Cell density-dependent induction of endogenous myogenin (myf4) gene expression by Myf5.

Transcription factors Myf5 and MyoD are critical for myoblast determination. Myogenin is a direct transcriptional target of these factors and its expression is associated with commitment to terminal differentiation. Here, we have used myogenic derivatives of human U20S cells expressing Myf5 or MyoD under control of a tetracycline-sensitive promoter to study expression of endogenous myogenin (myf4). We find that Myf5-mediated induction of myogenin shows striking dependence on cell density. At high cell density, Myf5 is a potent inducer of myogenin expression. At low cell density, Myf5 (unlike MyoD) is a poor inducer of myogenin expression, whilst retaining the capacity to direct expression of other muscle-specific genes. The permissive influence of high cell density on myogenin induction by Myf5 is not a consequence of serum depletion or cell cycle arrest, but is mimicked by a disruption adjacent to the basic region of Myf5 (Myf5/mt) which reduces its DNA binding affinity for E-boxes without compromising its ability to transactivate a reporter gene driven by the myogenin promoter. Coculture of cells expressing wild-type Myf5 and Myf5/mt leads to reduced myogenin induction in Myf5/mt cells. We propose that at low cell density Myf5 inhibits induction of myogenin.

Constitutive instability of muscle regulatory factor Myf5 is distinct from its mitosis-specific disappearance, which requires a D-box-like motif overlapping the basic domain.

Transcription factors Myf5 and MyoD play critical roles in controlling myoblast identity and differentiation. In the myogenic cell line C2, we have found that Myf5 expression, unlike that of MyoD, is restricted to cycling cells and regulated by proteolysis at mitosis. In the present study, we have examined Myf5 proteolysis through stable transfection of myogenically convertible U20S cells with Myf5 derivatives under the control of a tetracycline-sensitive promoter. A motif within the basic helix-loop-helix domain of Myf5 (R93 to Q101) resembles the "destruction box" characteristic of substrates of mitotic proteolysis and thought to be recognized by the anaphase-promoting complex or cyclosome (APC). Mutation of this motif in Myf5 stabilizes the protein at mitosis but does not affect its constitutive turnover. Conversely, mutation of a serine residue (S158) stabilizes Myf5 in nonsynchronized cultures but not at mitosis. Thus, at least two proteolytic pathways control Myf5 levels in cycling cells. The mitotic proteolysis of Myf5 is unlike that which has been described for other destruction box-dependent substrates: down-regulation of Myf5 at mitosis appears to precede that of known targets of the APC and is not affected by a dominant-negative version of the ubiquitin carrier protein UbcH10, implicated in the APC-mediated pathway. Finally, we find that induction of Myf5 perturbs the passage of cells through mitosis, suggesting that regulation of Myf5 levels at mitosis may influence cell cycle progression of Myf5-expressing muscle precursor cells.

DNA replication progresses on the periphery of nuclear aggregates formed by the BCL6 transcription factor.

The BCL6 proto-oncogene, frequently alterated in non-Hodgkin lymphoma, encodes a POZ/zinc finger protein that localizes into discrete nuclear subdomains. Upon prolonged BCL6 overexpression in cells bearing an inducible BCL6 allele (UTA-L cells), these subdomains apparently coincide with sites of DNA synthesis. Here, we explore the relationship between BCL6 and replication by both electron and confocal laser scanning microscopy. First, by electron microscope analyses, we found that endogenous BCL6 is associated with replication foci. Moreover, we show that a relatively low expression level of BCL6 reached after a brief induction in UTA-L cells is sufficient to observe its targeting to mid, late, and at least certain early replication foci visualized by a pulse-labeling with bromodeoxyuridine (BrdU). In addition, when UTA-L cells are simultaneously induced for BCL6 expression and exposed to BrdU for a few hours just after the release from a block in mitosis, a nuclear diffuse BCL6 staining indicates cells in G(1), while cells in S show a more punctate nuclear BCL6 distribution associated with replication foci. Finally, ultrastructural analyses in UTA-L cells exposed to BrdU for various times reveal that replication progresses just around, but not within, BCL6 subdomains. Thus, nascent DNA is localized near, but not colocalized with, BCL6 subdomains, suggesting that they play an architectural role influencing positioning and/or assembly of replication foci. Together with its previously function as transcription repressor recruiting a histone deacetylase complex, BCL6 may therefore contribute to link nuclear organization, replication, and chromatin-mediated regulation.

Cultured myf5 null and myoD null muscle precursor cells display distinct growth defects.

Myf-5 and MyoD are the two muscle regulatory factors expressed from the myoblast stage to maintain the identity and to promote the subsequent differentiation of muscle precursor cells. To get insight into their role we have studied the capacity to proliferate and to differentiate of myf-5 and myoD null myoblasts in primary cultures and in the subsequent passages. Our results indicate that myf-5 null myoblasts differ from wild type (wt) myoblasts in that they undergo precocious differentiation: they become myogenin- and troponin T-positive and fail to incorporate bromodeoxyuridine (BrdU) under culture conditions and at a time when wt cells are not yet differentiated and continue to proliferate. In primary cultures of myoD null cells, up to 60% of the cells were scored as myoblasts on the basis of the expression of myf-5. These myoD-deficient myoblasts, unlike myoD-expressing cells, were poorly differentiating and displayed a severe growth defect that led to their elimination from the cultures: within a few passages myoblasts were absent from myoD-deficient cultures, which mostly consisted of senescent cells. That a null mutation in either gene reduces the proliferative potential of cultured myoblasts raises the possibility that Myf-5 and MyoD serve proliferation of muscle precursor cells.

Overexpressed BCL6 (LAZ3) oncoprotein triggers apoptosis, delays S phase progression and associates with replication foci.

One of the most frequent genetic abnormalities associated with non Hodgkin lymphoma is the structural alteration of the 5' non coding/regulatory region of the BCL6 (LAZ3) protooncogene. BCL6 encodes a POZ/Zn finger protein, a structure similar to that of many Drosophila developmental regulators and to another protein involved in a human hematopoietic malignancy, PLZF. BCL6 is a sequence specific transcriptional repressor controlling germinal center formation and T cell dependent immune response. Although the expression of BCL6 negatively correlates with cellular proliferation in different cell types, the influence of BCL6 on cell growth and survival is currently unknown so that the way its deregulation may contribute to cancer remains elusive. To directly address this issue, we used a tetracycline-regulated system in human U2OS osteosarcoma cells and thus found that BCL6 mediates growth suppression associated with impaired S phase progression and apoptosis. Interestingly, overexpressed BCL6 can colocalize with sites of ongoing DNA synthesis, suggesting that it may directly interfere with S phase initiation and/or progression. In contrast, the isolated Zn finger region of BCL6, which binds BCL6 target sequence but lacks transcriptional repression activity, slows, but does not suppress, U2OS cell growth, is less efficient at delaying S phase progression, and does not trigger apoptosis. Thus, for a large part, the effects of BCL6 overexpression on cell growth and survival depend on its ability to engage protein/protein interactions with itself and/or its transcriptional corepressors. That BCL6 restricts cell growth suggests that its deregulation upon structural alterations may alleviate negative controls on the cell cycle and cell survival.

Cell cycle-regulated expression of the muscle determination factor Myf5 in proliferating myoblasts.

Myf5 is the earliest-known muscle-specific factor to be expressed in vivo and its expression is associated with determination of the myoblast lineage. In C2 cells, we show by immunocytolocalization that Myf5 disappears rapidly from cells in which the differentiation program has been initiated. In proliferating myoblasts, the levels of Myf5 and MyoD detected from cell to cell are very heterogeneous. We find that some of the heterogeneity of Myf5 expression arises from a posttranscriptional regulation of Myf5 by the cell cycle. Immunoblotting of extracts from synchronized cultures reveals that Myf5 undergoes periodic fluctuations during the cell cycle and is absent from cells blocked early in mitosis by use of nocodazole. The disappearance of Myf5 from mitotic cells involves proteolytic degradation of a phosphorylated form of Myf5 specific to this phase of the cell cycle. In contrast, MyoD levels are not depleted in mitotic C2 cells. The mitotic destruction of Myf5 is the first example of a transcription factor showing cell cycle-regulated degradation. These results may be significant in view of the possible role of Myf5 in maintaining the determination of proliferating cells and in timing the onset of differentiation.

The glucocorticoid receptor and AP-1 are involved in a positive regulation of the muscle regulatory gene myf5 in cultured myoblasts.

The muscle regulatory factor, myf5, is involved in the establishment of skeletal muscle precursor cells. Little is known, however, about the control of the expression of the gene encoding this basic helix-loop-helix (bHLH) factor. We have addressed this question in the mouse myogenic cell line, C2, and in a derivative of this cell line where the myf5 gene is the only muscle-specific bHLH factor to be expressed at the myoblast stage. We present evidence that the synthetic glucocorticoid dexamethasone, and the pharmacological agent anisomycin, act synergistically to rapidly up-regulate the levels of myf5 transcript and protein. The glucocorticoid antagonist RU 486 abolishes this synergy, demonstrating the involvement of the glucocorticoid receptor. The expression of a dominant negative mutant of c-jun which interferes with the transactivating properties of all AP-1 family members also blocks the induction of myf5 by anisomycin and dexamethasone. An activator of protein kinase C (PKCs), 12-O-tetradecanoyl phorbol 13-acetate (TPA), abolishes the up-regulation of myf5 gene expression by dexamethasone and anisomycin, and its effect is counteracted by an inhibitor of PKCs, GF 109203X. These results point to the possible involvement of PKCs in the negative control of myf5. Evidence that both positive and negative regulation of myf5 transcripts, described here, does not require the fresh synthesis of transcription factors suggests that myf5 may behave like an immediate early gene.