No abstract

CCAAT/enhancer-binding protein ␣ (C/EBP␣) is one of the key transcription factors that mediate lineage specification and differentiation of multipotent myeloid progenitors into mature granulocytes. Although C/EBP␣ is known to induce granulopoiesis while suppressing monocyte differentiation, it is unclear how C/EBP␣ regulates this cell fate choice at the mechanistic level. Here we report that inducers of monocyte differentiation inhibit the alternate cell fate choice, that of granulopoiesis, through inhibition of C/EBP␣. This inhibition is mediated by extracellular signal-regulated kinases 1 and/or 2 (ERK1/2), which interact with C/EBP␣ through an FXFP docking site and phosphorylate serine 21. As a consequence of C/EBP␣ phosphorylation, induction of granulocyte differentiation by C/EBP␣ or retinoic acid is inhibited. Our analysis of C/EBP␣ by fluorescent resonance energy transfer revealed that phosphorylation induces conformational changes in C/EBP␣, increasing the distance between the amino termini of C/EBP␣ dimers. Thus, myeloid development is partly regulated by an ERK1/2-mediated change in the conformation of C/EBP␣ that favors monocyte differentiation by blocking granulopoiesis.

Higher eukaryote genomes contain repetitive DNAs, often concentrated in transcriptionally inactive heterochromatin. Although repetitive DNAs are not typically considered as regulatory elements that directly affect transcription, they can contain binding sites for some transcription factors. Here, we demonstrate that binding of the transcription factor CCAAT/ enhancer-binding protein ␣ (C/EBP␣) to the mouse major ␣-satellite repetitive DNA sequesters C/EBP␣ in the transcriptionally inert pericentromeric heterochromatin. We find that this sequestration reduces the transcriptional capacity of C/EBP␣. Functional sequestration of C/EBP␣ was demonstrated by experimentally reducing C/EBP␣ binding to the major ␣-satellite DNA, which elevated the concentration of C/EBP␣ in the non-heterochromatic subcompartment of the cell nucleus. The reduction in C/EBP␣ binding to ␣-satellite DNA was induced by the co-expression of the transcription factor Pit-1, which removes C/EBP␣ from the heterochromatic compartment, and by the introduction of an altered-specificity mutation into C/EBP␣ that reduces binding to ␣-satellite DNA but permits normal binding to sites in some gene promoters. In both cases the loss of ␣-satellite DNA binding coincided with an elevation in the binding of C/EBP␣ to a promoter and an increased transcriptional output from that promoter. Thus, the binding of C/EBP␣ to this highly repetitive DNA reduced the amount of C/EBP␣ available for binding to and regulation of this promoter. The functional sequestration of some transcription factors through binding to repetitive DNAs may represent an underappreciated mechanism controlling transcription output.

The established in vitro tool used for testing the absorption and penetration of chemicals through skin in pharmacology, toxicology and cosmetic science is the static Franz diffusion cell. While widespread, Franz cells are relatively costly, low-throughput and results may suffer from poor reproducibility. Microfluidics has the potential to overcome these drawbacks. In this paper, we present a novel microfluidic skin permeation platform and validate it rigorously against the Franz cell by comparing the transport of 3 model chemicals of varying lipophilicity: caffeine, salicylic acid and testosterone. Permeation experiments through silicone membranes show that the chip yields higher sensitivity in permeant cumulative amounts and comparable or lower coefficients of variation. Using a skin organotypic culture, we show that the chip decreases the effect of unstirred water layers that can occur in static Franz cells. The validation reported herein sets the stage for efficient skin permeation and toxicity screening and further development of microfluidic skin-on-chip devices.