Beta-catenin inhibits TR4-mediated lipid accumulation in 3T3-L1 adipocytes via induction of Slug

Background TR4, an orphan nuclear receptor plays a key role in glucose and lipid metabolism by regulating the expression of genes involved in energy metabolism. We previously reported that overexpression of TR4 in 3T3-L1 adipocytes promotes lipid accumulation in part by facilitating fatty acid uptake and synthesis, indicating that TR4 tightly regulates lipid homeostasis during adipogenesis. Here, we report that β-catenin suppresses TR4 transcriptional activity and that this inhibition is achieved through induction of Slug gene, a well-known transcription repressor in a variety of cells Methods To generate the stable cell line, 3T3-L1 cells were transfected with plasmids then cultured in presence of geneticin and/or blasticidin for 2 weeks. The lipid accumulation was measured by Oil Red O. The TR4-Slug and TR4-β-catenin interactions were checked by GST pull-down and mammalian two-hybrid assay. The TR4 transcriptional activities on various promoters were measured by luciferase activity. To check the binding affinity of TR4, we performed the gel shift and chromatin immunoprecipitation (ChIP) assay. Gene expression was detected by RT-qPCR at the mRNA level and western blotting at the protein level. Results Stable overexpression of Slug gene in 3T3-L1 preadipocytes strongly inhibited differentiation of 3T3-L1 preadipocytes. Using GST pull-down, gel shift and ChIP assays, we found that Slug abolished the formation of TR4 homodimers through direct interaction with TR4 and reduced the binding affinity of TR4 for its response elements located in TR4 target gene promoters such as fatty acid transport protein 1 and pyruvate carboxylase. Consistently, Slug inhibited TR4 target gene expression and was accompanied by repression of TR4-induced lipid accumulation in 3T3-L1 adipocytes. Conclusions Our results demonstrated that Slug inhibits 3T3-L1 adipogenesis through suppression of TR4 transcriptional activity.

Transcriptional activation of CBFβ by CDK11p110 is necessary to promote osteosarcoma cell proliferation

Background Aberrant expression of cyclin-dependent protein kinases (CDK) is a hallmark of cancer. CDK11 plays a crucial role in cancer cell growth and proliferation. However, the molecular mechanisms of CDK11 and CDK11 transcriptionally regulated genes are largely unknown. Methods In this study, we performed a global transcriptional analysis using gene array technology to investigate the transcriptional role of CDK11 in osteosarcoma. The promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay were used to identify direct transcriptional targets of CDK11. Clinical relevance and function of core-binding factor subunit beta (CBFβ) were further accessed in osteosarcoma. Results We identified a transcriptional role of protein-DNA interaction for CDK11p110, but not CDK11p58, in the regulation of CBFβ expression in osteosarcoma cells. The CBFβ promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay confirmed that CBFβ is a direct transcriptional target of CDK11. High expression of CBFβ is associated with poor outcome in osteosarcoma patients. Expression of CBFβ contributes to the proliferation and metastatic behavior of osteosarcoma cells. Conclusions These data establish CBFβ as a mediator of CDK11p110 dependent oncogenesis and suggest that targeting the CDK11- CBFβ pathway may be a promising therapeutic strategy for osteosarcoma treatment. Graphical Abstract

HMGB1 Interacts with the MLL-AF4 Fusion Complex to Regulate Pro-Leukemic Gene Transcription in Infant Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) in infants carries a poor prognosis and is characterized by cytogenetic rearrangements producing abnormal MLL fusion genes. Clinically effective targeting of the MLL fusion heterocomplex remains challenging, and therapeutic options remain limited. We have observed that the reduced isoform of HMGB1, a chromatin architectural protein that stabilizes DNA and facilitates transcription, is selectively over-expressed in the nuclei of infant MLL-ALL cells. In this study, we generated an HMGB1 siRNA knockdown in primary MLL-ALL cells from 3 infants to test our hypothesis that HMGB1-MLL interactions regulate pro-leukemic gene expression and represent a rational therapeutic target. CD19-selected leukemic blasts were isolated from the cryopreserved bone marrow or peripheral blood specimens of 3 infants with cytogenetically confirmed MLL-AF4 rearrangements. HMGB1 knockdown was confirmed by comparing HMGB1 mRNA and protein expression, by qPCR and Western Blot, in cells transfected with HMGB1 vs. control sequence siRNA. First, determined whether HMGB1 knockdown affected expression of the MLL fusion gene itself, by comparing MLL-AF4 mRNA and protein levels 72 hours after siRNA transfection. HMGB1 knockdown produced a 2.8 (± 0.55)- fold decrease in MLL-AF4 mRNA expression by qPCR (p<0.05), with a corresponding decrease in MLL-AF4 fusion protein expression by Western Blot, in each of the 3 specimens. Next, we determined whether HMGB1 binds functionally relevant regions of the MLL gene. We developed an electrophoretic mobility assay (EMSA) to compare the mobility of lysates from control vs. HMGB1 siRNA-treated infant MLL-ALL cells when mixed with biotinylated oligonucleotides spanning the transcriptionally active domains of MLL1. In each of 3 primary infant MLL-ALL cells, we detected a consistent gel-shift pattern on SDS-PAGE, in wild-type and control siRNA lysates, with oligonucleotides spanning exons 6-9- where many MLL-AF4 fusions occur. The gel-shift was completely abrogated in HMGB1 siRNA lysates. We then compared the expression of MLL target genes involved in leukemic transformation, by qPCR, in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. We observed a significant (p<0.01) reduction in expression of MEIS1 (5.8 ± 2.2-fold decrease), HOXA7 (4.3 ± 0.4-fold decrease) and HOXA9 (3.7 ± 1.5-fold decrease) in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. These data confirmed a role for HMGB1 in MLL gene/target gene regulation at the DNA level. Finally, we considered whether HMGB1, as a scaffold protein, could interact directly with the MLL fusion heterocomplex at the protein level. We immunoprecipitated HMGB1 from the nuclear fraction of wild-type primary infant MLL-ALL cells (n=3 patients), then probed the pull-down for N-terminal MLL (MLLn), C-terminal MLL (MLLc), the MLLn-AF4 fusion, the MLLn-ENL fusion, and the MLL-associated histone 3 methyltransferase DOT1L. MLLn and MLLn-AF4 were strongly detected in all HMGB1 immunoprecipitates. Individual and sequential co-immunoprecipitation of HMGB1 with MLL-AF4 and DOT1L in revealed loss of known complex formation between MLL-AF4 and DOT1L following HMGB1 knockdown. This was accompanied by a 3.4 (± 0.9)-fold decrease in DOT1L mRNA expression (p<0.001) by qPCR and a complete loss of histone 3k79me2 protein expression by Western blot. Taken together, these data suggest a central role for the fully reduced isoform of HMGB1, found in high abundance in infant ALL nuclei, in the formation of the MLL-AF4 transcription complex- including for the stable recruitment of DOT1L and H3K79me2, and in the regulation of MLL target genes such as HOXA9 and MEIS1. We are currently conducting chromatin immunoprecipitation and sequencing studies to identify methylation marks, particularly at H3K79me2, impacted by HMGB1 knockdown in infant ALL cells. We hope these studies will directly inform the development of small molecule inhibitors that specifically disrupt the binding sites and capacities of HMGB1 with MLL, which could synergize with the effects of methyltransferase inhibitors to more completely silence leukemic gene expression in infant ALL and improve the prognosis of this devastating disease. Disclosures No relevant conflicts of interest to declare.

Rns regulates nonpilus adhesin EtpA in human EnterotoxigenicE.coli(ETEC)

Rns, an araC family of transcriptional activator (AFTR) is known to regulate many of the known pili in human ETEC. Apart from pili, Rns is also known to regulate some nonpilus genes believed to have role in virulence. EtpA is a nonpilus adhesin, encoded with inetpBACoperon in ETEC genome. Using a combination of qRT-PCR and gel shift assay, we show that Rns binds to upstream of etpBAC operon and upregulates the expression of EtpA. This is the first report of Rns regulating a known virulence factor in ETEC.

Evaluating Commercially Available Antibodies for Rat α7 Nicotinic Acetylcholine Receptors

Alpha7 nicotinic acetylcholine receptors (α7 nAChRs) are important drug targets in neurological disorders and inflammation, making their detection and localization by validated antibodies highly desirable. However, tests in knockout animals raised questions about specificity of antibodies to mouse α7 nAChRs. To date, methods for validating antibodies for rat or human α7 nAChR have not been reported. We developed a gel-shift assay for western blots using GH4C1 cells expressing either native rat receptors or α7 nAChR-green fluorescent protein (GFP) chimeras to evaluate seven commercially available α7 nAChR antibodies. Blots with anti-GFP antibody detected GFP or α7 nAChR-GFP expressed in GH4C1 cells, and 125I-α-bungarotoxin binding and RNA analysis demonstrated α7 nAChR expression. Validated samples were used to evaluate α7 nAChR antibodies by western blot and immunofluorescence studies. These methods confirmed that two of seven α7 nAChR antibodies identify gel-shifts for α7 nAChR/nAChR-GFP but only one antibody demonstrated low background and significant immunofluorescence differences between wild-type and α7 nAChR expressing GH4C1 cells. However, that polyclonal antibody displayed lot-to-lot variability. Our findings suggest that careful validation methods are required for all α7 nAChR receptor species and antibody lots and that the gel-shift assay may allow for relatively rapid antibody screening.