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.

No abstract

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.

No abstract

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.