Chromatin: receiver and quarterback for cellular signals.

Signal transduction pathways converge upon sequence-specific DNA binding factors to reprogram gene expression. Transcription factors, in turn, team up with chromatin modifying activities. However, chromatin is not simply an endpoint for signaling pathways. Histone modifications relay signals to other proteins to trigger more immediate responses than can be achieved through altered gene transcription, which might be especially important to time-urgent processes such as the execution of cell-cycle check points, chromosome segregation, or exit from mitosis. In addition, histone-modifying enzymes often have multiple nonhistone substrates, and coordination of activity toward different targets might direct signals both to and from chromatin.

Loss of the methylarginine reader function of SND1 confers resistance to hepatocellular carcinoma.

Staphylococcal nuclease Tudor domain containing 1 (SND1) protein is an oncogene that 'reads' methylarginine marks through its Tudor domain. Specifically, it recognizes methylation marks deposited by protein arginine methyltransferase 5 (PRMT5), which is also known to promote tumorigenesis. Although SND1 can drive hepatocellular carcinoma (HCC), it is unclear whether the SND1 Tudor domain is needed to promote HCC. We sought to identify the biological role of the SND1 Tudor domain in normal and tumorigenic settings by developing two genetically engineered SND1 mouse models, an Snd1 knockout (Snd1 KO) and an Snd1 Tudor domain-mutated (Snd1 KI) mouse, whose mutant SND1 can no longer recognize PRMT5-catalyzed methylarginine marks. Quantitative PCR analysis of normal, KO, and KI liver samples revealed a role for the SND1 Tudor domain in regulating the expression of genes encoding major acute phase proteins, which could provide mechanistic insight into SND1 function in a tumor setting. Prior studies indicated that ectopic overexpression of SND1 in the mouse liver dramatically accelerates the development of diethylnitrosamine (DEN)-induced HCC. Thus, we tested the combined effects of DEN and SND1 loss or mutation on the development of HCC. We found that both Snd1 KO and Snd1 KI mice were partially protected against malignant tumor development following exposure to DEN. These results support the development of small molecule inhibitors that target the SND1 Tudor domain or the use of upstream PRMT5 inhibitors, as novel treatments for HCC.

RB localizes to DNA double-strand breaks and promotes DNA end resection and homologous recombination through the recruitment of BRG1.

The retinoblastoma (RB) tumor suppressor is recognized as a master regulator that controls entry into the S phase of the cell cycle. Its loss leads to uncontrolled cell proliferation and is a hallmark of cancer. RB works by binding to members of the E2F family of transcription factors and recruiting chromatin modifiers to the promoters of E2F target genes. Here we show that RB also localizes to DNA double-strand breaks (DSBs) dependent on E2F1 and ATM kinase activity and promotes DSB repair through homologous recombination (HR), and its loss results in genome instability. RB is necessary for the recruitment of the BRG1 ATPase to DSBs, which stimulates DNA end resection and HR. A knock-in mutation of the ATM phosphorylation site on E2F1 (S29A) prevents the interaction between E2F1 and TopBP1 and recruitment of RB, E2F1, and BRG1 to DSBs. This knock-in mutation also impairs DNA repair, increases genomic instability, and renders mice hypersensitive to IR. Importantly, depletion of RB in osteosarcoma and breast cancer cell lines results in sensitivity to DNA-damaging drugs, which is further exacerbated by poly-ADP ribose polymerase (PARP) inhibitors. We uncovered a novel, nontranscriptional function for RB in HR, which could contribute to genome instability associated with RB loss.

The Retinoblastoma (RB) Tumor Suppressor: Pushing Back against Genome Instability on Multiple Fronts.

The retinoblastoma (RB) tumor suppressor is known as a master regulator of the cell cycle. RB is mutated or functionally inactivated in the majority of human cancers. This transcriptional regulator exerts its function in cell cycle control through its interaction with the E2F family of transcription factors and with chromatin remodelers and modifiers that contribute to the repression of genes important for cell cycle progression. Over the years, studies have shown that RB participates in multiple processes in addition to cell cycle control. Indeed, RB is known to interact with over 200 different proteins and likely exists in multiple complexes. RB, in some cases, acts through its interaction with E2F1, other members of the pocket protein family (p107 and p130), and/or chromatin remodelers and modifiers. RB is a tumor suppressor with important chromatin regulatory functions that affect genomic stability. These functions include the role of RB in DNA repair, telomere maintenance, chromosome condensation and cohesion, and silencing of repetitive regions. In this review we will discuss recent advances in RB biology related to RB, partner proteins, and their non-transcriptional functions fighting back against genomic instability.

Structure of Amido Pyridinium Betaines: Persistent Intermolecular C-H⋠⋠⋠N Hydrogen Bonding in Solution.

A hydrogen bond of the type C-H⋅⋅⋅X (X=O or N) is known to influence the structure and function of chemical and biological systems in solution. C-H⋅⋅⋅O hydrogen bonding in solution has been extensively studied, both experimentally and computationally, whereas the equivalent thermodynamic parameters have not been enumerated experimentally for C-H⋅⋅⋅N hydrogen bonds. This is, in part, due to the lack of systems that exhibit persistent C-H⋅⋅⋅N hydrogen bonds in solution. Herein, a class of molecule based on a biologically active norharman motif that exhibits unsupported intermolecular C-H⋅⋅⋅N hydrogen bonds in solution has been described. A pairwise interaction leads to dimerisation to give bond strengths of about 7 kJ mol-1 per hydrogen bond, which is similar to chemically and biologically relevant C-H⋅⋅⋅O hydrogen bonding. The experimental data is supported by computational work, which provides additional insight into the hydrogen bonding by consideration of electrostatic and orbital interactions and allowed a comparison between calculated and extrapolated NMR chemical shifts.

Chirality Transfer in Gold(I)-Catalysed Direct Allylic Etherifications of Unactivated Alcohols: Experimental and Computational Study.

Gold(I)-catalysed direct allylic etherifications have been successfully carried out with chirality transfer to yield enantioenriched, γ-substituted secondary allylic ethers. Our investigations include a full substrate-scope screen to ascertain substituent effects on the regioselectivity, stereoselectivity and efficiency of chirality transfer, as well as control experiments to elucidate the mechanistic subtleties of the chirality-transfer process. Crucially, addition of molecular sieves was found to be necessary to ensure efficient and general chirality transfer. Computational studies suggest that the efficiency of chirality transfer is linked to the aggregation of the alcohol nucleophile around the reactive π-bound Au-allylic ether complex. With a single alcohol nucleophile, a high degree of chirality transfer is predicted. However, if three alcohols are present, alternative proton transfer chain mechanisms that erode the efficiency of chirality transfer become competitive.

Gold-Catalyzed Proto- and Deuterodeboronation.

A mild gold-catalyzed protodeboronation reaction, which does not require acid or base additives and can be carried out in "green" solvents, is described. As a result, the reaction is very functional-group-tolerant, even to acid- and base-sensitive functional groups, and should allow for the boronic acid group to be used as an effective traceless directing or blocking group. The reaction has also been extended to deuterodeboronations for regiospecific ipso-deuterations of aryls and heteroaryls from the corresponding organoboronic acid. Based on density functional theory calculations, a mechanism is proposed that involves nucleophilic attack of water at boron followed by rate-limiting B-C bond cleavage and facile protonolysis of a Au-σ-phenyl intermediate.

Identification of prohibitin and prohibiton as novel factors binding to the p53 induced gene 3 (PIG3) promoter (TGYCC)(15) motif.

The promoter of p53 induced gene 3 (PIG3) contains a variable number of tandem repeats (VNTRs) of pentanucleotides (TGYCC)n that is known as a p53 binding site. In this study, we investigated whether other potential molecules could bind to this PIG3 promoter (TGYCC)n motif. Ligand-chromatography combined with liquid chromatography-tandem mass spectrometry analyses indicated direct interactions of prohibitin and/or prohibiton with the (TGYCC)15 motif, which was confirmed by electrophoretic mobility shift assay and super-gel shift analysis with anti-prohibitin and anti-prohibiton antibodies. Using the chromatin immunopercipipation assay, we further demonstrated that prohibitin and prohibiton associated with the (TGYCC)15 motif in vivo regardless of the p53 status and apoptotic stress. We also found that prohibitin and prohibiton up-regulated PIG3 transcription independent of p53, although p53 obviously enhanced this process, and that the knock-down of prohibitin and prohibiton inhibited camptothecin-induced apoptosis. Taken together, our findings suggest that prohibitin and prohibiton contribute to PIG3-mediated apoptosis by binding to the PIG3 promoter (TGYCC)15 motif.

Modeling gene-environment interactions in oral cavity and esophageal cancers demonstrates a role for the p53 R72P polymorphism in modulating susceptibility.

A large number of epidemiological studies have linked a common single-nucleotide polymorphism (SNP) in the human p53 gene to risk for developing a variety of cancers. This SNP encodes either an arginine or proline at position 72 (R72P) of the p53 protein, which can alter the apoptotic activity of p53 via transcriptional and non-transcriptional mechanisms. This SNP has also been reported to modulate the development of human papilloma virus (HPV)-driven cancers through differential targeting of the p53 variant proteins by the E6 viral oncoprotein. Mouse models for the p53 R72P polymorphism have recently been developed but a role for this SNP in modifying cancer risk in response to viral and chemical carcinogens has yet to be established experimentally. Here, we demonstrate that the p53 R72P polymorphism modulates the hyperprolferative, apoptotic and inflammatory phenotypes caused by expression of the HPV16 E6 and E7 oncoproteins. Moreover, the R72P SNP also modifies the carcinogenic response to the chemical carcinogen 4NQO, in the presence and absence of the HPV16 transgene. Our findings confirm several human epidemiological studies associating the codon 72 proline variant with increased risk for certain cancers but also suggest that there are tissue-specific differences in how the R72P polymorphism influences the response to environmental carcinogens.

Gold(I)-catalysed direct thioetherifications using allylic alcohols: an experimental and computational study.

A gold(I)-catalysed direct thioetherification reaction between allylic alcohols and thiols is presented. The reaction is generally highly regioselective (S(N)2'). This dehydrative allylation procedure is very mild and atom economical, producing only water as the by-product and avoiding any unnecessary waste/steps associated with installing a leaving or activating group on the substrate. Computational studies are presented to gain insight into the mechanism of the reaction. Calculations indicate that the regioselectivity is under equilibrium control and is ultimately dictated by the thermodynamic stability of the products.

Far-infrared spectroscopy of the troposphere: calibration with a cold background.

The far-infrared spectroscopy of the troposphere (FIRST) instrument is a Fourier-transform spectrometer developed to measure the Earth's thermal emission spectrum with a particular emphasis on the far-infrared. FIRST has observed the atmosphere from both the ground looking up and from a high-altitude balloon looking down. A recent absolute laboratory calibration of FIRST under ground-like operating conditions showed accuracy to better than 0.3 K at near-ambient temperatures (270-325 K) but reduced accuracy at lower temperatures. This paper presents calibration results for balloon-flight conditions using a cold blackbody to simulate the space view used for on-board calibration. An unusual detector nonlinearity was discovered and corrected, and stray light was measured and removed. Over most of the range of Earth scene temperatures (205-300 K), the accuracy of FIRST is 0.35-0.15 K (one sigma).

Palladium-catalyzed direct C-H functionalization of benzoquinone.

A direct Pd-catalyzed C-H functionalization of benzoquinone (BQ) can be controlled to give either mono- or disubstituted BQ, including the installation of two different groups in a one-pot procedure. BQ can now be directly functionalized with aryl, heteroaryl, cycloalkyl, and cycloalkene groups and, moreover, the reaction is conducted in environmentally benign water or acetone as solvents.

Ruthenium-mediated C-H functionalization of pyridine: the role of vinylidene and pyridylidene ligands.

A combined experimental and theoretical study has demonstrated that [Ru(η(5)-C(5)H(5))(py)(2)(PPh(3))](+) is a key intermediate, and active catalyst for, the formation of 2-substituted E-styrylpyridines from pyridine and terminal alkynes HC≡CR (R = Ph, C(6)H(4)-4-CF(3)) in a 100% atom efficient manner under mild conditions. A catalyst deactivation pathway involving formation of the pyridylidene-containing complex [Ru(η(5)-C(5)H(5))(κ(3)-C(3)-C(5)H(4)NCH═CHR)(PPh(3))](+) and subsequently a 1-ruthanaindolizine complex has been identified. Mechanistic studies using (13)C- and D-labeling and DFT calculations suggest that a vinylidene-containing intermediate [Ru(η(5)-C(5)H(5))(py)(═C═CHR)(PPh(3))](+) is formed, which can then proceed to the pyridylidene-containing deactivation product or the desired product depending on the reaction conditions. Nucleophilic attack by free pyridine at the α-carbon in this complex subsequently leads to formation of a C-H agostic complex that is the branching point for the productive and unproductive pathways. The formation of the desired products relies on C-H bond cleavage from this agostic complex in the presence of free pyridine to give the pyridyl complex [Ru(η(5)-C(5)H(5))(C(5)H(4)N)(═C═CHR)(PPh(3))]. Migration of the pyridyl ligand (or its pyridylidene tautomer) to the α-carbon of the vinylidene, followed by protonation, results in the formation of the 2-styrylpyridine. These studies demonstrate that pyridylidene ligands play an important role in both the productive and nonproductive pathways in this catalyst system.

Far-infrared spectroscopy of the troposphere: instrument description and calibration performance.

The far-infrared spectroscopy of the troposphere (FIRST) instrument is a Fourier transform spectrometer developed to measure the Earth's thermal emission spectrum with a particular emphasis on far-infrared (far-IR) wavelengths greater than 15 µm. FIRST was developed under NASA's Instrument Incubator Program to demonstrate technology for providing measurements from 10 to 100 µm (1000 to 100 cm(-1)) on a single focal plane with a spectral resolution finer than 1 cm(-1). Presently no spectrometers in orbit are capable of directly observing the Earth's far-IR spectrum. This fact, coupled with the fundamental importance of the far-IR to Earth's climate system, provided the impetus for the development of FIRST. In this paper the FIRST instrument is described and results of a detailed absolute laboratory calibration are presented. Specific channels in FIRST are shown to be accurate in the far-IR to better than 0.3 K at 270 K scene temperature, 0.5 K at 247 K, and 1 K at 225 K.

GCN5 and E2F1 stimulate nucleotide excision repair by promoting H3K9 acetylation at sites of damage.

Chromatin structure is known to be a barrier to DNA repair and a large number of studies have now identified various factors that modify histones and remodel nucleosomes to facilitate repair. In response to ultraviolet (UV) radiation several histones are acetylated and this enhances the repair of DNA photoproducts by the nucleotide excision repair (NER) pathway. However, the molecular mechanism by which UV radiation induces histone acetylation to allow for efficient NER is not completely understood. We recently discovered that the E2F1 transcription factor accumulates at sites of UV-induced DNA damage and directly stimulates NER through a non-transcriptional mechanism. Here we demonstrate that E2F1 associates with the GCN5 acetyltransferase in response to UV radiation and recruits GCN5 to sites of damage. UV radiation induces the acetylation of histone H3 lysine 9 (H3K9) and this requires both GCN5 and E2F1. Moreover, as previously observed for E2F1, knock down of GCN5 results in impaired recruitment of NER factors to sites of damage and inefficient DNA repair. These findings demonstrate a direct role for GCN5 and E2F1 in NER involving H3K9 acetylation and increased accessibility to the NER machinery.

INO80 chromatin remodeling complex promotes the removal of UV lesions by the nucleotide excision repair pathway.

The creation of accessible DNA in the context of chromatin is a key step in many DNA functions. To reveal how ATP-dependent chromatin remodeling activities impact DNA repair, we constructed mammalian genetic models for the INO80 chromatin remodeling complex and investigated the impact of loss of INO80 function on the repair of UV-induced photo lesions. We showed that deletion of two core components of the INO80 complex, INO80 and ARP5, significantly hampered cellular removal of UV-induced photo lesions but had no significant impact on the transcription of nucleotide excision repair (NER) factors. Loss of INO80 abolished the assembly of NER factors, suggesting that prior chromatin relaxation is important for the NER incision process. Ino80 and Arp5 are enriched to UV-damaged DNA in an NER-incision-independent fashion, suggesting that recruitment of the remodeling activity likely takes place during the initial stage of damage recognition. These results demonstrate a critical role of INO80 in creating DNA accessibility for the NER pathway and provide direct evidence that repair of UV lesions and perhaps most bulky adduct lesions requires chromatin reconfiguration.

Combined effects of E2F1 and E2F2 polymorphisms on risk and early onset of squamous cell carcinoma of the head and neck.

Deregulated expression of most members of the E2F family has been detected in many human cancers. We examined the association of common single nucleotide polymorphisms (SNPs) of E2F transcription factors 1 and 2 (E2F1 and E2F2) with risk of squamous cell carcinoma of the head and neck (SCCHN) in 1,096 SCCHN patients and 1,090 cancer-free controls. We genotyped 10 selected SNPs in E2F1 and E2F2, including those at the near 5'-untranslated region (UTR), microRNA (miRNA)-binding sites at the near 3'-UTR and tagSNPs according to bioinformatics analysis. Although none of the selected SNPs alone was significantly associated with risk of SCCHN, there was a statistically significantly increased risk of SCCHN associated with the combined risk genotypes (i.e., rs3213182 AA, rs3213183 GG, rs3213180 GG, rs321318121 GG, rs2742976 GT+TT, rs6667575 GA+AA, rs3218203 CC, rs3218148 AA, rs3218211 CC, and rs3218123 GT+TT). Compared with those with 0-4 risk genotypes, an increased risk was observed for those who carried 5-8 risk genotypes (adjusted OR = 1.04; 95% CI = 0.86-1.26) and 9-10 risk genotypes (adjusted OR = 1.62; 95% CI = 1.14-2.30) in a dose-response manner (P = 0.045). Furthermore, the joint effect was more pronounced among patients with oropharyngeal cancer, younger adults (≤57 yr old), men, non-smokers, non-drinkers, and individuals with family history of cancer in first-degree relatives. Additionally, we also observed that those with 5-10 risk genotypes had an earlier SCCHN onset than those with 0-4 risk genotypes, particularly for non-smokers and/or non-drinkers. We concluded that E2F1 and E2F2 genetic variants may jointly play important roles in head and neck carcinogenesis.

Charged behaviour from neutral ligands: synthesis and properties of N-heterocyclic pseudo-amides.

Deprotonation of the 1-isopropyl-3-(phenylamino)pyridin-1-ium iodide gives the corresponding neutral betaine, which is formalised as a pyridinium-amido ligand when coordinated to a metal. Spectroscopic, structural and theoretical methods have been used to investigate the metal-ligand bonding, ligand dynamics and electron distribution. Collectively, the data show that the ligand can be characterised as a pseudo-amide and is a strong donor akin to alkyl phosphines and N-heterocyclic carbenes. Furthermore, rotation about both N substituent C-N bonds occurs, which is in contrast to the two alternative pyridinium positional isomers that exhibit neutral resonance structures. For comparison, compounds and complexes derived from norharman were prepared, which contain an additional C-C bond supporting conjugation and the accessibility of a neutral resonance structure. Notwithstanding the formal neutral structure, norharman-derived ligands are comparably strong donors, and have the additional advantage of exhibiting stability to dioxygen and water.

Absolute linearity measurement of photodetectors using sinusoidal modulated radiation.

A method is presented for characterizing the linearity of photodetectors based on time-domain analysis of response to sinusoidal excitation. Nonlinearity is quantified solely from the output distortion. Relative response is converted to absolute response by including two calibration points. For low signal level, one calibration point is required, while using dark current as the second point. The response is mapped over a wider range using a series of overlapping sinusoids for calibration transfer. The method is demonstrated with a relatively linear photodiode and a nonlinear phototransistor. A Michelson interferometer is used to generate sinusoidal modulation of a laser source. Results demonstrate the potential of the proposed technique.

E2F1 and p53 transcription factors as accessory factors for nucleotide excision repair.

Many of the biochemical details of nucleotide excision repair (NER) have been established using purified proteins and DNA substrates. In cells however, DNA is tightly packaged around histones and other chromatin-associated proteins, which can be an obstacle to efficient repair. Several cooperating mechanisms enhance the efficiency of NER by altering chromatin structure. Interestingly, many of the players involved in modifying chromatin at sites of DNA damage were originally identified as regulators of transcription. These include ATP-dependent chromatin remodelers, histone modifying enzymes and several transcription factors. The p53 and E2F1 transcription factors are well known for their abilities to regulate gene expression in response to DNA damage. This review will highlight the underappreciated, transcription-independent functions of p53 and E2F1 in modifying chromatin structure in response to DNA damage to promote global NER.