Uta-Maria Bauer

Rb targets histone H3 methylation and HP1 to promoters

In eukaryotic cells the histone methylase SUV39H1 and the methyl-lysine binding protein HP1 functionally interact to repress transcription at heterochromatic sites. Lysine 9 of histone H3 is methylated by SUV39H1 (ref. 2), creating a binding site for the chromo domain of HP1 (refs 3, 4). Here we show that SUV39H1 and HP1 are both involved in the repressive functions of the retinoblastoma (Rb) protein. Rb associates with SUV39H1 and HP1 in vivo by means of its pocket domain. SUV39H1 cooperates with Rb to repress the cyclin E promoter, and in fibroblasts that are disrupted for SUV39, the activity of the cyclin E and cyclin A2 genes are specifically elevated. Chromatin immunoprecipitations show that Rb is necessary to direct methylation of histone H3, and is necessary for binding of HP1 to the cyclin E promoter. These results indicate that the SUV39H1–HP1 complex is not only involved in heterochromatic silencing but also has a role in repression of euchromatic genes by Rb and perhaps other co-repressor proteins.

Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence

The yeast Sir2 protein mediates chromatin silencing through an intrinsic NAD‐dependent histone deacetylase activity. Sir2 is a conserved protein and was recently shown to regulate lifespan extension both in budding yeast and worms. Here, we show that SIRT1, the human Sir2 homolog, is recruited to the promyelocytic leukemia protein (PML) nuclear bodies of mammalian cells upon overexpression of either PML or oncogenic Ras (Ha‐rasV12). SIRT1 binds and deacetylates p53, a component of PML nuclear bodies, and it can repress p53‐mediated transactivation. Moreover, we show that SIRT1 and p53 co‐localize in nuclear bodies upon PML upregulation. When overexpressed in primary mouse embryo fibroblasts (MEFs), SIRT1 antagonizes PML‐induced acetylation of p53 and rescues PML‐mediated premature cellular senescence. Taken together, our data establish the SIRT1 deacetylase as a novel negative regulator of p53 function capable of modulating cellular senescence.

Regulation of E2F1 activity by acetylation.

During the G1 phase of the cell cycle, an E2F–RB complex represses transcription, via the recruitment of histone deacetylase activity. Phosphorylation of RB at the G1/S boundary generates a pool of ‘free’ E2F, which then stimulates transcription of S‐phase genes. Given that E2F1 activity is stimulated by p300/CBP acetylase and repressed by an RB‐associated deacetylase, we asked if E2F1 was subject to modification by acetylation. We show that the p300/CBP‐associated factor P/CAF, and to a lesser extent p300/CBP itself, can acetylate E2F1 in vitro and that intracellular E2F1 is acetylated. The acetylation sites lie adjacent to the E2F1 DNA‐binding domain and involve lysine residues highly conserved in E2F1, 2 and 3. Acetylation by P/CAF has three functional consequences on E2F1 activity: increased DNA‐binding ability, activation potential and protein half‐life. These results suggest that acetylation stimulates the functions of the non‐RB bound ‘free’ form of E2F1. Consistent with this, we find that the RB‐associated histone deacetylase can deacetylate E2F1. These results identify acetylation as a novel regulatory modification that stimulates E2F1s activation functions.

Methylation at arginine 17 of histone H3 is linked to gene activation

The nuclear hormone receptor co‐activator CARM1 has the potential to methylate histone H3 at arginine residues in vitro. The methyltransferase activity of CARM1 is necessary for its co‐activator functions in transient transfection assays. However, the role of this methyltransferase in vivo is unclear, given that methylation of arginines is not easily detectable on histones. We have raised an antibody that specifically recognizes methylated arginine 17 (R17) of histone H3, the major site of methylation by CARM1. Using this antibody we show that methylated R17 exists in vivo. Chromatin immunoprecipitation analysis shows that R17 methylation on histone H3 is dramatically upregulated when the estrogen receptor‐regulated pS2 gene is activated. Coincident with the appearance of methylated R17, CARM1 is found associated with the histones on the pS2 gene. Together these results demonstrate that CARM1 is recruited to an active promoter and that CARM1‐mediated R17 methylation on histone H3 takes place in vivo during this active state.

Crosstalk between CARM1 Methylation and CBP Acetylation on Histone H3

BACKGROUND Dynamic changes in the modification pattern of histones, such as acetylation, phosphorylation, methylation, and ubiquitination, are thought to provide a code for the correct regulation of gene expression mostly by affecting chromatin structure and interactions of non-histone regulatory factors with chromatin. Recent studies have suggested the existence of an interplay between histone modifications during transcription. The CBP/p300 acetylase and the CARM1 methyltransferase can positively regulate the expression of estrogen-responsive genes, but the existence of a crosstalk between lysine acetylation and arginine methylation on chromatin has not yet been established in vivo. RESULTS By following the in vivo pattern of modifications on histone H3, following estrogen stimulation of the pS2 promoter, we show that arginine methylation follows prior acetylation of H3. Within 15 min after estrogen stimulation, CBP is bound to chromatin, and acetylation of K18 takes place. Following these events, K23 is acetylated, CARM1 associates with chromatin, and methylation at R17 takes place. Exogenous expression of CBP is sufficient to drive the association of CARM1 with chromatin and methylation of R17 in vivo, whereas an acetylase-deficient CBP mutant is unable to induce these events. A mechanism for the observed cooperation between acetylation and arginine methylation comes from the finding that acetylation at K18 and K23, but not K14, tethers recombinant CARM1 to the H3 tail and allows it to act as a more efficient arginine methyltransferase. CONCLUSION These results reveal an ordered and interdependent deposition of acetylation and arginine methylation during estrogen-regulated transcription and provide support for a combinatorial role of histone modifications in gene expression.

Epigenetic regulation in murine offspring as a novel mechanism for transmaternal asthma protection induced by microbes

BACKGROUND Bronchial asthma is a chronic inflammatory disease resulting from complex gene-environment interactions. Natural microbial exposure has been identified as an important environmental condition that provides asthma protection in a prenatal window of opportunity. Epigenetic regulation is an important mechanism by which environmental factors might interact with genes involved in allergy and asthma development. OBJECTIVE This study was designed to test whether epigenetic mechanisms might contribute to asthma protection conferred by early microbial exposure. METHODS Pregnant maternal mice were exposed to the farm-derived gram-negative bacterium Acinetobacter lwoffii F78. Epigenetic modifications in the offspring were analyzed in T(H)1- and T(H)2-relevant genes of CD4(+) T cells. RESULTS Prenatal administration of A lwoffii F78 prevented the development of an asthmatic phenotype in the progeny, and this effect was IFN-γ dependent. Furthermore, the IFNG promoter of CD4(+) T cells in the offspring revealed a significant protection against loss of histone 4 (H4) acetylation, which was closely associated with IFN-γ expression. Pharmacologic inhibition of H4 acetylation in the offspring abolished the asthma-protective phenotype. Regarding T(H)2-relevant genes only at the IL4 promoter, a decrease could be detected for H4 acetylation but not at the IL5 promoter or the intergenic T(H)2 regulatory region conserved noncoding sequence 1 (CNS1). CONCLUSION These data support the hygiene concept and indicate that microbes operate by means of epigenetic mechanisms. This provides a new mechanism in the understanding of gene-environment interactions in the context of allergy protection.

The protein arginine methyltransferases CARM1 and PRMT1 cooperate in gene regulation

Protein arginine methyltransferases (PRMT) have been implicated in the regulation of transcription. They are recruited to promoters via interaction with transcription factors and exert their coactivator function by methylating arginine residues in histones and other chromatin proteins. Here, we employ an unbiased approach to identify novel target genes, which are under the control of two members of the enzyme family, PRMT1 and CARM1/PRMT4 (coactivator associated arginine methyltransferase 1). By using cDNA microarray analysis, we find that the siRNA-mediated single knockdown of neither CARM1 nor PRMT1 causes significant changes in gene expression. In contrast, double knockdown of both enzymes results in the deregulated expression of a large group of genes, among them the CITED2 gene. Cytokine-stimulated expression analysis indicates that transcriptional activation of CITED2 depends on STAT5 and the coactivation of both PRMTs. ChIP analysis identifies the CITED2 gene as a direct target gene of STAT5, CARM1 and PRMT1. In reporter gene assays, we show that STAT5-mediated transcription is cooperatively enhanced by CARM1 and PRMT1. Interaction assays reveal a cytokine-induced association of STAT5 and the two PRMTs. Our data demonstrate a widespread cooperation of CARM1 and PRMT1 in gene activation as well as repression and that STAT5-dependent transcription of the CITED2 gene is a novel pathway coactivated by the two methyltransferases.

Arginine Methylation Provides Epigenetic Transcription Memory for Retinoid-Induced Differentiation in Myeloid Cells

ABSTRACT Cellular differentiation is governed by changes in gene expression, but at the same time, a cells identity needs to be maintained through multiple cell divisions during maturation. In myeloid cell lines, retinoids induce gene expression and a well-characterized two-step lineage-specific differentiation. To identify mechanisms that contribute to cellular transcriptional memory, we analyzed the epigenetic changes taking place on regulatory regions of tissue transglutaminase, a gene whose expression is tightly linked to retinoid-induced differentiation. Here we report that the induction of an intermediary or “primed” state of myeloid differentiation is associated with increased H4 arginine 3 and decreased H3 lysine 4 methylation. These modifications occur before transcription and appear to prime the chromatin for subsequent hormone-regulated transcription. Moreover, inhibition of methyltransferase activity, preacetylation, or activation of the enzyme PAD4 attenuated retinoid-regulated gene expression, while overexpression of PRMT1, a methyltransferase, enhanced retinoid responsiveness. Taken together, our results suggest that H4 arginine 3 methylation is a bona fide positive epigenetic marker and regulator of transcriptional responsiveness as well as a signal integration mechanism during cell differentiation and, as such, may provide epigenetic memory.

SET-mediated Promoter Hypoacetylation Is a Prerequisite for Coactivation of the Estrogen-responsive pS2 Gene by PRMT1

Induction of transcription requires an ordered recruitment of coregulators and specific combinations of histone modifications at the promoter. Occurrence of histone H4 arginine (Arg) 3 methylation by protein arginine methyltransferase 1 (PRMT1) represents an early promoter event in ER (estrogen receptor)-regulated gene activation. However, its in vivo significance in ER signaling and the prerequisites for PRMT1 recruitment to promoters have not been established yet. We show here that endogenous PRMT1 is a crucial and non-redundant coactivator of ER-mediated pS2 gene induction in MCF7 cells. By investigating promoter requirements for PRMT1 recruitment we find that the patient SE translocation (SET) protein, which was reported to protect histone tails from acetylation, associates with the uninduced pS2 gene promoter and dissociates early upon estrogen treatment. Knockdown of SET or trichostatin A (TSA) treatment causes premature acetylation of H4 and abrogation of H4 Arg3 methylation at the pS2 gene promoter resulting in diminished transcriptional induction. Thus, SET prevents promoter acetylation and is a prerequisite for the initial acetylation-sensitive steps of pS2 gene activation, namely PRMT1 function. Similar to pS2 we identify lactoferrin as a PRMT1-dependent and TSA-sensitive ER target gene. In contrast, we find that the C3 gene, another ER target, is activated in a PRMT1-independent manner and that SET is involved in C3 gene repression. These findings establish the existence of PRMT1-dependent and -independent ER target genes and show that proteins guarding promoter hypoacetylation, like SET, execute a key function in the coactivation process by PRMT1.

PRMT1-mediated arginine methylation of PIAS1 regulates STAT1 signaling

To elucidate the function of the transcriptional coregulator PRMT1 (protein arginine methyltranferase 1) in interferon (IFN) signaling, we investigated the expression of STAT1 (signal transducer and activator of transcription) target genes in PRMT1-depleted cells. We show here that PRMT1 represses a subset of IFNgamma-inducible STAT1 target genes in a methyltransferase-dependent manner. These genes are also regulated by the STAT1 inhibitor PIAS1 (protein inhibitor of activated STAT1). PIAS1 is arginine methylated by PRMT1 in vitro as well as in vivo upon IFN treatment. Mutational and mass spectrometric analysis of PIAS1 identifies Arg 303 as the single methylation site. Using both methylation-deficient and methylation-mimicking mutants, we find that arginine methylation of PIAS1 is essential for the repressive function of PRMT1 in IFN-dependent transcription and for the recruitment of PIAS1 to STAT1 target gene promoters in the late phase of the IFN response. Methylation-dependent promoter recruitment of PIAS1 results in the release of STAT1 and coincides with the decline of STAT1-activated transcription. Accordingly, knockdown of PRMT1 or PIAS1 enhances the anti-proliferative effect of IFNgamma. Our findings identify PRMT1 as a novel and crucial negative regulator of STAT1 activation that controls PIAS1-mediated repression by arginine methylation.