The nucleosomal response refers to the rapid phosphorylation of histone H3 on serine 10 and HMG-14 on serine 6 that occurs concomitantly with immediateearly (IE) gene induction in response to a wide variety of stimuli. Using antibodies against the phosphorylated residues, we show that H3 and HMG-14 phosphorylation is mediated via different MAP kinase (MAPK) cascades, depending on the stimulus. The nucleosomal response elicited by TPA is ERK-dependent, whereas that elicited by anisomycin is p38 MAPK-dependent. In intact cells, the nucleosomal response can be selectively inhibited using the protein kinase inhibitor H89. MAPK activation and phosphorylation of transcription factors are largely unaffected by H89, whereas induction of IE genes is inhibited and its characteristics markedly altered. MSK1 is considered the most likely kinase to mediate this response because (i) it is activated by both ERK and p38 MAPKs; (ii) it is an extremely efficient kinase for HMG-14 and H3, utilizing the physiologically relevant sites; and (iii) its activity towards H3/HMG-14 is uniquely sensitive to H89 inhibition. Thus, the nucleosomal response is an invariable consequence of ERK and p38 but not JNK/SAPK activation, and MSK1 potentially provides a link to complete the circuit between cell surface and nucleosome.

The induction of immediate‐early (IE) genes, including proto‐oncogenes c‐fos and c‐jun, correlates well with a nucleosomal response, the phosphorylation of histone H3 and HMG‐14 mediated via extracellular signal regulated kinase or p38 MAP kinase cascades. Phosphorylation is targeted to a minute fraction of histone H3, which is also especially susceptible to hyperacetylation. Here, we provide direct evidence that phosphorylation and acetylation of histone H3 occur on the same histone H3 tail on nucleosomes associated with active IE gene chromatin. Chromatin immunoprecipitation (ChIP) assays were performed using antibodies that specifically recognize the doubly‐modified phosphoacetylated form of histone H3. Analysis of the associated DNA shows that histone H3 on c‐fos‐ and c‐jun‐associated nucleosomes becomes doubly‐modified, the same H3 tails becoming both phosphorylated and acetylated, only upon gene activation. This study reveals potential complications of occlusion when using site‐specific antibodies against modified histones, and shows also that phosphorylated H3 is more sensitive to trichostatin A (TSA)‐induced hyperacetylation than non‐phosphorylated H3. Because MAP kinase‐mediated gene induction is implicated in controlling diverse biological processes, histone H3 phosphoacetylation is likely to be of widespread significance.

Acetylated histones are generally associated with active chromatin. The bromodomain has recently been identified as a protein module capable of binding to acetylated lysine residues, and hence is able to mediate the recruitment of factors to acetylated chromatin. Functional studies of bromodomain-containing proteins indicate how this domain contributes to the activity of a number of nuclear factors including histone acetyltransferases and chromatin remodelling complexes. Here, we review the characteristics of acetyllysine-binding by bromodomains, discuss associated domains found in these proteins, and address the function of the bromodomain in the context of chromatin. Finally, the modulation of bromodomain binding by neighbouring post-translational modifications within histone tails might provide a mechanism through which combinations of covalent marks could exert control on chromatin function.

Perturbation of the normal functioning of the endoplasmic reticulum (ER) increases the expression of lumenal proteins, such as grp78, and calreticulin. These proteins are retained within the compartment by a salvage mechanism involving the recognition of a C-terminal tetra-peptide sequence by the KDEL receptor. We have investigated whether disrupting normal ER function concomitantly increases the expression of the mRNAs encoding the two mammalian isoforms of the receptor, erd2.1 and erd2.2. Inhibition of N-linked glycosylation of proteins by tunicamycin had no effect upon the levels of the single mRNA species recognized by the erd2.1 probe, or the multiple transcripts detected with the erd2.2 cDNA probe. ER Ca2+ store depletion by thapsigargin did not increase erd2.1 mRNA, but actually caused a decrease in erd2.2 mRNA. Both thapsigargin, and tunicamycin, increased calreticulin secretion from the cells, although this might be due to more than simply saturation of KDEL receptor binding.