Functional Analysis of Histone ADP-Ribosylation In Vitro and in Cells.

Gene regulation in the nucleus requires precise control of the molecular processes that dictate how, when, and which genes are transcribed. The posttranslational modification (PTM) of histones in chromatin is an effective means to link cellular signaling to gene expression outcomes. The repertoire of histone PTMs includes phosphorylation, acetylation, methylation, ubiquitylation, and ADP-ribosylation (ADPRylation). ADPRylation is a reversible PTM that results in the covalent transfer of ADP-ribose units derived from NAD+ to substrate proteins on glutamate, aspartate, serine, and other amino acids. Histones were the first substrate proteins identified for ADPRylation, over five decades ago. Since that time, histone ADPRylation has been shown to be a widespread and critical regulator of chromatin structure and function during transcription, DNA repair, and replication. Here, we describe a set of protocols that allow the user to investigate site-specific histone ADPRylation and its functional consequences in biochemical assays and in cells in a variety of biological systems. With the recent discovery that some cancer-causing histone mutations (i.e., oncohistone mutations) occur at functional sites of regulatory ADPRylation, these protocols may have additional utility in studies of oncology.

Oncohistone Mutations Occur at Functional Sites of Regulatory ADP-Ribosylation.

Recent studies have identified cancer-associated mutations in histone genes that lead to the expression of mutant versions of core histones called oncohistones. Many oncohistone mutations occur at Asp and Glu residues, two amino acids known to be ADP-ribosylated (ADPRylated) by PARP1. We screened 25 Glu or Asp oncohistone mutants for their effects on cell growth in breast and ovarian cancer cells. Ectopic expression of six mutants of three different core histones (H2B, H3, and H4) altered cell growth in at least two different cell lines. Two of these sites, H2B-D51 and H4-D68, were indeed sites of ADPRylation in wild-type (unmutated) histones, and mutation of these sites inhibited ADPRylation. Mutation of H2B-D51 dramatically altered chromatin accessibility at enhancers and promoters, as well as gene expression outcomes, whereas mutation of H4-D68 did not. Additional biochemical, cellular, proteomic, and genomic analyses demonstrated that ADPRylation of H2B-D51 inhibits p300-mediated acetylation of H2B at many Lys residues. In breast cancer cell xenografts in mice, H2B-D51A promoted tumor growth, but did not confer resistance to the cytotoxic effects of PARP inhibition. Collectively, these results demonstrate that functional Asp and Glu ADPRylation sites on histones are mutated in cancers, allowing cancer cells to escape the growth-regulating effects of post-translational modifications via distinct mechanisms. SIGNIFICANCE: This study identifies cancer-driving mutations in histones as sites of PARP1-mediated ADP-ribosylation in breast and ovarian cancers, providing a molecular pathway by which cancers may subvert the growth-regulating effects of PARP1.

G-quadruplex DNA inhibits unwinding activity but promotes liquid-liquid phase separation by the DEAD-box helicase Ded1p.

G-quadruplex DNA interacts with the N-terminal intrinsically disordered domain of the DEAD-box helicase Ded1p, diminishing RNA unwinding activity but enhancing liquid-liquid phase separation of Ded1p in vitro and in cells. The data highlight multifaceted effects of quadruplex DNA on an enzyme with intrinsically disordered domains.

G-Quadruplex loops regulate PARP-1 enzymatic activation.

G-Quadruplexes are non-B form DNA structures present at regulatory regions in the genome, such as promoters of proto-oncogenes and telomeres. The prominence in such sites suggests G-quadruplexes serve an important regulatory role in the cell. Indeed, oxidized G-quadruplexes found at regulatory sites are regarded as epigenetic elements and are associated with an interlinking of DNA repair and transcription. PARP-1 binds damaged DNA and non-B form DNA, where it covalently modifies repair enzymes or chromatin-associated proteins respectively with poly(ADP-ribose) (PAR). PAR serves as a signal in regulation of transcription, chromatin remodeling, and DNA repair. PARP-1 is known to bind G-quadruplexes with stimulation of enzymatic activity. We show that PARP-1 binds several G-quadruplex structures with nanomolar affinities, but only a subset promote PARP-1 activity. The G-quadruplex forming sequence found in the proto-oncogene c-KIT promoter stimulates enzymatic activity of PARP-1. The loop-forming characteristics of the c-KIT G-quadruplex sequence regulate PARP-1 catalytic activity, whereas eliminating these loop features reduces PARP-1 activity. Oxidized G-quadruplexes that have been suggested to form unique, looped structures stimulate PARP-1 activity. Our results support a functional interaction between PARP-1 and G-quadruplexes. PARP-1 enzymatic activation by G-quadruplexes is dependent on the loop features and the presence of oxidative damage.

DEAD-box RNA helicases Dbp2, Ded1 and Mss116 bind to G-quadruplex nucleic acids and destabilize G-quadruplex RNA.

We identified 29 G-quadruplex binding proteins by affinity purification and quantitative LC-MS/MS. We demonstrated that the DEAD-box RNA helicases Dbp2, Ded1 and Mss116 preferentially bind to G-quadruplex nucleic acids in vitro and destabilize RNA quadruplexes, suggesting new potential roles for these helicases in disruption of quadruplex structures in RNA.

Hemodynamic response to burst-suppressed and discontinuous electroencephalography activity in infants with hypoxic ischemic encephalopathy.

Burst suppression (BS) is an electroencephalographic state associated with a profound inactivation of the brain. BS and pathological discontinuous electroencephalography (EEG) are often observed in term-age infants with neurological injury and can be indicative of a poor outcome and lifelong disability. Little is known about the neurophysiological mechanisms of BS or how the condition relates to the functional state of the neonatal brain. We used simultaneous EEG and diffuse optical tomography (DOT) to investigate whether bursts of EEG activity in infants with hypoxic ischemic encephalopathy are associated with an observable cerebral hemodynamic response. We were able to identify significant changes in concentration of both oxy and deoxyhemoglobin that are temporally correlated with EEG bursts and present a relatively consistent morphology across six infants. Furthermore, DOT reveals patient-specific spatial distributions of this hemodynamic response that may be indicative of a complex pattern of cortical activation underlying discontinuous EEG activity that is not readily apparent in scalp EEG.

Errata: Hemodynamic response to burst-suppressed and discontinuous electroencephalography activity in infants with hypoxic ischemic encephalopathy.

[This corrects the article DOI: 10.1117/1.NPh.3.3.031408.].

Interactive neonatal gastrointestinal magnetic resonance imaging using fruit juice as an oral contrast media.

BACKGROUND: The objective was to evaluate the use of fruit juice with an interactive inversion recovery (IR) MR pulse sequence to visualise the gastrointestinal tract. METHODS: We investigated the relaxation properties of 12 different natural fruit juices in vitro, to identify which could be used as oral contrast. We then describe our initial experience using an interactive MR pulse sequence to allow optimal visualisation after administering pineapple juice orally, and suppressing pre-existing bowel fluid contents, with variable TI in three adult and one child volunteer. RESULTS: Pineapple juice (PJ) had both the shortest T1 (243 ms) and shortest T2 (48 ms) of the fruit juices tested. Optimal signal differentiation between pre-existing bowel contents and oral PJ administration was obtained with TIs of between 900 and 1100 ms. CONCLUSION: The use of an inversion recovery preparation allowed long T1 pre-existing bowel contents to be suppressed whilst the short T1 of fruit juice acts as a positive contrast medium. Pineapple juice could be used as oral contrast agent for neonatal gastrointestinal magnetic resonance imaging.

Interactive magnetic resonance imaging for paediatric vesicoureteric reflux (VUR).

OBJECTIVES: The current gold standard for diagnosing vesicoureteric reflux in unsedated infants is the X-ray-based Micturating CystoUrethroGram (MCUG). The aim of this study was to assess the diagnostic performance of interactive MRI for voiding cysto-urethrography (iMRVC). METHODS: 25 infants underwent conventional MCUG followed by iMRVC. In iMRVC, patients were examined using a real-time MR technique, which allows interactive control of image contrast and imaging plane location, before, during and after micturition. Images were assessed for presence and grade of VUR. Parental feedback on both procedures was evaluated. RESULTS: iMRVC gave a sensitivity of 100%, specificity of 90.5% (95% CI: 81.6-99.4%), PPV of 66.7% and NPV of 100% in this population. There was 88% concordance (44/50 renal units) according to the presence of VUR between the two methods, with iMRVC up-grading VUR in 6 units (12%). There was very good agreement regarding VUR grade: Kappa=0.66±0.11 (95% CI 0.43-0.88). 60% of parents preferred the MRI, but did not score the two tests differently. CONCLUSION: Interactive MRI allows dynamic imaging of the whole urinary tract without ionising radiation exposure. iMRVC gives comparable results to the MCUG, and is acceptable to parents.

Paediatric MRI under sedation: is it necessary? What is the evidence for the alternatives?

To achieve diagnostic images during MRI examinations, small children need to lie still to avoid movement artefact. To reduce patient motion, obviate the need for voluntary immobilisation or breath-holding and therefore obtain high-quality images, MRI of infants is frequently carried out under sedation or general anaesthesia, but this is not without risk and expense. However, many other techniques are available for preparing children for MRI, which have not been fully evaluated. Here, we evaluate the advantages and disadvantage of sedation and anaesthesia for MRI. We then evaluate the alternatives, which include neonatal comforting techniques, sleep manipulation, and appropriate adaptation of the physical environment. We summarize the evidence for their use according to an established hierarchy. Lastly, we discuss several factors that will influence the choice of imaging preparation, including patient factors, imaging factors and service provision. The choice of approach to paediatric MRI is multi-factorial, with limited scientific evidence for many of the current approaches. These considerations may enable others to image children using MRI under different circumstances.

Interactive magnetic resonance voiding cystourethrography (iMRVC) for vesicoureteric reflux (VUR) in unsedated infants: a feasibility study.

OBJECTIVES: The current reference standard for diagnosing vesicoureteric reflux is the X-ray-based Micturating CystoUrethroGram (MCUG). The aim of this study was to evaluate the feasibility of performing interactive Magnetic Resonance voiding cysto-urethrography (iMRVC) in un-sedated infants. METHODS: Twelve infants underwent conventional single-cycle MCUG followed by iMRVC. In iMRVC, patients were examined using an in-house developed fluoroscopic pulse sequence, which allows on-the-fly control of image contrast and geometry. A single acquisition was performed during bladder filling, during and after micturition, with interactive control over imaging parameters. Images were assessed for diagnostic quality and presence of VUR. RESULTS: Every case of reflux identified with MCUG was identified on iMRVC (100% sensitivity). Over 24 renal units, there was 88% concordance (21/24) according to the presence of reflux between the two methods. There were three "false positives" detected by MRI, giving a specificity of 83.3%, PPV of 66.7% and NPV of 100%. CONCLUSION: iMRVC is a feasible method for evaluating the renal tract in infants without the need for radiation or sedation. A formal evaluation is required to establish its diagnostic potential.