Decoding chromatin states by proteomic profiling of nucleosome readers.

DNA and histone modifications combine into characteristic patterns that demarcate functional regions of the genome1,2. While many 'readers' of individual modifications have been described3-5, how chromatin states comprising composite modification signatures, histone variants and internucleosomal linker DNA are interpreted is a major open question. Here we use a multidimensional proteomics strategy to systematically examine the interaction of around 2,000 nuclear proteins with over 80 modified dinucleosomes representing promoter, enhancer and heterochromatin states. By deconvoluting complex nucleosome-binding profiles into networks of co-regulated proteins and distinct nucleosomal features driving protein recruitment or exclusion, we show comprehensively how chromatin states are decoded by chromatin readers. We find highly distinctive binding responses to different features, many factors that recognize multiple features, and that nucleosomal modifications and linker DNA operate largely independently in regulating protein binding to chromatin. Our online resource, the Modification Atlas of Regulation by Chromatin States (MARCS), provides in-depth analysis tools to engage with our results and advance the discovery of fundamental principles of genome regulation by chromatin states.

CpG island reconfiguration for the establishment and synchronization of polycomb functions upon exit from naive pluripotency.

Polycomb group (PcG) proteins are essential for post-implantation development by depositing repressive histone modifications at promoters, mainly CpG islands (CGIs), of developmental regulator genes. However, promoter PcG marks are erased after fertilization and de novo established in peri-implantation embryos, coinciding with the transition from naive to primed pluripotency. Nevertheless, the molecular basis for this establishment remains unknown. In this study, we show that the expression of the long KDM2B isoform (KDM2BLF), which contains the demethylase domain, is specifically induced at peri-implantation and that its H3K36me2 demethylase activity is required for PcG enrichment at CGIs. Moreover, KDM2BLF interacts with BRG1/BRM-associated factor (BAF) and stabilizes BAF occupancy at CGIs for subsequent gain of accessibility, which precedes PcG enrichment. Consistently, KDM2BLF inactivation results in significantly delayed post-implantation development. In summary, our data unveil dynamic chromatin configuration of CGIs during exit from naive pluripotency and provide a conceptual framework for the spatiotemporal establishment of PcG functions.

Complex-dependent histone acetyltransferase activity of KAT8 determines its role in transcription and cellular homeostasis.

Acetylation of lysine 16 on histone H4 (H4K16ac) is catalyzed by histone acetyltransferase KAT8 and can prevent chromatin compaction in vitro. Although extensively studied in Drosophila, the functions of H4K16ac and two KAT8-containing protein complexes (NSL and MSL) are not well understood in mammals. Here, we demonstrate a surprising complex-dependent activity of KAT8: it catalyzes H4K5ac and H4K8ac as part of the NSL complex, whereas it catalyzes the bulk of H4K16ac as part of the MSL complex. Furthermore, we show that MSL complex proteins and H4K16ac are not required for cell proliferation and chromatin accessibility, whereas the NSL complex is essential for cell survival, as it stimulates transcription initiation at the promoters of housekeeping genes. In summary, we show that KAT8 switches catalytic activity and function depending on its associated proteins and that, when in the NSL complex, it catalyzes H4K5ac and H4K8ac required for the expression of essential genes.

Phosphorylation of the alpha-I motif in SYMRK drives root nodule organogenesis.

Symbiosis receptor-like kinase SYMRK is required for root nodule symbiosis between legume plants and nitrogen-fixing bacteria. To understand symbiotic signaling from SYMRK, we determined the crystal structure to 1.95 Å and mapped the phosphorylation sites onto the intracellular domain. We identified four serine residues in a conserved "alpha-I" motif, located on the border between the kinase core domain and the flexible C-terminal tail, that, when phosphorylated, drives organogenesis. Substituting the four serines with alanines abolished symbiotic signaling, while substituting them with phosphorylation-mimicking aspartates induced the formation of spontaneous nodules in the absence of bacteria. These findings show that the signaling pathway controlling root nodule organogenesis is mediated by SYMRK phosphorylation, which may help when engineering this trait into non-legume plants.

Saccades are locked to the phase of alpha oscillations during natural reading.

We saccade 3 to 5 times per second when reading. However, little is known about the neuronal mechanisms coordinating the oculomotor and visual systems during such rapid processing. Here, we ask if brain oscillations play a role in the temporal coordination of the visuomotor integration. We simultaneously acquired MEG and eye-tracking data while participants read sentences silently. Every sentence was embedded with a target word of either high or low lexical frequency. Our key finding demonstrated that saccade onsets were locked to the phase of alpha oscillations (8 to 13 Hz), and in particular, for saccades towards low frequency words. Source modelling demonstrated that the alpha oscillations to which the saccades were locked, were generated in the right-visual motor cortex (BA 7). Our findings suggest that the alpha oscillations serve to time the processing between the oculomotor and visual systems during natural reading, and that this coordination becomes more pronounced for demanding words.

Suppression of distracting inputs by visual-spatial cues is driven by anticipatory alpha activity.

A growing body of research demonstrates that distracting inputs can be proactively suppressed via spatial cues, nonspatial cues, or experience, which are governed by more than one top-down mechanism of attention. However, how the neural mechanisms underlying spatial distractor cues guide proactive suppression of distracting inputs remains unresolved. Here, we recorded electroencephalography signals from 110 participants in 3 experiments to identify the role of alpha activity in proactive distractor suppression induced by spatial cues and its influence on subsequent distractor inhibition. Behaviorally, we found novel changes in the spatial proximity of the distractor: Cueing distractors far away from the target improves search performance for the target, while cueing distractors close to the target hampers performance. Crucially, we found dynamic characteristics of spatial representation for distractor suppression during anticipation. This result was further verified by alpha power increased relatively contralateral to the cued distractor. At both the between- and within-subjects levels, we found that these activities further predicted the decrement of the subsequent PD component, which was indicative of reduced distractor interference. Moreover, anticipatory alpha activity and its link with the subsequent PD component were specific to the high predictive validity of distractor cue. Together, our results reveal the underlying neural mechanisms by which cueing the spatial distractor may contribute to reduced distractor interference. These results also provide evidence supporting the role of alpha activity as gating by proactive suppression.

Top-down ion mobility/mass spectrometry reveals enzyme specificity: Separation and sequencing of isomeric proteoforms.

Enzymatic catalysis is one of the fundamental processes that drives the dynamic landscape of post-translational modifications (PTMs), expanding the structural and functional diversity of proteins. Here, we assessed enzyme specificity using a top-down ion mobility spectrometry (IMS) and tandem mass spectrometry (MS/MS) workflow. We successfully applied trapped IMS (TIMS) to investigate site-specific N-ε-acetylation of lysine residues of full-length histone H4 catalyzed by histone lysine acetyltransferase KAT8. We demonstrate that KAT8 exhibits a preference for N-ε-acetylation of residue K16, while also adding acetyl groups on residues K5 and K8 as the first degree of acetylation. Achieving TIMS resolving power values of up to 300, we fully separated mono-acetylated regioisomers (H4K5ac, H4K8ac, and H4K16ac). Each of these separated regioisomers produce unique MS/MS fragment ions, enabling estimation of their individual mobility distributions and the exact localization of the N-ε-acetylation sites. This study highlights the potential of top-down TIMS-MS/MS for conducting enzymatic assays at the intact protein level and, more generally, for separation and identification of intact isomeric proteoforms and precise PTM localization.

Statistical Learning of Distractor Suppression Downregulates Prestimulus Neural Excitability in Early Visual Cortex.

Visual attention is highly influenced by past experiences. Recent behavioral research has shown that expectations about the spatial location of distractors within a search array are implicitly learned, with expected distractors becoming less interfering. Little is known about the neural mechanism supporting this form of statistical learning. Here, we used magnetoencephalography (MEG) to measure human brain activity to test whether proactive mechanisms are involved in the statistical learning of distractor locations. Specifically, we used a new technique called rapid invisible frequency tagging (RIFT) to assess neural excitability in early visual cortex during statistical learning of distractor suppression while concurrently investigating the modulation of posterior alpha band activity (8-12 Hz). Male and female human participants performed a visual search task in which a target was occasionally presented alongside a color-singleton distractor. Unbeknown to the participants, the distracting stimuli were presented with different probabilities across the two hemifields. RIFT analysis showed that early visual cortex exhibited reduced neural excitability in the prestimulus interval at retinotopic locations associated with higher distractor probabilities. In contrast, we did not find any evidence of expectation-driven distractor suppression in alpha band activity. These findings indicate that proactive mechanisms of attention are involved in predictive distractor suppression and that these mechanisms are associated with altered neural excitability in early visual cortex. Moreover, our findings indicate that RIFT and alpha band activity might subtend different and possibly independent attentional mechanisms.SIGNIFICANCE STATEMENT What we experienced in the past affects how we perceive the external world in the future. For example, an annoying flashing light might be better ignored if we know in advance where it usually appears. This ability of extracting regularities from the environment is called statistical learning. In this study, we explore the neuronal mechanisms allowing the attentional system to overlook items that are unequivocally distracting based on their spatial distribution. By recording brain activity using MEG while probing neural excitability with a novel technique called RIFT, we show that the neuronal excitability in early visual cortex is reduced in advance of stimulus presentation for locations where distracting items are more likely to occur.

Spatiotemporal Properties of Common Semantic Categories for Words and Pictures.

The timing of semantic processing during object recognition in the brain is a topic of ongoing discussion. One way of addressing this question is by applying multivariate pattern analysis to human electrophysiological responses to object images of different semantic categories. However, although multivariate pattern analysis can reveal whether neuronal activity patterns are distinct for different stimulus categories, concerns remain on whether low-level visual features also contribute to the classification results. To circumvent this issue, we applied a cross-decoding approach to magnetoencephalography data from stimuli from two different modalities: images and their corresponding written words. We employed items from three categories and presented them in a randomized order. We show that if the classifier is trained on words, pictures are classified between 150 and 430 msec after stimulus onset, and when training on pictures, words are classified between 225 and 430 msec. The topographical map, identified using a searchlight approach for cross-modal activation in both directions, showed left lateralization, confirming the involvement of linguistic representations. These results point to semantic activation of pictorial stimuli occurring at ≈150 msec, whereas for words, the semantic activation occurs at ≈230 msec.

Multiplatform High-Definition Ion Mobility Separations of the Largest Epimeric Peptides.

Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) has become a versatile tool to fractionate complex mixtures, distinguish structural isomers, and elucidate molecular geometries. Along with the whole MS field, IMS/MS advances to ever larger species. A topical proteomic problem is the discovery and characterization of d-amino acid-containing peptides (DAACPs) that are critical to neurotransmission and toxicology. Both linear IMS and FAIMS previously disentangled d/l epimers with up to ∼30 residues. In the first study using all three most powerful IMS methodologies─trapped IMS, cyclic IMS, and FAIMS─we demonstrate baseline resolution of the largest known d/l peptides (CHH from Homarus americanus with 72 residues) with a dynamic range up to 100. This expands FAIMS analyses of isomeric modified peptides, especially using hydrogen-rich buffers, to the ∼50-100 residue range of small proteins. The spectra for d and l are unprecedentedly strikingly similar except for a uniform shift of the separation parameter, indicating the conserved epimer-specific structural elements across multiple charge states and conformers. As the interepimer resolution tracks the average for smaller DAACPs, the IMS approaches could help search for yet larger DAACPs. The a priori method to calibrate cyclic (including multipass) IMS developed here may be broadly useful.

Assessing the influence of dopamine and mindfulness on the formation of routines in visual search.

Given experience in cluttered but stable visual environments, our eye-movements form stereotyped routines that sample task-relevant locations, while not mixing-up routines between similar task-settings. Both dopamine signaling and mindfulness have been posited as factors that influence the formation of such routines, yet quantification of their impact remains to be tested in healthy humans. Over two sessions, participants searched through grids of doors to find hidden targets, using a gaze-contingent display. Within each session, door scenes appeared in either one of two colors, with each color signaling a differing set of likely target locations. We derived measures for how well target locations were learned (target-accuracy), how routine were sets of eye-movements (stereotypy), and the extent of interference between the two scenes (setting-accuracy). Participants completed two sessions, where they were administered either levodopa (dopamine precursor) or placebo (vitamin C), under double-blind counterbalanced conditions. Dopamine and trait mindfulness (assessed by questionnaire) interacted to influence both target-accuracy and stereotypy. Increasing dopamine improved accuracy and reduced stereotypy for high mindfulness scorers, but induced the opposite pattern for low mindfulness scorers. Dopamine also disrupted setting-accuracy invariant to mindfulness. Our findings show that mindfulness modulates the impact of dopamine on the target-accuracy and stereotypy of eye-movement routines, whereas increasing dopamine promotes interference between task-settings, regardless of mindfulness. These findings provide a link between non-human and human models regarding the influence of dopamine on the formation of task-relevant eye-movement routines and provide novel insights into behavior-trait factors that modulate the use of experience when building adaptive repertoires.

Brain areas associated with visual spatial attention display topographic organization during auditory spatial attention.

Spatially selective modulation of alpha power (8-14 Hz) is a robust finding in electrophysiological studies of visual attention, and has been recently generalized to auditory spatial attention. This modulation pattern is interpreted as reflecting a top-down mechanism for suppressing distracting input from unattended directions of sound origin. The present study on auditory spatial attention extends this interpretation by demonstrating that alpha power modulation is closely linked to oculomotor action. We designed an auditory paradigm in which participants were required to attend to upcoming sounds from one of 24 loudspeakers arranged in a circular array around the head. Maintaining the location of an auditory cue was associated with a topographically modulated distribution of posterior alpha power resembling the findings known from visual attention. Multivariate analyses allowed the prediction of the sound location in the horizontal plane. Importantly, this prediction was also possible, when derived from signals capturing saccadic activity. A control experiment on auditory spatial attention confirmed that, in absence of any visual/auditory input, lateralization of alpha power is linked to the lateralized direction of gaze. Attending to an auditory target engages oculomotor and visual cortical areas in a topographic manner akin to the retinotopic organization associated with visual attention.

Modulation in alpha band activity reflects syntax composition: an MEG study of minimal syntactic binding.

Successful sentence comprehension requires the binding, or composition, of multiple words into larger structures to establish meaning. Using magnetoencephalography, we investigated the neural mechanisms involved in binding at the syntax level, in a task where contributions from semantics were minimized. Participants were auditorily presented with minimal sentences that required binding (pronoun and pseudo-verb with the corresponding morphological inflection; "she grushes") and pseudo-verb wordlists that did not require binding ("cugged grushes"). Relative to no binding, we found that syntactic binding was associated with a modulation in alpha band (8-12 Hz) activity in left-lateralized language regions. First, we observed a significantly smaller increase in alpha power around the presentation of the target word ("grushes") that required binding (-0.05 to 0.1 s), which we suggest reflects an expectation of binding to occur. Second, during binding of the target word (0.15-0.25 s), we observed significantly decreased alpha phase-locking between the left inferior frontal gyrus and the left middle/inferior temporal cortex, which we suggest reflects alpha-driven cortical disinhibition serving to strengthen communication within the syntax composition neural network. Altogether, our findings highlight the critical role of rapid spatial-temporal alpha band activity in controlling the allocation, transfer, and coordination of the brain's resources during syntax composition.

Relating neural oscillations to laminar fMRI connectivity in visual cortex.

Laminar functional magnetic resonance imaging (fMRI) holds the potential to study connectivity at the laminar level in humans. Here we analyze simultaneously recorded electroencephalography (EEG) and high-resolution fMRI data to investigate how EEG power modulations, induced by a task with an attentional component, relate to changes in fMRI laminar connectivity between and within brain regions in visual cortex. Our results indicate that our task-induced decrease in beta power relates to an increase in deep-to-deep layer coupling between regions and to an increase in deep/middle-to-superficial layer connectivity within brain regions. The attention-related alpha power decrease predominantly relates to reduced connectivity between deep and superficial layers within brain regions, since, unlike beta power, alpha power was found to be positively correlated to connectivity. We observed no strong relation between laminar connectivity and gamma band oscillations. These results indicate that especially beta band, and to a lesser extent, alpha band oscillations relate to laminar-specific fMRI connectivity. The differential effects for alpha and beta bands indicate that they relate to different feedback-related neural processes that are differentially expressed in intra-region laminar fMRI-based connectivity.

Trypanosoma brucei histones are heavily modified with combinatorial post-translational modifications and mark Pol II transcription start regions with hyperacetylated H2A.

Trypanosomes diverged from the main eukaryotic lineage about 600 million years ago, and display some unusual genomic and epigenetic properties that provide valuable insight into the early processes employed by eukaryotic ancestors to regulate chromatin-mediated functions. We analysed Trypanosoma brucei core histones by high mass accuracy middle-down mass spectrometry to map core histone post-translational modifications (PTMs) and elucidate cis-histone combinatorial PTMs (cPTMs). T. brucei histones are heavily modified and display intricate cPTMs patterns, with numerous hypermodified cPTMs that could contribute to the formation of non-repressive euchromatic states. The Trypanosoma brucei H2A C-terminal tail is hyperacetylated, containing up to five acetylated lysine residues. MNase-ChIP-seq revealed a striking enrichment of hyperacetylated H2A at Pol II transcription start regions, and showed that H2A histones that are hyperacetylated in different combinations localised to different genomic regions, suggesting distinct epigenetic functions. Our genomics and proteomics data provide insight into the complex epigenetic mechanisms used by this parasite to regulate a genome that lacks the transcriptional control mechanisms found in later-branched eukaryotes. The findings further demonstrate the complexity of epigenetic mechanisms that were probably shared with the last eukaryotic common ancestor.

Phosphorylation of the FACT histone chaperone subunit SPT16 affects chromatin at RNA polymerase II transcriptional start sites in Arabidopsis.

The heterodimeric histone chaperone FACT, consisting of SSRP1 and SPT16, contributes to dynamic nucleosome rearrangements during various DNA-dependent processes including transcription. In search of post-translational modifications that may regulate the activity of FACT, SSRP1 and SPT16 were isolated from Arabidopsis cells and analysed by mass spectrometry. Four acetylated lysine residues could be mapped within the basic C-terminal region of SSRP1, while three phosphorylated serine/threonine residues were identified in the acidic C-terminal region of SPT16. Mutational analysis of the SSRP1 acetylation sites revealed only mild effects. However, phosphorylation of SPT16 that is catalysed by protein kinase CK2, modulates histone interactions. A non-phosphorylatable version of SPT16 displayed reduced histone binding and proved inactive in complementing the growth and developmental phenotypes of spt16 mutant plants. In plants expressing the non-phosphorylatable SPT16 version we detected at a subset of genes enrichment of histone H3 directly upstream of RNA polymerase II transcriptional start sites (TSSs) in a region that usually is nucleosome-depleted. This suggests that some genes require phosphorylation of the SPT16 acidic region for establishing the correct nucleosome occupancy at the TSS of active genes.

Utilizing the Banana S-Adenosyl-L-Homocysteine Hydrolase Allergen to Identify Cross-Reactive IgE in Ryegrass-, Latex-, and Kiwifruit-Allergic Individuals.

Food allergies mediated by specific IgE (sIgE) have a significant socioeconomic impact on society. Evaluating the IgE cross-reactivity between allergens from different allergen sources can enable the better management of these potentially life-threatening adverse reactions to food proteins and enhance food safety. A novel banana fruit allergen, S-adenosyl-L-homocysteine hydrolase (SAHH), has been recently identified and its recombinant homolog was heterologously overproduced in E. coli. In this study, we performed a search in the NCBI (National Center for Biotechnology Information) for SAHH homologs in ryegrass, latex, and kiwifruit, all of which are commonly associated with pollen-latex-fruit syndrome. In addition, Western immunoblot analysis was utilized to identify the cross-reactive IgE to banana SAHH in the sera of patients with a latex allergy, kiwifruit allergy, and ryegrass allergy. ClustalOmega analysis showed more than 92% amino acid sequence identity among the banana SAHH homologs in ryegrass, latex, and kiwifruit. In addition to five B-cell epitopes, in silico analysis predicted eleven T-cell epitopes in banana SAHH, seventeen in kiwifruit SAHH, twelve in ryegrass SAHH, and eight in latex SAHH, which were related to the seven-allele HLA reference set (HLA-DRB1*03:01, HLA-DRB1*07:01, HLA-DRB1*15:01, HLA-DRB3*01:01, HLA-DRB3*02:02, HLA-DRB4*01:01, HLA-DRB5*01:01). Four T-cell epitopes were identical in banana and kiwifruit SAHH (positions 328, 278, 142, 341), as well as banana and ryegrass SAHH (positions 278, 142, 96, and 341). All four SAHHs shared two T-cell epitopes (positions 278 and 341). In line with the high amino acid sequence identity and B-cell epitope homology among the analyzed proteins, the cross-reactive IgE to banana SAHH was detected in three of three latex-allergic patients, five of six ryegrass-allergic patients, and two of three kiwifruit-allergic patients. Although banana SAHH has only been studied in a small group of allergic individuals, it is a novel cross-reactive food allergen that should be considered when testing for pollen-latex-fruit syndrome.

Sleep-Specific Processing of Auditory Stimuli Is Reflected by Alpha and Sigma Oscillations.

Recent research revealed a surprisingly large range of cognitive operations to be preserved during sleep in humans. The new challenge is therefore to understand functions and mechanisms of processes, which so far have been mainly investigated in awake subjects. The current study focuses on dynamic changes of brain oscillations and connectivity patterns in response to environmental stimulation during non-REM sleep. Our results indicate that aurally presented names were processed and neuronally differentiated across the wake-sleep spectrum. Simultaneously recorded EEG and MEG signals revealed two distinct clusters of oscillatory power increase in response to the stimuli: (1) vigilance state-independent θ synchronization occurring immediately after stimulus onset, followed by (2) sleep-specific α/σ synchronization peaking after stimulus offset. We discuss the possible role of θ, α, and σ oscillations during non-REM sleep, and work toward a unified theory of brain rhythms and their functions during sleep.SIGNIFICANCE STATEMENT Previous research has revealed (residual) capacity of the sleeping human brain to interact with the environment. How sensory processing is realized by the neural assemblies in different stages of sleep is however unclear. To tackle this question, we examined simultaneously recorded MEG and EEG data. We discuss the possible role of θ, α, and σ oscillations during non-REM sleep. In contrast to versatile θ band response that reflected early stimulus processing step, succeeding α and σ band activity was sensitive to the saliency of the incoming information, and contingent on the sleep stage. Our findings suggest that the specific reorganization of mechanisms involved in later stages of sensory processing takes place upon falling asleep.

Perceptual echoes as travelling waves may arise from two discrete neuronal sources.

Growing evidence suggests that travelling waves are functionally relevant for cognitive operations in the brain. Several electroencephalography (EEG) studies report on a perceptual alpha-echo, representing the brain response to a random visual flicker, propagating as a travelling wave across the cortical surface. In this study, we ask if the propagating activity of the alpha-echo is best explained by a set of discrete sources mixing at the sensor level rather than a cortical travelling wave. To this end, we presented participants with gratings modulated by random noise and simultaneously acquired the ongoing MEG. The perceptual alpha-echo was estimated using the temporal response function linking the visual input to the brain response. At the group level, we observed a spatial decay of the amplitude of the alpha-echo with respect to the sensor where the alpha-echo was the largest. Importantly, the propagation latencies consistently increased with the distance. Interestingly, the propagation of the alpha-echoes was predominantly centro-lateral, while EEG studies reported mainly posterior-frontal propagation. Moreover, the propagation speed of the alpha-echoes derived from the MEG data was around 10 m/s, which is higher compared to the 2 m/s reported in EEG studies. Using source modelling, we found an early component in the primary visual cortex and a phase-lagged late component in the parietal cortex, which may underlie the travelling alpha-echoes at the sensor level. We then simulated the alpha-echoes using realistic EEG and MEG forward models by placing two sources in the parietal and occipital cortices in accordance with our empirical findings. The two-source model could account for both the direction and speed of the observed alpha-echoes in the EEG and MEG data. Our results demonstrate that the propagation of the perceptual echoes observed in EEG and MEG data can be explained by two sources mixing at the scalp level equally well as by a cortical travelling wave. Importantly, these findings should not be directly extrapolated to intracortical recordings, where travelling waves gradually propagate at a sub-millimetre scale.