Distinct roles of DBHS family members in the circadian transcriptional feedback loop

Factors interacting with core circadian clock components are essential to achieve transcriptional feedback necessary for metazoan clocks. Here we show that all three members of the Drosophila Behavior Human Splicing (DBHS) family of RNA-binding proteins play a role in the mammalian circadian oscillator, abrogating or altering clock function when overexpressed or depleted in cells. Although these proteins are members of so-called nuclear paraspeckles, depletion of paraspeckles themselves via silencing of the structural non-coding RNA (ncRNA) Neat1 did not affect overall clock function, suggesting that paraspeckles are not required for DBHS-mediated circadian effects. Instead, we show that the proteins bound to circadian promoter DNA in a fashion that required the PERIOD (PER) proteins, and potently repressed E box-mediated transcription but not CMV promoter-mediated transcription when exogenously recruited. Nevertheless, mice with one or both copies of these genes deleted show only small changes in period length or clock gene expression in vivo. Data from transient transfections show that each of these proteins can either repress or activate depending on the context. Taken together, our data suggest that all of the DBHS family members serve overlapping or redundant roles as transcriptional cofactors at circadian clock-regulated genes

Building block libraries and structural considerations in the self-assembly of polyoxometalate and polyoxothiometalate systems

Inorganic metal-oxide clusters form a class of compounds that are unique in their topological and electronic versatility and are becoming increasingly more important in a variety of applications. Namely, Polyoxometalates (POMs) have shown an unmatched range of physical properties and the ability to form structures that can bridge several length scales. The formation of these molecular clusters is often ambiguous and is governed by self-assembly processes that limit our ability to rationally design such molecules. However, recent years have shown that by considering new building block principles the design and discovery of novel complex clusters is aiding our understanding of this process. Now with current progress in thiometalate chemistry, specifically polyoxothiometalates (POTM), the field of inorganic molecular clusters has further diversified allowing for the targeted development of molecules with specific functionality. This chapter discusses the main differences between POM and POTM systems and how this affects synthetic methodologies and reactivities. We will illustrate how careful structural considerations can lead to the generation of novel building blocks and further deepen our understanding of complex systems

Affinity-restricted memory B cells dominate recall responses to heterologous flaviviruses

Memory B cells (MBCs) can respond to heterologous antigens either by molding new specificities through secondary germinal centers (GCs) or selecting pre-existing clones without further affinity maturation. To distinguish these mechanisms in flavivirus infections and immunizations, we studied recall responses to envelope protein domain III (DIII). Conditional deletion of activation induced cytidine deaminase (AID) between heterologous challenges of West Nile, Japanese encephalitis, Zika, and Dengue viruses did not affect recall responses. DIII-specific MBCs were contained mostly within the plasma cell-biased CD80(+) subset and few GCs arose following heterologous boosters, demonstrating that recall responses are confined by pre-existing clonal diversity. Measurement of monoclonal antibody binding affinity to DIII proteins, timed AID deletion, single cell RNA-sequencing, and lineage tracing experiments point to selection of relatively low affinity MBCs as a mechanism to promote diversity. Engineering immunogens to avoid this MBC diversity may facilitate flavivirus type-specific vaccines with minimized potential for infection enhancement

Silicate weathering and carbon cycle controls on the Oligocene-Miocene transition glaciation

Changes in both silicate weathering rates and organic carbon burial have been proposed as drivers of the transient “Mi-1” glaciation event at the Oligocene-Miocene transition (OMT; ~23 Ma). However detailed geochemical proxy data are required to test these hypotheses. Here we present records of Li/Ca, Mg/Ca, Cd/Ca, U/Ca, δ18O, δ13C, and shell weight in planktonic foraminifera from marine sediments spanning the OMT in the equatorial Atlantic Ocean. Li/Ca values increase by 1 μmol/mol across this interval. We interpret this to indicate a ~20% increase in silicate weathering rates, which would have lowered atmospheric CO2, potentially forcing the Antarctic glaciation circa 23 Ma. δ13C of thermocline dwelling planktonic foraminifera track the global increase in seawater δ13C across the OMT and during the Mi-1 event, hence supporting a hypothesized global increase in organic carbon burial rates. High δ13C previously measured in epipelagic planktonic foraminifera and high Cd/Ca ratios during Mi-1 are interpreted to represent locally enhanced primary productivity, stimulated by increased nutrients supply to surface waters. The fingerprint of high export production and associated organic carbon burial at this site is found in reduced bottom water oxygenation (inferred from high foraminiferal U/Ca), and enhanced respiratory dissolution of carbonates, characterised by reduced foraminiferal shell weight. Replication of our results elsewhere would strengthen the case that weathering-induced CO2 sequestration preconditioned climate for Antarctic ice sheet growth across the OMT and increased burial of organic carbon acted as a feedback that intensified cooling at this time

CHD1 Remodels Chromatin and Influences Transient DNA Methylation at the Clock Gene frequency

Circadian-regulated gene expression is predominantly controlled by a transcriptional negative feedback loop, and it is evident that chromatin modifications and chromatin remodeling are integral to this process in eukaryotes. We previously determined that multiple ATP–dependent chromatin-remodeling enzymes function at frequency (frq). In this report, we demonstrate that the Neurospora homologue of chd1 is required for normal remodeling of chromatin at frq and is required for normal frq expression and sustained rhythmicity. Surprisingly, our studies of CHD1 also revealed that DNA sequences within the frq promoter are methylated, and deletion of chd1 results in expansion of this methylated domain. DNA methylation of the frq locus is altered in strains bearing mutations in a variety of circadian clock genes, including frq, frh, wc-1, and the gene encoding the frq antisense transcript (qrf). Furthermore, frq methylation depends on the DNA methyltransferase, DIM-2. Phenotypic characterization of Δdim-2 strains revealed an approximate WT period length and a phase advance of approximately 2 hours, indicating that methylation plays only an ancillary role in clock-regulated gene expression. This suggests that DNA methylation, like the antisense transcript, is necessary to establish proper clock phasing but does not control overt rhythmicity. These data demonstrate that the epigenetic state of clock genes is dependent on normal regulation of clock components

Small-molecule inhibition of METTL3 as a strategy against myeloid leukaemia.

N6-methyladenosine (m6A) is an abundant internal RNA modification1,2 that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex3,4. The m6A methyltransferase METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown5-7. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m6A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy

Mutation screening of the RNF8, UBC13 and MMS2 genes in Northern Finnish breast cancer families

<p>Abstract</p> <p>Background</p> <p>Currently known susceptibility genes such as <it>BRCA1 </it>and <it>BRCA2 </it>explain less than 25% of familial aggregation of breast cancer, which suggests the involvement of additional susceptibility genes. RNF8, UBC13 and MMS2 are involved in the DNA damage response pathway and play important roles in BRCA1-mediated DNA damage recognition. Based on the evidence that several players in the ubiquitin-mediated BRCA1-dependent DDR seem to contribute to breast cancer predisposition, <it>RNF8, UBC13 </it>and <it>MMS2 </it>were considered plausible candidate genes for susceptibility to breast cancer.</p> <p>Methods</p> <p>The entire coding region and splice junctions of <it>RNF8, UBC13 </it>and <it>MMS2 </it>genes were screened for mutations in affected index cases from 123 Northern Finnish breast cancer families by using conformation sensitive gel electrophoresis, high resolution melting (HRM) analysis and direct sequencing.</p> <p>Results</p> <p>Mutation analysis revealed several changes in <it>RNF8 </it>and <it>UBC13</it>, whereas no aberrations were observed in <it>MMS2</it>. None of the found sequence changes appeared to associate with breast cancer susceptibility.</p> <p>Conclusions</p> <p>The present data suggest that mutations in <it>RNF8, UBC13 </it>and <it>MMS2 </it>genes unlikely make any sizeable contribution to breast cancer predisposition in Northern Finland.</p

Screening for BRCA1, BRCA2, CHEK2, PALB2, BRIP1, RAD50, and CDH1 mutations in high-risk Finnish BRCA1/2-founder mutation-negative breast and/or ovarian cancer individuals

Two major high-penetrance breast cancer genes, BRCA1 and BRCA2, are responsible for approximately 20% of hereditary breast cancer (HBC) cases in Finland. Additionally, rare mutations in several other genes that interact with BRCA1 and BRCA2 increase the risk of HBC. Still, a majority of HBC cases remain unexplained which is challenging for genetic counseling. We aimed to analyze additional mutations in HBC-associated genes and to define the sensitivity of our current BRCA1/2 mutation analysis protocol used in genetic counseling