The activity of the immunoglobulin heavy-chain (IgH) enhancer is restricted to B cells, although it binds both B-cell-restricted and ubiquitous transcription factors. Activation of the enhancer in non-B cells upon overexpression of the basic helix-loop-helix (bHLH) protein E2A appears to be mediated not only by the binding of E2A to its cognate E box but also by the resulting displacement of a repressor from that same site.We have identified a "two-handed" zinc finger protein, denoted ZEB, the DNA-binding specificity of which mimics that of the cellular repressor. By employing a derivative E box that binds ZEB but not E2A, we have shown that the repressor is active in B cells and the IgH enhancer is silenced in the absence of binding competition by bHLH proteins. Hence, we propose that a necessary prerequisite of enhancer activity is the B-cell-specific displacement of a ZEB-like repressor by bHLH proteins.
MyoD is a muscle-specific transcriptional activator; £12 is a B-cell activator. An IgH enhancer is activated almost 100-fold by E12 but not at all by MyoD; an MCK enhancer is activated almost 1000-fold by MyoD and not at all by £12. MyoD and £12 are both basic helix-loop-helix proteins that bind to similar E-box sequences (CANNTG); the IgH enhancer contains the same £ boxes as the MCK enhancer, yet each retains exclusive specificity for either £12 or MyoD, respectively. We show that the IgH enhancer contains a cis-acting negative element that is directed at MyoD, but not at £12. This repression requires the jjiES £ box within the IgH enhancer; however, the specificity for repression, as opposed to activation, is associated with 2 bp flanking each side of the (JLE5 E box. The target for repression of MyoD in the IgH enhancer is the bHLH region of MyoD. Our results suggest that MyoD only activates myogenic genes because nonmuscle enhancers that contain £ boxes also contain negative elements that prevent MyoD activity.
Autophagy may contribute to ischemia-induced cell death in the brain, but the regulation of autophagic cell death is largely unknown. Nuclear factor kappa B (NF-κB) is a regulator of apoptosis in cerebral ischemia. We examined the hypothesis that autophagy-like cell death could contribute to ischemia-induced brain damage and the process was regulated by NF-κB. In adult wild-type (WT) and NF-κB p50 knockout (p50−/−) mice, focal ischemia in the barrel cortex was induced by ligation of distal branches of the middle cerebral artery. Twelve to 24 h later, autophagic activity increased as indicated by enhanced expression of Beclin-1 and LC3 in the ischemic core and/or penumbra regions. This increased autophagy contributed to cell injury, evidenced by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) co-staining and a protective effect achieved by the autophagy inhibitor 3-methyladenine. The number of Beclin-1/TUNEL-positive cells was significantly more in p50−/− mice than in WT mice. Neuronal and vascular cell death, as determined by TUNEL-positive cells co-staining with NeuN or Collagen IV, was more abundant in p50−/− mice. Immunostaining of the endothelial cell tight junction marker occludin revealed more damage to the blood–brain barrier in p50−/− mice. Western blotting of the peri-infarct tissue showed a reduction of Akt-the mammalian target of rapamycin (mTOR) signaling in p50−/− mice after ischemia. These findings provide the first evidence that cerebral ischemia induced autophagy-like injury is regulated by the NF-κB pathway, which may suggest potential treatments for ischemic stroke.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.