Mutations in the CEBPA gene are present in 7%-10% of human patients with acute myeloid leukemia (AML). However, no genetic models exist that demonstrate their etiological relevance. To mimic the most common mutations affecting CEBPA-that is, those leading to loss of the 42 kDa C/EBPalpha isoform (p42) while retaining the 30kDa isoform (p30)-we modified the mouse Cebpa locus to express only p30. p30 supported the formation of granulocyte-macrophage progenitors. However, p42 was required for control of myeloid progenitor proliferation, and p42-deficient mice developed AML with complete penetrance. p42-deficient leukemia could be transferred by a Mac1+c-Kit+ population that gave rise only to myeloid cells in recipient mice. Expression profiling of this population against normal Mac1+c-Kit+ progenitors revealed a signature shared with MLL-AF9-transformed AML.

The transcriptional regulators that couple interfollicular basal keratinocyte proliferation arrest to commitment and differentiation are yet to be identified. Here we report that the basic region leucine zipper transcription factors C/EBPalpha and C/EBPbeta are co-expressed in basal keratinocytes, and are coordinately upregulated as keratinocytes exit the basal layer and undergo terminal differentiation. Mice lacking both C/EBPalpha and beta in the epidermis showed increased proliferation of basal keratinocytes and impaired commitment to differentiation. This led to ectopic expression of keratin 14 (K14) and DeltaNp63 in suprabasal cells, decreased expression of spinous and granular layer proteins, parakeratosis and defective epidermal water barrier function. Knock-in mutagenesis revealed that C/EBP-E2F interaction was required for control of interfollicular epidermis (IFE) keratinocyte proliferation, but not for induction of spinous and granular layer markers, whereas C/EBP DNA binding was required for DeltaNp63 downregulation and K1/K10 induction. Finally, loss of C/EBPalpha/beta induced stem cell gene expression signatures in the epidermis. C/EBPs, therefore, couple basal keratinocyte cell cycle exit to commitment to differentiation through E2F repression and DNA binding, respectively, and may act to restrict the epidermal stem cell compartment.

T cells detect with their T cell antigen receptors (TCRs) the presence of rare agonist peptide/MHC complexes (pMHCs) on the surface of antigen-presenting cells (APCs). How extracellular ligand binding triggers intracellular signaling is poorly understood, yet spatial antigen arrangement on the APC surface has been suggested to be a critical factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering. When targeting TCRs via stably binding monovalent antibody fragments, we found the minimum signaling unit promoting efficient T cell activation to consist of two antibody-ligated TCRs within a distance of 20 nm. In contrast, transiently engaging antigenic pMHCs stimulated T cells robustly as well-isolated entities. These results identify pairs of antibody-bound TCRs as minimal receptor entities for effective TCR triggering yet validate the exceptional stimulatory potency of single isolated pMHC molecules.

The C/EBPa transcription factor regulates hepatic nitrogen, glucose, lipid and iron metabolism. However, how it is able to independently control these processes is not known. Here, we use mouse knock-in mutagenesis to identify C/EBPa domains that specifically regulate hepatic gluconeogenesis and lipogenesis. In vivo deletion of a proline-histidine rich domain (PHR), dephosphorylated at S193 by insulin signaling, dysregulated genes involved in the generation of acetyl-CoA and NADPH for triglyceride synthesis and led to increased hepatic lipogenesis. These promoters bound SREBP-1 as well as C/EBPa, and the PHR was required for C/EBPa-SREBP transcriptional synergy. In contrast, the highly conserved C/EBPa CR4 domain was found to undergo liver-specific dephosphorylation of residues T222 and T226 upon fasting, and alanine mutation of these residues upregulated the hepatic expression of the gluconeogenic G6Pase and PEPCK mRNAs, but not PGC-1a, leading to glucose intolerance. Our results show that pathway-specific metabolic regulation can be achieved through a single transcription factor containing context-sensitive regulatory domains, and indicate C/EBPa phosphorylation as a PGC-1a-independent mechanism for regulating hepatic gluconeogenesis. IntroductionThe liver is central to metabolic control, and processes, stores and releases glucose, nitrogen metabolites and lipids. It is also central to detoxification and participates in iron metabolism. Adjusting the production and consumption of metabolites to the influx provided by nutrition requires coordinated regulation of groups of genes (regulons) involved in a given metabolic pathway. Thus, sugar intake leads to the suppression of hepatic glucose production and initiation of energy storage in glycogen and lipids. This process is controlled by pancreatic insulin release, and involves the transcriptional suppression of hepatic enzymes rate-limiting for gluconeogenesis (phosphoenol pyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pc)) through insulin response elements (IREs) in their promoters (O'Brien et al, 2001). This pathway can be activated during fasting by cAMP/CREB-mediated induction of PGC-1a, which acts as a coactivator for Foxo1 to upregulate a gluconeogenic regulon consisting of PEPCK, G6Pc and fructose-1,6-bisphosphatase (FBP-1) . Similarly, lipogenesis is regulated by dedicated transcription factors, the sterol regulatory element binding proteins (SREBPs), which coordinately regulate a large group of genes required for lipid biosynthesis through their cognate binding sites (SREs). SREBP-2 primarily activates cholesterol biosynthetic genes, whereas SREBP-1 preferentially upregulates genes involved in fatty acid production (Horton et al, 2003). SREBPs are activated by proteolysis in the endoplasmatic reticulum, a process that is inhibited by sterols, providing negative feedback regulation. In addition, SREBP-1 is transcriptionally induced by insulin signaling, and repressed by the glucagon/cAMP pathway, providing a means to control energy storage i...

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