Galnt17 loss-of-function leads to developmental delay and abnormal coordination, activity, and social interactions with cerebellar vermis pathology.

GALNT17 encodes a N-acetylgalactosaminyltransferase (GalNAc-T) protein specifically involved in mucin-type O-linked glycosylation of target proteins, a process important for cell adhesion, cell signaling, neurotransmitter activity, neurite outgrowth, and neurite sensing. GALNT17, also known as WBSCR17, is located at the edge of the Williams-Beuren Syndrome (WBS) critical region and adjacent to the AUTS2 locus, genomic regions associated with neurodevelopmental phenotypes that are thought to be co-regulated. Although previous data have implicated Galnt17 in neurodevelopment, the in vivo functions of this gene have not been investigated. In this study, we have analyzed behavioral, brain pathology, and molecular phenotypes exhibited by Galnt17 knockout (Galnt17-/-) mice. We show that Galnt17-/- mutants exhibit developmental neuropathology within the cerebellar vermis, along with abnormal activity, coordination, and social interaction deficits. Transcriptomic and protein analysis revealed reductions in both mucin type O-glycosylation and heparan sulfate synthesis in the developing mutant cerebellum along with disruption of pathways central to neuron differentiation, axon pathfinding, and synaptic signaling, consistent with the mutant neuropathology. These brain and behavioral phenotypes and molecular data confirm a specific role for Galnt17 in brain development and suggest new clues to factors that could contribute to phenotypes in certain WBS and AUTS2 syndrome patients.

Behavior-related gene regulatory networks: A new level of organization in the brain.

Neuronal networks are the standard heuristic model today for describing brain activity associated with animal behavior. Recent studies have revealed an extensive role for a completely distinct layer of networked activities in the brain-the gene regulatory network (GRN)-that orchestrates expression levels of hundreds to thousands of genes in a behavior-related manner. We examine emerging insights into the relationships between these two types of networks and discuss their interplay in spatial as well as temporal dimensions, across multiple scales of organization. We discuss properties expected of behavior-related GRNs by drawing inspiration from the rich literature on GRNs related to animal development, comparing and contrasting these two broad classes of GRNs as they relate to their respective phenotypic manifestations. Developmental GRNs also represent a third layer of network biology, playing out over a third timescale, which is believed to play a crucial mediatory role between neuronal networks and behavioral GRNs. We end with a special emphasis on social behavior, discuss whether unique GRN organization and cis-regulatory architecture underlies this special class of behavior, and review literature that suggests an affirmative answer.

An extended regulatory landscape drives Tbx18 activity in a variety of prostate-associated cell lineages.

The evolutionarily conserved transcription factor, Tbx18, is expressed in a dynamic pattern throughout embryonic and early postnatal life and plays crucial roles in the development of multiple organ systems. Previous studies have indicated that this dynamic function is controlled by an expansive regulatory structure, extending far upstream and downstream of the gene. With the goal of identifying elements that interact with the Tbx18 promoter in developing prostate, we coupled chromatin conformation capture (4C) and ATAC-seq from embryonic day 18.5 (E18.5) mouse urogenital sinus (UGS), where Tbx18 is highly expressed. The data revealed dozens of active chromatin elements distributed throughout a 1.5 million base pair topologically associating domain (TAD). To identify cell types contributing to this chromatin signal, we used lineage tracing methods with a Tbx18 Cre "knock-in" allele; these data show clearly that Tbx18-expressing precursors differentiate into wide array of cell types in multiple tissue compartments, most of which have not been previously reported. We also used a 209 kb Cre-expressing Tbx18 transgene, to partition enhancers for specific precursor types into two rough spatial domains. Within this central 209 kb compartment, we identified ECR1, previously described to regulate Tbx18 expression in ureter, as an active regulator of UGS expression. Together these data define the diverse fates of Tbx18+ precursors in prostate-associated tissues for the first time, and identify a highly active TAD controlling the gene's essential function in this tissue.

Transcriptional regulatory dynamics drive coordinated metabolic and neural response to social challenge in mice.

Agonistic encounters are powerful effectors of future behavior, and the ability to learn from this type of social challenge is an essential adaptive trait. We recently identified a conserved transcriptional program defining the response to social challenge across animal species, highly enriched in transcription factor (TF), energy metabolism, and developmental signaling genes. To understand the trajectory of this program and to uncover the most important regulatory influences controlling this response, we integrated gene expression data with the chromatin landscape in the hypothalamus, frontal cortex, and amygdala of socially challenged mice over time. The expression data revealed a complex spatiotemporal patterning of events starting with neural signaling molecules in the frontal cortex and ending in the modulation of developmental factors in the amygdala and hypothalamus, underpinned by a systems-wide shift in expression of energy metabolism-related genes. The transcriptional signals were correlated with significant shifts in chromatin accessibility and a network of challenge-associated TFs. Among these, the conserved metabolic and developmental regulator ESRRA was highlighted for an especially early and important regulatory role. Cell-type deconvolution analysis attributed the differential metabolic and developmental signals in this social context primarily to oligodendrocytes and neurons, respectively, and we show that ESRRA is expressed in both cell types. Localizing ESRRA binding sites in cortical chromatin, we show that this nuclear receptor binds both differentially expressed energy-related and neurodevelopmental TF genes. These data link metabolic and neurodevelopmental signaling to social challenge, and identify key regulatory drivers of this process with unprecedented tissue and temporal resolution.

Temporal dynamics of neurogenomic plasticity in response to social interactions in male threespined sticklebacks.

Animals exhibit dramatic immediate behavioral plasticity in response to social interactions, and brief social interactions can shape the future social landscape. However, the molecular mechanisms contributing to behavioral plasticity are unclear. Here, we show that the genome dynamically responds to social interactions with multiple waves of transcription associated with distinct molecular functions in the brain of male threespined sticklebacks, a species famous for its behavioral repertoire and evolution. Some biological functions (e.g., hormone activity) peaked soon after a brief territorial challenge and then declined, while others (e.g., immune response) peaked hours afterwards. We identify transcription factors that are predicted to coordinate waves of transcription associated with different components of behavioral plasticity. Next, using H3K27Ac as a marker of chromatin accessibility, we show that a brief territorial intrusion was sufficient to cause rapid and dramatic changes in the epigenome. Finally, we integrate the time course brain gene expression data with a transcriptional regulatory network, and link gene expression to changes in chromatin accessibility. This study reveals rapid and dramatic epigenomic plasticity in response to a brief, highly consequential social interaction.

Site-Specific Phosphorylation of Histone H1.4 Is Associated with Transcription Activation.

Core histone variants, such as H2A.X and H3.3, serve specialized roles in chromatin processes that depend on the genomic distributions and amino acid sequence differences of the variant proteins. Modifications of these variants alter interactions with other chromatin components and thus the protein's functions. These inferences add to the growing arsenal of evidence against the older generic view of those linker histones as redundant repressors. Furthermore, certain modifications of specific H1 variants can confer distinct roles. On the one hand, it has been reported that the phosphorylation of H1 results in its release from chromatin and the subsequent transcription of HIV-1 genes. On the other hand, recent evidence indicates that phosphorylated H1 may in fact be associated with active promoters. This conflict suggests that different H1 isoforms and modified versions of these variants are not redundant when together but may play distinct functional roles. Here, we provide the first genome-wide evidence that when phosphorylated, the H1.4 variant remains associated with active promoters and may even play a role in transcription activation. Using novel, highly specific antibodies, we generated the first genome-wide view of the H1.4 isoform phosphorylated at serine 187 (pS187-H1.4) in estradiol-inducible MCF7 cells. We observe that pS187-H1.4 is enriched primarily at the transcription start sites (TSSs) of genes activated by estradiol treatment and depleted from those that are repressed. We also show that pS187-H1.4 associates with 'early estrogen response' genes and stably interacts with RNAPII. Based on the observations presented here, we propose that phosphorylation at S187 by CDK9 represents an early event required for gene activation. This event may also be involved in the release of promoter-proximal polymerases to begin elongation by interacting directly with the polymerase or other parts of the transcription machinery. Although we focused on estrogen-responsive genes, taking into account previous evidence of H1.4's enrichment of promoters of pluripotency genes, and its involvement with rDNA activation, we propose that H1.4 phosphorylation for gene activation may be a more global observation.

Neuromolecular responses to social challenge: common mechanisms across mouse, stickleback fish, and honey bee.

Certain complex phenotypes appear repeatedly across diverse species due to processes of evolutionary conservation and convergence. In some contexts like developmental body patterning, there is increased appreciation that common molecular mechanisms underlie common phenotypes; these molecular mechanisms include highly conserved genes and networks that may be modified by lineage-specific mutations. However, the existence of deeply conserved mechanisms for social behaviors has not yet been demonstrated. We used a comparative genomics approach to determine whether shared neuromolecular mechanisms could underlie behavioral response to territory intrusion across species spanning a broad phylogenetic range: house mouse (Mus musculus), stickleback fish (Gasterosteus aculeatus), and honey bee (Apis mellifera). Territory intrusion modulated similar brain functional processes in each species, including those associated with hormone-mediated signal transduction and neurodevelopment. Changes in chromosome organization and energy metabolism appear to be core, conserved processes involved in the response to territory intrusion. We also found that several homologous transcription factors that are typically associated with neural development were modulated across all three species, suggesting that shared neuronal effects may involve transcriptional cascades of evolutionarily conserved genes. Furthermore, immunohistochemical analyses of a subset of these transcription factors in mouse again implicated modulation of energy metabolism in the behavioral response. These results provide support for conserved genetic "toolkits" that are used in independent evolutions of the response to social challenge in diverse taxa.

A distant downstream enhancer directs essential expression of Tbx18 in urogenital tissues.

The vertebrate T-box transcription factor gene Tbx18 performs a vital role in development of multiple organ systems. Tbx18 insufficiency manifests as recessive phenotypes in the upper urinary system, cardiac venous pole, inner ear, and axial skeleton; homozygous null mutant animals die perinatally. Here, we report a new regulatory mutation of Tbx18, a reciprocal translocation breaking 78kbp downstream of the gene. 12Gso homozygotes present urinary and vertebral defects very similar to those associated with Tbx18-null mutations, but 12Gso is clearly not a global null allele since homozygotes survive into adulthood. We show that 12Gso down-regulates Tbx18 expression in a manner that is both spatially- and temporally-specific; combined with other data, the mutation points particularly to the presence of an essential urogenital enhancer located near the translocation breakpoint site. In support of this hypothesis, we identify a distal enhancer element, ECR1, which is active in developing urogenital and other tissues; we propose that disruption of this element leads to premature loss of Tbx18 function in 12Gso mutant mice. These data reveal a long-range regulatory architecture extending far downstream of Tbx18, identify a novel and likely essential urogenital enhancer, and introduce a new tool for dissecting postnatal phenotypes associated with dysregulation of Tbx18.

Isolated loss of the AUTS2 long isoform, brain-wide or targeted to Calbindin-lineage cells, generates a specific suite of brain, behavioral, and molecular pathologies.

Rearrangements within the AUTS2 region are associated with a rare syndromic disorder with intellectual disability, developmental delay, and behavioral abnormalities as core features. In addition, smaller regional variants are linked to wide range of neuropsychiatric disorders, underscoring the gene's essential role in brain development. Like many essential neurodevelopmental genes, AUTS2 is large and complex, generating distinct long (AUTS2-l) and short (AUTS2-s) protein isoforms from alternative promoters. Although evidence suggests unique isoform functions, the contributions of each isoform to specific AUTS2-linked phenotypes have not been clearly resolved. Furthermore, Auts2 is widely expressed across the developing brain, but cell populations most central to disease presentation have not been determined. In this study, we focused on the specific roles of AUTS2-l in brain development, behavior, and postnatal brain gene expression, showing that brain-wide AUTS2-l ablation leads to specific subsets of the recessive pathologies associated with mutations in 3' exons (exons 8-19) that disrupt both major isoforms. We identify downstream genes that could explain expressed phenotypes including hundreds of putative direct AUTS2-l target genes. Furthermore, in contrast to 3' Auts2 mutations which lead to dominant hypoactivity, AUTS2-l loss-of-function is associated with dominant hyperactivity and repetitive behaviors, phenotypes exhibited by many human patients. Finally, we show that AUTS2-l ablation in Calbindin 1-expressing cell lineages is sufficient to yield learning/memory deficits and hyperactivity with abnormal dentate gyrus granule cell maturation, but not other phenotypic effects. These data provide new clues to in vivo AUTS2-l functions and novel information relevant to genotype-phenotype correlations in the human AUTS2 region.

A reciprocal translocation dissects roles of Pax6 alternative promoters and upstream regulatory elements in the development of pancreas, brain, and eye.

Pax6 encodes a transcription factor with key roles in the development of the pancreas, central nervous system, and eye. Gene expression is orchestrated by several alternative promoters and enhancer elements that are distributed over several hundred kilobases. Here, we describe a reciprocal translocation, called 1Gso, which disrupts the integrity of transcripts arising from the 5'-most promoter, P0, and separates downstream promoters from enhancers active in pancreas and eye. Despite this fact, 1Gso animals exhibit none of the dominant Pax6 phenotypes, and the translocation complements recessive brain and craniofacial phenotypes. However, 1Gso fails to complement Pax6 recessive effects in lacrimal gland, conjunctiva, lens, and pancreas. The 1Gso animals also express a corneal phenotype that is related to but distinct from that expressed by Pax6 null mutants, and an abnormal density and organization of retinal ganglion cell axons; these phenotypes may be related to a modest upregulation of Pax6 expression from downstream promoters that we observed during development. Our investigation maps the activities of Pax6 alternative promoters including a novel one in developing tissues, confirms the phenotypic consequences of upstream enhancer disruption, and limits the likely effects of the P0 transcript null mutation to recessive abnormalities in the pancreas and specific structures of the eye.

SEAMoD: A fully interpretable neural network for cis-regulatory analysis of differentially expressed genes.

A common way to investigate gene regulatory mechanisms is to identify differentially expressed genes using transcriptomics, find their candidate enhancers using epigenomics, and search for over-represented transcription factor (TF) motifs in these enhancers using bioinformatics tools. A related follow-up task is to model gene expression as a function of enhancer sequences and rank TF motifs by their contribution to such models, thus prioritizing among regulators. We present a new computational tool called SEAMoD that performs the above tasks of motif finding and sequence-to-expression modeling simultaneously. It trains a convolutional neural network model to relate enhancer sequences to differential expression in one or more biological conditions. The model uses TF motifs to interpret the sequences, learning these motifs and their relative importance to each biological condition from data. It also utilizes epigenomic information in the form of activity scores of putative enhancers and automatically searches for the most promising enhancer for each gene. Compared to existing neural network models of non-coding sequences, SEAMoD uses far fewer parameters, requires far less training data, and emphasizes biological interpretability. We used SEAMoD to understand regulatory mechanisms underlying the differentiation of neural stem cell (NSC) derived from mouse forebrain. We profiled gene expression and histone modifications in NSC and three differentiated cell types and used SEAMoD to model differential expression of nearly 12,000 genes with an accuracy of 81%, in the process identifying the Olig2, E2f family TFs, Foxo3, and Tcf4 as key transcriptional regulators of the differentiation process.

Isolated loss of the AUTS2 long isoform, brain-wide or targeted to Calbindin -lineage cells, generates a specific suite of brain, behavioral and molecular pathologies.

Rearrangements within the AUTS2 region are associated with a rare syndromic disorder with intellectual disability, developmental delay and behavioral abnormalities as core features. In addition, smaller regional variants are linked to wide range of neuropsychiatric disorders, underscoring the gene's essential role in brain development. Like many essential neurodevelopmental genes, AUTS2 is large and complex, generating distinct long (AUTS2-l) and short (AUTS2-s) protein isoforms from alternative promoters. Although evidence suggests unique isoform functions, the contributions of each isoform to specific AUTS2- linked phenotypes have not been clearly resolved. Furthermore, Auts2 is widely expressed across the developing brain, but cell populations most central to disease presentation have not been determined. In this study, we focused on the specific roles of AUTS2-l in brain development, behavior, and postnatal brain gene expression, showing that brain-wide AUTS2-l ablation leads to specific subsets of the recessive pathologies associated with C-terminal mutations that disrupt both isoforms. We identify downstream genes that could explain expressed phenotypes including hundreds of putative direct AUTS2- l target genes. Furthermore, in contrast to C-terminal Auts2 mutations which lead to dominant hypoactivity, AUTS2-l loss-of-function is associated with dominant hyperactivity, a phenotype exhibited by many human patients. Finally, we show that AUTS2-l ablation in Calbindin 1 -expressing cell lineages is sufficient to yield learning/memory deficits and hyperactivity with abnormal dentate gyrus granule cell maturation, but not other phenotypic effects. These data provide new clues to in vivo AUTS2-l functions and novel information relevant to genotype-phenotype correlations in the human AUTS2 region.

Function and Evolution of C2H2 Zinc Finger Arrays.

Krüppel-type or C2H2 zinc fingers represent a dominant DNA-binding motif in eukaryotic transcription factor (TF) proteins. In Krüppel-type (KZNF) TFs, KZNF motifs are arranged in arrays of three to as many as 40 tandem units, which cooperate to define the unique DNA recognition properties of the protein. Each finger contains four amino acids located at specific positions, which are brought into direct contact with adjacent nucleotides in the DNA sequence as the KZNF array winds around the major groove of the alpha helix. This arrangement creates an intimate and potentially predictable relationship between the amino acid sequence of KZNF arrays and the nucleotide sequence of target binding sites. The large number of possible combinations and arrangements of modular KZNF motifs, and the increasing lengths of KZNF arrays in vertebrate species, has created huge repertoires of functionally unique TF proteins. The properties of this versatile DNA-binding motif have been exploited independently many times over the course of evolution, through attachment to effector motifs that confer activating, repressing or other activities to the proteins. Once created, some of these novel inventions have expanded in specific evolutionary clades, creating large families of TFs that are lineage- or species-unique. This chapter reviews the properties and their remarkable evolutionary history of eukaryotic KZNF TF proteins, with special focus on large families that dominate the TF landscapes in different metazoan species.

Differences in human and chimpanzee gene expression patterns define an evolving network of transcription factors in brain.

Humans differ from other primates by marked differences in cognitive abilities and a significantly larger brain. These differences correlate with metabolic changes, as evidenced by the relative up-regulation of energy-related genes and metabolites in human brain. While the mechanisms underlying these evolutionary changes have not been elucidated, altered activities of key transcription factors (TFs) could play a pivotal role. To assess this possibility, we analyzed microarray data from five tissues from humans and chimpanzees. We identified 90 TF genes with significantly different expression levels in human and chimpanzee brain among which the rapidly evolving KRAB-zinc finger genes are markedly over-represented. The differentially expressed TFs cluster within a robust regulatory network consisting of two distinct but interlinked modules, one strongly associated with energy metabolism functions, and the other with transcription, vesicular transport, and ubiquitination. Our results suggest that concerted changes in a relatively small number of interacting TFs may coordinate major gene expression differences in human and chimpanzee brain.

An epigenomic shift in amygdala marks the transition to maternal behaviors in alloparenting virgin female mice.

Adults of many species will care for young offspring that are not their own, a phenomenon called alloparenting. However, in many cases, nonparental adults must be sensitized by repeated or extended exposures to newborns before they will robustly display parental-like behaviors. To capture neurogenomic events underlying the transition to active parental caring behaviors, we analyzed brain gene expression and chromatin profiles of virgin female mice co-housed with pregnant dams during pregnancy and after birth. After an initial display of antagonistic behaviors and a surge of defense-related gene expression, we observed a dramatic shift in the chromatin landscape specifically in amygdala of the pup-exposed virgin females compared to females co-housed with mother before birth, accompanied by a dampening of anxiety-related gene expression. This epigenetic shift coincided with hypothalamic expression of the oxytocin gene and the emergence of behaviors and gene expression patterns classically associated with maternal care. The results outline a neurogenomic program associated with dramatic behavioral changes and suggest molecular networks relevant to human postpartum mental health.

Rapid sequence and expression divergence suggest selection for novel function in primate-specific KRAB-ZNF genes.

Recent segmental duplications (SDs), arising from duplication events that occurred within the past 35-40 My, have provided a major resource for the evolution of proteins with primate-specific functions. KRAB zinc finger (KRAB-ZNF) transcription factor genes are overrepresented among genes contained within these recent human SDs. Here, we examine the structural and functional diversity of the 70 human KRAB-ZNF genes involved in the most recent primate SD events including genes that arose in the hominid lineage. Despite their recent advent, many parent-daughter KRAB-ZNF gene pairs display significant differences in zinc finger structure and sequence, expression, and splicing patterns, each of which could significantly alter the regulatory functions of the paralogous genes. Paralogs that emerged on the lineage to humans and chimpanzees have undergone more evolutionary changes per unit of time than genes already present in the common ancestor of rhesus macaques and great apes. Taken together, these data indicate that a substantial fraction of the recently evolved primate-specific KRAB-ZNF gene duplicates have acquired novel functions that may possibly define novel regulatory pathways and suggest an active ongoing selection for regulatory diversity in primates.

Allele-specific enhancer interaction at the Peg3 imprinted domain.

The parental allele specificity of mammalian imprinted genes has been evolutionarily well conserved, although its functional constraints and associated mechanisms are not fully understood. In the current study, we generated a mouse mutant with switched active alleles driving the switch from paternal-to-maternal expression for Peg3 and the maternal-to-paternal expression for Zim1. The expression levels of Peg3 and Zim1, but not the spatial expression patterns, within the brain showed clear differences between wild type and mutant animals. We identified putative enhancers localized upstream of Peg3 that displayed allele-biased DNA methylation, and that also participate in allele-biased chromosomal conformations with regional promoters. Most importantly, these data suggest for the first time that long-distance enhancers may contribute to allelic expression within imprinted domains through allele-biased interactions with regional promoters.

A Mouse Mutation That Dysregulates Neighboring Galnt17 and Auts2 Genes Is Associated with Phenotypes Related to the Human AUTS2 Syndrome.

AUTS2 was originally discovered as the gene disrupted by a translocation in human twins with Autism spectrum disorder, intellectual disability, and epilepsy. Since that initial finding, AUTS2-linked mutations and variants have been associated with a very broad array of neuropsychiatric disorders, sugg esting that AUTS2 is required for fundamental steps of neurodevelopment. However, genotype-phenotype correlations in this region are complicated, because most mutations could also involve neighboring genes. Of particular interest is the nearest downstream neighbor of AUTS2, GALNT17, which encodes a brain-expressed N-acetylgalactosaminyltransferase of unknown brain function. Here we describe a mouse (Mus musculus) mutation, T(5G2;8A1)GSO (abbreviated 16Gso), a reciprocal translocation that breaks between Auts2 and Galnt17 and dysregulates both genes. Despite this complex regulatory effect, 16Gso homozygotes model certain human AUTS2-linked phenotypes very well. In addition to abnormalities in growth, craniofacial structure, learning and memory, and behavior, 16Gso homozygotes display distinct pathologies of the cerebellum and hippocampus that are similar to those associated with autism and other types of AUTS2-linked neurological disease. Analyzing mutant cerebellar and hippocampal transcriptomes to explain this pathology, we identified disturbances in pathways related to neuron and synapse maturation, neurotransmitter signaling, and cellular stress, suggesting possible cellular mechanisms. These pathways, coupled with the translocation's selective effects on Auts2 isoforms and coordinated dysregulation of Galnt17, suggest novel hypotheses regarding the etiology of the human "AUTS2 syndrome" and the wide array of neurodevelopmental disorders linked to variance in this genomic region.

Honey bee neurogenomic responses to affiliative and agonistic social interactions.

Social interactions can be divided into two categories, affiliative and agonistic. How neurogenomic responses reflect these opposing valences is a central question in the biological embedding of experience. To address this question, we exposed honey bees to a queen larva, which evokes nursing, an affiliative alloparenting interaction, and measured the transcriptomic response of the mushroom body brain region at different times after exposure. Hundreds of genes were differentially expressed at distinct time points, revealing a dynamic temporal patterning of the response. Comparing these results to our previously published research on agonistic aggressive interactions, we found both shared and unique transcriptomic responses to each interaction. The commonly responding gene set was enriched for nuclear receptor signaling, the set specific to nursing was enriched for olfaction and neuron differentiation, and the set enriched for aggression was enriched for cytoskeleton, metabolism, and chromosome organization. Whole brain histone profiling after the affiliative interaction revealed few changes in chromatin accessibility, suggesting that the transcriptomic changes derive from already accessible areas of the genome. Although only one stimulus of each type was studied, we suggest that elements of the observed transcriptomic responses reflect molecular encoding of stimulus valence, thus priming individuals for future encounters. This hypothesis is supported by behavioral analyses showing that bees responding to either the affiliative or agonistic stimulus exhibited a higher probability of repeating the same behavior but a lower probability of performing the opposite behavior. These findings add to our understanding of the biological embedding at the molecular level.