Novel mechanism of C/EBPbeta (NF-M) transcriptional control: activation through derepression

Phosphorylation of transcription factors is regarded as a major mechanism to control their activity in regulation of gene expression. C/EBP beta is a transcription factor that becomes activated after phosphorylation to induce genes involved in inflammation, acute-phase response, cytokine expression, cell growth, and differentiation. The chicken homolog NF-M collaborates with Myb and various kinase oncogenes in normal myeloid differentiation as well as in the leukemic transformation of myelomonocytic cells. Here, we examined the structure of NF-M and its mechanism of activation. We show that NF-M is a repressed transcription factor with concealed activation potential. Derepressed NF-M exhibits enhanced transcriptional efficacy in reporter assays. More importantly, NF-M activates resident chromatin-embedded, myelomonocyte-specific target genes, even in heterologous cell types such as fibroblasts or erythroblasts. We identified two regions within NF-M that act to repress trans-activation. Repression is abolished by deletion of these regions, activation of signal transduction kinases including v-erbB, polyoma middle T, ras and mil/raf, or point mutation of a critical phosphorylation site for MAP kinases. We provide evidence that phosphorylation plays a unique role to derepress rather than to enhance the trans-activation domain as a novel mechanism to regulate gene expression by NF-M/C/EBP beta

Crosstalk between C/EBPbeta phosphorylation, arginine methylation, and SWI/SNF/Mediator implies an indexing transcription factor code

Cellular signalling cascades regulate the activity of transcription factors that convert extracellular information into gene regulation. C/EBPbeta is a ras/MAPkinase signal-sensitive transcription factor that regulates genes involved in metabolism, proliferation, differentiation, immunity, senescence, and tumourigenesis. The protein arginine methyltransferase 4 PRMT4/CARM1 interacts with C/EBPbeta and dimethylates a conserved arginine residue (R3) in the C/EBPbeta N-terminal transactivation domain, as identified by mass spectrometry of cell-derived C/EBPbeta. Phosphorylation of the C/EBPbeta regulatory domain by ras/MAPkinase signalling abrogates the interaction between C/EBPbeta and PRMT4/CARM1. Differential proteomic screening, protein interaction studies, and mutational analysis revealed that methylation of R3 constraines interaction with SWI/SNF and Mediator complexes. Mutation of the R3 methylation site alters endogenous myeloid gene expression and adipogenic differentiation. Thus, phosphorylation of the transcription factor C/EBPbeta couples ras signalling to arginine methylation and regulates the interaction of C/EBPbeta with epigenetic gene regulatory protein complexes during cell differentiation

Metabolism of 5'-nucleotide monosaccharides in inflammatory connective tissue

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33223/1/0000613.pd

C/EBPβ regulates homeostatic and oncogenic gastric cell proliferation

Cancer of the stomach is among the leading causes of death from cancer worldwide. The transcription factor C/EBPβ is frequently overexpressed in gastric cancer and associated with the suppression of the differentiation marker TFF1. We show that the murine C/EBP{beta} knockout stomach displays unbalanced homeostasis and reduced cell proliferation and that tumorigenesis of human gastric cancer xenograft is inhibited by knockdown of C/EBPβ. Cross-species comparison of gene expression profiles between C/EBPβ-deficient murine stomach and human gastric cancer revealed a subset of tumors with a C/EBPβ signature. Within this signature, the RUNX1t1 tumor suppressor transcript was down-regulated in 38% of gastric tumor samples. The RUNX1t1 promoter was frequently hypermethylated and ectopic expression of RUNX1t1 in gastric cancer cells inhibited proliferation and enhanced TFF1 expression. These data suggest that the tumor suppressor activity of both RUNX1t1 and TFF1 are mechanistically connected to C/EBPβ and that cross-regulation between C/EBPβ-RUNX1t1-TFF1 plays an important role in gastric carcinogenesis

Early patterning of the chorion leads to the trilaminar trophoblast cell structure in the placental labyrinth

The labyrinth of the rodent placenta contains villi that are the site of nutrient exchange between mother and fetus. They are covered by three trophoblast cell types that separate the maternal blood sinusoids from fetal capillaries - a single mononuclear cell that is a subtype of trophoblast giant cell (sinusoidal or S-TGC) with endocrine function and two multinucleated syncytiotrophoblast layers, each resulting from cell-cell fusion, that function in nutrient transport. The developmental origins of these cell types have not previously been elucidated. We report here the discovery of cell-layer-restricted genes in the mid-gestation labyrinth (E12.5-14.5) including Ctsq in S-TGCs (also Hand1-positive), Syna in syncytiotrophoblast layer I (SynT-1), and Gcm1, Cebpa and Synb in syncytiotrophoblast layer II (SynT-II). These genes were also expressed in distinct layers in the chorion as early as E8.5, prior to villous formation. Specifically, Hand1 was expressed in apical cells lining maternal blood spaces (Ctsq is not expressed until E12.5), Syna in a layer immediately below, and Gcm1, Cebpa and Synb in basal cells in contact with the allantois. Cebpa and Synb were co-expressed with Gcm1 and were reduced in Gcm1 mutants. By contrast, Hand1 and Syna expression was unaltered in Gcm1 mutants, suggesting that Gcm1-positive cells are not required for the induction of the other chorion layers. These data indicate thatthe three differentiated trophoblast cell types in the abyrinth arise from distinct and autonomous precursors in the chorion that are patterned before morphogenesis begins

Stable conditional expression and effect of C/EBPβ-LIP in adipocytes using the pSLIK system

Murine 3T3-L1 adipocytes are widely used as a cellular model of obesity. However, whereas transfection of 3T3-L1 preadipocytes is straightforward, ectopic gene expression in mature 3T3-L1 adipocytes has proved challenging. Here, we used the pSLIK vector system to generate stable doxycycline-inducible expression of the liver-enriched inhibitor protein isoform of CCAAT/enhancer binding protein (C/EBP) {beta} (C/EBP{beta}-LIP) in fully differentiated 3T3-L1 adipocytes. Because overexpression of C/EBP{beta}-LIP impairs adipocyte differentiation, the C/EBP{beta}-LIP construct was first integrated in 3T3-L1 preadipocytes but expression was induced only when adipocytes were fully differentiated. Increased C/EBP{beta}-LIP in mature adipocytes down-regulated C/EBP{beta} target genes including 11{beta}-hydroxysteroid dehydrogenase type 1, phosphoenolpyruvate carboxykinase and fatty acid binding protein 4, but had no effect on asparagine synthetase, demonstrating that transcriptional down-regulation by C/EBP{beta}-LIP in 3T3-L1 adipocytes is not a general effect. Importantly, these genes were modulated in a similar manner in adipose tissue of mice with genetically increased C/EBP{beta}-LIP levels. The use of the pSLIK system to conditionally express transgenes in 3T3-L1 cells could be a valuable tool to dissect adipocyte physiology

Distinct Mechanisms for Induction and Tolerance Regulate the Immediate Early Genes Encoding Interleukin 1β and Tumor Necrosis Factor α

Interleukin-1β and Tumor Necrosis Factor α play related, but distinct, roles in immunity and disease. Our study revealed major mechanistic distinctions in the Toll-like receptor (TLR) signaling-dependent induction for the rapidly expressed genes (IL1B and TNF) coding for these two cytokines. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), hallmarks of poised immediate-early (IE) genes. In contrast, unstimulated IL1B displayed very low levels of both TBP and paused Pol II, requiring the lineage-specific Spi-1/PU.1 (Spi1) transcription factor as an anchor for induction-dependent interaction with two TLR-activated transcription factors, C/EBPβ and NF-κB. Activation and DNA binding of these two pre-expressed factors resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of elongation factor P-TEFb. This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Furthermore, the dependence on phosphoinositide 3-kinase for P-TEFb recruitment to IL1B paralleled a greater sensitivity to the metabolic state of the cell and a lower sensitivity to the phenomenon of endotoxin tolerance than was evident for TNF. Such differences in induction mechanisms argue against the prevailing paradigm that all IE genes possess paused Pol II and may further delineate the specific roles played by each of these rapidly expressed immune modulators. © 2013 Adamik et al