The ability of somatic stem cells to self-renew and differentiate into downstream lineages is dependent on specialized chromatin environments that keep stem cell-specific genes active and key differentiation factors repressed but poised for activation. The epigenetic factors that provide this type of regulation remain ill-defined. Here we provide the first evidence that the SNF2-like ATPase Mi-2␤ of the Nucleosome Remodeling Deacetylase (NuRD) complex is required for maintenance of and multilineage differentiation in the early hematopoietic hierarchy. Shortly after conditional inactivation of Mi-2␤, there is an increase in cycling and a decrease in quiescence in an HSC (hematopoietic stem cell)-enriched bone marrow population. These cycling mutant cells readily differentiate into the erythroid lineage but not into the myeloid and lymphoid lineages. Together, these effects result in an initial expansion of mutant HSC and erythroid progenitors that are later depleted as more differentiated proerythroblasts accumulate at hematopoietic sites exhibiting features of erythroid leukemia. Examination of gene expression in the mutant HSC reveals changes in the expression of genes associated with self-renewal and lineage priming and a pivotal role of Mi-2␤ in their regulation. Thus, Mi-2␤ provides the hematopoietic system with immune cell capabilities as well as with an extensive regenerative capacity.[Keywords: Mi-2␤; chromatin; HSC; multipotency; self-renewal; lineage priming] Supplemental material is available at http://www.genesdev.org. Received December 13, 2007; revised version accepted March 4, 2008. The defining properties of somatic stem cells, their ability to self-renew and to progress through available differentiation pathways, are critical for the life-long tissue integrity of multicellular organisms (Weissman 2000;Lemischka and Moore 2003). A balance between stem cell quiescence and activation is required to sustain the stem cell pool and to provide adequate numbers of mature cells to meet normal homeostatic conditions. Both the self-renewal and differentiation properties of stem cells can be altered dramatically in order to meet demands imposed by stress conditions.In hematopoietic tissue, the most primitive stem cells are thought to be in a comparatively quiescent state. They cycle with slow kinetics that strongly correlate with their long-term self-renewing potential (LT-HSC) (Morrison and Weissman 1994;Cheshier et al. 1999). Thus, maintenance of hematopoietic stem cell (HSC) activity can be compromised in two ways. On the one hand, a cell cycle block can prevent self-renewing divisions. On the other hand, prolonged cell cycle activation can lead to HSC exhaustion. This has been corroborated by studies on components of the cell cycle machinery and on signaling pathways that modulate their activity. An increase in expression of the cell cycle inhibitors p16 Ink4A, p19Ink4D/Arf , or p18Ink4C has an adverse effect on the HSC's self-renewal, presumably by restricting its entry into the cell cycle (Park et a...

The mechanism for apical growth during hyphal morphogenesis in Candida albicans is unknown. Studies from Saccharomyces cerevisiae indicate that cell morphogenesis may involve cell cycle regulation by cyclin-dependent kinase. To examine whether this is the mechanism for hyphal morphogenesis, the temporal appearance of different spindle pole body and spindle structures, the cell cycle-regulated rearrangements of the actin cytoskeleton, and the phosphorylation state of the conserved Tyr19 of Cdc28 during the cell cycle were compared and found to be similar between yeast and serum-induced hyphal apical cells. These data suggest that hyphal elongation is not mediated by altering cell cycle progression or through phosphorylation of Tyr19 of Cdc28. We have also shown that germ tubes can evaginate before spindle pole body duplication, chitin ring formation, and DNA replication. Similarly, tip-associated actin polarization in each hypha occurs before the events of the G 1 /S transition and persists throughout the cell cycle, whereas cell cycle-regulated actin assemblies come and go. We have also shown that cells in phases other than G 1 can be induced to form hyphae. Hyphae induced from G 1 cells have no constrictions, and the first chitin ring is positioned in the germ tube at various distances from the base. Hyphae induced from budded cells have a constriction and a chitin ring at the bud neck, beyond which the hyphae continue to elongate with no further constrictions. Our data suggest that hyphal elongation and cell cycle morphogenesis programs are uncoupled, and each contributes to different aspects of cell morphogenesis. INTRODUCTIONCandida albicans is a polymorphic fungal pathogen that undergoes reversible morphogenetic transitions among budding, pseudohyphal, and hyphal growth forms (Odds, 1985). Its ability to switch between yeast and hyphal growth forms is directly related to its virulence, because mutants defective in hyphal growth are less virulent in mouse models than are their wild-type counterparts (Leberer et al., 1997;Lo et al., 1997;Gale et al., 1998). Hyphae may be suited to breach barriers in the host, whereas the yeast form is more easily disseminated within the host. Therefore, understanding the mechanisms for this morphogenetic switch should provide insight into the pathogenicity of this fungus.During hyphal growth in C. albicans, cell surface expansion is restricted to a small region at the hyphal tip. This apical growth zone is active during the entire hyphal growth period (Staebell and Soll, 1985). In contrast, yeast-form cells expand from a small area in a mostly apical manner only at the initial stage of budding. When the bud has reached a critical size, apical growth shuts down and general (isotropic) expansion takes place (Staebell and Soll, 1985). The localization of the actin cytoskeleton in yeast and hyphal cells reflects these differences in morphogenesis. Polarization of the actin cytoskeleton to the hyphal tip is observed in all hyphal cells (Anderson and Soll, 1986). However, in yeast-...

Candida albicans undergoes a dramatic morphological transition in response to various growth conditions. This ability to switch from a yeast form to a hyphal form is required for its pathogenicity. The intractability of Candida to traditional genetic approaches has hampered the study of the molecular mechanism governing this developmental switch. Our approach is to use the more genetically tractable yeast Saccharomyces cerevisiae to yield clues about the molecular control of filamentation for further studies in Candida. G 1 cyclins Cln1 and Cln2 have been implicated in the control of morphogenesis in S. cerevisiae. We show that C. albicans CLN1 (CaCLN1) has the same cell cycle-specific expression pattern as CLN1 and CLN2 of S. cerevisiae. To investigate whether G 1 cyclins are similarly involved in the regulation of cell morphogenesis during the yeast-to-hypha transition of C. albicans, we mutated CaCLN1. Cacln1/Cacln1 cells were found to be slower than wild-type cells in cell cycle progression. The Cacln1/Cacln1 mutants were also defective in hyphal colony formation on several solid media. Furthermore, while mutant strains developed germ tubes under several hypha-inducing conditions, they were unable to maintain the hyphal growth mode in a synthetic hypha-inducing liquid medium and were deficient in the expression of hypha-specific genes in this medium. Our results suggest that CaCln1 may coordinately regulate hyphal development with signal transduction pathways in response to various environmental cues.

The rho-type GTPase Cdc42 is important for the establishment and maintenance of eukaryotic cell polarity. To examine whether Cdc42 is regulated during the yeast-to-hypha transition in Candida albicans, we constructed a green fluorescence protein (GFP)-Cdc42 fusion under the ACT1 promoter and observed its localization in live C. albicans cells. As in Saccharomyces cerevisiae, GFP-Cdc42 was observed around the entire periphery of the cell. In yeast-form cells of C. albicans, it clustered to the tips and sides of small buds as well as to the mother-daughter neck region of large-budded cells. Upon hyphal induction, GFP-Cdc42 clustered to the site of hyphal evagination and remained at the tips of the hyphae. This temporal and spatial localization of Cdc42 suggests that its activity is regulated during the yeast-to-hypha transition. In addition to the accumulation at the hyphal tip, GFP-Cdc42 was also seen as a band within the hyphal tube in cells that had undergone nuclear separation. With the F-actin-assembly inhibitor latrunculin A, we found that GFP-Cdc42 accumulation at the bud site in yeast-form cells is F-actin independent, whereas GFP-Cdc42 accumulation at the hyphal tip requires F-actin. Furthermore, disruption of the F-actin cytoskeleton impaired the transcriptional induction of hypha-specific genes. Therefore, hypha formation resembles mating in Saccharomyces cerevisiae in that both require F-actin for GFP-Cdc42 localization and efficient signaling.

Graphical Abstract Highlights d Physiological DSBs are enriched at highly active oncogenic super-enhancers (SEs) d RAD51 co-localizes with transcription factors at SE in various cells d TOP1 mediates DSBs at SEs that are repaired by a RAD51dependent mechanism d Depletion of RAD51 increases DSBs at SEs and decreases expression of related oncogenes. SUMMARY DNA double-strand breaks (DSBs) are deleterious and tumorigenic but could also be essential for DNA-based processes. Yet the landscape of physiological DSBs and their role and repair are still elusive.Here, we mapped DSBs at high resolution in cancer and non-tumorigenic cells and found a transcription-coupled repair mechanism at oncogenic superenhancers. At these super-enhancers the transcription factor TEAD4, together with various transcription factors and co-factors, co-localizes with the repair factor RAD51 of the homologous recombination pathway. Depletion of TEAD4 or RAD51 increases DSBs at RAD51/TEAD4 common binding sites within super-enhancers and decreases expression of related genes, which are mostly oncogenes. Colocalization of RAD51 with transcription factors at super-enhancers occurs in various cell types, suggesting a broad phenomenon. Together, our findings uncover a coupling between transcription and repair mechanisms at oncogenic super-enhancers, to control the hyper-transcription of multiple cancer drivers.