👤 Fred Sablitzky

Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Ppargamma2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Ppargamma2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis. Show less

RNA-binding motif (RBM) proteins comprise a large family of RNA-binding proteins whose functions are poorly understood. Since some RBM proteins are candidate alternative splicing factors we examined whether one such member of the family, RBM6, exhibited a pattern of nuclear distribution and targeting consistent with this role. Using antibodies raised against mouse RBM6 to immunostain mammalian cell lines we found that the endogenous protein was both distributed diffusely in the nucleus and concentrated in a small number of nuclear foci that corresponded to splicing speckles/interchromatin granule clusters (IGCs). Tagged RBM6 was also targeted to IGCs, although it accumulated in large bodies confined to the IGC periphery. The basis of this distribution pattern was suggested by the targeting of tagged RBM6 in the giant nuclei (or germinal vesicles (GVs)) of Xenopus oocytes. In spread preparations of GV contents RBM6 was localized both to lampbrush chromosomes and to the surface of many oocyte IGCs, where it was confined to up to 50 discrete patches. Each patch of RBM6 labelling corresponded to a bead-like structure of 0.5-1 microm diameter that assembled de novo on the IGC surface. Assembly of these novel structures depended on the repetitive N-terminal region of RBM6, which acts as a multimerization domain. Without this domain, RBM6 was no longer excluded from the IGC interior but accumulated homogeneously within it. Assembly of IGC-surface structures in mammalian cell lines also depended on the oligomerization domain of RBM6. Oligomerization of RBM6 also had morphological effects on its other major target in GVs, namely the arrays of nascent transcripts visible in lampbrush chromosome transcription units. The presence of oligomerized RBM6 on many lampbrush loops caused them to appear as dense structures with a spiral morphology that appeared quite unlike normal, extended loops. This distribution pattern suggests a new role for RBM6 in the co-transcriptional packaging or processing of most nascent transcripts. Show less

DEF-3(g16/NY-LU-12) encodes a novel RNA binding protein isolated by positional cloning from an SCLC homozygous deletion region in 3p21.3 and, in parallel, as a differentially expressed gene during myelopoiesis from FDCPmix-A4 cells. DEF-3(g16/NY-LU-12) is ubiquitously expressed during mouse embryogenesis and in adult organs while human hematopoietic tissues showed differential expression. The mouse and human proteins are highly conserved containing two RNA recognition motifs (RRMs) and other domains associated with RNA binding and protein-protein interactions. A database search identified related proteins in human, rat, C. elegans and S. pombe including the 3p21.3 co-deleted gene, LUCA15. Recombinant proteins containing the RRMs of DEF-3(g16/NY-LU-12) and LUCA15 specifically bound poly(G) RNA homopolymers in vitro. These RRMs also show similarity to those of the Hu protein family. Since anti-Hu RRM domain antibodies are associated with an anti-tumor effect and paraneoplastic encephalomyelitis, we tested sera from Hu syndrome patients with the RRMs of DEF-3(g16/NY-LU-12) and LUCA15. These were non-reactive. Thus, DEF-3(g16/NY-LU-12) and LUCA15 represent members of a novel family of RNA binding proteins with similar expression patterns and in vitro RNA binding characteristics. They are co-deleted in some lung cancers and immunologically distinct from the Hu proteins. Show less