A Novel RNAi Lethality Rescue Screen to Identify Regulators of Adipogenesis

Adipogenesis, the differentiation of fibroblast-like mesenchymal stem cells into mature adipocytes, is tightly regulated by a complex cascade of transcription factors, including the nuclear receptor Peroxisome proliferator activator receptor γ (PPARγ). RNAi-mediated knock down libraries may present an atractive method for the identification of additional adipogenic factors. However, using in vitro adipogenesis model systems for high-throughput screening with siRNA libraries is limited since (i) differentiation is not homogeneous, but results in mixed cell populations, and (ii) the expression levels (and activity) of adipogenic regulators is highly dynamic during differentiation, indicating that the timing of RNAi-mediated knock down during differentiation may be extremely critical. Here we report a proof-of-principle for a novel RNAi screening method to identify regulators of adipogenesis that is based on lethality rescue rather than differentiation, using microRNA expression driven by a PPARγ responsive RNA polymerase II promoter. We validated this novel method through screening of a dedicated deubiquitinase knock down library, resulting in the identification of UCHL3 as an essential deubiquitinase in adipogenesis. This system therefore enables the identification of novel genes regulating PPARγ-mediated adipogenesis in a high-throughput setting

The Effect of Robotics Education on Gender Differences in STEM Attitudes among Dutch 7th and 8th Grade Students

Because of its hands-on and integrative approach to STEM, educational robotics has become increasingly popular in recent years. Yet, a gender gap still exists in attitudes towards STEM studies and careers, especially among middle and high school students, potentially resulting in a lack of women in the STEM workforce. This study explores the effect of a robotics curriculum on Dutch 7th- and 8th-grade students’ attitude towards STEM subjects and careers, as assessed by the S-STEM survey. The results revealed no difference between the pre-test and post-test in attitudes toward STEM for both boys and girls. However, boys scored significantly higher than girls on attitude towards technology, engineering and future STEM studies on the post-test. A post hoc analysis revealed a significant difference between boys and girls on their attitude towards engineering and technology during the pre-test. These results demonstrate the difference between boys and girls in their attitudes towards STEM subjects and careers within the context of robotics education. Considering the lack of research on educational robotics among young teenagers, this field needs to be further studied to assess its effect on gender differences within attitudes towards STEM

A siRNA-mediated knock down of PPARγ rescues miRNA FF2 mediated loss of blasticidin resistance.

<p>Different PPARγ specific siRNA vectors were generated and tested for their ability to rescue the loss of blasticidin resistance. The functional siRNA vector #4 rescues the miRNA FF2 induced loss of resistance. B. The U2OS 3×PPRE miRNA FF2 cells were partially rescued with PPAR#4 siRNA expressing GFP virus. The percentage of GFP positive cells was determined using FACS analysis after 1 week of blasticidin selection at various concentrations.</p

Schematic model of miRNA mediated siRNA screening.

<p>The upper panel illustrates the situation of cells with active PPARγ2-mediated gene expression. PPARγ drives miRNA FF2 expression, resulting in repression of the Blasticidin S deaminase expression cassette via the target repeat FF2 sequences present in the 3′ UTR. The lower panel depicts the situation of cells with an siRNA mediated knock-down of a PPARγ2 activating protein. Expression of the FF2 miRNA is diminished and Blasticidin S deaminase expression is no longer repressed, resulting in blasticidin resistance.</p

A RNA polymerase II and RNA polymerase III driven miRNA FF2 expression results in loss of Blasticidine resistance.

<p>U2OS cells expressing the Blasticidin 3×FF2 expression cassette were transduced with miRNA FF2 expressing virus and selected (50 µg/ml Blasticidin S) for 1 week. As a control either empty virus or not FF2 specific miRNA FF3 virus was used, the latter expressing miRNA not targetting the modified 3′UTR of the blasticidin S deaminase gene. B. PPARγ2 expression compared to empty vector control as detected in the stably transfected U2OS cell line also expressing the blasticidin 3×FF2 expression cassette C. U2OS cells stably transduced with 3×PPRE miRNA FF2 virus were retransduced with blasticidin 3×FF2 virus and PPARγ2 virus at a ration of 1∶10 or 1∶25 respectively. Cells transduced at a ratio of 1∶25 show loss of resistance when incubated in 1 µM rosiglitazone and increasing amounts of Blasticidin S.</p

A, Small siRNA DUB screen perfomed in U2OS 3×PPRE miRNA FF2 cells.

<p>A partial siRNA library against 24 different deubiquitinating enzymes was tested for the ability to rescue the miRNA FF2 induced loss of Blasticidin resistance. Knock down of UCHL3 and UCHL5, but not UCHL1, resulted in colony formation. B. UCHL3 expression increases during 3T3-L1 adipocyte differentiation. Mouse 3T3L1 preadipocytes were differentiated into mature adipocytes and samples were taken at different time points during differentiation. Protein expression levels of UCHL3 were determined by Western blot analysis. As control for differentiation Fabp4 protein levels were analysed. C. UCHL3 activity in 3T3-L1 adipocytes. Cell lysates of differentiated 3T3-L1 cells (day 6) were incubated with or without HA-Ub probe, DUB activities were immunprecipitated (anti-HA agarose) and UCHL3 was detected by Western blotting. Note the difference in mobility between unmodified UCHL3 (input lane) and UCHL3 covalently bound to the DUB probe. An aspecific band is indicated (*). D. Localization of UCHL3 in differentiated 3T3-L1 cells. Nuclei were stained with DAPI, UCHL3 was visualized by indirect immunofluorescence. Merged pictures demonstrate the predominant cytoplasmic localization of UCHL3. As a control, the primary antibody was ommited. Bar, 10 µm. E. 3T3-L1 cells, stably transduced with lentiviral constructs expressing short hairpin RNAs directed against UCHL3 or control shRNA, were subjected to differentiation conditions. At day 10 of differentiation, cells were fixed and stained for triglycerides using Oil-red-O. Pictures are representative for three independent experiments. F. 3T3-L1 cells were stably transduced with either control or UCHL3 shRNA and differentiated as described under E. Cell lysates were subjected to Western blot analysis, using antibodies against UCHL3, PPARγ, FABP4 and tubulin.</p

Regulation of Treg functionality by acetylation-mediated Foxp3 protein stabilization

Regulatory T cells (Tregs) are a specific subset of lymphocytes that are critical for the maintenance of self-tolerance. Expression levels of the transcription factor Foxp3 have been causally associated with Treg differentiation and function. Recent studies show that Foxp3 can also be transiently expressed in effector T cells; however, stable Foxp3 expression is required for development of a functional Treg suppressor phenotype. Here, we demonstrate that Foxp3 is acetylated, and this can be reciprocally regulated by the histone acetyltransferase p300 and the histone deacetylase SIRT1. Hyperacetylation of Foxp3 prevented polyubiquitination and proteasomal degradation, therefore dramatically increasing stable Foxp3 protein levels. Moreover, using mouse splenocytes, human peripheral blood mononuclear cells, T cell clones, and skin-derived T cells, we demonstrate that treatment with histone deacetylase inhibitors resulted in significantly increased numbers of functional Treg cells. Taken together, our data demonstrate that modulation of the acetylation state of Foxp3 provides a novel molecular mechanism for assuring rapid temporal control of Foxp3 levels in T cells, thereby regulating Treg numbers and functionality. Manipulating Foxp3 acetylation levels could therefore provide a new therapeutic strategy to control inappropriate (auto)immune responses

The Multiple Endocrine Neoplasia Type 1 (MEN1) Tumor Suppressor Regulates Peroxisome Proliferator-Activated Receptor γ-Dependent Adipocyte Differentiation▿

Menin, the product of the MEN1 (multiple endocrine neoplasia type 1) tumor suppressor gene, is involved in activation of gene transcription as part of an MLL1 (mixed-lineage leukemia 1)/MLL2 (KMT2A/B)-containing protein complex which harbors methyltransferase activity for lysine 4 of histone H3 (H3K4). As MEN1 patients frequently develop lipomas and peroxisome proliferator-activated receptor γ (PPARγ) is expressed in several MEN1-related tumor types, we investigated regulation of PPARγ activity by menin. We found that menin is required for adipocyte differentiation of murine 3T3-L1 cells and PPARγ-expressing mouse embryonic fibroblasts. Menin augments PPARγ target gene expression through recruitment of H3K4 methyltransferase activity. Menin interacts directly with the activation function 2 transcription activation domain of PPARγ in a ligand-independent fashion. Ligand-dependent coactivation, however, is dependent on the LXXLL motif of menin and the intact helix 12 of PPARγ. We propose that menin is an important factor in PPARγ-mediated adipogenesis and that loss of PPARγ function may contribute to lipoma development in MEN1 patients