukemia (APL) is characterized by the accumulation of abnormal promyelocytes in the bone marrow (BM), and by the presence of a reciprocal chromosomal translocation involving retinoic acid receptor alpha (RARα). To date, five RARα partner genes have been identified in APL. NuMA-RARα was identified in a pediatric case of APL carrying a translocation t(11;17)(q13;q21). Using a construct containing the NuMA-RARα fusion gene driven by the human cathepsin G promoter (hCG-NuMA-RARα), two transgenic mouse lines were generated. Transgenic mice were observed to have a genetic myeloproliferation (increased granulopoiesis in BM) at an early age, and rapidly developed a myeloproliferative disease-like myeloid leukemia. This leukemia was morphologically and immunophenotypically indistinguishable from human APL, with a penetrance of 100%. The phenotype of transgenic mice was consistent with a blockade of neutrophil differentiation. NuMA-RARα is therefore sufficient for disease development in this APL model.

Blastocyst Injection of Wild Type Embryonic Stem Cells Induces Global Corrections in Mdx Mice

Duchenne muscular dystrophy (DMD) is an incurable neuromuscular degenerative disease, caused by a mutation in the dystrophin gene. Mdx mice recapitulate DMD features. Here we show that injection of wild-type (WT) embryonic stem cells (ESCs) into mdx blastocysts produces mice with improved pathology and function. A small fraction of WT ESCs incorporates into the mdx mouse nonuniformly to upregulate protein levels of dystrophin in the skeletal muscle. The chimeric muscle shows reduced regeneration and restores dystrobrevin, a dystrophin-related protein, in areas with high and with low dystrophin content. WT ESC injection increases the amount of fat in the chimeras to reach WT levels. ESC injection without dystrophin does not prevent the appearance of phenotypes in the skeletal muscle or in the fat. Thus, dystrophin supplied by the ESCs reverses disease in mdx mice globally in a dose-dependent manner.

Male Fertility Defect Associated with Disrupted BRCA1-PALB2 Interaction in Mice

Background: BRCA1 and PALB2 interact with each other to promote homologous recombination and DNA double strand break repair. Results: Mice with abrogated PALB2-BRCA1 interaction show male fertility defect. Conclusion: PALB2 and BRCA1 function together to ensure normal male meiosis. Significance: This work demonstrates the importance of the PALB2-BRCA1 interaction in vivo and reveals a novel role of PALB2 in sex chromosome synapsis. PALB2 links BRCA1 and BRCA2 in homologous recombinational repair of DNA double strand breaks (DSBs). Mono-allelic mutations in PALB2 increase the risk of breast, pancreatic, and other cancers, and biallelic mutations cause Fanconi anemia (FA). Like Brca1 and Brca2, systemic knock-out of Palb2 in mice results in embryonic lethality. In this study, we generated a hypomorphic Palb2 allele expressing a mutant PALB2 protein unable to bind BRCA1. Consistent with an FA-like phenotype, cells from the mutant mice showed hypersensitivity and chromosomal breakage when treated with mitomycin C, a DNA interstrand crosslinker. Moreover, mutant males showed reduced fertility due to impaired meiosis and increased apoptosis in germ cells. Interestingly, mutant meiocytes showed a significant defect in sex chromosome synapsis, which likely contributed to the germ cell loss and fertility defect. Our results underscore the in vivo importance of the PALB2-BRCA1 complex formation in DSB repair and male meiosis.

Essential Roles of BCCIP in Mouse Embryonic Development and Structural Stability of Chromosomes

BCCIP is a BRCA2- and CDKN1A(p21)-interacting protein that has been implicated in the maintenance of genomic integrity. To understand the in vivo functions of BCCIP, we generated a conditional BCCIP knockdown transgenic mouse model using Cre-LoxP mediated RNA interference. The BCCIP knockdown embryos displayed impaired cellular proliferation and apoptosis at day E7.5. Consistent with these results, the in vitro proliferation of blastocysts and mouse embryonic fibroblasts (MEFs) of BCCIP knockdown mice were impaired considerably. The BCCIP deficient mouse embryos die before E11.5 day. Deletion of the p53 gene could not rescue the embryonic lethality due to BCCIP deficiency, but partially rescues the growth delay of mouse embryonic fibroblasts in vitro. To further understand the cause of development and proliferation defects in BCCIP-deficient mice, MEFs were subjected to chromosome stability analysis. The BCCIP-deficient MEFs displayed significant spontaneous chromosome structural alterations associated with replication stress, including a 3.5-fold induction of chromatid breaks. Remarkably, the BCCIP-deficient MEFs had a ∼20-fold increase in sister chromatid union (SCU), yet the induction of sister chromatid exchanges (SCE) was modestly at 1.5 fold. SCU is a unique type of chromatid aberration that may give rise to chromatin bridges between daughter nuclei in anaphase. In addition, the BCCIP-deficient MEFs have reduced repair of irradiation-induced DNA damage and reductions of Rad51 protein and nuclear foci. Our data suggest a unique function of BCCIP, not only in repair of DNA damage, but also in resolving stalled replication forks and prevention of replication stress. In addition, BCCIP deficiency causes excessive spontaneous chromatin bridges via the formation of SCU, which can subsequently impair chromosome segregations in mitosis and cell division.

Heme oxygenase-1-dependent central cardiorespiratory adaptations to chronic hypoxia in mice

Adaptations to chronic hypoxia (CH) could reflect cellular changes within the cardiorespiratory regions of the rostral ventrolateral medulla (RVLM), the C1 region, and the pre-Bötzinger complex (pre-BötC). Previous studies have shown that the hypoxic chemosensitivity of these regions are heme oxygenase (HO) dependent and that CH induces HO-1. To determine the time course of HO-1 induction within these regions and explore its relevance to the respiratory and sympathetic responses during CH, the expression of HO-1 mRNA and protein in the RVLM and measures of respiration, sigh frequency, and sympathetic activity (spectral analysis of heart rate) were examined during 10 days of CH. Respiratory and sympathetic responses to acute hypoxia were obtained in chronically instrumented awake wild-type (WT) and HO-1 null mice. After 4 days of CH, there was a significant induction of HO-1 within the C1 region and pre-BötC. WT mice acclimated to CH by increasing peak diaphragm EMG after 10 days of CH but had no change in the respiratory response to acute hypoxia. There were no significant differences between WT and HO-1 null mice. In WT mice, hypoxic sigh frequency and hypoxic sensitivity of sympathetic activity initially declined before returning toward baseline after 5 days of CH, correlating with the induction of HO-1. In contrast, HO-1 null mice had a persistent decline in hypoxic sigh frequency and hypoxic sensitivity of sympathetic activity. We conclude that induction of HO-1 in these RVLM cardiorespiratory regions may be important for the hypoxic sensitivity of sighs and sympathetic activity during CH.

Inhibition of type I interferon signalling prevents TLR ligand-mediated proteinuria

The mechanisms by which inflammation or autoimmunity causes proteinuric kidney disease remain elusive. Yet proteinuria is a hallmark and a prognostic indicator of kidney disease, and also an independent risk factor for cardiovascular morbidity and mortality. Podocytes are an integral component of the kidney filtration barrier and podocyte injury leads to proteinuria. Here we show that podocytes, which receive signals from the vascular space including circulating antigens, constitutively express TLR1–6 and TLR8. We find that podocytes can respond to TLR ligands including staphylococcal enterotoxin B (SEB), poly I:C, or lipopolysaccharide (LPS) with pro‐inflammatory cytokine release and activation of type I interferon (IFN) signalling. This in turn stimulates podocyte B7‐1 expression and actin remodelling in vitro and transient proteinuria in vivo. Importantly, the treatment of mice with a type I IFN receptor‐blocking antibody (Ab) prevents LPS‐induced proteinuria. These results significantly extend our understanding of podocyte response to immune stimuli and reveal a novel mechanism for infection‐ or inflammation‐induced transient proteinuria. Dysregulation or aberrant activation of this response may result in persistent proteinuria and progressive glomerular disease. In summary, the inhibition of glomerular type I IFN signalling with anti‐IFN Abs may be a novel therapy for proteinuric kidney diseases. Copyright

Deficiency of the dual ubiquitin/SUMO ligase Topors results in genetic instability and an increased rate of malignancy in mice

BackgroundTopors is a nuclear protein that co-localizes with promyelocytic leukemia bodies and has both ubiquitin and SUMO E3 ligase activity. Expression studies implicated Topors as a tumor suppressor in various malignancies. To gain insight into the function of Topors, we generated a Topors-deficient mouse strain.ResultsMice homozygous for a mutant Topors allele exhibited a high rate of perinatal mortality and decreased lifespan. In addition, heterozygotes were found to have an increased incidence of malignancy, involving a variety of tissues. Consistent with this finding, primary embryonic fibroblasts lacking Topors exhibited an increased rate of malignant transformation, associated with aneuploidy and defective chromosomal segregation. While loss of Topors did not alter sensitivity to DNA-damaging or microtubule-targeting agents, cells lacking Topors exhibited altered pericentric heterochromatin, manifested by mislocalization of HP1α and an increase in transcription from pericentric major satellite DNA. Topors-deficient cells exhibited a transcriptional profile similar to that of cells treated with histone deacetylase inhibitors, and were resistant to the anti-proliferative effects of the histone deacetylase inhibitor trichostatin A.ConclusionThese results indicate a unique role for Topors in the maintenance of genomic stability and pericentric heterochromatin, as well as in cellular sensitivity to histone deacetylase inhibitors.

Injection of wild type embryonic stem cells into Mst1 transgenic blastocysts prevents adult-onset cardiomyopathy.

Embryonic stem cells have the capacity to differentiate into a wide range of cell types. We previously described that blastocyst injection of wild type (WT) embryonic stem cells (ESCs) into various knockout (KO) mouse models of human disease prevents disease from occurring. In this study we ask if the blastocyst approach can also correct defects in a mouse model of transgenic (Tg) overexpression of a pro-apoptotic factor. We injected ROSA26 (LacZ-marked) WT ESCs into human mammalian sterile 20 like-kinase 1 (Mst1) Tg blastocysts. Mst1 Tg mice overexpress Mst1, a pro-apoptotic factor, in a cardiac-specific manner. As a result, Mst1 Tg mice develop adult dilated cardiomyopathy driven by apoptosis, reduction in cell density and no hypertrophic compensation. Incorporation of WT ESCs generated WT/Mst1 chimeric mice with normal hearts at histological and functional levels. Accordingly, apoptosis and cell density parameters were normalized. The experiments suggest that an adult-onset cardiac myopathy induced by overexpression of the pro-apoptotic Mst1 can be reversed by developmental incorporation of WT ESCs. The findings also suggest that since forced expression of the Mst1 transgene is not abolished in the rescued chimeras, the WT ES-derived cells normalize pathways that lie downstream of Mst1. The results expand the therapeutic capability of the ESCs to mouse models that overproduce detrimental proteins.

Differential Requirement for Utrophin in the Induced Pluripotent Stem Cell Correction of Muscle versus Fat in Muscular Dystrophy Mice

Duchenne muscular dystrophy (DMD) is an incurable degenerative muscle disorder. We injected WT mouse induced pluripotent stem cells (iPSCs) into mdx and mdx∶utrophin mutant blastocysts, which are predisposed to develop DMD with an increasing degree of severity (mdx <<< mdx∶utrophin). In mdx chimeras, iPSC-dystrophin was supplied to the muscle sarcolemma to effect corrections at morphological and functional levels. Dystrobrevin was observed in dystrophin-positive and, at a lesser extent, utrophin-positive areas. In the mdx∶utrophin mutant chimeras, although iPSC-dystrophin was also supplied to the muscle sarcolemma, mice still displayed poor skeletal muscle histopathology, and negligible levels of dystrobrevin in dystrophin- and utrophin-negative areas. Not only dystrophin-expressing tissues are affected by iPSCs. Mdx and mdx∶utrophin mice have reduced fat/body weight ratio, but iPSC injection normalized this parameter in both mdx and mdx∶utrophin chimeras, despite the fact that utrophin was compromised in the mdx∶utrophin chimeric fat. The results suggest that the presence of utrophin is required for the iPSC-corrections in skeletal muscle. Furthermore, the results highlight a potential (utrophin-independent) non-cell autonomous role for iPSC-dystrophin in the corrections of non-muscle tissue like fat, which is intimately related to the muscle.