Scaffold attachment factor B: distribution and interaction with ERα in the rat brain.

Scaffold attachment factor (SAFB) 1 and its homologue SAFB2 are multifunctional proteins that are involved in various cellular mechanisms, including chromatin organization and transcriptional regulation, and are also corepressors of estrogen receptor alpha (ERα). Both SAFBs are expressed at high levels in the brain. However, the distributions of SAFB1 and SAFB2 have yet to be characterized in detail and it is unclear whether both proteins interact with ERα in the brain. In this study, we investigated the expression and distribution of both SAFBs and their interaction with ERα in adult male rat brain. Immunohistochemical staining showed that SAFB1 and SAFB2 have a similar distribution pattern and are widely expressed throughout the brain. Double-fluorescence immunohistochemical and immunocytochemical analyses in primary cultures showed that the two SAFB proteins are localized in nuclei of neurons, astrocytes, and oligodendrocytes. Of note, SAFB2 was also found in cytoplasmic regions in these cell lineages. Both SAFB proteins were also expressed in ERα-positive cells in the medial preoptic area (MPOA) and arcuate and ventromedial hypothalamic nuclei. Co-immunoprecipitation experiments revealed that both SAFB proteins from the MPOA reciprocally interact with endogenous ERα. These results indicate that, in addition to a role in basal cellular function in the brain, the SAFB proteins may serve as ERα corepressors in hormone-sensitive regions.

The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex.

Understanding the molecular pathways disrupted in motor neuron diseases is urgently needed. Here, we employed CRISPR knockout (KO) to investigate the functions of four ALS-causative RNA/DNA binding proteins (FUS, EWSR1, TAF15 and MATR3) within the RNAP II/U1 snRNP machinery. We found that each of these structurally related proteins has distinct roles with FUS KO resulting in loss of U1 snRNP and the SMN complex, EWSR1 KO causing dissociation of the tRNA ligase complex, and TAF15 KO resulting in loss of transcription factors P-TEFb and TFIIF. However, all four ALS-causative proteins are required for association of the ASC-1 transcriptional co-activator complex with the RNAP II/U1 snRNP machinery. Remarkably, mutations in the ASC-1 complex are known to cause a severe form of Spinal Muscular Atrophy (SMA), and we show that an SMA-causative mutation in an ASC-1 component or an ALS-causative mutation in FUS disrupts association between the ASC-1 complex and the RNAP II/U1 snRNP machinery. We conclude that ALS and SMA are more intimately tied to one another than previously thought, being linked via the ASC-1 complex.

Mice Deficient in CIZ/NMP4 Develop an Attenuated Form of K/BxN-Serum Induced Arthritis.

CIZ/NMP4 (Cas interacting zinc finger protein, Nmp4, Zfp384) is a transcription factor that is known to regulate matrix related-proteins. To explore the possible pathophysiological role of CIZ/NMP4 in arthritis, we examined CIZ/NMP4 expression in articular cartilage in arthritis model. CIZ/NMP4 was expressed in the articular chondrocytes of mice at low levels while its expression was enhanced when arthritis was induced. Arthritis induction increased clinical score in wild type mice. In contrast, CIZ/NMP4 deficiency suppressed such rise in the levels of arthritis score and swelling of soft tissue. CIZ/NMP4 deficiency also reduced invasion of inflammatory cells in joint tissue. Quantitative PCR analyses of mRNA from joints revealed that arthritis-induced increase in expressions of IL-1ß was suppressed by CIZ/NMP4 deficiency. CIZ/NMP4 bound to IL-1ß promoter and activated its transcription. The increase in CIZ/NMP4 in arthritis was also associated with enhancement in bone resorption and cartilage matrix degradation. In fact, RANKL, a signaling molecule prerequisite for osteoclastogenesis and, MMP-3, a clinical marker for arthritis were increased in joints upon arthritis induction. In contrast, CIZ/NMP4 deficiency suppressed the arthritis-induced increase in bone resorption, expression of RANKL and MMP-3 mRNA. Thus, CIZ/NMP4 plays a role in the development of arthritis at least in part through regulation of key molecules related to the arthritis.

RAE-1, a novel PHR binding protein, is required for axon termination and synapse formation in Caenorhabditis elegans.

Previous studies in Caenorhabditis elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. To understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam (protein associated with Myc). rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development.

Matrin 3 in neuromuscular disease: physiology and pathophysiology.

RNA-binding proteins (RBPs) are essential factors required for the physiological function of neurons, muscle, and other tissue types. In keeping with this, a growing body of genetic, clinical, and pathological evidence indicates that RBP dysfunction and/or gene mutation leads to neurodegeneration and myopathy. Here, we summarize the current understanding of matrin 3 (MATR3), a poorly understood RBP implicated not only in ALS and frontotemporal dementia but also in distal myopathy. We begin by reviewing MATR3's functions, its regulation, and how it may be involved in both sporadic and familial neuromuscular disease. We also discuss insights gleaned from cellular and animal models of MATR3 pathogenesis, the links between MATR3 and other disease-associated RBPs, and the mechanisms underlying RBP-mediated disorders.

Early embryonic lethality of mice lacking the essential protein SNEV.

SNEV (Prp19, Pso4, NMP200) is a nuclear matrix protein known to be involved in pre-mRNA splicing, ubiquitylation, and DNA repair. In human umbilical vein endothelial cells, SNEV overexpression delayed the onset of replicative senescence. Here we analyzed the function of the mouse SNEV gene in vivo by employing homologous recombination in mice and conclude that SNEV is indispensable for early mouse development. Mutant preimplantation embryos initiated blastocyst formation but died shortly thereafter. Outgrowth of SNEV-null blastocysts showed a lack of proliferation of cells of the inner cell mass, which subsequently underwent cell death. While SNEV-heterozygous mice showed no overt phenotype, heterozygous mouse embryonic fibroblast cell lines with reduced SNEV levels displayed a decreased proliferative potential in vitro. Our experiments demonstrate that the SNEV protein is essential, functionally nonredundant, and indispensable for mouse development.

Nmp4/CIZ suppresses parathyroid hormone-induced increases in trabecular bone.

The nucleocytoplasmic shuttling transcription factor Nmp4/CIZ (nuclear matrix protein 4/cas interacting zinc finger protein) is a ubiquitously expressed protein that regulates both cytoplasmic and nuclear activities. In the nucleus, Nmp4/CIZ represses transcription of genes crucial to osteoblast differentiation and genes activated by various anabolic stimuli, including parathyroid hormone (PTH). We investigated the role of Nmp4/CIZ in the PTH-induced increase in bone by engineering mice with loss-of-function mutations in the Nmp4/CIZ gene, and treating 10-week-old female mice with anabolic doses of human PTH (1-34) at 30 microg/kg/day, 7 day/week, for 7 weeks or vehicle control. The untreated, baseline phenotype of the Nmp4-null mice between 8 and 16 weeks of age included a modest but significant increase in bone mineral density (BMD) and bone mineral content (BMC) compared to wild-type (WT) mice. Type I collagen mRNA expression was moderately elevated in the femurs of the Nmp4-null mice. The Nmp4 mutant alleles decreased body weight by 4% when expressed on a mixed background but the same alleles on a pure B6 background yielded a significant, 15% increase in body weight among the KO mice, compared to their WT controls. Hormone treatment equally enhanced BMD and BMC over vehicle-treated mice in both the WT and Nmp4-null groups but Nmp4-KO mice exhibited a significantly greater PTH-induced acquisition of femoral trabecular bone as compared to WT mice. These data support our hypothesis that Nmp4/CIZ is a transcriptional attenuator that suppresses osteoid synthesis and PTH-mediated acquisition of cancellous bone. J. Cell. Physiol. 219: 734-743, 2009. (c) 2009 Wiley-Liss, Inc.

Rae1 is an essential mitotic checkpoint regulator that cooperates with Bub3 to prevent chromosome missegregation.

The WD-repeat proteins Rae1 and Bub3 show extensive sequence homology, indicative of functional similarity. However, previous studies have suggested that Rae1 is involved in the mRNA export pathway and Bub3 in the mitotic checkpoint. To determine the in vivo roles of Rae1 and Bub3 in mammals, we generated knockout mice that have these genes deleted individually or in combination. Here we show that haplo-insufficiency of either Rae1 or Bub3 results in a similar phenotype involving mitotic checkpoint defects and chromosome missegregation. We also show that overexpression of Rae1 can correct for Rae1 haplo-insufficiency and, surprisingly, Bub3 haplo-insufficiency. Rae1-null and Bub3-null mice are embryonic lethal, although cells from these mice did not have a detectable defect in nuclear export of mRNA. Unlike null mice, compound haplo-insufficient Rae1/Bub3 mice are viable. However, cells from these mice exhibit much greater rates of premature sister chromatid separation and chromosome missegregation than single haplo-insufficient cells. Finally, we show that mice with mitotic checkpoint defects are more susceptible to dimethylbenzanthrene-induced tumorigenesis than wild-type mice. Thus, our data demonstrate a novel function for Rae1 and characterize Rae1 and Bub3 as related proteins with essential, overlapping, and cooperating roles in the mitotic checkpoint.

Deficiency of CIZ, a nucleocytoplasmic shuttling protein, prevents unloading-induced bone loss through the enhancement of osteoblastic bone formation in vivo.

Disuse osteoporosis is a major cause to increase the risk of fractures in bed-ridden patients whose numbers are increasing in our modern society. However, the mechanisms underlying the sensing of mechanical stress in bone are largely unknown. CIZ localizes at cell adhesion plaque and transfers into nuclear compartments and activates promoters of the genes encoding enzymes, which degrade matrix proteins to link signals from the cell adhesion site to nuclear events. We examined whether this nucleocytoplasmic shuttling protein would be involved in mediation of mechanical stress signaling. Unloading based on tail suspension reduced bone volume in wild-type mice. In contrast, CIZ-deficient mice revealed suppression in such reduction of bone mass due to unloading. Histomorphometric analysis revealed that unloading suppressed the levels of osteoblastic bone formation parameters, and such suppression of bone formation parameters was blocked by CIZ-deficiency. Osteoclastic bone resorption parameters were similar regardless of CIZ-deficiency after 2-week unloading. Mineralized nodule formation in the cultures of bone marrow cells obtained from the bone of mice subjected to unloading was suppressed in wild-type mice. CIZ deficiency blocked such reduction in nodule formation induced by unloading. These data indicated that nucleocytoplasmic shuttling protein, CIZ, plays a pivotal role in the response of bone mass in unloading condition.