Hepatitis B doubly spliced protein (HBDSP) promotes hepatocellular carcinoma cell apoptosis via ETS1/GATA2/YY1-mediated p53 transcription.

IMPORTANCE: Hepatitis B virus (HBV) spliced variants are associated with viral persistence or pathogenicity. Hepatitis B doubly spliced protein (HBDSP), which has been previously reported as a pleiotropic transactivator protein, can potentially serve as an HBV virulence factor. However, the underlying mechanisms of HBDSP in HBV-associated liver diseases remain to be elucidated. In this study, we revealed that HBDSP promotes cellular apoptosis and induces wt-p53-dependent apoptotic signaling pathway in wt-p53 hepatocellular cells by transactivating p53 transcription, and increases the release of HBV progeny. Therefore, HBDSP may promote the HBV particles release through wt-p53-dependent hepatocellular apoptosis. Our findings suggest that blocking HBDSP-induced wt-p53-dependent apoptosis might have therapeutic values for chronic hepatitis B.

DAB2IP regulates intratumoral testosterone synthesis and CRPC tumor growth by ETS1/AKR1C3 signaling.

The intratumoral androgen synthesis is one of the mechanisms by which androgen receptor (AR) is aberrantly re-activated in castration-resistant prostate cancer (CRPC) after androgen ablation. However, pathways controlling steroidogenic enzyme expression and de novo androgen synthesis in prostate cancer (PCa) cells are largely unknown. In this study, we explored the potential roles of DAB2IP in testosterone synthesis and CRPC tumor growth. Indeed, DAB2IP loss could maintain AR transcriptional activity, PSA re-expression and tumor growth under castrated condition in vitro and in vivo, and reprogram the expression profiles of steroidogenic enzymes, including AKR1C3. Mechanistically, DAB2IP could dramatically inhibit the AKR1C3 promoter activity and the conversion from androgen precursors (i.e., DHEA) to testosterone through PI3K/AKT/mTOR/ETS1 signaling. Consistently, there was a high co-expression of ETS1 and AKR1C3 in PCa tissues and xenografts, and their expression in prostate tissues could also restore AR nuclear staining in castrated DAB2IP-/- mice after DHEA supplement. Together, this study reveals a novel regulation of intratumoral de novo androgen synthesis in CRPC, and provides the DAB2IP/ETS1/AKR1C3 signaling as a potential therapeutic target.

ETS1 is coexpressed with ZEB2 and mediates ZEB2-induced epithelial-mesenchymal transition in human tumors.

Epithelial-mesenchymal transition (EMT) is an embryonic program that is reactivated in cancer and regulates the invasion and metastasis of tumor cells. Zinc finger E-box binding homeobox 2 (ZEB2) induces EMT by upregulating matrix metalloproteinases (MMP), yet MMP genes lack ZEB2 binding motif in their promoters. Recently, expression of MMPs was associated to the activation of ETS1 transcription factor; however, a link between ZEB2 and ETS proto-oncogene 1, transcription factor (ETS1) remains to be elucidated. Hence, we investigated the transcriptional regulation of ETS1 by ZEB2 after our initial observation that ZEB2 and ETS1 are coexpressed in hepatocellular carcinoma cells (HCCs). Chromatin immunoprecipitation and luciferase reporter assays clearly showed that ZEB2 binds to E-box sequences on the promoter of ETS1. Elevated expression of ETS1 was found in DLD-ZEB2 and A431-ZEB2 inducible systems, and knockdown of ZEB2 caused an explicit downregulation of ETS1 in shZEB2-SNU398 and shZEB2-SK-HEP-1 cells. Repression of ETS1 expression in ZEB2-induced conditions substantially impaired the migration and invasive capacities of DLD1 cells. Mechanistically, knockdown of ETS1 in ZEB2-expressing cells resulted in the downregulation of established ZEB2 targets TWIST and MMP9. Correlation analyses in HCC lines, cancer complementary DNA arrays, and The Cancer Genome Atlas RNA-sequencing data set revealed that ZEB2 and ETS1 are coexpressed, and their expressions in human tumors show a highly significant positive correlation. Our results demonstrated that ZEB2 acts as an upstream regulator of ETS1 and, in turn, ETS1 maintains ZEB2-induced EMT. These findings add another level of complexity to the understanding of ZEB2 in the invasion and metastasis of cancer cells, and put ZEB2/ETS1 axis as a novel therapeutic target in human malignancies.

Synergy of aromatic residues and phosphoserines within the intrinsically disordered DNA-binding inhibitory elements of the Ets-1 transcription factor.

The E26 transformation-specific (Ets-1) transcription factor is autoinhibited by a conformationally disordered serine-rich region (SRR) that transiently interacts with its DNA-binding ETS domain. In response to calcium signaling, autoinhibition is reinforced by calmodulin-dependent kinase II phosphorylation of serines within the SRR. Using mutagenesis and quantitative DNA-binding measurements, we demonstrate that phosphorylation-enhanced autoinhibition requires the presence of phenylalanine or tyrosine (ϕ) residues adjacent to the SRR phosphoacceptor serines. The introduction of additional phosphorylated Ser-ϕ-Asp, but not Ser-Ala-Asp, repeats within the SRR dramatically reinforces autoinhibition. NMR spectroscopic studies of phosphorylated and mutated SRR variants, both within their native context and as separate trans-acting peptides, confirmed that the aromatic residues and phosphoserines contribute to the formation of a dynamic complex with the ETS domain. Complementary NMR studies also identified the SRR-interacting surface of the ETS domain, which encompasses its positively charged DNA-recognition interface and an adjacent region of neutral polar and nonpolar residues. Collectively, these studies highlight the role of aromatic residues and their synergy with phosphoserines in an intrinsically disordered regulatory sequence that integrates cellular signaling and gene expression.

Berberine represses DAXX gene transcription and induces cancer cell apoptosis.

Death-domain-associated protein (DAXX) is a multifunctional protein that regulates a wide range of cellular signaling pathways for both cell survival and apoptosis. Regulation of DAXX gene expression remains largely obscure. We recently reported that berberine (BBR), a natural product derived from a plant used in Chinese herbal medicine, downregulates DAXX expression at the transcriptional level. Here, we further investigate the mechanisms underlying the transcriptional suppression of DAXX by BBR. By analyzing and mapping the putative DAXX gene promoter, we identified the core promoter region (from -161 to -1), which contains consensus sequences for the transcriptional factors Sp1 and Ets1. We confirmed that Sp1 and Ets1 bound to the core promoter region of DAXX and stimulated DAXX transcriptional activity. In contrast, BBR bound to the DAXX core promoter region and suppressed its transcriptional activity. Following studies demonstrated a possible mechanism that BBR inhibited the DAXX promoter activity through blocking or disrupting the association of Sp1 or Ets1 and their consensus sequences in the promoter. Downregulation of DAXX by BBR resulted in inhibition of MDM2 and subsequently, activation of p53, leading to cancer cell death. Our results reveal a novel possible mechanism: by competitively binding to the Sp1 and Ets1 consensus sequences, BBR inhibits the transcription of DAXX, thus inducing cancer cell apoptosis through a p53-dependent pathway.

Cell-type specific properties of pyramidal neurons in neocortex underlying a layout that is modifiable depending on the cortical area.

To understand sensory representation in cortex, it is crucial to identify its constituent cellular components based on cell-type-specific criteria. With the identification of cell types, an important question can be addressed: to what degree does the cellular properties of neurons depend on cortical location? We tested this question using pyramidal neurons in layer 5 (L5) because of their role in providing major cortical output to subcortical targets. Recently developed transgenic mice with cell-type-specific enhanced green fluorescent protein labeling of neuronal subtypes allow reliable identification of 2 cortical cell types in L5 throughout the entire neocortex. A comprehensive investigation of anatomical and functional properties of these 2 cell types in visual and somatosensory cortex demonstrates that, with important exceptions, most properties appear to be cell-type-specific rather than dependent on cortical area. This result suggests that although cortical output neurons share a basic layout throughout the sensory cortex, fine differences in properties are tuned to the cortical area in which neurons reside.

Yin yang 1 regulates the expression of snail through a distal enhancer.

Expression of the Snail gene is required for the epithelial-mesenchymal transitions that accompany mammalian gastrulation, neural crest migration, and organ formation. Pathologic expression of Snail contributes to the migratory capacity of invasive tumors, including melanomas. To investigate the mechanism of Snail up-regulation in human melanoma cells, a conserved enhancer located 3' of the Snail gene was analyzed. An overlapping Ets and yin yang 1 (YY1) consensus sequence, in addition to a SOX consensus sequence, was required for full enhancer activity. Proteins specifically binding these sequences were detected by electrophoretic mobility shift assay. The Ets/YY1 binding activity was purified by DNA-affinity chromatography and identified as YY1. Although ubiquitously expressed, YY1 was bound at the Snail 3' enhancer in vivo in Snail-expressing cells but not in cells that did not express Snail. Knockdown of YY1 in A375 cells led to decreased Snail expression. These results identify a role for YY1 in regulating transcription of Snail in melanoma cells through binding to the Snail 3' enhancer.