Autophagy is required for necrotic cell death in Caenorhabditis elegans.

Autophagy is the main process for bulk protein and organelle recycling in cells under extracellular or intracellular stress. Deregulation of autophagy has been associated with pathological conditions such as cancer, muscular disorders and neurodegeneration. Necrotic cell death underlies extensive neuronal loss in acute neurodegenerative episodes such as ischemic stroke. We find that excessive autophagosome formation is induced early during necrotic cell death in C. elegans. In addition, autophagy is required for necrotic cell death. Impairment of autophagy by genetic inactivation of autophagy genes or by pharmacological treatment suppresses necrosis. Autophagy synergizes with lysosomal catabolic mechanisms to facilitate cell death. Our findings demonstrate that autophagy contributes to cellular destruction during necrosis. Thus, interfering with the autophagic process may protect neurons against necrotic damage in humans.

The Gcn5 bromodomain co-ordinates nucleosome remodelling.

The access of transcription factors to eukaryotic promoters often requires modification of their chromatin structure, which is accomplished by the action of two general classes of multiprotein complexes. One class contains histone acetyltransferases (HATs), such as Gcn5 in the SAGA complex, which acetylate nucleosomal histones. The second class contains ATPases, such as Swi2 in the Swi/Snf complex, which provide the energy for nucleosome remodelling. In several promoters these two complexes cooperate but their functional linkage is unknown. A protein module that is present in all nuclear HATs, the bromodomain, could provide such a link. The recently reported in vitro binding of a HAT bromodomain with acetylated lysines within H3 and H4 amino-terminal peptides indicates that this interaction may constitute a targeting step for events that follow histone acetylation. Here we use a suitable promoter to show that bromodomain residues essential for acetyl-lysine binding are not required in vivo for Gcn5-mediated histone acetylation but are fundamental for the subsequent Swi2-dependent nucleosome remodelling and consequent transcriptional activation. We show that the Gcn5 bromodomain stabilizes the Swi/Snf complex on this promoter.

The Gcn5.Ada complex potentiates the histone acetyltransferase activity of Gcn5.

The Gcn5 histone acetyltransferase (HAT) is part of a large multimeric complex that is required for transcriptional activation in yeast. This complex can acetylate in vitro and in a Gcn5-dependent manner both nucleosomal and free core histones. For this reason it is believed that part of the function of the Gcn5.Ada complex is chromatin remodeling effected by histone acetylation. The roles of the other subunits of this complex are not yet known. We have generated mutated Gcn5 proteins with severely attenuated in vitro HAT activities. Despite their apparent loss in HAT activity, these GCN5 derivatives complemented all the defects of a gcn5 strain. We have shown that when these mutated proteins were produced in yeast cells in the absence of another component of the complex, Ada2, their activity was still compromised. By contrast, when produced in the wild type context, they were partially capable of acetylating free histones and were even more active when nucleosomal arrays were used as substrates. Kinetic enzymatic analyses showed that the rate of catalysis by Gcn5 was enhanced when the mutated proteins were produced in yeast in the presence of Ada2. Because Ada2 is required for the assembly of Gcn5, we conclude that one role for components of the Gcn5.Ada complex is the potentiation of its HAT activity.