The Mds1 and Evi1 complex locus (Mecom) gives rise to several alternative transcripts implicated in leukemogenesis. However, the contribution that Mecom-derived gene products make to normal hematopoiesis remains largely unexplored. To investigate the role of the upstream transcription start site of Mecom in adult hematopoiesis, we created a mouse model with a lacZ knock-in at this site, termed ME m1 , which eliminates Mds1-Evi1 (ME), the longer, PR-domaincontaining isoform produced by the gene (also known as PRDM3). ␤-galactosidasemarking studies revealed that, within hematopoietic cells, ME is exclusively expressed in the stem cell compartment. ME deficiency leads to a reduction in the number of HSCs and a complete loss of long-term repopulation capacity, whereas the stem cell compartment is shifted from quiescence to active cycling. Genetic exploration of the relative roles of endogenous ME and EVI1 isoforms revealed that ME preferentially rescues long-term HSC defects. RNA-seq analysis in Lin ؊ Sca-1 ؉ cKit ؉ cells (LSKs) of ME m1 documents near complete silencing of Cdkn1c, encoding negative cell-cycle regulator p57-Kip2. Reintroduction of ME into ME m1 LSKs leads to normalization of both p57-Kip2 expression and growth control. Our results clearly demonstrate a critical role of PR-domain-containing ME in linking p57-kip2 regulation to long-term HSC function. (Blood. 2011;118(14):3853-3861) IntroductionMaintenance of normal hematopoiesis depends on balanced selfrenewal of HSCs and differentiation of blood cell progenitors within a supportive BM environment. Defects in stem cell function can result in a wide range of phenotypes, including cytopenias, dysplasias, and leukemias. HSCs can be functionally divided into those that can reconstitute long-term (Ͼ 16 weeks) and those with short-term repopulating ability (3-6 weeks). 1 It was inferred early on from studies with 5-fluorouracil (5-FU) that the most primitive, long-term reconstituting cells within the marrow are largely quiescent, whereas the short-term repopulating cells are actively cycling. 2,3 Further studies showed that a second injection of 5-FU 2 days after the first can kill off long-term repopulating cells, 4 indicating that the stem cell pool enters the cell cycle after depletion of cycling progenitors by the first 5-FU injection. 5 More recent studies indicate that immunophenotypic HSCs, Lin Ϫ Sca-1 ϩ cKit ϩ cells (LSKs), can be separated into actively dividing HSCs and dormant HSCs, with the latter being in a quiescent state but able to be recruited to the active state during stress. 6 HSCs with the ability to long-term engraft irradiated recipients reside in the dormant pool. These are uniquely marked by the cell-surface phenotype CD150 ϩ /CD48 Ϫ LSKs or by CD135 Ϫ /CD34 Ϫ LSKs. There is good evidence that the relative quiescence of HSCs is due to negative regulation of the cell cycle, 7 metabolic activity, 8,9 and differentiation 10 ; pathways involved in aging, 11,12 response to oxidative stress, 13 and apoptosis 14 play important roles as we...

p204, an interferon-inducible p200 family protein, inhibits rRNA synthesis in fibroblasts by blocking the binding of the upstream binding factor transcription factor to DNA. Here we report that among 10 adult mouse tissues tested, the level of p204 was highest in heart and skeletal muscles. In cultured C2C12 skeletal muscle myoblasts, p204 was nucleoplasmic and its level was low. During myoblast fusion this level strongly increased, p204 became phosphorylated, and the bulk of p204 appeared in the cytoplasm of the myotubes. Leptomycin B, an inhibitor of nuclear export that blocked myoblast fusion, inhibited the nuclear export signal-dependent translocation of p204 to the cytoplasm. The increase in the p204 level during myoblast fusion was a consequence of MyoD transcription factor binding to several MyoD-specific sequences in the gene encoding p204, followed by transcription. Overexpression of p204 (in C2C12 myoblasts carrying an inducible p204 expression plasmid) accelerated the fusion of myoblasts to myotubes in differentiation medium and induced the fusion even in growth medium. The level of p204 in mouse heart muscle strongly increased during differentiation; it was barely detectable in 10.5-day-old embryos, reached the peak level in 16.5-day-old embryos, and remained high thereafter. p204 is the second p200 family protein (after p202a) found to be involved in muscle differentiation.

Voltage-gated sodium channels initiate electrical signaling in excitable cells such as muscle and neurons. They also are expressed in non-excitable cells such as macrophages and neoplastic cells. Previously, in macrophages, we demonstrated expression of SCN8A, the gene that encodes the channel NaV1.6, and intracellular localization of NaV1.6 to regions near F-actin bundles, particularly at areas of cell attachment. Here we show that a splice variant of NaV1.6 regulates cellular invasion through its effects on podosome and invadopodia formation in macrophages and melanoma cells. cDNA sequence analysis of SCN8A from THP-1 cells, a human monocyte-macrophage cell line, confirmed the expression of a full-length splice variant that lacks exon 18. Immunoelectron microscopy demonstrated NaV1.6-positive staining within the electron dense podosome rosette structure. Pharmacologic antagonism with tetrodotoxin (TTX) in differentiated THP-1 cells or absence of functional NaV1.6 through a naturally occurring mutation (med) in mouse peritoneal macrophages inhibited podosome formation. Agonistmediated activation of the channel with veratridine caused release of sodium from cationic vesicular compartments, uptake by mitochondria, and mitochondrial calcium release through the Na/Ca exchanger. Invasion by differentiated THP-1 and HTB-66 cells, an invasive melanoma cell line, through extracellular matrix was inhibited by TTX. THP-1 invasion also was inhibited by small hairpin RNA knockdown of SCN8A. These results demonstrate that a variant of NaV1.6 participates in the control of podosome and invadopodia formation and suggest that intracellular sodium release mediated by NaV1.6 may regulate cellular invasion of macrophages and melanoma cells.

BRCA2 is a multi-faceted protein critical for the proper regulation of homology-directed repair of DNA double-strand breaks. Elucidating the mechanistic features of BRCA2 is crucial for understanding homologous recombination and how patient-derived mutations impact future cancer risk. Eight centrally located BRC repeats in BRCA2 mediate binding and regulation of RAD51 on resected DNA substrates. Herein, we dissect the biochemical and cellular features of the BRC repeats tethered to the DNA binding domain of BRCA2. To understand how the BRC repeats and isolated domains of BRCA2 contribute to RAD51 binding, we analyzed both the biochemical and cellular properties of these proteins. In contrast to the individual BRC repeat units, we find that the BRC5–8 region potentiates RAD51-mediated DNA strand pairing and provides complementation functions exceeding those of BRC repeats 1–4. Furthermore, BRC5–8 can efficiently repair nuclease-induced DNA double-strand breaks and accelerate the assembly of RAD51 repair complexes upon DNA damage. These findings highlight the importance of the BRC5–8 domain in stabilizing the RAD51 filament and promoting homology-directed repair under conditions of cellular DNA damage.

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