HES6 enhances the motility of alveolar rhabdomyosarcoma cells.

HES6, a member of the hairy-enhancer-of-split family of transcription factors, plays multiple roles in myogenesis. It is a direct target of the myogenic transcription factor MyoD and has been shown to regulate the formation of the myotome in development, myoblast cell cycle exit and the organization of the actin cytoskeleton during terminal differentiation. Here we investigate the expression and function of HES6 in rhabdomyosarcoma, a soft tissue tumor which expresses myogenic genes but fails to differentiate into muscle. We show that HES6 is expressed at high levels in the subset of alveolar rhabdomyosarcomas expressing PAX/FOXO1 fusion genes (ARMSp). Knockdown of HES6 mRNA in the ARMSp cell line RH30 reduces proliferation and cell motility. This phenotype is rescued by expression of mouse Hes6 which is insensitive to HES6 siRNA. Furthermore, expression microarray analysis indicates that the HES6 knockdown is associated with a decrease in the levels of Transgelin, (TAGLN), a regulator of the actin cytoskeleton. Knockdown of TAGLN decreases cell motility, whilst TAGLN overexpression rescues the motility defect resulting from HES6 knockdown. These findings indicate HES6 contributes to the pathogenesis of ARMSp by enhancing both proliferation and cell motility.

Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts.

Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, but F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.

p53 mutation in normal esophagus promotes multiple stages of carcinogenesis but is constrained by clonal competition.

Aging normal human oesophagus accumulates TP53 mutant clones. These are the origin of most oesophageal squamous carcinomas, in which biallelic TP53 disruption is almost universal. However, how p53 mutant clones expand and contribute to cancer development is unclear. Here we show that inducing the p53R245W mutant in single oesophageal progenitor cells in transgenic mice confers a proliferative advantage and clonal expansion but does not disrupt normal epithelial structure. Loss of the remaining p53 allele in mutant cells results in genomically unstable p53R245W/null epithelium with giant polyaneuploid cells and copy number altered clones. In carcinogenesis, p53 mutation does not initiate tumour formation, but tumours developing from areas with p53 mutation and LOH are larger and show extensive chromosomal instability compared to lesions arising in wild type epithelium. We conclude that p53 has distinct functions at different stages of carcinogenesis and that LOH within p53 mutant clones in normal epithelium is a critical step in malignant transformation.

A single-progenitor model as the unifying paradigm of epidermal and esophageal epithelial maintenance in mice.

In adult skin epidermis and the epithelium lining the esophagus cells are constantly shed from the tissue surface and replaced by cell division. Tracking genetically labelled cells in transgenic mice has given insight into cell behavior, but conflicting models appear consistent with the results. Here, we use an additional transgenic assay to follow cell division in mouse esophagus and the epidermis at multiple body sites. We find that proliferating cells divide at a similar rate, and place bounds on the distribution cell cycle times. By including these results in a common analytic approach, we show that data from eight lineage tracing experiments is consistent with tissue maintenance by a single population of proliferating cells. The outcome of a given cell division is unpredictable but, on average, the likelihood of producing proliferating and differentiating cells is equal, ensuring cellular homeostasis. These findings are key to understanding squamous epithelial homeostasis and carcinogenesis.

Role for amplification and expression of glypican-5 in rhabdomyosarcoma.

Overexpression of genes, through genomic amplification and other mechanisms, can critically affect the behavior of tumor cells. Genomic amplification of the 13q31-32 region is reported in many tumors, including rhabdomyosarcomas that are primarily pediatric sarcomas resembling developing skeletal muscle. The minimum overlapping region of amplification at 13q31-32 in rhabdomyosarcomas was defined as containing two genes: Glypican-5 (GPC5) encoding a cell surface proteoglycan and C13orf25 encompassing the miR-17-92 micro-RNA cluster. Genomic copy number and gene expression analyses of rhabdomyosarcomas indicated that GPC5 was the only gene consistently expressed and up-regulated in all cases with amplification. Constitutive overexpression and knockdown of GPC5 expression in rhabdomyosarcoma cell lines increased and decreased cell proliferation, respectively. A correlation between expression levels of nascent pre-rRNA and GPC5 (P = 0.001), but not a C13orf25 transcript containing miR-17-92, in primary samples supports an association of GPC5 with proliferative capacity in vivo. We show that GPC5 increases proliferation through potentiating the action of the growth factors fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and Wnt1A. GPC5 enhanced the intracellular signaling of FGF2 and HGF and altered the cellular distribution of FGF2. The mesoderm-inducing effect of FGF2 and FGF4 in Xenopus blastocysts was also enhanced. Our data are consistent with a role of GPC5, in the context of sarcomagenesis, in enhancing FGF signaling that leads to mesodermal cell proliferation without induction of myogenic differentiation. Furthermore, the properties of GPC5 make it an attractive target for therapeutic intervention in rhabdomyosarcomas and other tumors that amplify and/or overexpress the gene.

Outcompeting p53-Mutant Cells in the Normal Esophagus by Redox Manipulation.

As humans age, normal tissues, such as the esophageal epithelium, become a patchwork of mutant clones. Some mutations are under positive selection, conferring a competitive advantage over wild-type cells. We speculated that altering the selective pressure on mutant cell populations may cause them to expand or contract. We tested this hypothesis by examining the effect of oxidative stress from low-dose ionizing radiation (LDIR) on wild-type and p53 mutant cells in the transgenic mouse esophagus. We found that LDIR drives wild-type cells to stop proliferating and differentiate. p53 mutant cells are insensitive to LDIR and outcompete wild-type cells following exposure. Remarkably, combining antioxidant treatment and LDIR reverses this effect, promoting wild-type cell proliferation and p53 mutant differentiation, reducing the p53 mutant population. Thus, p53-mutant cells can be depleted from the normal esophagus by redox manipulation, showing that external interventions may be used to alter the mutational landscape of an aging tissue.

Hes6 is required for MyoD induction during gastrulation.

The specification of mesoderm into distinct compartments sharing the same lineage restricted fates is a crucial step occurring during gastrulation, and is regulated by morphogenic signals such as the FGF/MAPK and activin pathways. One target of these pathways is the transcription factor XmyoD, which in early gastrulation is expressed in the lateral and ventral mesoderm. Expression of the hairy/enhancer of split transcription factor hes6, is also restricted to lateral and ventral mesoderm in gastrula stage Xenopus embryos, leading us to investigate whether it has a role in XmyoD regulation. In vivo, Xhes6 is required for FGF-mediated induction of XmyoD expression but not for induction of early mesoderm. The WRPW domain of Xhes6, which binds Groucho family transcriptional co-regulators, is essential for the XmyoD-inducing activity of Xhes6. Two Groucho proteins, Xgrg2 and Xgrg4, are expressed in lateral and ventral mesoderm, and inhibit expression of XmyoD. Xhes6 binds both Xgrg2 and Xgrg4 and relieves their inhibition of XmyoD expression. We also find that lowering Xhes6 expression levels blocks normal myogenic differentiation at tail bud stage. We conclude that Xhes6 is essential for XmyoD induction and acts by relieving Groucho-mediated repression of gene expression.

Epidermal Tissue Adapts to Restrain Progenitors Carrying Clonal p53 Mutations.

Aging human tissues, such as sun-exposed epidermis, accumulate a high burden of progenitor cells that carry oncogenic mutations. However, most progenitors carrying such mutations colonize and persist in normal tissue without forming tumors. Here, we investigated tissue-level constraints on clonal progenitor behavior by inducing a single-allele p53 mutation (Trp53R245W; p53∗/wt), prevalent in normal human epidermis and squamous cell carcinoma, in transgenic mouse epidermis. p53∗/wt progenitors initially outcompeted wild-type cells due to enhanced proliferation, but subsequently reverted toward normal dynamics and homeostasis. Physiological doses of UV light accelerated short-term expansion of p53∗/wt clones, but their frequency decreased with protracted irradiation, possibly due to displacement by UV-induced mutant clones with higher competitive fitness. These results suggest multiple mechanisms restrain the proliferation of p53∗/wt progenitors, thereby maintaining epidermal integrity.

Somatic mutant clones colonize the human esophagus with age.

The extent to which cells in normal tissues accumulate mutations throughout life is poorly understood. Some mutant cells expand into clones that can be detected by genome sequencing. We mapped mutant clones in normal esophageal epithelium from nine donors (age range, 20 to 75 years). Somatic mutations accumulated with age and were caused mainly by intrinsic mutational processes. We found strong positive selection of clones carrying mutations in 14 cancer genes, with tens to hundreds of clones per square centimeter. In middle-aged and elderly donors, clones with cancer-associated mutations covered much of the epithelium, with NOTCH1 and TP53 mutations affecting 12 to 80% and 2 to 37% of cells, respectively. Unexpectedly, the prevalence of NOTCH1 mutations in normal esophagus was several times higher than in esophageal cancers. These findings have implications for our understanding of cancer and aging.

Interaction of serum response factor (SRF) with the Elk-1 B box inhibits RhoA-actin signaling to SRF and potentiates transcriptional activation by Elk-1.

Serum response factor (SRF) is a transcription factor which regulates many immediate-early genes. Rho GTPases regulate SRF activity through changes in actin dynamics, but some SRF target genes, such as c-fos, are insensitive to this pathway. At the c-fos promoter, SRF recruits members of the ternary complex factor (TCF) family of Ets domain proteins through interactions with the TCF B-box region. Analysis of c-fos promoter mutations demonstrates that the TCF and ATF/AP1 sites adjoining the SRF binding site inhibit activation of the promoter by RhoA-actin signaling. The presence of the TCF binding site is sufficient for inhibition, and experiments with an altered-specificity Elk-1 derivative demonstrate that inhibition can be mediated by the Elk-1 TCF. Using Elk-1 fusion proteins that can bind DNA autonomously, we show that inhibition of RhoA-actin signaling requires physical interaction between the Elk-1 B box and SRF. These results account for the insensitivity of c-fos to RhoA-actin signaling. Interaction of the B box with SRF also potentiates transcriptional activation by the Elk-1 C-terminal activation domain. Combinatorial interactions between SRF and TCF proteins are thus likely to play an important role in determining the relative sensitivity of SRF target genes to Ras- and Rho-controlled signal transduction pathways.

Human keratinocytes have two interconvertible modes of proliferation.

Single stem cells, including those in human epidermis, have a remarkable ability to reconstitute tissues in vitro, but the cellular mechanisms that enable this are ill-defined. Here we used live imaging to track the outcome of thousands of divisions in clonal cultures of primary human epidermal keratinocytes. Two modes of proliferation were seen. In 'balanced' mode, similar proportions of proliferating and differentiating cells were generated, achieving the 'population asymmetry' that sustains epidermal homeostasis in vivo. In 'expanding' mode, an excess of cycling cells was produced, generating large expanding colonies. Cells in expanding mode switched their behaviour to balanced mode once local confluence was attained. However, when a confluent area was wounded in a scratch assay, cells near the scratch switched back to expanding mode until the defect was closed. We conclude that the ability of a single epidermal stem cell to reconstitute an epithelium is explained by two interconvertible modes of proliferation regulated by confluence.

Hes6 is required for the neurogenic activity of neurogenin and NeuroD.

In the embryonic neural plate, a subset of precursor cells with neurogenic potential differentiates into neurons. This process of primary neurogenesis requires both the specification of cells for neural differentiation, regulated by Notch signaling, and the activity of neurogenic transcription factors such as neurogenin and NeuroD which drive the program of neural gene expression. Here we study the role of Hes6, a member of the hairy enhancer of split family of transcription factors, in primary neurogenesis in Xenopus embryos. Hes6 is an atypical Hes gene in that it is not regulated by Notch signaling and promotes neural differentiation in mouse cell culture models. We show that depletion of Xenopus Hes6 (Xhes6) by morpholino antisense oligonucleotides results in a failure of neural differentiation, a phenotype rescued by both wild type Xhes6 and a Xhes6 mutant unable to bind DNA. However, an Xhes6 mutant that lacks the ability to bind Groucho/TLE transcriptional co-regulators is only partly able to rescue the phenotype. Further analysis reveals that Xhes6 is essential for the induction of neurons by both neurogenin and NeuroD, acting via at least two distinct mechanisms, the inhibition of antineurogenic Xhairy proteins and by interaction with Groucho/TLE family proteins. We conclude Xhes6 is essential for neurogenesis in vivo, acting via multiple mechanisms to relieve inhibition of proneural transcription factor activity within the neural plate.

Characterization of a megakaryocyte-specific enhancer of the key hemopoietic transcription factor GATA1.

Specification and differentiation of the megakaryocyte and erythroid lineages from a common bipotential progenitor provides a well studied model to dissect binary cell fate decisions. To understand how the distinct megakaryocyte- and erythroid-specific gene programs arise, we have examined the transcriptional regulation of the megakaryocyte erythroid transcription factor GATA1. Hemopoietic-specific mouse (m)GATA1 expression requires the mGata1 enhancer mHS-3.5. Within mHS-3.5, the 3' 179 bp of mHS-3.5 are required for megakaryocyte but not red cell expression. Here, we show mHS-3.5 binds key hemopoietic transcription factors in vivo and is required to maintain histone acetylation at the mGata1 locus in primary megakaryocytes. Analysis of GATA1-LacZ reporter gene expression in transgenic mice shows that a 25-bp element within the 3'-179 bp in mHS-3.5 is critical for megakaryocyte expression. In vitro three DNA binding activities A, B, and C bind to the core of the 25-bp element, and these binding sites are conserved through evolution. Activity A is the zinc finger transcription factor ZBP89 that also binds to other cis elements in the mGata1 locus. Activity B is of particular interest as it is present in primary megakaryocytes but not red cells. Furthermore, mutation analysis in transgenic mice reveals activity B is required for megakaryocyte-specific enhancer function. Bioinformatic analysis shows sequence corresponding to the binding site for activity B is a previously unrecognized motif, present in the cis elements of the Fli1 gene, another important megakaryocyte-specific transcription factor. In summary, we have identified a motif and a DNA binding activity likely to be important in directing a megakaryocyte gene expression program that is distinct from that in red cells.