Hormone-dependent, CARM1-directed, arginine-specific methylation of histone H3 on a steroid-regulated promoter

AbstractActivation of gene transcription involves chromatin remodeling by coactivator proteins that are recruited by DNA-bound transcription factors. Local modification of chromatin structure at specific gene promoters by ATP-dependent processes and by posttranslational modifications of histone N-terminal tails provides access to RNA polymerase II and its accompanying transcription initiation complex [1, 2]. While the roles of lysine acetylation, serine phosphorylation, and lysine methylation of histones in chromatin remodeling are beginning to emerge [2–5], low levels of arginine methylation of histones have only recently been documented [4, 6–9], and its physiological role is unknown. The coactivator CARM1 methylates histone H3 at Arg17 and Arg26 in vitro [7] and cooperates synergistically with p160-type coactivators (e.g., GRIP1, SRC-1, ACTR) and coactivators with histone acetyltransferase activity (e.g., p300, CBP) to enhance gene activation by steroid and nuclear hormone receptors (NR) in transient transfection assays [10, 11]. In the current study, CARM1 cooperated with GRIP1 to enhance steroid hormone-dependent activation of stably integrated mouse mammary tumor virus (MMTV) promoters, and this coactivator function required the methyltransferase activity of CARM1. Chromatin immunoprecipitation assays and immunofluorescence studies indicated that CARM1 and the CARM1-methylated form of histone H3 specifically associated with a large tandem array of MMTV promoters in a hormone-dependent manner. Thus, arginine-specific histone methylation by CARM1 is an important part of the transcriptional activation process

Isoaspartylation appears to trigger small cell lung cancer-associated autoimmunity against neuronal protein ELAVL4

Autoantibodies against SCLC-associated neuronal antigen ELAVL4 (HuD) have been linked to smaller tumors and improved survival, but the antigenic epitope and mechanism of autoimmunity have never been solved. We report that recombinant human ELAVL4 protein incubated under physiological conditions acquires isoaspartylation, a type of immunogenic protein damage. Specifically, the N-terminal region of ELAVL4, previously implicated in SCLC-associated autoimmunity, undergoes isoaspartylation in vitro, is recognized by sera from anti-ELAVL4 positive SCLC patients and is highly immunogenic in subcutaneously injected mice and in vitro stimulated human lymphocytes. Our data suggest that isoaspartylated ELAVL4 is the trigger for the SCLC-associated anti-ELAVL4 autoimmune response

Isoaspartyl formation in creatine kinase B is associated with loss of enzymatic activity; implications for the linkage of isoaspartate accumulation and neurological dysfunction in the PIMT knockout mouse.

Isoaspartate (isoAsp) formation is a common type of spontaneous protein damage that is normally kept in check by the repair enzyme protein-L-isoaspartyl methyltransferase (PIMT). PIMT-KO (knockout) mice exhibit a pronounced neuropathology highlighted by death from an epileptic seizure at 30 to 60 days after birth. The mechanisms by which isoaspartyl damage disrupts normal brain function are incompletely understood. Proteomic analysis of the PIMT-KO mouse brain has shown that a number of key neuronal proteins accumulate high levels of isoAsp, but the extent to which their cellular functions is altered has yet to be determined. One of the major neuronal targets of PIMT is creatine kinase B (CKB), a well-characterized enzyme whose activity is relatively easy to assay. We show here that (1) the specific activity of CKB is significantly reduced in the brains of PIMT-deficient mice, (2) that in vitro aging of recombinant CKB results in significant accumulation of isoAsp sites with concomitant loss of enzymatic activity, and (3) that incubation of in vitro aged CKB with PIMT and its methyl donor S-adenosyl-L-methionine substantially repairs the aged CKB with regard to both its isoAsp content and its enzymatic activity. These results, combined with similarity in phenotypes of PIMT-KO and CKB-KO mice, suggests that loss of normal CKB structure and function contributes to the mechanisms by which isoAsp accumulation leads to CNS dysfunction in the PIMT-KO mouse

The d-isoAsp-25 variant of histone H2B is highly enriched in active chromatin: potential role in the regulation of gene expression?

Approximately 12 % of histone H2B in mammalian brain contains an unusual D-aspartate residue in its N-terminal tail. Most of this D-aspartate is linked to the C-flanking glycine via an isopeptide bond. To explore the possible significance of these modifications, we generated an antibody to the D-isoaspartyl form of H2B, and used it to assess its levels in H2B associated with "active" vs. "silent" chromatin. We found that the D-isoaspartyl form of H2B appears to be highly enriched in the former. This irreversible modification could serve a novel regulatory function in gene expression

Characterization of a polyclonal antibody that is highly selective for the d-isoAsp-25 variant of mammalian histone H2B

Approximately 12% of histone H2B molecules in mammalian brain contain a modification wherein Asp25 is present as the D-enantiomer, and is mostly linked to Gly26 via the side-chain carboxyl. Here we (1) demonstrate the high specificity of a polyclonal antibody to this modification, and (2) use this Ab to demonstrate that this modification is enriched in brain relative to liver, thymus, and HeLa cells

Isoaspartyl Protein Damage and Repair in Mouse RetinaIsoaspartyl Protein Damage and Repair in Mouse Retina

PurposeTo determine the propensity of retinal proteins for spontaneous damage via formation of isoaspartyl sites, a common type of protein damage that could contribute to retinal disease.MethodsTissue extracts were obtained from retinas and brains of control mice and from mice in which the gene for protein L-isoaspartate O-methyltransferase (PIMT; an enzyme that repairs isoaspartyl protein damage) was knocked out. PIMT expression in these extracts was measured by Western blot, and its specific activity was assayed by monitoring the rate of [(3)H]methyl transfer from S-adenosyl-[methyl-(3)H]L-methionine to γ-globulin. Isoaspartate levels in extracts were measured by their capacity to accept [(3)H]methyl groups via the PIMT-catalyzed methylation reaction. To compare molecular weight distributions of isoaspartyl-rich proteins in retina versus brain, proteins from PIMT knockout (KO) and control mice were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF). Isoaspartyl proteins were (3)H-labeled on-blot using a PIMT overlay and imaged by autoradiography.ResultsWhen normalized to the β-actin content of each tissue, retina was found to be nearly identical to brain with regard to expression and activity of PIMT and its propensity to accumulate isoaspartyl sites when PIMT is absent. The two tissues show distinct differences in the molecular weight distribution of isoaspartyl proteins.ConclusionsThe retina is rich in PIMT activity and contains a wide range of proteins that are highly susceptible to this type of protein damage. Recoverin may be one such protein. Isoaspartate formation, along with oxidation, should be considered as a potential source of protein dysfunction and autoimmunity in retinal disease

Sequence alignment of chicken (CHK); mouse (MUS) and human (HUM) CKB.

<p>Six canonical hotspots for isoAsp formation are indicated with carets (∧). Color-coding indicates regions of β-sheet (blue), α-helix (magenta), and turns (yellow) as deduced from crystal structure data according to the ExPASY (<a href="http://www.expasy.org" target="_blank">www.expasy.org</a>) alignment application. Mouse is not color-coded because there is no crystal structure for mouse CKB. The dashed box encloses the acidic EED triplet essential for enzymatic activity.</p

Determination of CKB specific activity in brain extracts from 4-week mice.

<p>CKB activity and protein concentrations were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100622#s2" target="_blank">Materials and Methods</a>. Data are expressed as means ± S.D (n = 5 for each genotype). **P<0.01.</p