Cells respond to a wide variety of stresses through the transcriptional activation of genes that harbour stress elements within their promoters. While many of these elements are shared by genes encoding proteins representative of all subcellular compartments, cells can also respond to stresses that are speci®c to individual organelles, such as the endoplasmic reticulum unfolded protein response. Here we report on the discovery and characterization of a mitochondrial stress response in mammalian cells. We ®nd that the accumulation of unfolded protein within the mitochondrial matrix results in the transcriptional upregulation of nuclear genes encoding mitochondrial stress proteins such as chaperonin 60, chaperonin 10, mtDnaJ and ClpP, but not those encoding stress proteins of the endoplasmic reticulum. Analysis of the chaperonin 60/ 10 bidirectional promoter identi®ed a CHOP element as the mitochondrial stress response element. Dominant-negative mutant forms of CHOP and overexpression of CHOP revealed that this transcription factor, in association with C/EBPb, regulates expression of mitochondrial stress genes in response to the accumulation of unfolded proteins.

Hypoxia-inducible factor-1α (HIF-1α) 1 is a global transcriptional regulator of the hypoxic response. Under normoxic conditions, HIF-1α is recognized by the von Hippel-Lindau tumor-suppressor protein (VHL), a component of an E3 ubiquitin ligase complex. This interaction thereby promotes the rapid degradation of HIF-1α. Under hypoxic conditions, HIF-1α is stabilized. We have previously shown that VHL binds in a hypoxia-sensitive manner to a 27-aa segment of HIF-1α, and that this regulation depends on a posttranslational modification of HIF-1α. Through a combination of in vivo coimmunoprecipitation assays using VHL and a panel of point mutants of HIF-1α in this region, as well as MS and in vitro binding assays, we now provide evidence that this modification, which occurs under normoxic conditions, is hydroxylation of Pro-564 of HIF-1α. The data furthermore show that this proline hydroxylation is the primary regulator of VHL binding.

Mammalian gene silencing is established through methylation of histones and DNA, although the order in which these modifications occur remains contentious. Using the human β-globin locus as a model, we demonstrate that symmetric methylation of histone H4 arginine 3 (H4R3me2s) by the protein arginine methyltransferase PRMT5 is required for subsequent DNA methylation. H4R3me2s serves as a direct binding target for the DNA methyltransferase DNMT3A, which interacts through the ADD domain containing the PHD motif. Loss of the H4R3me2s mark through short hairpin RNA–mediated knockdown of PRMT5 leads to reduced DNMT3A binding, loss of DNA methylation and gene activation. In primary erythroid progenitors from adult bone marrow, H4R3me2s marks the inactive methylated globin genes coincident with localization of PRMT5. Our findings define DNMT3A as both a reader and a writer of repressive epigenetic marks, thereby directly linking histone and DNA methylation in gene silencing.