Snail is a multifunctional transcriptional factor that has been described as a repressor in many different contexts. It is also proposed as an activator in a few cases relevant to tumor progression and cell‐cycle arrest. This study investigated the detailed mechanisms by which Snail upregulates gene expression of the CDK inhibitor p15^INK4b in HepG2 induced by the tumor promoter tetradecanoyl phorbol acetate (TPA). Using deletion mapping, the TPA‐responsive element on the p15^INK4b promoter was located between 77 and 228 bp upstream of the transcriptional initiation site, within which the putative binding regions of early growth response gene 1 (EGR‐1) and stimulatory protein 1 (SP‐1) were found. Gene expression of EGR‐1, Snail and SP‐1 can be induced by TPA within 0.5–6 h. In addition, basal levels of SP‐1, but not of the other two transcriptional factors, were observed. Blockade of TPA‐induced gene expression of Snail, EGR‐1 or SP‐1 suppressed activation of the p15–pro228 reporter plasmid harboring the TPA‐responsive element. More detailed deletion mapping and site‐directed mutagenesis further concluded that the overlapping EGR‐1/SP‐1‐binding site was required for TPA‐induced p15–pro228 activation. In an EMSA, a DNA–protein complex was elevated by TPA, which can be blocked by antibodies against EGR‐1, SP‐1 or Snail at 6 h. Immunoprecipitation/western blotting demonstrated that TPA could trigger the association of EGR‐1 with Snail or SP‐1. Furthermore, a double chromatin immunoprecipitation assay verified that EGR‐1 could form a complex with Snail or SP‐1 on the TPA‐responsive element after treatment with TPA for 2–6 h. Finally, we demonstrated a novel Snail‐target region which could be bound by Snail and was also required for TPA‐induced p15–pro228 activation. In conclusion, Snail associates with EGR‐1 and SP‐1 to mediate TPA‐induced transcriptional upregulation of p15^INK4b in HepG2.Structured digital abstract MINT‐7384899: Snail (uniprotkb:O95863) physically interacts (MI:0915) with EGR‐1 (uniprotkb:P18146) by anti bait coimmunoprecipitation (MI:0006) MINT‐7384908: SP‐1 (uniprotkb:P08047) physically interacts (MI:0915) with EGR‐1 (uniprotkb:P18146) by anti bait coimmunoprecipitation (MI:0006)

Radiotherapy can induce various adverse effects including fibrosis in cancer patients. Radiation‐induced aberrant expression of profibrotic genes has been associated with dysregulated epigenetic mechanisms. Pan‐BET (bromodomain and extraterminal domain) inhibitors, such as JQ1 and I‐BET151, have been reported to attenuate the profibrotic response after irradiation. Despite their profound preclinical efficacy, the clinical utility of pan‐inhibitors is limited due to observed cytotoxicicities. Recently, inhibitors were developed that selectively target the first (BD1) and second (BD2) bromodomain of the BET proteins (iBET‐BD1 [GSK778] and iBET‐BD2 [GSK046]). Here, their potential to attenuate radiation‐induced fibroblast activation with low‐toxicity was investigated. Our results indicated that cell proliferation and cell cycle progression in fibroblasts from BJ cells and six donors were reduced when treated with I‐BET151 and iBET‐BD1, but not with iBET‐BD2. After irradiation, induction of DGKA and profibrotic markers, especially COL1A1 and ACTA2, was attenuated with all BET inhibitors. H3K27ac enrichment was similar at the DGKA enhancer region after I‐BET151 treatment and irradiation, but was reduced at the COL1A1 transcription start site and the ACTA2 enhancer site. iBET‐BD2 did not change H3K27ac levels in these regions. BRD4 occupancy at these regions was not altered by any of the compounds. Cell migration activity was measured as a characteristic independent of extracellular matrix production and was unchanged in fibroblasts after irradiation and BET inhibitor‐treatment. In conclusion, iBET‐BD2 efficiently suppressed radiation‐induced expression of DGKA and profibrotic markers without showing cytotoxicity. Thus BD2‐selective targeting is a promising new therapeutic avenue for further investigations to prevent or attenuate radiotherapy‐induced fibrosis.

JAK2 activation is crucial for cytokine receptor signal transduction and leukemogenesis. However, the underlying processes that lead to full activation of JAK2 are unclear. Here, we report a positive role for ubiquitination of JAK2 during GM-CSF-induced activation. Upon GM-CSF stimulation, JAK2 ubiquitination is significantly enhanced through K63-linked poly-ubiquitination. Studies employing both knockout and overexpression of Cbl, an E3 ubiquitin ligase, led to the conclusion that Cbl specifically promotes JAK2 ubiquitination, and this was further confirmed in vitro using a Cbl ubiquitination assay. Moreover, following GM-CSF stimulation, the levels of phospho-JAK2 and -STAT5 and a STAT5 luciferase reporter assay were all reduced in Cbl knockout cells and this effect could be rescued by Cbl expression. Mechanistically, Cbl can interact with, and ubiquitinate JAK2 FERM and kinase domains via the Cbl TKB domain. Using lysine-to-arginine site-directed mutagenesis, K970 in the kinase domain of JAK2 was identified as the ubiquitination site important for promoting full JAK2 activation by Cbl via K63-conjugated poly-ubiquitination. Our study suggests that GM-CSF-induced JAK2 activation is enhanced by Cbl-mediated ubiquitination of JAK2. Targeting ubiquitination of JAK2 might offer a novel therapeutic strategy against JAK2-mediated disorders.

Radiotherapy, a common component in cancer treatment, can induce adverse effects including fibrosis in co-irradiated tissues. We previously showed that differential DNA methylation at an enhancer of diacylglycerol kinase alpha (DGKA) in normal dermal fibroblasts is associated with radiation-induced fibrosis. After irradiation, the transcription factor EGR1 is induced and binds to the hypomethylated enhancer, leading to increased DGKA and pro-fibrotic marker expression. We now modulated this DGKA induction by targeted epigenomic and genomic editing of the DGKA enhancer and administering epigenetic drugs. Targeted DNA demethylation of the DGKA enhancer in HEK293T cells resulted in enrichment of enhancer-related histone activation marks and radiation-induced DGKA expression. Mutations of the EGR1-binding motifs decreased radiation-induced DGKA expression in BJ fibroblasts and caused dysregulation of multiple fibrosis-related pathways. EZH2 inhibitors (GSK126, EPZ6438) did not change radiation-induced DGKA increase. Bromodomain inhibitors (CBP30, JQ1) suppressed radiation-induced DGKA and pro-fibrotic marker expression. Similar drug effects were observed in donor-derived fibroblasts with low DNA methylation. Overall, epigenomic manipulation of DGKA expression may offer novel options for a personalized treatment to prevent or attenuate radiotherapy-induced fibrosis.

It is well known that ligand binding to the high-affinity GM-CSF receptor (GMR) activates JAK2. However, how and where this event occurs in a cellular environment remains unclear. Here, we demonstrate that clathrin- but not lipid raft-mediated endocytosis is crucial for GMR signaling. Knockdown expression of clathrin heavy chain or intersectin 2 (ITSN2) attenuated GMR-mediated activation of JAK2, whereas inhibiting clathrin-coated pits or plagues to bud off the membrane by the dominant-negative mutant of dynamin enhanced such event. Moreover, unlike the wild-type receptor, an ITSN2-non-binding mutant of GMR defective in targeting to clathrin-coated pits or plagues [collectively referred to as clathrin-coated structures (CCSs) here] failed to activate JAK2 at such locations. Additional experiments demonstrate that ligand treatment not only enhanced JAK2/GMR association at CCSs, but also induced a conformational change of JAK2 which is required for JAK2 to be activated by CCS-localized CK2. Interestingly, ligand-independent activation of the oncogenic mutant of JAK2 (JAK2V617F) also requires the targeting of this mutant to CCSs. But JAK2V617F seems to be constitutively in an open conformation for CK2 activation. Together, this study reveals a novel functional role of CCSs in GMR signaling and the oncogenesis of JAK2V617F.