Local chromatin microenvironment determines DNMT activity: from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase

Insights on active DNA demethylation disproved the original assumption that DNA methylation is a stable epigenetic modification. Interestingly, mammalian DNA methyltransferases 3A and 3B (DNMT-3A and -3B) have also been reported to induce active DNA demethylation, in addition to their well-known function in catalyzing methylation. In situations of extremely low levels of S-adenosyl methionine (SAM), DNMT-3A and -3B might demethylate C-5 methyl cytosine (5mC) via deamination to thymine, which is subsequently replaced by an unmodified cytosine through the base excision repair (BER) pathway. Alternatively, 5mC when converted to 5- hydroxymethylcytosine (5hmC) by TET enzymes, might be further modified to an unmodified cytosine by DNMT-3A and -3B under oxidized redox conditions, although exact pathways are yet to be elucidated. Interestingly, even direct conversion of 5mC to cytosine might be catalyzed by DNMTs. Here, we summarize the evidence on the DNA dehydroxymethylase and demethylase activity of DNMT-3A and -3B. Although physiological relevance needs to be demonstrated, the current indications on the 5mC- and 5hmC-modifying activities of de novo DNA C-5 methyltransferases shed a new light on these enzymes. Despite the extreme circumstances required for such unexpected reactions to occur, we here put forward that the chromatin microenvironment can be locally exposed to extreme conditions, and hypothesize that such waves of extremes allow enzymes to act in differential ways.     

Deciphering the oxygen isotope composition of nitrous oxide produced by nitrification

The ability to use δ¹⁸O values of nitrous oxide (N₂O) to apportion environmental emissions is currently hindered by a poor understanding of the controls on δ¹⁸O–N₂O from nitrification (hydroxylamine oxidation to N₂O and nitrite reduction to N₂O). In this study fertilized agricultural soils and unfertilized temperate forest soils were aerobically incubated with different ¹⁸O/¹⁶O waters, and conceptual and mathematical models were developed to systematically explain the δ¹⁸O–N₂O formed by nitrification. Modeling exercises used a set of defined input parameters to emulate the measured soil δ¹⁸O–N₂O data (Monte Carlo approach). The Monte Carlo simulations implied that abiotic oxygen (O) exchange between nitrite (NO₂^?) and H₂O is important in all soils, but that biological, enzyme‐controlled O‐exchange does not occur during the reduction of NO₂^? to N₂O (nitrifier‐denitrification). Similarly, the results of the model simulations indicated that N₂O consumption is not characteristic of aerobic N₂O formation. The results of this study and a synthesis of the published literature data indicate that δ¹⁸O–N₂O formed in aerobic environments is constrained between +13‰ and +35‰ relative to Vienna Standard Mean Ocean Water (VSMOW). N₂O formed via hydroxylamine oxidation and nitrifier‐denitrification cannot be separated using δ¹⁸O unless ¹⁸O tracers are employed. The natural range of nitrifier δ¹⁸O–N₂O is discussed and explained in terms of our conceptual model, and the major and minor controls that define aerobically produced δ¹⁸O–N₂O are identified. Despite the highly complex nature of δ¹⁸O–N₂O produced by nitrification this δ¹⁸O range is narrow. As a result, in many situations δ¹⁸O values may be used in conjunction with δ¹⁵N–N₂O data to apportion nitrifier‐ and denitrifier‐derived N₂O. However, when biological O‐exchange during denitrification is high and N₂O consumption is low, there may be too much overlap in δ¹⁸O values to distinguish N₂O formed by these pathways.     

Biological Nitrogen Fixation Prevents the Response of a Eutrophic Lake to Reduced Loading of Nitrogen: Evidence from a 46-Year Whole-Lake Experiment

A whole-ecosystem experiment in Lake 227 (L227) at the Experimental Lakes Area, ongoing since 1969, examined the roles of carbon (C), nitrogen (N), and phosphorus (P) in controlling eutrophication. During 2011, we conducted a series of sub-experiments and more intensive monitoring to improve estimates of N fixation and its ability to meet algal growth demands in the decades following the cessation of artificial N loading, while maintaining long-term high artificial P loading. Stoichiometric nutrient ratios indicated both moderate N and P limitation of the phytoplankton during spring, preceding a shift in phytoplankton community structure toward dominance by N fixing cyanobacteria. During bloom development, and for the remainder of the stratified period, stoichiometric nutrient ratios indicated moderate to strong P limitation. N fixation rates, corrected using ¹⁵N₂ methods, increased 2× after 1990, when N loading ceased. Ambient dissolved inorganic nitrogen prior to the bloom represented less than 3% of N demands of the phytoplankton. N fixation accounted for between 69–86% of total N loading to the epilimnion during the period of rapid bloom development, and 72–86% of total N loading during the May–October period. Phytoplankton biomass did not decline in L227 during the 40 years since artificial N loading was reduced, or the nearly 25 years since artificial N loads ceased entirely (1990–2013), and remained approximately 20× higher than four nearby reference lakes. These results suggest that despite constraints on biological N fixation, it retains a large capacity to offset potential N loading reductions in freshwaters.     

From the Ground Up: Global Nitrous Oxide Sources are Constrained by Stable Isotope Values

Rising concentrations of nitrous oxide (N₂O) in the atmosphere are causing widespread concern because this trace gas plays a key role in the destruction of stratospheric ozone and it is a strong greenhouse gas. The successful mitigation of N₂O emissions requires a solid understanding of the relative importance of all N₂O sources and sinks. Stable isotope ratio measurements (δ¹⁵N-N₂O and δ¹⁸O-N₂O), including the intramolecular distribution of ¹⁵N (site preference), are one way to track different sources if they are isotopically distinct. ‘Top-down’ isotope mass-balance studies have had limited success balancing the global N₂O budget thus far because the isotopic signatures of soil, freshwater, and marine sources are poorly constrained and a comprehensive analysis of global N₂O stable isotope measurements has not been done. Here we used a robust analysis of all available in situ measurements to define key global N₂O sources. We showed that the marine source is isotopically distinct from soil and freshwater N₂O (the continental source). Further, the global average source (sum of all natural and anthropogenic sources) is largely controlled by soils and freshwaters. These findings substantiate past modelling studies that relied on several assumptions about the global N₂O cycle. Finally, a two-box-model and a Bayesian isotope mixing model revealed marine and continental N₂O sources have relative contributions of 24–26% and 74–76% to the total, respectively. Further, the Bayesian modeling exercise indicated the N₂O flux from freshwaters may be much larger than currently thought.     

Metabolic regimes of three mid-order streams in southern Ontario,Canada exposed to contrasting sources of nutrients

Light, temperature, and discharge control stream metabolism, but the response of gross primary production (GPP) and ecosystem respiration (ER) to seasonal variation in these physical drivers may differ in accordance with the types of human activities present in the catchment. Our study examined three mid-order streams in southern Ontario, Canada that differed in anthropogenic nutrient sources (i.e., sewage treatment plant effluent, sewage lagoon effluent, and agriculture), but had comparable light, temperature, and discharge regimes. For each stream, GPP and ER were estimated daily from June through November. Comparisons of paired daily metabolic rates revealed pairwise differences among all streams, with streams receiving sewage effluent having greater rates and variability of GPP and ER than the stream draining agricultural land. The two sewage influenced streams differed only in ER. Temporal patterns of GPP and ER were correlated for all streams throughout the study period and were most affected by seasonal variation in temperature, whereby effluent receiving streams responded more rapidly to increases in temperature. Our findings suggest that managers may need to balance effects of human activities with regional environmental constraints on stream metabolism to maintain and enhance the ecological condition and services of stream ecosystems.

     

Antisense recognition of the HER-2 mRNA: effects of phosphorothioate substitution and polyamines on DNA.RNA, RNA.RNA, and DNA.DNA duplex stability

The HER-2 gene is overexpressed in a subset of breast, ovarian, lung, and pancreatic cancers. Antisense oligonucleotides suppress gene expression depending on the stability of the DNA.RNA hybrids formed at the target site. Polyamines, the cellular cations that interact with DNA and RNA, may influence hybrid stability in the cell. Therefore, we studied the ability of natural polyamines (putrescine, spermidine, and spermine) and a series of their structural analogues to stabilize DNA.RNA and RNA.RNA duplexes using melting temperature (T(m)) measurements and circular dichroism (CD) spectroscopy. Phosphodiester (PO) and phosphorothioate (PS) oligonucleotides (ODNs) (15 nucleotides, 5'-CTCCATGGTGCTCAC-3') targeted to the initiation codon region of the HER-2 mRNA, and complementary RNA and DNA ODNs, were used in this study. The relative order of thermal stability was as follows: RNA.RNA > PO-DNA.RNA > PO-DNA.PO-DNA > PS-DNA.RNA > PS-DNA.PO-DNA > PS-DNA.PS-DNA. The ability of polyamines to stabilize the duplexes improved with the cationicity of the polyamine, with hexamines being more effective than pentamines, which in turn were more effective than tetramines and triamines. However, chemical structural effects were clearly evident with isovalent homologues of spermidine and spermine. CD spectra showed B and A conformations, respectively, for the DNA and RNA helices. DNA.RNA hybrids adopted an intermediate structure between the B and A forms. These data help us to understand the role of endogenous polyamines in DNA.RNA hybrid stabilization, and provide information for designing novel polyamines to facilitate the use of antisense ODNs for controlling HER-2 gene expression.     

Large Carbon Dioxide Fluxes from Headwater Boreal and Sub-Boreal Streams

Half of the world''s forest is in boreal and sub-boreal ecozones, containing large carbon stores and fluxes. Carbon lost from headwater streams in these forests is underestimated. We apply a simple stable carbon isotope idea for quantifying the CO₂ loss from these small streams; it is based only on in-stream samples and integrates over a significant distance upstream. We demonstrate that conventional methods of determining CO₂ loss from streams necessarily underestimate the CO₂ loss with results from two catchments. Dissolved carbon export from headwater catchments is similar to CO₂ loss from stream surfaces. Most of the CO₂ originating in high CO₂ groundwaters has been lost before typical in-stream sampling occurs. In the Harp Lake catchment in Canada, headwater streams account for 10% of catchment net CO₂ uptake. In the Krycklan catchment in Sweden, this more than doubles the CO₂ loss from the catchment. Thus, even when corrected for aquatic CO₂ loss measured by conventional methods, boreal and sub-boreal forest carbon budgets currently overestimate carbon sequestration on the landscape.     

Carbon Dioxide and Methane Production in Small Reservoirs Flooding Upland Boreal Forest

The FLooded Uplands Dynamics EXperiment (FLUDEX) was designed to assess the impact of reservoir creation on carbon cycling in boreal forests by (a) determining whether production of the greenhouse gases (GHG) carbon dioxide (CO₂) and methane (CH₄) in reservoirs is related to the amount of organic carbon (OC) stored in the flooded landscape, (b) examining temporal trends in GHG production during initial stages of flooding, and (c) considering the net difference between GHG fluxes before and after flooding to estimate the true effect of reservoir creation on atmospheric GHG levels. Three forested sites that varied in the amount of OC stored in soils and vegetation (30,870–45,860 kg C ha^–1) were experimentally flooded from June to September in 1999–2001. Throughout the study, net CO₂ and CH₄ production in all three reservoirs was not related to overall site OC storage. During the 1st flooding season, net CO₂ production in the three reservoirs was 703–797 kg C ha^–1, but it decreased during the 2nd and 3rd flooding seasons to between 408 and 479 kg C ha^–1. However, CH₄ production increased in all reservoirs with each flooding season, from about 3.2–4.6 kg C ha^–1 in 1999 to 12.8–24.9 kg C ha^–1 in 2000 and 29.7–35.2 kg C ha^–1 in 2001. Over the long term, effects of boreal reservoir creation on atmospheric GHG levels may be largely due to net changes in CH₄ cycling between the undisturbed and flooded ecosystems.