Lipotype acquisition during neural development is not recapitulated in stem cell-derived neurons.

During development, different tissues acquire distinct lipotypes that are coupled to tissue function and homeostasis. In the brain, where complex membrane trafficking systems are required for neural function, specific glycerophospholipids, sphingolipids, and cholesterol are highly abundant, and defective lipid metabolism is associated with abnormal neural development and neurodegenerative disease. Notably, the production of specific lipotypes requires appropriate programming of the underlying lipid metabolic machinery during development, but when and how this occurs is unclear. To address this, we used high-resolution MSALL lipidomics to generate an extensive time-resolved resource of mouse brain development covering early embryonic and postnatal stages. This revealed a distinct bifurcation in the establishment of the neural lipotype, whereby the canonical lipid biomarkers 22:6-glycerophospholipids and 18:0-sphingolipids begin to be produced in utero, whereas cholesterol attains its characteristic high levels after birth. Using the resource as a reference, we next examined to which extent this can be recapitulated by commonly used protocols for in vitro neuronal differentiation of stem cells. Here, we found that the programming of the lipid metabolic machinery is incomplete and that stem cell-derived cells can only partially acquire a neural lipotype when the cell culture media is supplemented with brain-specific lipid precursors. Altogether, our work provides an extensive lipidomic resource for early mouse brain development and highlights a potential caveat when using stem cell-derived neuronal progenitors for mechanistic studies of lipid biochemistry, membrane biology and biophysics, which nonetheless can be mitigated by further optimizing in vitro differentiation protocols.

Direct observation of motor protein stepping in living cells using MINFLUX.

Dynamic measurements of molecular machines can provide invaluable insights into their mechanism, but these measurements have been challenging in living cells. Here, we developed live-cell tracking of single fluorophores with nanometer spatial and millisecond temporal resolution in two and three dimensions using the recently introduced super-resolution technique MINFLUX. Using this approach, we resolved the precise stepping motion of the motor protein kinesin-1 as it walked on microtubules in living cells. Nanoscopic tracking of motors walking on the microtubules of fixed cells also enabled us to resolve the architecture of the microtubule cytoskeleton with protofilament resolution.

Comparative chromatin accessibility upon BDNF stimulation delineates neuronal regulatory elements.

Neuronal stimulation induced by the brain-derived neurotrophic factor (BDNF) triggers gene expression, which is crucial for neuronal survival, differentiation, synaptic plasticity, memory formation, and neurocognitive health. However, its role in chromatin regulation is unclear. Here, using temporal profiling of chromatin accessibility and transcription in mouse primary cortical neurons upon either BDNF stimulation or depolarization (KCl), we identify features that define BDNF-specific chromatin-to-gene expression programs. Enhancer activation is an early event in the regulatory control of BDNF-treated neurons, where the bZIP motif-binding Fos protein pioneered chromatin opening and cooperated with co-regulatory transcription factors (Homeobox, EGRs, and CTCF) to induce transcription. Deleting cis-regulatory sequences affect BDNF-mediated Arc expression, a regulator of synaptic plasticity. BDNF-induced accessible regions are linked to preferential exon usage by neurodevelopmental disorder-related genes and the heritability of neuronal complex traits, which were validated in human iPSC-derived neurons. Thus, we provide a comprehensive view of BDNF-mediated genome regulatory features using comparative genomic approaches to dissect mammalian neuronal stimulation.

Multi-omic profiling of histone variant H3.3 lysine 27 methylation reveals a distinct role from canonical H3 in stem cell differentiation.

Histone variants, such as histone H3.3, replace canonical histones within the nucleosome to alter chromatin accessibility and gene expression. Although the biological roles of selected histone post-translational modifications (PTMs) have been extensively characterized, the potential differences in the function of a given PTM on different histone variants is almost always elusive. By applying proteomics and genomics techniques, we investigate the role of lysine 27 tri-methylation specifically on the histone variant H3.3 (H3.3K27me3) in the context of mouse embryonic stem cell pluripotency and differentiation as a model system for development. We demonstrate that while the steady state overall levels of methylation on both H3K27 and H3.3K27 decrease during differentiation, methylation dynamics studies indicate that methylation on H3.3K27 is maintained more than on H3K27. Using a custom-made antibody, we identify a unique enrichment of H3.3K27me3 at lineage-specific genes, such as olfactory receptor genes, and at binding motifs for the transcription factors FOXJ2/3. REST, a predicted FOXJ2/3 target that acts as a transcriptional repressor of terminal neuronal genes, was identified with H3.3K27me3 at its promoter region. H3.3K27A mutant cells confirmed an upregulation of FOXJ2/3 targets upon the loss of methylation at H3.3K27. Thus, while canonical H3K27me3 has been characterized to regulate the expression of transcription factors that play a general role in differentiation, our work suggests H3.3K27me3 is essential for regulating distinct terminal differentiation genes. This work highlights the importance of understanding the effects of PTMs not only on canonical histones but also on specific histone variants, as they may exhibit distinct roles.

High-throughput functional characterization of protein phosphorylation sites in yeast.

Phosphorylation is a critical post-translational modification involved in the regulation of almost all cellular processes. However, fewer than 5% of thousands of recently discovered phosphosites have been functionally annotated. In this study, we devised a chemical genetic approach to study the functional relevance of phosphosites in Saccharomyces cerevisiae. We generated 474 yeast strains with mutations in specific phosphosites that were screened for fitness in 102 conditions, along with a gene deletion library. Of these phosphosites, 42% exhibited growth phenotypes, suggesting that these are more likely functional. We inferred their function based on the similarity of their growth profiles with that of gene deletions and validated a subset by thermal proteome profiling and lipidomics. A high fraction exhibited phenotypes not seen in the corresponding gene deletion, suggestive of a gain-of-function effect. For phosphosites conserved in humans, the severity of the yeast phenotypes is indicative of their human functional relevance. This high-throughput approach allows for functionally characterizing individual phosphosites at scale.

Zonally asymmetric phytoplankton response to the Southern annular mode in the marginal sea of the Southern ocean.

Antarctic marine biological variability modulates climate systems via the biological pump. However, the knowledge of biological response in the Southern Ocean to climate variability still has been lack of understanding owing to limited ocean color data in the high latitude region. We investigated the surface chlorophyll concentration responses to the Southern annular mode (SAM) in the marginal sea of the Southern ocean using satellite observation and reanalysis data focusing on the austral summer. The positive phase of SAM is associated with enhanced and poleward-shifted westerly winds, leading to physical and biogeochemical responses over the Southern ocean. Our result indicates that chlorophyll has strong zonally asymmetric responses to SAM owing to different limiting factors of phytoplankton growth per region. For the positive SAM phase, chlorophyll tends to increase in the western Amundsen-Ross Sea but decreases in the D'Urville Sea. It is suggested that the distinct limiting factors are associated with the seasonal variability of sea ice and upwelling per region.

Donor cell memory confers a metastable state of directly converted cells.

Generation of induced oligodendrocyte progenitor cells (iOPCs) from somatic fibroblasts is a strategy for cell-based therapy of myelin diseases. However, iOPC generation is inefficient, and the resulting iOPCs exhibit limited expansion and differentiation competence. Here we overcome these limitations by transducing an optimized transcription factor combination into a permissive donor phenotype, the pericyte. Pericyte-derived iOPCs (PC-iOPCs) are stably expandable and functionally myelinogenic with high differentiation competence. Unexpectedly, however, we found that PC-iOPCs are metastable so that they can produce myelination-competent oligodendrocytes or revert to their original identity in a context-dependent fashion. Phenotypic reversion of PC-iOPCs is tightly linked to memory of their original transcriptome and epigenome. Phenotypic reversion can be disconnected from this donor cell memory effect, and in vivo myelination can eventually be achieved by transplantation of O4+ pre-oligodendrocytes. Our data show that donor cell source and memory can contribute to the fate and stability of directly converted cells.

Histone Variant H3.3 Mutations in Defining the Chromatin Function in Mammals.

The systematic mutation of histone 3 (H3) genes in model organisms has proven to be a valuable tool to distinguish the functional role of histone residues. No system exists in mammalian cells to directly manipulate canonical histone H3 due to a large number of clustered and multi-loci histone genes. Over the years, oncogenic histone mutations in a subset of H3 have been identified in humans, and have advanced our understanding of the function of histone residues in health and disease. The oncogenic mutations are often found in one allele of the histone variant H3.3 genes, but they prompt severe changes in the epigenetic landscape of cells, and contribute to cancer development. Therefore, mutation approaches using H3.3 genes could be relevant to the determination of the functional role of histone residues in mammalian development without the replacement of canonical H3 genes. In this review, we describe the key findings from the H3 mutation studies in model organisms wherein the genetic replacement of canonical H3 is possible. We then turn our attention to H3.3 mutations in human cancers, and discuss H3.3 substitutions in the N-terminus, which were generated in order to explore the specific residue or associated post-translational modification.

Genomic Rewiring of SOX2 Chromatin Interaction Network during Differentiation of ESCs to Postmitotic Neurons.

Cellular differentiation requires dramatic changes in chromatin organization, transcriptional regulation, and protein production. To understand the regulatory connections between these processes, we generated proteomic, transcriptomic, and chromatin accessibility data during differentiation of mouse embryonic stem cells (ESCs) into postmitotic neurons and found extensive associations between different molecular layers within and across differentiation time points. We observed that SOX2, as a regulator of pluripotency and neuronal genes, redistributes from pluripotency enhancers to neuronal promoters during differentiation, likely driven by changes in its protein interaction network. We identified ATRX as a major SOX2 partner in neurons, whose co-localization correlated with an increase in active enhancer marks and increased expression of nearby genes, which we experimentally confirmed for three loci. Collectively, our data provide key insights into the regulatory transformation of SOX2 during neuronal differentiation, and we highlight the significance of multi-omic approaches in understanding gene regulation in complex systems.

Lysine 4 of histone H3.3 is required for embryonic stem cell differentiation, histone enrichment at regulatory regions and transcription accuracy.

Mutations in enzymes that modify histone H3 at lysine 4 (H3K4) or lysine 36 (H3K36) have been linked to human disease, yet the role of these residues in mammals is unclear. We mutated K4 or K36 to alanine in the histone variant H3.3 and showed that the K4A mutation in mouse embryonic stem cells (ESCs) impaired differentiation and induced widespread gene expression changes. K4A resulted in substantial H3.3 depletion, especially at ESC promoters; it was accompanied by reduced remodeler binding and increased RNA polymerase II (Pol II) activity. Regulatory regions depleted of H3.3K4A showed histone modification alterations and changes in enhancer activity that correlated with gene expression. In contrast, the K36A mutation did not alter H3.3 deposition and affected gene expression at the later stages of differentiation. Thus, H3K4 is required for nucleosome deposition, histone turnover and chromatin remodeler binding at regulatory regions, where tight regulation of Pol II activity is necessary for proper ESC differentiation.

The functional landscape of the human phosphoproteome.

Protein phosphorylation is a key post-translational modification regulating protein function in almost all cellular processes. Although tens of thousands of phosphorylation sites have been identified in human cells, approaches to determine the functional importance of each phosphosite are lacking. Here, we manually curated 112 datasets of phospho-enriched proteins, generated from 104 different human cell types or tissues. We re-analyzed the 6,801 proteomics experiments that passed our quality control criteria, creating a reference phosphoproteome containing 119,809 human phosphosites. To prioritize functional sites, we used machine learning to identify 59 features indicative of proteomic, structural, regulatory or evolutionary relevance and integrate them into a single functional score. Our approach identifies regulatory phosphosites across different molecular mechanisms, processes and diseases, and reveals genetic susceptibilities at a genomic scale. Several regulatory phosphosites were experimentally validated, including identifying a role in neuronal differentiation for phosphosites in SMARCC2, a member of the SWI/SNF chromatin-remodeling complex.

Haploinsufficiency of the intellectual disability gene SETD5 disturbs developmental gene expression and cognition.

SETD5 gene mutations have been identified as a frequent cause of idiopathic intellectual disability. Here we show that Setd5-haploinsufficient mice present developmental defects such as abnormal brain-to-body weight ratios and neural crest defect-associated phenotypes. Furthermore, Setd5-mutant mice show impairments in cognitive tasks, enhanced long-term potentiation, delayed ontogenetic profile of ultrasonic vocalization, and behavioral inflexibility. Behavioral issues are accompanied by abnormal expression of postsynaptic density proteins previously associated with cognition. Our data additionally indicate that Setd5 regulates RNA polymerase II dynamics and gene transcription via its interaction with the Hdac3 and Paf1 complexes, findings potentially explaining the gene expression defects observed in Setd5-haploinsufficient mice. Our results emphasize the decisive role of Setd5 in a biological pathway found to be disrupted in humans with intellectual disability and autism spectrum disorder.

Systematic analysis of protein turnover in primary cells.

A better understanding of proteostasis in health and disease requires robust methods to determine protein half-lives. Here we improve the precision and accuracy of peptide ion intensity-based quantification, enabling more accurate protein turnover determination in non-dividing cells by dynamic SILAC-based proteomics. This approach allows exact determination of protein half-lives ranging from 10 to >1000 h. We identified 4000-6000 proteins in several non-dividing cell types, corresponding to 9699 unique protein identifications over the entire data set. We observed similar protein half-lives in B-cells, natural killer cells and monocytes, whereas hepatocytes and mouse embryonic neurons show substantial differences. Our data set extends and statistically validates the previous observation that subunits of protein complexes tend to have coherent turnover. Moreover, analysis of different proteasome and nuclear pore complex assemblies suggests that their turnover rate is architecture dependent. These results illustrate that our approach allows investigating protein turnover and its implications in various cell types.

CTCF-Mediated Chromatin Loops between Promoter and Gene Body Regulate Alternative Splicing across Individuals.

The CCCTC-binding factor (CTCF) is known to establish long-range DNA contacts that alter the three-dimensional architecture of chromatin, but how the presence of CTCF influences nearby gene expression is still poorly understood. Here, we analyze CTCF chromatin immunoprecipitation sequencing, RNA sequencing, and Hi-C data, together with genotypes from a healthy human cohort, and measure statistical associations between inter-individual variability in CTCF binding and alternative exon usage. We demonstrate that CTCF-mediated chromatin loops between promoters and intragenic regions are prevalent and that when exons are in physical proximity with their promoters, CTCF binding correlates with exon inclusion in spliced mRNA. Genome-wide, CTCF-bound exons are enriched for genes involved in signaling and cellular stress-response pathways. Structural analysis of three specific examples, checkpoint kinase 2 (CHK2), CDC-like kinase 3 (CLK3), and euchromatic histone-lysine N-methyltransferase (EHMT1), suggests that CTCF-mediated exon inclusion is likely to downregulate enzyme activity by disrupting annotated protein domains. In total, our study suggests that alternative exon usage is regulated by CTCF-dependent chromatin structure.

Intragenic CpG islands play important roles in bivalent chromatin assembly of developmental genes.

CpG, 5'-C-phosphate-G-3', islands (CGIs) have long been known for their association with enhancers, silencers, and promoters, and for their epigenetic signatures. They are maintained in embryonic stem cells (ESCs) in a poised but inactive state via the formation of bivalent chromatin containing both active and repressive marks. CGIs also occur within coding sequences, where their functional role has remained obscure. Intragenic CGIs (iCGIs) are largely absent from housekeeping genes, but they are found in all genes associated with organ development and cell lineage control. In this paper, we investigated the epigenetic status of iCGIs and found that they too reside in bivalent chromatin in ESCs. Cell type-specific DNA methylation of iCGIs in differentiated cells was linked to the loss of both the H3K4me3 and H3K27me3 marks, and disruption of physical interaction with promoter regions, resulting in transcriptional activation of key regulators of differentiation such as PAXs, HOXs, and WNTs. The differential epigenetic modification of iCGIs appears to be mediated by cell type-specific transcription factors distinct from those bound by promoter, and these transcription factors may be involved in the hypermethylation of iCGIs upon cell differentiation. iCGIs thus play a key role in the cell type-specific regulation of transcription.

Reading between the Lines: "ADD"-ing Histone and DNA Methylation Marks toward a New Epigenetic "Sum".

Covalent modifications of both DNA and histones act in concert to define the landscape of our epigenome. In this review, we explore the interconnections between histone and DNA modifications by focusing on a conserved chromatin-binding regulatory domain, the ATRX-DNMT3-DNMT3L (ADD) domain. New studies show that the ADD domain is capable of sensing, and therefore integrating, the status of multiple histone modifications. This in turn dictates the in vivo localization or allosteric regulation of the full-length ADD-containing protein and its ability to function in downstream chromatin remodeling events. Strategies to re-engineer the ADD "reader pocket" in the de novo DNA methyltransferase DNMT3A such that it redirects this "writer" to new genomic loci proved useful in understanding important biological downstream consequences of mis-targeting of DNA methylation via altered reading of histone marks. Combined with genome-editing tools, this approach stands as a poof-of-principle and will be broadly applicable to the elucidation of epigenetic networks that have been altered by "reader" mutations, either artificially or as naturally occurs in some human diseases.

Critical Role of Histone Turnover in Neuronal Transcription and Plasticity.

Turnover and exchange of nucleosomal histones and their variants, a process long believed to be static in post-replicative cells, remains largely unexplored in brain. Here, we describe a novel mechanistic role for HIRA (histone cell cycle regulator) and proteasomal degradation-associated histone dynamics in the regulation of activity-dependent transcription, synaptic connectivity, and behavior. We uncover a dramatic developmental profile of nucleosome occupancy across the lifespan of both rodents and humans, with the histone variant H3.3 accumulating to near-saturating levels throughout the neuronal genome by mid-adolescence. Despite such accumulation, H3.3-containing nucleosomes remain highly dynamic-in a modification-independent manner-to control neuronal- and glial-specific gene expression patterns throughout life. Manipulating H3.3 dynamics in both embryonic and adult neurons confirmed its essential role in neuronal plasticity and cognition. Our findings establish histone turnover as a critical and previously undocumented regulator of cell type-specific transcription and plasticity in mammalian brain.

Engineering of a Histone-Recognition Domain in Dnmt3a Alters the Epigenetic Landscape and Phenotypic Features of Mouse ESCs.

Histone modification and DNA methylation are associated with varying epigenetic "landscapes," but detailed mechanistic and functional links between the two remain unclear. Using the ATRX-DNMT3-DNMT3L (ADD) domain of the DNA methyltransferase Dnmt3a as a paradigm, we apply protein engineering to dissect the molecular interactions underlying the recruitment of this enzyme to specific regions of chromatin in mouse embryonic stem cells (ESCs). By rendering the ADD domain insensitive to histone modification, specifically H3K4 methylation or H3T3 phosphorylation, we demonstrate the consequence of dysregulated Dnmt3a binding and activity. Targeting of a Dnmt3a mutant to H3K4me3 promoters decreases gene expression in a subset of developmental genes and alters ESC differentiation, whereas aberrant binding of another mutant to H3T3ph during mitosis promotes chromosome instability. Our studies support the general view that histone modification "reading" and DNA methylation are closely coupled in mammalian cells, and suggest an avenue for the functional assessment of chromatin-associated proteins.