Recombinant expression and purification of AF1q and its interaction with T-cell Factor 7.

The protein ALL1 fused from chromosome 1q (AF1q) is overexpressed in a variety of cancers and acts to activate several signaling pathways that lead to oncogenesis. For example, AF1q has been shown to interact with T-cell Factor 7 (TCF7; also known as TCF1) from the Wnt/ß-catenin pathway resulting in the transcriptional activation of the CD44 and the enhancement of breast cancer metastasis. Despite the importance of AF1q in facilitating oncogenesis and metastasis, the structural and biophysical properties of AF1q remain largely unexplored due to the absence of a viable method for producing recombinant protein. Here, we report the overexpression of AF1q in E. coli as a fusion to a N-terminal His6-tag, which forms inclusion bodies (IBs) during expression. The AF1q protein was purified from IBs under denaturing conditions by immobilized metal affinity chromatography followed by a successful one-step dialysis refolding. Refolded AF1q was further purified to homogeneity by gel filtration chromatography resulting in an overall yield of 35 mg/L culture. Our nuclear magnetic resonance (NMR) and analytical ultracentrifugation (AUC) measurements reveal AF1q interacts with TCF7, specifically with TCF7's high-mobility group (HMG) domain (residues 154-237), which is, to our knowledge, the first biophysical characterization of the AF1q and TCF7 interaction.

The heterothallic sugarbeet pathogen Cercospora beticola contains exon fragments of both MAT genes that are homogenized by concerted evolution.

Dothideomycetes is one of the most ecologically diverse and economically important classes of fungi. Sexual reproduction in this group is governed by mating type (MAT) genes at the MAT1 locus. Self-sterile (heterothallic) species contain one of two genes at MAT1 (MAT1-1-1 or MAT1-2-1) and only isolates of opposite mating type are sexually compatible. In contrast, self-fertile (homothallic) species contain both MAT genes at MAT1. Knowledge of the reproductive capacities of plant pathogens are of particular interest because recombining populations tend to be more difficult to manage in agricultural settings. In this study, we sequenced MAT1 in the heterothallic Dothideomycete fungus Cercospora beticola to gain insight into the reproductive capabilities of this important plant pathogen. In addition to the expected MAT gene at MAT1, each isolate contained fragments of both MAT1-1-1 and MAT1-2-1 at ostensibly random loci across the genome. When MAT fragments from each locus were manually assembled, they reconstituted MAT1-1-1 and MAT1-2-1 exons with high identity, suggesting a retroposition event occurred in a homothallic ancestor in which both MAT genes were fused. The genome sequences of related taxa revealed that MAT gene fragment pattern of Cercospora zeae-maydis was analogous to C. beticola. In contrast, the genome of more distantly related Mycosphaerella graminicola did not contain MAT fragments. Although fragments occurred in syntenic regions of the C. beticola and C. zeae-maydis genomes, each MAT fragment was more closely related to the intact MAT gene of the same species. Taken together, these data suggest MAT genes fragmented after divergence of M. graminicola from the remaining taxa, and concerted evolution functioned to homogenize MAT fragments and MAT genes in each species.

Sox transcription factors require selective interactions with Oct4 and specific transactivation functions to mediate reprogramming.

The unique ability of Sox2 to cooperate with Oct4 at selective binding sites in the genome is critical for reprogramming somatic cells into induced pluripotent stem cells (iPSCs). We have recently demonstrated that Sox17 can be converted into a reprogramming factor by alteration of a single amino acid (Sox17EK) within its DNA binding HMG domain. Here we expanded this study by introducing analogous mutations to 10 other Sox proteins and interrogated the role of N-and C-termini on the reprogramming efficiency. We found that point-mutated Sox7 and Sox17 can convert human and mouse fibroblasts into iPSCs, but Sox4, Sox5, Sox6, Sox8, Sox9, Sox11, Sox12, Sox13, and Sox18 cannot. Next we studied regions outside the HMG domain and found that the C-terminal transactivation domain of Sox17 and Sox7 enhances the potency of Sox2 in iPSC assays and confers weak reprogramming potential to the otherwise inactive Sox4EK and Sox18EK proteins. These results suggest that the glutamate (E) to lysine (K) mutation in the HMG domain is necessary but insufficient to swap the function of Sox factors. Moreover, the HMG domain alone fused to the VP16 transactivation domain is able to induce reprogramming, albeit at low efficiency. By molecular dissection of the C-terminus of Sox17, we found that the ß-catenin interaction region contributes to the enhanced reprogramming efficiency of Sox17EK. To mechanistically understand the enhanced reprogramming potential of Sox17EK, we analyzed ChIP-sequencing and expression data and identified a subset of candidate genes specifically regulated by Sox17EK and not by Sox2.

SOX17 is a Critical Factor in Maintaining Endothelial Function in Pulmonary Hypertension by an Exosome-Mediated Autocrine Manner.

Endothelial dysfunction is considered a predominant driver for pulmonary vascular remodeling in pulmonary hypertension (PH). SOX17, a key regulator of vascular homoeostasis, has been found to harbor mutations in PH patients, which are associated with PH susceptibility. Here, this study explores whether SOX17 mediates the autocrine activity of pulmonary artery ECs to maintain endothelial function and vascular homeostasis in PH and its underlying mechanism. It is found that SOX17 expression is downregulated in the endothelium of remodeled pulmonary arteries in IPH patients and SU5416/hypoxia (Su/hypo)-induced PH mice as well as dysfunctional HPAECs. Endothelial knockdown of SOX17 accelerates the progression of Su/hypo-induced PH in mice. SOX17 overexpression in the pulmonary endothelium of mice attenuates Su/hypo-induced PH. SOX17-associated exosomes block the proliferation, apoptosis, and inflammation of HPAECs, preventing pulmonary arterial remodeling and Su/hypo-induced PH. Mechanistic analyses demonstrates that overexpressing SOX17 promotes the exosome-mediated release of miR-224-5p and miR-361-3p, which are internalized by injured HPAECs in an autocrine manner, ultimately repressing the upregulation of NR4A3 and PCSK9 genes and improving endothelial function. These results suggest that SOX17 is a key gene in maintaining endothelial function and vascular homeostasis in PH through regulating exosomal miRNAs in an autocrine manner.

The role of SOX family transcription factors in gastric cancer.

Gastric cancer (GC) is a leading cause of death worldwide. GC is the third-most common cause of cancer-related death after lung and colorectal cancer. It is also the fifth-most commonly diagnosed cancer. Accumulating evidence has revealed the role of signaling networks in GC progression. Identification of these molecular pathways can provide new insight into therapeutic approaches for GC. Several molecular factors involved in GC can play both onco-suppressor and oncogene roles. Sex-determining region Y (Sry)-box-containing (SOX) family members are transcription factors with a well-known role in cancer. SOX proteins can bind to DNA to regulate cellular pathways via a highly conserved domain known as high mobility group (HMG). In the present review, the roles of SOX proteins in the progression and/or inhibition of GC are discussed. The dual role of SOX proteins as tumor-promoting and tumor-suppressing factors is highlighted. SOX members can affect upstream mediators (microRNAs, long non-coding RNAs and NF-κB) and down-stream mediators (FAK, HIF-1α, CDX2 and PTEN) in GC. The possible role of anti-tumor compounds to target SOX pathway members in GC therapy is described. Moreover, SOX proteins may be used as diagnostic or prognostic biomarkers in GC.

Wnt/ß-catenin signaling regulates follicular development by modulating the expression of Foxo3a signaling components.

Wnt signaling is an evolutionarily conserved pathway that regulates cell proliferation, differentiation and apoptosis. To investigate the possible role of Wnt signaling in the regulation of ovarian follicular development, secondary follicles were isolated and cultured in vitro in the presence or absence of its activator (LiCl or Wnt3a) or inhibitor (IWR-1). We have demonstrated that activation of ß-catenin signals by activators dramatically suppressed follicular development by increasing granulosa cell apoptosis and inhibiting follicle steroidogenesis. In contrast, inhibition of Wnt signaling by IWR-1 was observed with better developed follicles and increased steroidogenesis. Further studies have shown that the transcription factor Forkhead box O3a (Foxo3a) and its downstream target molecules were modulated by the activators or the inhibitor. These findings provide evidence that Wnt signaling might negatively regulate follicular development potentially through Foxo3a signaling components.