An in vitro biochemical investigation into the conformation, binding and E3-ubiquitin ligase activity of mammalian UHRF1 with reconstituted chromatin

In the eukaryotic genome, DNA and histone modifications regulate chromatin function and mediate basic processes such as gene transcription, DNA repair and DNA replication. Maintaining chromatin modifications after DNA replication is essential for chromatin homeostasis, especially for regions of the genome that need to be kept silenced such as repetitive elements. The maintenance DNA methyltransferase, DNMT1, is responsible for ensuring that cytosine methylation at CpG dinucleotides, and thus proper transcriptional programmes, are propagated to the daughter cells. DNMT1 is specifically recruited to newly replicated, hemi-methylated DNA and the E3-ubiquitin ligase UHRF1 (Ubiquitin-like containing PHD- and RING-finger domains protein 1) plays a critical role for this. The mechanisms of the recruitment of DNMT1 to chromatin via UHRF1 are currently an area of active investigation. Several studies using modified nucleosomes, histone peptides and DNA oligonucleotides have identified UHRF1 to bind to hemi-methylated CpG dinucleotides and to histone H3 di- or tri-methylated at Lys-9. Since UHRF1 was also found to interact with DNMT1, it was postulated that UHRF1 acts as an adapter that directly recruits DNMT1 to newly replicated DNA. Additionally, it has recently been reported that the E3-ubiquitin ligase activity of its C-terminal RING-finger is required for the recruitment of DNMT1 to replication forks. Ubiquitylation of either K18 or K23 on histone H3 that is recognised by a ubiquitin-interacting motif within DNMT1 appears to be critical for DNMT1 targeting but the recruitment mechanism has so far not been completely elucidated. This study has investigated the binding and E3-ubiquitin ligase activity of UHRF1 in the context of physiologically relevant chromatin substrates. Using a fully reconstituted system, the chromatin binding and enzymatic activity of UHRF1 and how this is linked to its intra-molecular arrangement have been elucidated. In the context of modified nucleosome substrates, we observe an increase in binding of recombinant UHRF1 in the presence of hemi-methylated DNA whilst with histone H3K9me2/3, only a small increase in binding is detected. We also provide evidence that binding to nucleosome core particles is enhanced by a basic region between the SRA-domain and the RING-finger. This so called polybasic region or PBR has previously been implicated in the regulation of UHRF1 binding to H3K9me2/3 marks. Our findings therefore suggest that binding of UHRF1 to physiological chromatin substrates is more complex than previously thought. In-solution crosslinking/mass spectrometry experiments using the full-length protein confirm that UHRF1 exhibits complex intra-molecular contacts that can potentially regulate its interaction with chromatin or other factors. In addition to reported contacts between the PBR with the Tandem-Tudor domain and between the PHD-finger and the SRA-domain, the UBL-domain also makes extensive contacts to other regions within UHRF1. These appear to be weak and dynamic. Crucially, removal of the UBL-domain does not affect nucleosome binding but does result in a strong reduction in UHRF1 E3-ubiquitin ligase activity. Further experiments suggest that the UBL-domain is involved in establishing the enzyme/substrate complex between the E2-conjugating enzyme and the chromatin substrate and in stimulating the transfer of ubiquitin from the E2~Ub complex to histone H3. In summary, by combining a crosslinking/mass spectrometry approach to interrogate the intra-molecular arrangement of UHRF1 with fully reconstituted enzyme and chromatin-binding assays using physiologically relevant substrates, we have identified a function for the UBL-domain of UHRF1. Our results suggest that the UBL is highly flexible in solution and that it forms transient contacts with other parts of UHRF1 and the E2-conjugating enzyme that are required for the formation of the E2/E3/substrate complex in allosterically activating ubiquitin transfer from the E2~Ub to the histone target substrate. These findings assign, for the first time, a function for the UBL-domain and pave the way for further investigation of the involvement of this domain in the physiological role of UHRF1.Open Acces

Sampling and Inference for Beta Neutral-to-the-Left Models of Sparse Networks

Empirical evidence suggests that heavy-tailed degree distributions occurring in many real networks are well-approximated by power laws with exponents $\eta$ that may take values either less than and greater than two. Models based on various forms of exchangeability are able to capture power laws with $\eta < 2$, and admit tractable inference algorithms; we draw on previous results to show that $\eta > 2$ cannot be generated by the forms of exchangeability used in existing random graph models. Preferential attachment models generate power law exponents greater than two, but have been of limited use as statistical models due to the inherent difficulty of performing inference in non-exchangeable models. Motivated by this gap, we design and implement inference algorithms for a recently proposed class of models that generates $\eta$ of all possible values. We show that although they are not exchangeable, these models have probabilistic structure amenable to inference. Our methods make a large class of previously intractable models useful for statistical inference.Comment: Accepted for publication in the proceedings of Conference on Uncertainty in Artificial Intelligence (UAI) 201

Impact of non-audit assurance level (compilation versus review) on production management of private manufacturing companies

Private small companies have been the subject of much interest by accounting regulators recently. The AICPA issued SSARS 19 and the Financial Reporting Framework for Small- and Medium-Sized Entities in 2009 and 2013, respectively. The Financial Accounting Foundation issued the Blue Ribbon Panel Report on Standard Setting for Private Companies in 2011. This is apparently the first study to compare reporting practices of small private companies whose financial statements are compiled or reviewed. Overall results indicate that reviewed companies tend to exhibit positive abnormal production while compiled companies tend to exhibit negative abnormal production. However, for the companies most likely to engage in earnings management, only separately taxable reviewed entities tend to manage earnings higher to meet earnings benchmarks. External users of reviewed and compiled statements should be aware of the tendencies of the different types of entities to manage production and inventory levels

First Scarab Host for \u3ci\u3eStrongygaster Triangulifer\u3c/i\u3e (Diptera: Tachinidae): the Dung Beetle, \u3ci\u3eAphodius Fimetarius\u3c/i\u3e (Coleoptera: Scarabaeidae)

We report Strongygaster (=Hyalomyodes ) triangulifer as a solitary primary parasite of the adult introduced dung beetle, Aphodius fimetarius. This is the first record of this tachinid fly parastizing scarab

Ground Beetles From a Remnant Oak-Maple-Beech Forest and Its Surroundings in Northeastern Ohio (Coleoptera: Carabidae)

We report 66 ground beetle species in 14 tribes from a natural preserve in northeastern Ohio (Stark County). Six species are new state records. Data from pitfall trap transects across adjoining habitats suggest narrow habitat preferences in some species and broad tolerances in others. Trends toward flightlessness in forest species and macroptery in the fauna of disturbed agricultural sites are apparent

Alarm Pheromone in a Gregarious Poduromorph Collembolan (Collembola: Hypogastruridae)

We report an alarm pheromone in the gregarious poduromorph collembolan, Hypogastrura pannosa. Cuticular rupture results in emission of a rapidly vaporizing hexane-soluble material with an active space diameter of ca. 1 cm. Conspecifics encountering the vapor front respond with stereotypic aversion and dispersal behaviors. This is the first report on the presence of an alarm pheromone in the order Collembola

Material Internal Control Weaknesses And Earnings Management In The Post-SOX Environment

Prior studies found that companies with internal control deficiencies incorporate abnormal accounting accruals into their financial statements. However, these studies did not consider the materiality of abnormal accruals. Abnormal accruals should be within materiality when financial statements receive clean audit opinions. When material internal control weaknesses (MICW) exist, to compensate for additional risk, auditors should apply more audit effort to gain the quantity and quality of evidence necessary to obtain a reasonable degree of assurance to support their audit reports. We find evidence of this because audit fees are significantly higher for MICW companies than those for effective internal controls (EIC) companies in our sample. Accordingly, financial statements receiving clean audit opinions should not contain material abnormal accruals irrespective of whether controls are effective EIC or ineffective MICW. To examine this issue, we use post-SOX data to estimate abnormal accruals using a revenue-based accrual model for a matched sample of companies with clean audit opinions on their financial statements: one-half EIC and the other half with MICW. Then, we establish material abnormal revenue accruals (MARA), which is the difference between estimated abnormal revenue accruals and a quantitative materiality based on assets. Finally, we compare MARA between EIC and MICW companies. We find no significant difference in MARA between EIC and MICW companies. We provide a summary of important findings in Table 3, and conclude with suggestions to further improve audit and financial reporting quality