Concetta G.A. Marfella

Skeletal muscle specification by myogenin and Mef2D via the SWI/SNF ATPase Brg1

Myogenin is required not for the initiation of myogenesis but instead for skeletal muscle formation through poorly understood mechanisms. We demonstrate in cultured cells and, for the first time, in embryonic tissue, that myogenic late genes that specify the skeletal muscle phenotype are bound by MyoD prior to the initiation of gene expression. At the onset of muscle specification, a transition from MyoD to myogenin occurred at late gene loci, concomitant with loss of HDAC2, the appearance of both the Mef2D regulator and the Brg1 chromatin‐remodeling enzyme, and the opening of chromatin structure. We further demonstrated that ectopic expression of myogenin and Mef2D, in the absence of MyoD, was sufficient to induce muscle differentiation in a manner entirely dependent on Brg1. These results indicate that myogenin specifies the muscle phenotype by cooperating with Mef2D to recruit an ATP‐dependent chromatin‐remodeling enzyme that alters chromatin structure at regulatory sequences to promote terminal differentiation.

Deletion of p37Ing1 in Mice Reveals a p53-Independent Role for Ing1 in the Suppression of Cell Proliferation, Apoptosis, and Tumorigenesis

ING proteins have been proposed to alter chromatin structure and gene transcription to regulate numerous aspects of cell physiology, including cell growth, senescence, stress response, apoptosis, and transformation. ING1, the founding member of the inhibitor of growth family, encodes p37(Ing1), a plant homeodomain (PHD) protein that interacts with the p53 tumor suppressor protein and seems to be a critical cofactor in p53-mediated regulation of cell growth and apoptosis. In this study, we have generated and analyzed p37(Ing1)-deficient mice and primary cells to further explore the role of Ing1 in the regulation of cell growth and p53 activity. The results show that endogenous levels of p37(Ing1) inhibit the proliferation of p53-wild-type and p53-deficient fibroblasts, and that p53 functions are unperturbed in p37(Ing1)-deficient cells. In addition, loss of p37(Ing1) induces Bax expression and increases DNA damage-induced apoptosis in primary cells and mice irrespective of p53 status. Finally, p37(Ing1) suppresses the formation of spontaneous follicular B-cell lymphomas in mice. These results indicate that p53 does not require p37(Ing1) to negatively regulate cell growth and offers genetic proof that Ing1 suppresses cell growth and tumorigenesis. Furthermore, these data reveal that p37(Ing1) can negatively regulate cell growth and apoptosis in a p53-independent manner.

Mutation of the SNF2 family member Chd2 affects mouse development and survival.

The chromodomain helicase DNA‐binding domain (Chd) proteins belong to the SNF2‐like family of ATPases that function in chromatin remodeling and assembly. These proteins are characterized by the presence of tandem chromodomains and are further subdivided based on the presence or absence of additional structural motifs. The Chd1–Chd2 subfamily is distinguished by the presence of a DNA‐binding domain that recognizes AT‐rich sequence. Currently, there are no reports addressing the function of the Chd2 family member. Embryonic stem cells containing a retroviral gene‐trap inserted at the Chd2 locus were utilized to generate mice expressing a Chd2 protein lacking the DNA‐binding domain. This mutation in Chd2 resulted in a general growth delay in homozygous mutants late in embryogenesis and in perinatal lethality. Animals heterozygous for the mutation showed decreased neonatal viability and increased susceptibility to non‐neoplastic lesions affecting most primary organs. In particular, approximately 85% of the heterozygotes showed gross kidney abnormalities. Our results demonstrate that mutation of Chd2 dramatically affects mammalian development and long‐term survival. J. Cell. Physiol. 209: 162–171, 2006.

Myogenin and the SWI/SNF ATPase Brg1 Maintain Myogenic Gene Expression at Different Stages of Skeletal Myogenesis

Many studies have examined transcriptional regulation during the initiation of skeletal muscle differentiation; however, there is less information regarding transcriptional control during adult myogenesis and during the maintenance of the differentiated state. MyoD and the mammalian SWI/SNF chromatin-remodeling enzymes containing the Brg1 ATPase are necessary to induce myogenesis in cell culture models and in developing embryonic tissue, whereas myogenin and Brg1 are critical for the expression of the late genes that induce terminal muscle differentiation. Here, we demonstrate that myogenin also binds to its own promoter during the late stages of embryonic muscle development. As is the case during embryonic myogenesis, MyoD and Brg1 co-localize to the myogenin promoter in primary adult muscle satellite cells. However, in mature myofibers, myogenin and Brg1 are preferentially co-localized to the myogenin promoter. Thus, the myogenin promoter is occupied by different myogenic factors at different times of myogenesis. The relevance of myogenin in the continued expression from its own promoter is demonstrated in culture, where we show that myogenin, in the absence of MyoD, is capable of maintaining its own expression by recruiting the Brg1 ATPase to modify promoter chromatin structure and facilitate myogenin expression. Finally, we utilized in vivo electroporation to demonstrate that Brg1 is required for the continued production of the myogenin protein in newborn skeletal muscle tissue. These findings strongly suggest that the skeletal muscle phenotype is maintained by myogenin and the continuous activity of Brg1-based SWI/SNF chromatin-remodeling enzymes.

Chromatin Accessibility and Transcription Factor Binding at the PPARγ2 Promoter during Adipogenesis is Protein Kinase A-Dependent

The nuclear hormone receptor peroxisome proliferator‐activated receptor gamma (PPARγ) is a ligand‐activated transcription factor that specifies formation of the adipocyte lineage. PPARγ also serves as a primary target for the treatment of type 2 diabetes, illustrating both its medical relevance as well as the need to understand fundamental aspects of PPARγ expression and function. Here, we characterize molecular changes that occur at the PPARγ2 promoter within the first several hours of adipocyte differentiation in culture. Our results demonstrate that changes in chromatin accessibility at the PPARγ2 promoter and occupancy of the promoter by the c‐Fos transcription factor occur within an hour of the onset of differentiation, followed closely by the binding of the CCAAT/enhancer binding protein beta (C/EBPβ) transcription factor. All three events show a remarkable dependency on protein kinase A (PKA) activity. These results reflect novel requirements for the PKA signaling pathway and reinforce the importance of PKA function during the onset of adipocyte differentiation. J. Cell. Physiol. 226: 86–93, 2010.

A Mutation in the Mouse Chd2 Chromatin Remodeling Enzyme Results in a Complex Renal Phenotype

Background and Aims: Glomerular diseases are the third leading cause of kidney failure worldwide, behind only diabetes and hypertension. The molecular mechanisms underlying the cause of glomerular diseases are still largely unknown. The identification and characterization of new molecules associated with glomerular function should provide new insights into understanding the diverse group of glomerular diseases. The Chd2 protein belongs to a family of enzymes involved in ATP-dependent chromatin remodeling, suggesting that it likely functions as an epigenetic regulator of gene expression via the modification of chromatin structure. Methods: In this study, we present a detailed histomorphologic characterization of mice containing a mutation in the chromodomain helicase DNA-binding protein 2 (Chd2). Results: We show that Chd2-mutant mice present with glomerulopathy, proteinuria, and significantly impaired kidney function. Additionally, serum analysis revealed decreased hemoglobin and hematocrit levels in Chd2-mutant mice, suggesting that the glomerulopathy observed in these mice is associated with anemia. Conclusion: Collectively, the data suggest a role for the Chd2 protein in the maintenance of kidney function.

p37Ing1b Regulates B-Cell Proliferation and Cooperates with p53 to Suppress Diffuse Large B-Cell Lymphomagenesis

The Inhibitor of Growth (ING) gene family encodes structurally related proteins that alter chromatin to regulate gene expression and cell growth. The initial member, ING1, has also been proposed to function as a tumor suppressor in human cancer based on its ability to suppress cell growth and transformation in vitro. Mouse Ing1 produces two proteins (p31 and p37) from differentially spliced transcripts. We have recently generated p37(Ing1b)-null mice and observed spontaneous follicular B-cell lymphomagenesis in this model to show that ING proteins can function in vivo as tumor suppressors. In this present report, we examine the role of p37(Ing1b) in the regulation of B-cell growth and explore the relationship between p37(Ing1b) and p53-mediated tumor suppression. Our results indicate that p37(Ing1b) inhibits the proliferation of B cells and follicular B cells regardless of p53 status, and loss of p53 greatly accelerates the rate of B-cell lymphomagenesis in p37(Ing1b)-null mice. However, in contrast to the highly penetrant follicular B-cell lymphomas observed in p37(Ing1b)-null mice, mice lacking both p37(Ing1b) and p53 typically present with aggressive diffuse large B-cell lymphomas (DLBL). Analysis of marker gene expression in p37(Ing1b)/p53 null tumors indicates that the double-null mice develop both nongerminal center and germinal center B-cell-like DLBL, and also documents up-regulation of nuclear factor-kappaB activity in p37(Ing1b)/p53-null B cells and B-cell tumors. These results confirm that p53 mutation is an important mechanistic step in the formation of diffuse large B-cell lymphomas and reveals a p53-independent role for Ing1b in suppressing B-cell tumorigenesis.

Marfella CG, Ohkawa Y, Coles AH, Garlick DS, Jones SN, Imbalzano AN. 2006. Mutation of the SNF2 family member Chd2 affects mouse development and survival. J Cell Physiol 209:162–171.

The original article to which this erratum refers was published in J Cell Physiol 2006; 209:162–171.