ABSTRACT
We recently demonstrated that accelerating the relaxation of nonphotochemical quenching leads to higher soybean photosynthetic efficiency and yield. In response, Sinclair et al. assert that improved photosynthesis cannot improve crop yields and that there is only one valid experimental design for proving a genetic improvement in yield. We explain here why neither assertion is valid.
Subject(s)
Crops, Agricultural , Glycine max , Photosynthesis , Glycine max/genetics , Glycine max/physiology , Crops, Agricultural/genetics , Crops, Agricultural/physiologyABSTRACT
BACKGROUND: We and others have suggested that pioneer activity - a transcription factor's (TF's) ability to bind and open inaccessible loci - is not a qualitative trait limited to a select class of pioneer TFs. We hypothesize that most TFs display pioneering activity that depends on the TF concentration and the motif content at their target loci. RESULTS: Here, we present a quantitative in vivo measure of pioneer activity that captures the relative difference in a TF's ability to bind accessible versus inaccessible DNA. The metric is based on experiments that use CUT&Tag to measure the binding of doxycycline-inducible TFs. For each location across the genome, we determine the concentration of doxycycline required for a TF to reach half-maximal occupancy; lower concentrations reflect higher affinity. We propose that the relative difference in a TF's affinity between ATAC-seq labeled accessible and inaccessible binding sites is a measure of its pioneer activity. We estimate binding affinities at tens of thousands of genomic loci for the endodermal TFs FOXA1 and HNF4A and show that HNF4A has stronger pioneer activity than FOXA1. We show that both FOXA1 and HNF4A display higher binding affinity at inaccessible sites with more copies of their respective motifs. The quantitative analysis of binding suggests different modes of binding for FOXA1, including an anti-cooperative mode of binding at certain accessible loci. CONCLUSIONS: Our results suggest that relative binding affinities are reasonable measures of pioneer activity and support the model wherein most TFs have some degree of context-dependent pioneer activity.
Subject(s)
DNA , Doxycycline , Binding Sites , DNA/metabolism , Genome , GenomicsABSTRACT
Crop leaves in full sunlight dissipate damaging excess absorbed light energy as heat. This protective dissipation continues after the leaf transitions to shade, reducing crop photosynthesis. A bioengineered acceleration of this adjustment increased photosynthetic efficiency and biomass in tobacco in the field. But could that also translate to increased yield in a food crop? Here we bioengineered the same change into soybean. In replicated field trials, photosynthetic efficiency in fluctuating light was higher and seed yield in five independent transformation events increased by up to 33%. Despite increased seed quantity, seed protein and oil content were unaltered. This validates increasing photosynthetic efficiency as a much needed strategy toward sustainably increasing crop yield in support of future global food security.
Subject(s)
Crop Production , Glycine max , Photosynthesis , Bioengineering , Plant Leaves/metabolism , Glycine max/metabolism , Sunlight , Nicotiana/metabolismABSTRACT
The pioneer factor hypothesis (PFH) states that pioneer factors (PFs) are a subclass of transcription factors (TFs) that bind to and open inaccessible sites and then recruit non-pioneer factors (non-PFs) that activate batteries of silent genes. The PFH predicts that ectopic gene activation requires the sequential activity of qualitatively different TFs. We tested the PFH by expressing the endodermal PF FOXA1 and non-PF HNF4A in K562 lymphoblast cells. While co-expression of FOXA1 and HNF4A activated a burst of endoderm-specific gene expression, we found no evidence for a functional distinction between these two TFs. When expressed independently, both TFs bound and opened inaccessible sites, activated endodermal genes, and 'pioneered' for each other, although FOXA1 required fewer copies of its motif for binding. A subset of targets required both TFs, but the predominant mode of action at these targets did not conform to the sequential activity predicted by the PFH. From these results, we hypothesize an alternative to the PFH where 'pioneer activity' depends not on categorically different TFs but rather on the affinity of interaction between TF and DNA.
Cells only use a fraction of their genetic information to make the proteins they need. The rest is carefully packaged away and tightly bundled in structures called nucleosomes. This physically shields the DNA from being accessed by transcription factors the molecular actors that can read genes and kickstart the protein production process. Effectively, the genetic sequences inside nucleosomes are being silenced. However, during development, transcription factors must overcome this nucleosome barrier and activate silent genes to program cells. The pioneer factor hypothesis describes how this may be possible: first, 'pioneer' transcription factors can bind to and 'open up' nucleosomes to make target genes accessible. Then, non-pioneer factors can access the genetic sequence and recruit cofactors that begin copying the now-exposed genetic information. The widely accepted theory is based on studies of two proteins FOXA1, an archetypal pioneer factor, and HNF4A, a non-pioneer factor but the predictions of the pioneer factor hypothesis have yet to be explicitly tested. To do so, Hansen et al. expressed FOXA1 and HNF4A, separately and together, in cells which do not usually make these proteins. They then assessed how the proteins could bind to DNA and impact gene accessibility and transcription. The experiments demonstrate that FOXA1 and HNF4A do not necessarily follow the two-step activation predicted by the pioneer factor hypothesis. When expressed independently, both transcription factors bound and opened inaccessible sites, activated target genes, and 'pioneered' for each other. Similar patterns were observed across the genome. The only notable distinction between the two factors was that FOXA1, the archetypal pioneering factor, required fewer copies of its target sequence to bind DNA than HNF4A. These findings led Hansen et al. to propose an alternative theory to the pioneer factor hypothesis which eliminates the categorical distinction between pioneer and non-pioneer factors. Overall, this work has implications for how biologists understand the way that transcription factors activate silent genes during development.
Subject(s)
Ectopic Gene Expression , Hepatocyte Nuclear Factor 3-alpha/genetics , Hepatocyte Nuclear Factor 4/genetics , Liver/metabolism , Hepatocyte Nuclear Factor 3-alpha/metabolism , Hepatocyte Nuclear Factor 4/metabolism , Humans , K562 CellsABSTRACT
The physiology of respiration is a challenging subject for many medical students. To assist students, we have developed an active learning game that physically places students within a model outlining the respiratory control pathway. Participants were provided with a vodcast describing the physiology of respiratory control and instructed to view this before the activity. Once in the classroom, groups of students sat at tables marked to represent components of the respiratory control pathway (e.g., apneustic center, diaphragm etc.). Tables were connected with green and red ropes indicating excitatory or inhibitory effects, respectively. Students were presented with various scenarios (e.g., diabetic ketoacidosis) and asked to predict and illustrate the scenario's effect on subsequent steps in the respiratory pathway by waving the appropriate connecting rope. The next table would continue the pattern to simulate the collective physiological adaptation of the respiratory pathway. Thirty first-year medical students participated in this study. Following the activity, 25 out of the 30 participants completed an optional survey. The survey aimed to assess the benefits of adding this activity to our first-year medical curriculum to build a foundational understanding of the physiology of respiration. Responses were overwhelmingly favorable, and participants reported that playing the game significantly improved their perceived understanding of the physiology of respiratory control. All but one of the participants recommended using the activity in future classes. Because the small size of the study group may limit generalizability, future larger scale studies are planned.
Subject(s)
Education, Medical, Undergraduate , Students, Medical , Curriculum , Educational Measurement , Humans , Problem-Based Learning , Surveys and QuestionnairesABSTRACT
The review begins with a concise description of the principles of phase separation. This is followed by a comprehensive section on phase separation of chromatin, in which we recount the 60 years history of chromatin aggregation studies, discuss the evidence that chromatin aggregation intrinsically is a physiologically relevant liquid-solid phase separation (LSPS) process driven by chromatin self-interaction, and highlight the recent findings that under specific solution conditions chromatin can undergo liquid-liquid phase separation (LLPS) rather than LSPS. In the next section of the review, we discuss how certain chromatin-associated proteins undergo LLPS in vitro and in vivo. Some chromatin-binding proteins undergo LLPS in purified form in near-physiological ionic strength buffers while others will do so only in the presence of DNA, nucleosomes, or chromatin. The final section of the review evaluates the solid and liquid states of chromatin in the nucleus. While chromatin behaves as an immobile solid on the mesoscale, nucleosomes are mobile on the nanoscale. We discuss how this dual nature of chromatin, which fits well the concept of viscoelasticity, contributes to genome structure, emphasizing the dominant role of chromatin self-interaction.
Subject(s)
Chromatin , Nucleosomes , Cell Nucleus , DNASubject(s)
Polycythemia , Transgender Persons , Follow-Up Studies , Humans , Male , Polycythemia/chemically induced , Polycythemia/epidemiology , Prevalence , TestosteroneABSTRACT
The association of nuclear DNA with histones to form chromatin is essential for temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin in vitro and in vivo. Our in vitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create an elastic gel and provides a scaffold that supports liquid-liquid phase separation of chromatin binding proteins.
Subject(s)
Chromatin/metabolism , Acetylation/drug effects , Animals , Cell Line , Cell Survival/drug effects , Chromatin/drug effects , DNA Damage , Euchromatin/metabolism , Fluorescence , Heterochromatin/metabolism , Histone Deacetylase Inhibitors/pharmacology , Lasers , Mice , Models, Biological , Osmolar Concentration , PhotobleachingABSTRACT
Eukaryotic chromatin is a negatively charged polymer consisting of genomic DNA, histones, and various nonhistone proteins. Because of its highly charged character, the structure of chromatin varies greatly depending on the surrounding environment (i.e. cations etc.): from an extended 10-nm fiber, to a folded 30-nm fiber, to chromatin condensates/liquid-droplets. Over the last ten years, newly developed technologies have drastically shifted our view on chromatin from a static regular structure to a more irregular and dynamic one, locally like a fluid. Since no single imaging (or genomics) method can tell us everything and beautiful images (or models) can fool our minds, comprehensive analyses based on many technical approaches are important to capture actual chromatin organization inside the cell. Here we critically discuss our current view on chromatin and methodology used to support the view.
Subject(s)
Chromatin/metabolism , Adenosine Triphosphate/metabolism , Animals , Cell Survival , Chromatin/chemistry , DNA/genetics , Histones/metabolism , Humans , Nucleosomes/metabolismABSTRACT
Sulfur hexafluoride (SF6 ) is an established tracer for use in managed aquifer recharge projects. SF6 exsolves from groundwater when it encounters trapped air according to Henry's law. This results in its retardation relative to groundwater flow, which can help determine porous media saturation and flow dynamics. SF6 and the conservative, nonpartitioning tracer, bromide (Br- added as KBr), were introduced to recharge water infiltrated into stacked glacial aquifers in Thurston County, Washington, providing the opportunity to observe SF6 partitioning. Br- , which is assumed to travel at the same velocity as the groundwater, precedes SF6 at most monitoring wells (MWs). Average groundwater velocity in the unconfined aquifer in the study area ranges from 3.9 to 40 m/d, except in the southwestern corner where it is slower. SF6 in the shallow aquifer exhibits an average retardation factor of 2.5 ± 3.8, suggesting an air-to-water ratio on the order of 10-3 to 10-2 in the pore space. Notable differences in tracer arrival times at adjacent wells indicate very heterogeneous conductivity. One MW exhibits double peaks in concentrations of both tracers with different degrees of retardation for the first and second peaks. This suggests multiple flowpaths to the well with variable saturation. The confining layer between the upper two aquifers appears to allow intermittent connection between aquifers but serves as an aquitard in most areas. This study demonstrates the utility of SF6 partitioning for evaluating hydrologic conditions at prospective recharge sites.
Subject(s)
Groundwater , Bromides , Potassium Compounds , Prospective Studies , Sulfur Hexafluoride , WashingtonABSTRACT
The dynamic structure of chromatin is linked to gene regulation and many other biological functions. Consequently, it is of importance to understand the factors that regulate chromatin dynamics. While the in vivo analysis of chromatin has verified that histone post-translational modifications play a role in modulating DNA accessibility, the complex nuclear environment and multiplicity of modifications prevents clear conclusions as to how individual modifications influence chromatin dynamics in the cell. For this reason, in vitro analyses of model reconstituted nucleosomal arrays has been pivotal in understanding the dynamic nature of chromatin compaction and the affects that specific post-translational modifications can have on the higher order chromatin structure. In this mini-review, we briefly describe the dynamic chromatin structures that have been observed in vitro and the environmental conditions that give rise to these various conformational states. Our focus then turns to a discussion of the specific histone post-translational modifications that have been shown to alter formation of these higher order chromatin structures in vitro and how this may relate to the biological state and accessibility of chromatin in vivo.
Subject(s)
Histones/metabolism , Nucleosomes/metabolism , Protein Processing, Post-Translational , Acetylation , DNA/chemistry , DNA/metabolism , Histones/chemistry , Humans , Methylation , Nucleosomes/chemistry , Protein Folding , Protein Structure, Quaternary , SumoylationABSTRACT
Total dissolved solids (TDS) concentrations in groundwater tapped for beneficial uses (drinking water, irrigation, freshwater industrial) have increased on average by about 100â¯mg/L over the last 100â¯years in the San Joaquin Valley, California (SJV). During this period land use in the SJV changed from natural vegetation and dryland agriculture to dominantly irrigated agriculture with growing urban areas. Century-scale salinity trends were evaluated by comparing TDS concentrations and major ion compositions of groundwater from wells sampled in 1910 (Historic) to data from wells sampled in 1993-2015 (Modern). TDS concentrations in subregions of the SJV, the southern (SSJV), western (WSJV), northeastern (NESJV), and southeastern (SESJV) were calculated using a cell-declustering method. TDS concentrations increased in all regions, with the greatest increases found in the SSJV and SESJV. Evaluation of the Modern data from the NESJV and SESJV found higher TDS concentrations in recently recharged (post-1950) groundwater from shallow (<50â¯m) wells surrounded predominantly by agricultural land uses, while premodern (pre-1950) groundwater from deeper wells, and recently recharged groundwater from wells surrounded by mainly urban, natural, and mixed land uses had lower TDS concentrations, approaching the TDS concentrations in the Historic groundwater. For the NESJV and SESJV, inverse geochemical modeling with PHREEQC indicated that weathering of primary silicate minerals accounted for the majority of the increase in TDS concentrations, contributing more than nitrate from fertilizers and sulfate from soil amendments combined. Bicarbonate showed the greatest increase among major ions, resulting from enhanced silicate weathering due to recharge of irrigation water enriched in CO2 during the growing season. The results of this study demonstrate that large anthropogenic changes to the hydrologic regime, like massive development of irrigated agriculture in semi-arid areas like the SJV, can cause large changes in groundwater quality on a regional scale.
Subject(s)
Environmental Monitoring , Groundwater/chemistry , Salinity , Water Pollutants/analysis , Water Supply/statistics & numerical data , Agriculture , California , Water Pollutants, ChemicalABSTRACT
The process of transcriptional elongation by RNA polymerase II (RNAPII) in a chromatin context involves a large number of crucial factors. Spn1 is a highly conserved protein encoded by an essential gene and is known to interact with RNAPII and the histone chaperone Spt6. Spn1 negatively regulates the ability of Spt6 to interact with nucleosomes, but the chromatin binding properties of Spn1 are largely unknown. Here, we demonstrate that full length Spn1 (amino acids 1-410) binds DNA, histones H3-H4, mononucleosomes and nucleosomal arrays, and has weak nucleosome assembly activity. The core domain of Spn1 (amino acids 141-305), which is necessary and sufficient in Saccharomyces cerevisiae for growth under ideal growth conditions, is unable to optimally interact with histones, nucleosomes and/or DNA and fails to assemble nucleosomes in vitro. Although competent for binding with Spt6 and RNAPII, the core domain derivative is not stably recruited to the CYC1 promoter, indicating chromatin interactions are an important aspect of normal Spn1 functions in vivo. Moreover, strong synthetic genetic interactions are observed with Spn1 mutants and deletions of histone chaperone genes. Taken together, these results indicate that Spn1 is a histone binding factor with histone chaperone functions.
Subject(s)
Nucleosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Cytochromes c/genetics , DNA/metabolism , Histone Chaperones/metabolism , Histones/metabolism , Promoter Regions, Genetic , RNA Polymerase II/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Transcriptional Elongation Factors/metabolismABSTRACT
A chromosome is a single long DNA molecule assembled along its length with nucleosomes and proteins. During interphase, a mammalian chromosome exists as a highly organized supramolecular globule in the nucleus. Here, we discuss new insights into how genomic DNA is packaged and organized within interphase chromosomes. Our emphasis is on the structural principles that underlie chromosome organization, with a particular focus on the intrinsic contributions of the 10-nm chromatin fiber, but not the regular 30-nm fiber. We hypothesize that the hierarchical globular organization of an interphase chromosome is fundamentally established by the self-interacting properties of a 10-nm zig-zag array of nucleosomes, while histone post-translational modifications, histone variants, and chromatin-associated proteins serve to mold generic chromatin domains into specific structural and functional entities.
Subject(s)
Chromatin/metabolism , Chromosomes , Interphase , Animals , DNA Packaging , HeLa Cells , Humans , Nucleosomes/metabolism , Protein Processing, Post-TranslationalABSTRACT
The activation of a silent gene locus is thought to involve pioneering transcription factors that initiate changes in the local chromatin structure to increase promoter accessibility and binding of downstream effectors. To better understand the molecular requirements for the first steps of locus activation, we investigated whether acetylation of a single nucleosome is sufficient to alter DNA accessibility within a condensed 25-nucleosome array. We found that acetylation mimics within the histone H4 tail domain increased accessibility of the surrounding linker DNA, with the increased accessibility localized to the immediate vicinity of the modified nucleosome. In contrast, acetylation mimics within the H3 tail had little effect, but were able to synergize with H4 tail acetylation mimics to further increase accessibility. Moreover, replacement of the central nucleosome with a nucleosome free region also resulted in increased local, but not global DNA accessibility. Our results indicate that modification or disruption of only a single target nucleosome results in significant changes in local chromatin architecture and suggest that very localized chromatin modifications imparted by pioneer transcription factors are sufficient to initiate a cascade of events leading to promoter activation.
Subject(s)
DNA/metabolism , Histones/metabolism , Nucleosomes/metabolism , Acetylation , Animals , Chromatin/metabolism , Chromatin/ultrastructure , Deoxyribonucleases, Type II Site-Specific/genetics , Deoxyribonucleases, Type II Site-Specific/metabolism , Histones/genetics , Lysine/metabolism , Lytechinus/genetics , Nucleosomes/genetics , Templates, Genetic , Xenopus/geneticsABSTRACT
The existence of a 30-nm fiber as a basic folding unit for DNA packaging has remained a topic of active discussion. Here, we characterize the supramolecular structures formed by reversible Mg(2+)-dependent self-association of linear 12-mer nucleosomal arrays using microscopy and physicochemical approaches. These reconstituted chromatin structures, which we call "oligomers", are globular throughout all stages of cooperative assembly and range in size from ~50 nm to a maximum diameter of ~1,000 nm. The nucleosomal arrays were packaged within the oligomers as interdigitated 10-nm fibers, rather than folded 30-nm structures. Linker DNA was freely accessible to micrococcal nuclease, although the oligomers remained partially intact after linker DNA digestion. The organization of chromosomal fibers in human nuclei in situ was stabilized by 1 mM MgCl2, but became disrupted in the absence of MgCl2, conditions that also dissociated the oligomers in vitro These results indicate that a 10-nm array of nucleosomes has the intrinsic ability to self-assemble into large chromatin globules stabilized by nucleosome-nucleosome interactions, and suggest that the oligomers are a good in vitro model for investigating the structure and organization of interphase chromosomes.
Subject(s)
Nucleosomes/metabolism , DNA/metabolism , HeLa Cells , Humans , Magnesium Chloride/pharmacology , Micrococcal Nuclease/metabolism , Nucleosomes/drug effectsABSTRACT
Chromatin DNA must be read out for various cellular functions, and copied for the next cell division. These processes are targets of many anticancer agents. Platinum-based drugs, such as cisplatin, have been used extensively in cancer chemotherapy. The drug-DNA interaction causes DNA crosslinks and subsequent cytotoxicity. Recently, it was reported that an azolato-bridged dinuclear platinum(II) complex, 5-H-Y, exhibits a different anticancer spectrum from cisplatin. Here, using an interdisciplinary approach, we reveal that the cytotoxic mechanism of 5-H-Y is distinct from that of cisplatin. 5-H-Y inhibits DNA replication and also RNA transcription, arresting cells in the S/G2 phase, and are effective against cisplatin-resistant cancer cells. Moreover, it causes much less DNA crosslinking than cisplatin, and induces chromatin folding. 5-H-Y will expand the clinical applications for the treatment of chemotherapy-insensitive cancers.
Subject(s)
Antineoplastic Agents/pharmacology , Chromatin Assembly and Disassembly/drug effects , DNA Replication/drug effects , Organoplatinum Compounds/pharmacology , Tetrazoles/pharmacology , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cisplatin/pharmacology , DNA Damage , DNA Repair , Histones/metabolism , Humans , Organoplatinum Compounds/chemistry , Tetrazoles/chemistry , Transcription, Genetic/drug effectsABSTRACT
Telomerase biogenesis is a highly regulated process that solves the DNA end-replication problem. Recombinant expression has so far been accomplished only within a eukaryotic background. Towards structural and functional analyses, we developed bacterial expression of human telomerase. Positive activity by the telomerase repeat amplification protocol (TRAP) was identified in cell extracts of Escherichia coli expressing a sequence-optimized hTERT gene, the full-length hTR RNA with a self-splicing hepatitis delta virus ribozyme, and the human heat shock complex of Hsp90, Hsp70, p60/Hop, Hsp40, and p23. The Hsp90 inhibitor geldanamycin did not affect post-assembly TRAP activity. By various purification methods, TRAP activity was also obtained upon expression of only hTERT and hTR. hTERT was confirmed by tandem mass spectrometry in a â¼120 kDa SDS-PAGE fragment from a TRAP-positive purification fraction. TRAP activity was also supported by hTR constructs lacking the box H/ACA small nucleolar RNA domain. End-point TRAP indicated expression levels within 3-fold of that from HeLa carcinoma cells, which is several orders of magnitude below detection by the direct assay. These results represent the first report of TRAP activity from a bacterium and provide a facile system for the investigation of assembly factors and anti-cancer therapeutics independently of a eukaryotic setting.
Subject(s)
Escherichia coli/genetics , RNA/genetics , Telomerase/genetics , Amino Acid Sequence , Cloning, Molecular , DNA, Complementary/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Plasmids/genetics , RNA/chemistry , RNA/isolation & purification , RNA/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Telomerase/chemistry , Telomerase/isolation & purification , Telomerase/metabolism , Transformation, GeneticABSTRACT
Linker histones H1 are ubiquitous chromatin proteins that play important roles in chromatin compaction, transcription regulation, nucleosome spacing and chromosome spacing. H1 function in DNA and chromatin structure stabilization is well studied and established. The current paradigm of linker histone mode of function considers all other cellular roles of linker histones to be a consequence from H1 chromatin compaction and repression. Here we review the multiple processes regulated by linker histones and the emerging importance of protein interactions in H1 functioning. We propose a new paradigm which explains the multi functionality of linker histones through linker histones protein interactions as a way to directly regulate recruitment of proteins to chromatin.