Introduction and Aims: Chronic kidney disease (CKD) is a major risk factor for atherosclerotic cardiovascular diseases. Recently it has been reported that apoptosis of aortic smooth muscle cells (AoSMCs) may relate to the atherosclerosis with plaque formation or calcification. Therefore, in the present study, we examined the effect of indoxyl sulfate (IS), which is thought to be one of uremic toxin, for apoptosis in cultured rat AoSMCs. Methods: The induction of apoptosis was quantitated by assay of the caspase CPP32, which plays a direct role in the execution of cell death. And the activity of SAPK/JNK and P38 MAP kinase, which is known as apoptosis-inducing signal transduction, was assessed by standard immunoblot using phospho-specific antibodies. Results: Twenty five μg/ml of IS, which is compatible with the concentration of IS in the serum of end-stage renal failure patients, induced 3.9±2.7-fold increase in the caspase CPP32 activity responding to serum withdrawal for 12 hours. The blockade of organic anion transporter (OAT) by 0.5mM probenecid (Pb) abolished the effect of IS on the apoptosis in AoSMCs (relative increase in the caspase CPP32: Pb-, IS-; 1±0.1, Pb-, IS+; 2.4±0.2, Pb+, IS-; 0.9±0.1, Pb+, IS+; 1.2±0.1). Indoxyl sulfate activated SAPK/JNK in AoSMCs that was significantly elevated by 30 minutes and sustained for over 2 hours, although it did not affect the activation of P38 MAP kinase. Conclusions: These results indicate that IS accelerates apoptosis induced by serum withdrawal in rat AoSMCs, which is mediated by cellular transport of IS via the OAT and may also be related to the activation of SAPK/JNK pathway. The induction of apoptosis by the accumulation of IS in blood due to CKD may play an important role in atherosclerotic lesion formation.

Ribosomes are specialized entities that participate in regulation of gene expression through their rRNAs carrying ribozyme activity. Ribosome biogenesis is overactivated in p53-inactivated cancer cells, although involvement of p53 on ribosome quality is unknown. Here, we show that p53 represses expression of the rRNA methyl-transferase fibrillarin (FBL) by binding directly to FBL. High levels of FBL are accompanied by modifications of the rRNA methylation pattern, impairment of translational fidelity, and an increase of internal ribosome entry site (IRES)-dependent translation initiation of key cancer genes. FBL overexpression contributes to tumorigenesis and is associated with poor survival in patients with breast cancer. Thus, p53 acts as a safeguard of protein synthesis by regulating FBL and the subsequent quality and intrinsic activity of ribosomes.

Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2'-O-methylation (2'-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2'-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2'-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2'-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2'-O-Me, we identified a repertoire of 2'-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2'-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2'-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2'-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.

Histone H1 and the high-mobility group (HMG) proteins are chromatin binding proteins that regulate gene expression by modulating the compactness of the chromatin fiber and affecting the ability of regulatory factors to access their nucleosomal targets. Histone H1 stabilizes the higher-order chromatin structure and decreases nucleosomal access, while the HMG proteins decrease the compactness of the chromatin fiber and enhance the accessibility of chromatin targets to regulatory factors. Here we show that in living cells, each of the three families of HMG proteins weakens the binding of H1 to nucleosomes by dynamically competing for chromatin binding sites. The HMG families weaken H1 binding synergistically and do not compete among each other, suggesting that they affect distinct H1 binding sites. We suggest that a network of dynamic and competitive interactions involving HMG proteins and H1, and perhaps other structural proteins, constantly modulates nucleosome accessibility and the local structure of the chromatin fiber.The chromatin fiber is a dynamic, malleable structure that is targeted by numerous regulatory factors that modify the histones and the DNA and remodel the structure of the nucleosome. The dynamics of the chromatin fiber reflect the combined action of numerous chromatin modifiers, including architectural proteins such as histone H1 and members of the high-mobility group (HMG) protein superfamily. The interaction of histone H1 with nucleosomes stabilizes the higherorder, compact chromatin structure (57, 61), thereby restricting the ability of regulatory factors, nucleosome remodeling complexes, and histone modifiers to access their chromatin binding sites (17,27,28,30,32,34). Loss of H1 results in both up regulation and down regulation of specific gene expression (2,26,53,56), suggesting that the protein affects the action of both positive and negative transcriptional regulators. Experiments with H1 knockout mice demonstrate the existence of cellular mechanisms that strive to maintain a constant level of H1 and that reduction of H1 beyond a critical point is not compatible with normal embryonic development (21). Thus, factors that modulate the interaction of H1 with nucleosomes may affect a variety of chromatin-related processes and participate in genetic regulatory mechanisms.The HMG superfamily is composed of three families: HMGB, HMGA, and HMGN, each family being characterized by a distinct DNA or chromatin binding motif (12). These non-histones decompact the higher-order chromatin structure and promote the binding of nuclear regulatory factors (1,12,49,58). Footprinting analysis and in vitro binding assays (29,64) showed that the chromatin binding sites of the HMG proteins are similar to those of H1 and suggested that HMG proteins and H1 compete for the same binding sites, and recent photobleaching experiments demonstrated that HMGN proteins affect the binding of H1 to chromatin in living cells (16). Photobleaching techniques like fluorescence recovery after photobleaching (FRAP), which is used...

Over 80% of the nucleosomes in chromatin contain histone H1, a protein family known to affect the structure and activity of chromatin. Genetic studies and in vivo imaging experiments are changing the traditional view of H1 function and mechanism of action. H1 variants are partially redundant, mobile molecules that interact with nucleosomes as members of a dynamic protein network and serve as fine tuners of chromatin function.

Here we demonstrate that HMGN1, a nuclear protein that binds to nucleosomes and reduces the compaction of the chromatin fiber, modulates histone posttranslational modifications. In Hmgn1-/- cells, loss of HMGN1 elevates the steady-state levels of phospho-S10-H3 and enhances the rate of stress-induced phosphorylation of S10-H3. In vitro, HMGN1 reduces the rate of phospho-S10-H3 by hindering the ability of kinases to modify nucleosomal, but not free, H3. During anisomycin treatment, the phosphorylation of HMGN1 precedes that of H3 and leads to a transient weakening of the binding of HMGN1 to chromatin. We propose that the reduced binding of HMGN1 to nucleosomes, or the absence of the protein, improves access of anisomysin-induced kinases to H3. Thus, the levels of posttranslational modifications in chromatin are modulated by nucleosome binding proteins that alter the ability of enzymatic complexes to access and modify their nucleosomal targets.

The ability of regulatory factors to access their nucleosomal targets is modulated by nuclear proteins such as histone H1 and HMGN (previously named HMG-14/-17 family) that bind to nucleosomes and either stabilize or destabilize the higherorder chromatin structure. We tested whether HMGN proteins affect the interaction of histone H1 with chromatin. Using microinjection into living cells expressing H1-GFP and photobleaching techniques, we found that wild-type HMGN, but not HMGN point mutants that do not bind to nucleosomes, inhibits the binding of H1 to nucleosomes. HMGN proteins compete with H1 for nucleosome sites but do not displace statically bound H1 from chromatin. Our results provide evidence for in vivo competition among chromosomal proteins for binding sites on chromatin and suggest that the local structure of the chromatin fiber is modulated by a dynamic interplay between nucleosomal binding proteins.

Efficient and correct responses to double stranded breaks (DSB) in chromosomal DNA are critical for maintaining genomic stability and preventing chromosomal alterations leading to cancer1. The generation of DSB is associated with structural changes in chromatin and the activation of the protein kinase ataxia-telangiectasia mutated (ATM), a key regulator of the signaling network of the cellular response to DSB 2,3. The interrelationship between DSB-induced changes in chromatin architecture and the activation of ATM is unclear 3. Here we show that the nucleosome-binding protein HMGN1 modulates the interaction of ATM with chromatin both prior to and after DSB formation thereby optimizing its activation. Loss of HMGN1, or ablation of its ability to bind to chromatin, reduces the levels of IR-induced ATM autophosphorylation and the activation of several ATM targets. IR treatments lead to a global increase in the acetylation of Lys14 of histone H3 (H3K14) in an HMGN1 dependent manner and treatment of cells with a histone deacetylase inhibitor bypasses the HMGN1 requirement for efficient ATM activation. Thus, by regulating the levels of histone modifications, HMGN1 affects ATM activation. Our studies identify a new mediator of ATM activation and demonstrate a direct link between the steady-state intranuclear organization of ATM and the kinetics of its activation following DNA damage.