Id1 is an inhibitor of a group of basic helix-loop-helix transcription factors, collectively called E proteins, which includes E12, E47, E2-2, and HEB. We have generated transgenic mice in which Id1 is specifically expressed in T cells. The total number of thymocytes in these mice is less than 4% of that in wild-type mice. The majority of the transgenic thymocytes are CD4 and CD8 double negative and bear the cell surface markers of multipotent progenitor cells. A small number of thymocytes, however, differentiate into CD4 or CD8 single-positive T cells, which also display different characteristics from their wild-type counterparts. More importantly, apoptotic cells constitute about 50% of the total thymocytes. These apoptotic thymocytes have rearranged their T-cell receptor genes, suggesting that they are differentiating T cells. This finding has raised the possibility that the T-cell deficiency in Id1 transgenic mice is the result of a massive apoptosis of differentiating T cells triggered by Id1 expression as opposed to a developmental block at the earliest progenitor stage. The progenitor cells accumulated in the transgenic mice might have survived because they are not susceptible to the apoptotic signals. Despite the massive cell death of the thymocytes at young ages, Id1 transgenic mice frequently develop T-cell lymphoma later in their life span, and lymphomagenesis appears to occur at different stages of T-cell development. Taken together, our data suggest that E proteins, being the targets of Id1, are essential regulators for normal T-cell differentiation and tumor suppression.

E2A and HEB are basic helix-loop-helix transcription factors essential for T cell development. Complete inhibition of their activities through transgenic overexpression of their inhibitors Id1 and Tal1 leads to a dramatic loss of thymocytes. Here, we suggest that bHLH proteins play important roles in establishing thresholds for pre-TCR and TCR signaling. Inhibition of their function allows double-negative cells to differentiate without a functional pre-TCR, while anti-CD3 stimulation downregulates bHLH activities. We also find that the transcription factor NF-kappaB becomes activated in transgenic thymocytes. Further activation of NF-kappaB exacerbates the loss of thymocytes, whereas inhibition of NF-kappaB leads to the rescue of double-positive thymocytes. Therefore, we propose that E2A and HEB negatively regulate pre-TCR and TCR signaling and their removal causes hyperactivation and apoptosis of thymocytes.

Background In industrial fermentation, pH fluctuation resulted from microbial metabolism influences the strain performance and the final production. The common way to control pH is adding acid or alkali after probe detection, which is not a fine-tuned method and often leads to increased costs and complex downstream processing. Here, we constructed an intelligent pH-sensing and controlling genetic circuits called “Genetic pH Shooting (GPS)” to realize microbial self-regulation of pH. Results In order to achieve the self-regulation of pH, GPS circuits consisting of pH-sensing promoters and acid-/alkali-producing genes were designed and constructed. Designed pH-sensing promoters in the GPS can respond to high or low pHs and generate acidic or alkaline substances, achieving endogenously self-responsive pH adjustments. Base shooting circuit (BSC) and acid shooting circuit (ASC) were constructed and enabled better cell growth under alkaline or acidic conditions, respectively. Furthermore, the genetic circuits including GPS, BSC and ASC were applied to lycopene production with a higher yield without an artificial pH regulation compared with the control under pH values ranging from 5.0 to 9.0. In scale-up fermentations, the lycopene titer in the engineered strain harboring GPS was increased by 137.3% and ammonia usage decreased by 35.6%. Conclusions The pH self-regulation achieved through the GPS circuits is helpful to construct intelligent microbial cell factories and reduce the production costs, which would be much useful in industrial applications.

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