Protocol of the Double-Click Seq Method to Evaluate the Deposition Bias of Chromatin Proteins

Symmetrical deposition of parental and newly synthesized chromatin proteins over both sister chromatids is important for the maintenance of epigenetic integrity. However, the mechanisms to maintain equal distribution of parental and newly synthesized chromatid proteins over sister chromatids remains largely unknown. Here, we describe the protocol for the recently developed double-click seq method that enables mapping of asymmetry in the deposition of parental and newly synthesized chromatin proteins on both sister chromatids in DNA replication. The method involved metabolic labeling of new chromatin proteins with l-Azidohomoalanine (AHA) and newly synthesized DNA with Ethynyl-2′-deoxyuridine (EdU) followed by two subsequent click reactions for biotinylation and subsequently by corresponding separation steps. This enables isolation of parental DNA that was bound to nucleosomes containing new chromatin proteins. Sequencing of these DNA samples and mapping around origins of replication in the cellular DNA enables estimation of the asymmetry in deposition of chromatin proteins over the leading and lagging strand in DNA replication. Altogether, this method contributes to the toolbox to understand histone deposition in DNA replication.</p

Identification of a Bromodomain-like region in 15-Lipoxygenase-1 explains its Nuclear Localization

Lipoxygenase (LOX) activity provides oxidative lipid metabolites, which are involved in inflammatory disorders and tumorigenesis. Activity‐based probes to detect the activity of LOX enzymes in their cellular context provide opportunities to explore LOX biology and LOX inhibition. Here, we developed Labelox B as a potent covalent LOX inhibitor for one‐step activity‐based labeling of proteins with LOX activity. Labelox B was used to establish an ELISA‐based assay for affinity capture and antibody‐based detection of specific LOX isoenzymes. Moreover, Labelox B enabled efficient activity‐based labeling of endogenous LOXs in living cells. LOX proved to localize in the nucleus, which was rationalized by identification of a functional bromodomain‐like consensus motif in 15‐LOX‐1. This indicates that 15‐LOX‐1 is not only involved in oxidative lipid metabolism, but also in chromatin binding, which suggests a potential role in chromatin modifications

Induced protein degradation of histone deacetylases 3 (HDAC3) by proteolysis targeting chimera (PROTAC)

Histone deacetylases (HDACs) play important roles in inflammatory diseases like asthma and chronic obstructive pulmonary disease (COPD). Unravelling of and interfering with the functions of specific isoenzymes contributing to inflammation provides opportunities for drug development. Here we synthesize proteolysis targeting chimeras (PROTACs) for degradation of class I HDACs in which o-aminoanilide-based class I HDAC inhibitors are tethered to the cereblon ligand pomalidomide. One of these PROTACs, denoted HD-TAC7, showed promising degradation effects for HDAC3 with a DC50 value of 0.32 μM. In contrast to biochemical evidence using siRNA, HD-TAC7 showed a minimal effect on gene expression in LPS/IFNγ-stimulated RAW 264.7 macrophages. The lack of effect can be attributed to downregulation of the NF-κB subunit p65, which is a known side effect of pomalidomide treatment. Altogether, we describe a novel PROTAC that enables selective downregulation of HDAC3 levels, however we note that concomitant downregulation of the NF-κB subunit p65 can confound the biological outcome

Longitudinal associations of multiple physical symptoms with recurrence of depressive and anxiety disorders

Objective To examine longitudinal associations of multiple physical symptoms with recurrence of depressive and anxiety disorders. Methods Follow-up data of 584 participants with remitted depressive or anxiety disorders were used from the Netherlands Study of Depressive and Anxiety disorders. Multiple physical symptoms were measured at baseline (T1) and two-year follow-up (T2) by the Four-Dimensional Symptom Questionnaire (4DSQ) somatization subscale. Recurrence of depressive and anxiety disorders was assessed at two-year (T2) and four-year (T4) follow-up with the Composite International Diagnostic Interview. Logistic Generalized Estimating Equations were used to examine associations of multiple physical symptoms with recurrence of depressive and anxiety disorders. Depressive (IDS-SR) and anxiety symptoms (BAI), and other relevant covariates were taken into account. Results Multiple physical symptoms were significantly associated with recurrence of depression (OR = 1.04, 95%CI = 1.00–1.08), anxiety (OR = 1.07, 95%CI = 1.03–1.12), and depressive or anxiety disorders (OR = 1.06, 95%CI = 1.02–1.10), on average over time. Odds ratios did not change substantially when the IDS-SR mood-cognition and BAI subjective scale were included as covariates. Conclusion The presence of multiple physical symptoms was positively related to recurrence of depressive and anxiety disorders, independent of depressive and anxiety symptoms. Knowledge of risk factors for recurrence of depressive and anxiety disorders, such as the presence of multiple physical symptoms, could provide possibilities for better targeting interventions to prevent recurrence

4-Iodopyrimidine labeling reveals nuclear translocation and nuclease activity for both MIF and MIF2

Macrophage migration inhibitory factor (MIF) and its homolog MIF2 (also known as D-dopachrome tautomerase or DDT) play key roles in cell growth and immune responses. MIF and MIF2 expression is dysregulated in cancers and neurodegenerative diseases. Accurate and convenient detection of MIF and MIF2 will facilitate research on their roles in cancer and other diseases. Herein, we report the development and application of a 4-iodopyrimidine based probe 8 for the selective labeling of MIF and MIF2. Probe 8 incorporates a fluorophore that allows in situ imaging of these two proteins. This enabled visualization of the translocation of MIF2 from the cytoplasm to the nucleus upon methylnitronitrosoguanidine stimulation of HeLa cells. This observation, combined with literature on nuclease activity for MIF, enabled the identification of nuclease activity for MIF2 on human genomic DNA

HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity

AbstractThe increasing number of patients suffering from chronic obstructive pulmonary disease (COPD) represents a major and increasing health problem. Therefore, novel therapeutic approaches are needed. Class I HDACs 1, 2 and 3 play key roles in the regulation of inflammatory gene expression with a particular pro-inflammatory role for HDAC 3. HDAC 3 has been reported to be an important player in inflammation by deacetylating NF-κB p65, which has been implicated in the pathology of COPD. Here, we applied the pharmacological HDAC 3-selective inhibitor RGFP966, which attenuated pro-inflammatory gene expression in models for inflammatory lung diseases. Consistent with this, a robust decrease of the transcriptional activity of NF-κB p65 was observed. HDAC 3 inhibition affected neither the acetylation status of NF-κB p65 nor histone H3 or histone H4. This indicates that HDAC 3 inhibition does not inhibit NF-κB p65 transcriptional activity by affecting its deacetylation but rather by inhibiting enzymatic activity of HDAC 3. Taken together, our findings indicate that pharmacological HDAC 3-selective inhibition by inhibitors such as RGFP966 may provide a novel and effective approach toward development of therapeutics for inflammatory lung diseases

Deposition Bias of Chromatin Proteins Inverts under DNA Replication Stress Conditions

Following DNA replication, equal amounts of chromatin proteins are distributed over sister chromatids by re-deposition of parental chromatin proteins and deposition of newly synthesized chromatin proteins. Molecular mechanisms balancing the allocation of new and old chromatin proteins remain largely unknown. Here, we studied the genome-wide distribution of new chromatin proteins relative to parental DNA template strands and replication initiation zones using the double-click-seq. Under control conditions, new chromatin proteins were preferentially found on DNA replicated by the lagging strand machinery. Strikingly, replication stress induced by hydroxyurea or curaxin treatment and inhibition of ataxia telangiectasia and Rad3-related protein (ATR) or p53 inactivation inverted the observed chromatin protein deposition bias to the strand replicated by the leading strand polymerase in line with previously reported effects on replication protein A occupancy. We propose that asymmetric deposition of replication protein occupancy. propose asymmetric deposition newly synthesized chromatin proteins onto sister chromatids reflects differences in the processivity of leading and lagging strand synthesis

Light-controlled inhibition of BRAFV600E kinase

Metastatic melanoma is amongst the most difficult types of cancer to treat, with current therapies mainly relying on the inhibition of the BRAFV600E mutant kinase. However, systemic inhibition of BRAF by small molecule drugs in cancer patients results - paradoxically - in increased wild-type BRAF activity in healthy tissue, causing side-effects and even the formation of new tumors. Here we show the development of BRAFV600E kinase inhibitors of which the activity can be switched on and off reversibly with light, offering the possibility to overcome problems of systemic drug activity by selectively activating the drug at the desired site of action. Based on a known inhibitor, eight photoswitchable effectors containing an azobenzene photoswitch were designed, synthesized and evaluated. The most promising inhibitor showed an approximately 10-fold increase in activity upon light-activation. This research offers inspiration for the development of therapies for metastatic melanoma in which tumor tissue is treated with an active BRAFV600E inhibitor with high spatial and temporal resolution, thus limiting the damage to other tissues

Supported self-help to prevent relapse or recurrence of depression: who benefits most?

Objectives To identify subgroups for whom supported self-help preventive cognitive therapy (S-PCT) is more (cost)effective than treatment as usual (TAU) in preventing relapse and recurrence of major depression. Methods We conducted a randomized controlled trial in which 248 remitted, recurrently depressed participants were randomized to S-PCT (n=124) or TAU (n=124). Clinical outcome was relapse or recurrence of major depressive disorder (SCID-I). We tested the potential moderating effects on relapse or recurrence of age, gender, education level, residual depressive symptoms, number of previous episodes, age of onset, antidepressant medication, somatization, and self-efficacy with logistic regression analyses adjusted for baseline values of depressive symptoms. We examined moderating effects on costs using linear regression analyses adjusted for baseline costs. A stratified cost-effectiveness analysis was performed to tease out differences in cost-effectiveness between subgroups. Results We found no moderating effect on relapse or recurrence for any of the potential moderators. For costs, the number of previous depressive episodes was identified as a moderator. At a willingness-to-pay of 16,000€, the probability that S-PCT was cost-effective compared to TAU was 95% for participants with 2-3 previous episodes and 11% for participants with ≥4 episodes. Conclusions S-PCT was effective in preventing relapse or recurrence of depressive disorders in a broad range of participants, but is more likely to be cost-effective in participants with 2-3 episodes than with ≥4 episodes. This indicates that S-PCT can best be offered to participants with fewer previous depressive episodes

Thieno[2,3- d]pyrimidine-2,4(1 H,3 H)-dione Derivative Inhibits d -Dopachrome Tautomerase Activity and Suppresses the Proliferation of Non-Small Cell Lung Cancer Cells

The homologous cytokines macrophage migration inhibitory factor (MIF) and d-dopachrome tautomerase (d-DT or MIF2) play key roles in cancers. Molecules binding to the MIF tautomerase active site interfere with its biological activity. In contrast, the lack of potent MIF2 inhibitors hinders the exploration of MIF2 as a drug target. In this work, screening of a focused compound collection enabled the identification of a MIF2 tautomerase inhibitor R110. Subsequent optimization provided inhibitor 5d with an IC50 of 1.0 μM for MIF2 tautomerase activity and a high selectivity over MIF. 5d suppressed the proliferation of non-small cell lung cancer cells in two-dimensional (2D) and three-dimensional (3D) cell cultures, which can be explained by the induction of cell cycle arrest via deactivation of the mitogen-activated protein kinase (MAPK) pathway. Thus, we discovered and characterized MIF2 inhibitors (5d) with improved antiproliferative activity in cellular models systems, which indicates the potential of targeting MIF2 in cancer treatment.</p