SALL4 controls cell fate in response to DNA base composition.

Mammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4, which contains multiple zinc fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely upregulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4 null cells, including precocious differentiation of embryonic stem cells (ESCs) and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by downregulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive byproduct of genome evolution and constitutes a signal that aids control of cell fate.

Do short, frequent DNA sequence motifs mould the epigenome?

'Epigenome' refers to the panoply of chemical modifications borne by DNA and its associated proteins that locally affect genome function. Epigenomic patterns are thought to be determined by external constraints resulting from development, disease and the environment, but DNA sequence is also a potential influence. We propose that domains of relatively uniform DNA base composition may modulate the epigenome through cell type-specific proteins that recognize short, frequent sequence motifs. Differential recruitment of epigenomic modifiers may adjust gene expression in multigene blocks as an alternative to tuning the activity of each gene separately, thus simplifying gene expression programming.

Sphingosine 1-phosphate induces MKP-1 expression via p38 MAPK- and CREB-mediated pathways in airway smooth muscle cells.

Sphingosine 1-phosphate (S1P), a bioactive sphingolipid elevated in asthmatic airways, is increasingly recognized as playing an important role in respiratory disease. S1P activates receptor-mediated signaling to modulate diverse cellular functions and promote airway inflammation. Although many of the stimulatory pathways activated by S1P have been delineated, especially mitogen-activated protein kinases (MAPK), the question of whether S1P exerts negative feedback control on its own signaling cascade via upregulation of phosphatases remains unexplored. We show that S1P rapidly and robustly upregulates mRNA and protein expression of the MAPK deactivator-MAPK phosphatase 1 (MKP-1). Utilizing the pivotal airway structural cell, airway smooth muscle (ASM), we confirm that S1P activates all members of the MAPK family and, in part, S1P upregulates MKP-1 expression in a p38 MAPK-dependent manner. MKP-1 is a cAMP response element binding (CREB) protein-responsive gene and here, we reveal for the first time that an adenylate cyclase/PKA/CREB-mediated pathway also contributes to S1P-induced MKP-1. Thus, by increasing MKP-1 expression via parallel p38 MAPK- and CREB-mediated pathways, S1P temporally regulates MAPK signaling pathways by upregulating the negative feedback controller MKP-1. This limits the extent and duration of pro-inflammatory MAPK signaling and represses cytokine secretion in ASM cells. Taken together, our results demonstrate that S1P stimulates both kinases and the phosphatase MKP-1 to control inflammation in ASM cells and may provide a greater understanding of the molecular mechanisms responsible for the pro-asthmatic functions induced by the potent bioactive sphingolipid S1P in the lung.

Modulation of gene expression in U251 glioblastoma cells by binding of mutant p53 R273H to intronic and intergenic sequences.

Missense point mutations in the TP53 gene are frequent genetic alterations in human tumor tissue and cell lines derived thereof. Mutant p53 (mutp53) proteins have lost sequence-specific DNA binding, but have retained the ability to interact in a structure-selective manner with non-B DNA and to act as regulators of transcription. To identify functional binding sites of mutp53, we established a small library of genomic sequences bound by p53(R273H) in U251 human glioblastoma cells using chromatin immunoprecipitation (ChIP). Mutp53 binding to isolated DNA fragments confirmed the specificity of the ChIP. The mutp53 bound DNA sequences are rich in repetitive DNA elements, which are dispersed over non-coding DNA regions. Stable down-regulation of mutp53 expression strongly suggested that mutp53 binding to genomic DNA is functional. We identified the PPARGC1A and FRMD5 genes as p53(R273H) targets regulated by binding to intronic and intra-genic sequences. We propose a model that attributes the oncogenic functions of mutp53 to its ability to interact with intronic and intergenic non-B DNA sequences and modulate gene transcription via re-organization of chromatin.

Corticosteroids reduce IL-6 in ASM cells via up-regulation of MKP-1.

The mechanisms by which corticosteroids reduce airway inflammation are not completely understood. Traditionally, corticosteroids were thought to inhibit cytokines exclusively at the transcriptional level. Our recent evidence, obtained in airway smooth muscle (ASM), no longer supports this view. We have found that corticosteroids do not act at the transcriptional level to reduce TNF-alpha-induced IL-6 gene expression. Rather, corticosteroids inhibit TNF-alpha-induced IL-6 secretion by reducing the stability of the IL-6 mRNA transcript. TNF-alpha-induced IL-6 mRNA decays at a significantly faster rate in ASM cells pretreated with the corticosteroid dexamethasone (t(1/2) = 2.4 h), compared to vehicle (t(1/2) = 9.0 h; P < 0.05) (results are expressed as decay constants [k] [mean +/- SEM] and half-life [h]). Interestingly, the underlying mechanism of inhibition by corticosteroids is via the up-regulation of an endogenous mitogen-activated protein kinase (MAPK) inhibitor, MAPK phosphatase-1 (MKP-1). Corticosteroids rapidly up-regulate MKP-1 in a time-dependent manner (44.6 +/- 10.5-fold increase after 24 h treatment with dexamethasone; P < 0.05), and MKP-1 up-regulation was temporally related to the inhibition of TNF-alpha-induced p38 MAPK phosphorylation. Moreover, TNF-alpha acts via a p38 MAPK-dependent pathway to stabilize the IL-6 mRNA transcript (TNF-alpha, t(1/2) = 9.6 h; SB203580 + TNF-alpha, t(1/2) = 1.5 h), exogenous expression of MKP-1 significantly inhibits TNF-alpha-induced IL-6 secretion and MKP-1 siRNA reverses the inhibition of TNF-alpha-induced IL-6 secretion by dexamethasone. Taken together, these results suggest that corticosteroid-induced MKP-1 contributes to the repression of IL-6 secretion in ASM cells.

Synthetic CpG islands reveal DNA sequence determinants of chromatin structure.

The mammalian genome is punctuated by CpG islands (CGIs), which differ sharply from the bulk genome by being rich in G + C and the dinucleotide CpG. CGIs often include transcription initiation sites and display 'active' histone marks, notably histone H3 lysine 4 methylation. In embryonic stem cells (ESCs) some CGIs adopt a 'bivalent' chromatin state bearing simultaneous 'active' and 'inactive' chromatin marks. To determine whether CGI chromatin is developmentally programmed at specific genes or is imposed by shared features of CGI DNA, we integrated artificial CGI-like DNA sequences into the ESC genome. We found that bivalency is the default chromatin structure for CpG-rich, G + C-rich DNA. A high CpG density alone is not sufficient for this effect, as A + T-rich sequence settings invariably provoke de novo DNA methylation leading to loss of CGI signature chromatin. We conclude that both CpG-richness and G + C-richness are required for induction of signature chromatin structures at CGIs.

Aberrant Proliferation of Differentiating Alveolar Cells Induces Hyperplasia in Resting Mammary Glands of SV40-TAg Transgenic Mice.

WAP-T1 transgenic mice express SV40-TAg under control of the whey acidic protein (WAP) promoter, which directs activity of this strong viral oncogene to luminal cells of the mammary gland. Resting uniparous WAP-T1 glands develop hyperplasia composed of TAg positive cells prior to appearance of advanced tumor stages. We show that cells in hyperplasia display markers of alveolar differentiation, suggesting that TAg targets differentiating cells of the alveolar compartment. The glands show significant expression of Elf5 and milk genes (Lalba, Csn2, and Wap). TAg expressing cells largely co-stain with antibodies to Elf5, lack the epithelial marker Sca1, and are hormone receptor negative. High expression levels of Elf5 but not of milk genes are also seen in resting glands of normal BALB/c mice. This indicates that expression of Elf5 in resting WAP-T1 glands is not specifically induced by TAg. CK6a positive luminal cells lack TAg. These cells co-express the markers prominin-1, CK6a, and Sca1, and are positive for hormone receptors. These hormone sensitive cells localize to ducts and seem not to be targeted by TAg. Despite reaching an advanced stage in alveolar differentiation, the cells in hyperplasia do not exit the cell cycle. Thus, expression of TAg in conjunction with regular morphogenetic processes of alveologenesis seem to provide the basis for a hormone independent, unscheduled proliferation of differentiating cells in resting glands of WAP-T1 transgenic mice, leading to the formation of hyperplastic lesions.

Mutant p53 is a transcriptional co-factor that binds to G-rich regulatory regions of active genes and generates transcriptional plasticity.

The molecular mechanisms underlying mutant p53 (mutp53) "gain-of-function" (GOF) are still insufficiently understood, but there is evidence that mutp53 is a transcriptional regulator that is recruited by specialized transcription factors. Here we analyzed the binding sites of mutp53 and the epigenetic status of mutp53-regulated genes that had been identified by global expression profiling upon depletion of endogenous mutp53 (R273H) expression in U251 glioblastoma cells. We found that mutp53 preferentially and autonomously binds to G/C-rich DNA around transcription start sites (TSS) of many genes characterized by active chromatin marks (H3K4me3) and frequently associated with transcription-competent RNA polymerase II. Mutp53-bound regions overlap predominantly with CpG islands and are enriched in G4-motifs that are prone to form G-quadruplex structures. In line, mutp53 binds and stabilizes a well-characterized G-quadruplex structure in vitro. Hence, we assume that binding of mutp53 to G/C-rich DNA regions associated with a large set of cancer-relevant genes is an initial step in their regulation by mutp53. Using GAS1 and HTR2A as model genes, we show that mutp53 affects several parameters of active transcription. Finally, we discuss a dual mode model of mutp53 GOF, which includes both stochastic and deterministic components.