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Developmental and epileptic encephalopathy 56 (DEE-56) is caused by pathogenic variants in YWHAG and is characterized by early-onset epilepsy and neurodevelopmental delay. This study reports on a cohort of DEE-56 individuals, correlating antiseizure medication usage and comorbidities, to aid in understanding disease evolution. We analyzed data from thirty-nine individuals aged 3–40 years with YWHAG variants, including 12 previously unreported individuals (2 of these with recurrent distal 7q11.23 deletions) and 27 previously published cases (21 families, including 3 adult individuals reported in a family case). Our assessments encompassed clinical, radiological, and genetic evaluations. All procedures adhered to standardized protocols for patient approvals, registrations, and data collection. Individuals with YWHAG variants exhibited variable psychomotor delay, with the majority experiencing mild intellectual disability. Early-onset seizures, particularly febrile seizures, were common, with various seizure types reported. Valproic acid has emerged as an effective antiseizure medication. Movement disorders were present in a subset of individuals, primarily manifesting as ataxia and tremor. Comorbidities such as autism spectrum disorders and attention deficit-hyperreactivity disorder were observed in a proportion of individuals. We identified a novel YWHAG variant (c.634_645del/p.Asn212_Ser215del) and expanded the genotypic spectrum of the disease. We provide insights into the clinical, radiological, and genetic features of YWHAG -related epileptic encephalopathy. Despite mild clinical symptoms, affected individuals face challenges in daily functioning, underscoring the need for comprehensive care. Valproic acid has been used for seizure control with variable results. • Early epilepsy onset often with febrile seizures in DEE-56 individuals. • DEE-56 individuals show variable psychomotor delay, mostly mild. • DEE-56 and 7q11.23del show broad developmental delay/intellectual disability with ataxia, autism, and ADHD. • Novel YWHAG variant c.634_645del/p.Asn212_Ser215del added to genotype spectrum. [ABSTRACT FROM AUTHOR]

Transcriptional enhancers, including super-enhancers (SEs), form physical interactions with promoters to regulate cell-type-specific gene expression. SEs are characterized by high transcription factor occupancy and large domains of active chromatin, and they are commonly assigned to target promoters using computational predictions. How promoter-SE interactions change upon cell state transitions, and whether transcription factors maintain SE interactions, have not been reported. Here, we used promoter-capture Hi-C to identify promoters that interact with SEs in mouse embryonic stem cells (ESCs). We found that SEs form complex, spatial networks in which individual SEs contact multiple promoters, and a rewiring of promoter-SE interactions occurs between pluripotent states. We also show that long-range promoter-SE interactions are more prevalent in ESCs than in epiblast stem cells (EpiSCs) or Nanog-deficient ESCs. We conclude that SEs form cell-type-specific interaction networks that are partly dependent on core transcription factors, thereby providing insights into the gene regulatory organization of pluripotent cells. [ABSTRACT FROM AUTHOR]

To produce a diverse antibody repertoire, immunoglobulin heavy-chain (Igh) loci undergo large-scale alterations in structure to facilitate juxtaposition and recombination of spatially separated variable (V H), diversity (D H), and joining (J H) genes. These chromosomal alterations are poorly understood. Uncovering their patterns shows how chromosome dynamics underpins antibody diversity. Using tiled Capture Hi-C, we produce a comprehensive map of chromatin interactions throughout the 2.8-Mb Igh locus in progenitor B cells. We find that the Igh locus folds into semi-rigid subdomains and undergoes flexible looping of the V H genes to its 3′ end, reconciling two views of locus organization. Deconvolution of single Igh locus conformations using polymer simulations identifies thousands of different structures. This heterogeneity may underpin the diversity of V(D)J recombination events. All three immunoglobulin loci also participate in a highly specific, developmentally regulated network of interchromosomal interactions with genes encoding B cell-lineage factors. This suggests a model of interchromosomal coordination of B cell development. [Display omitted] • Capture Hi-C maps intra- and interchromosomal contacts of the immunoglobulin heavy chain • Polymer modeling shows thousands of different Igh structures that underpin diversity • The Ig loci participate in a stage-specific interchromosomal network with B lineage genes Mielczarek et al. use Capture Hi-C to obtain a detailed map of chromosomal interactions within the immunoglobulin heavy-chain locus. Polymer modeling shows that each Igh structure is unique, enabling antibody diversity. The Ig loci associate with key B lineage genes in a developmental-stage-specific interchromosomal network. [ABSTRACT FROM AUTHOR]

Summary Global DNA demethylation is an integral part of reprogramming processes in vivo and in vitro, but whether it occurs in the derivation of induced pluripotent stem cells (iPSCs) is not known. Here, we show that iPSC reprogramming involves both global and targeted demethylation, which are separable mechanistically and by their biological outcomes. Cells at intermediate-late stages of reprogramming undergo transient genome-wide demethylation, which is more pronounced in female cells. Global demethylation requires activation-induced cytidine deaminase (AID)-mediated downregulation of UHRF1 protein, and abolishing demethylation leaves thousands of hypermethylated regions in the iPSC genome. Independently of AID and global demethylation, regulatory regions, particularly ESC enhancers and super-enhancers, are specifically targeted for hypomethylation in association with transcription of the pluripotency network. Our results show that global and targeted DNA demethylation are conserved and distinct reprogramming processes, presumably because of their respective roles in epigenetic memory erasure and in the establishment of cell identity. [ABSTRACT FROM AUTHOR]

Summary: The small G protein family Rac has numerous regulators that integrate extracellular signals into tight spatiotemporal maps of its activity to promote specific cell morphologies and responses. Here, we have generated a mouse strain, Rac-FRET, which ubiquitously expresses the Raichu-Rac biosensor. It enables FRET imaging and quantification of Rac activity in live tissues and primary cells without affecting cell properties and responses. We assessed Rac activity in chemotaxing Rac-FRET neutrophils and found enrichment in leading-edge protrusions and unexpected longitudinal shifts and oscillations during protruding and stalling phases of migration. We monitored Rac activity in normal or disease states of intestinal, liver, mammary, pancreatic, and skin tissue, in response to stimulation or inhibition and upon genetic manipulation of upstream regulators, revealing unexpected insights into Rac signaling during disease development. The Rac-FRET strain is a resource that promises to fundamentally advance our understanding of Rac-dependent responses in primary cells and native environments. [Copyright &y& Elsevier]

Collagen I is a major tendon protein whose polypeptide chains are linked by covalent crosslinks. It is unknown how the crosslinking contributes to the mechanical properties of tendon or whether crosslinking changes in response to stretching or relaxation. Since their discovery, imine bonds within collagen have been recognized as being important in both crosslink formation and collagen structure. They are often described as acidic or thermally labile, but no evidence is available from direct measurements of crosslink levels whether these bonds contribute to the mechanical properties of collagen. Here, we used MS to analyze these imine bonds after reduction with sodium borohydride while under tension and found that their levels are altered in stretched tendon. We studied the changes in crosslink bonding in tail tendon from 11-week-old C57Bl/6 mice at 4% physical strain, at 10% strain, and at breaking point. The crosslinks hydroxy-lysino-norleucine (HLNL), dihydroxylysino- norleucine (DHLNL), and lysino-norleucine (LNL) increased or decreased depending on the specific crosslink and amount of mechanical strain. We also noted a decrease in glycated lysine residues in collagen, indicating that the imine formed between circulating glucose and lysine is also stress labile. We also carried out mechanical testing, including cyclic testing at 4% strain, stress relaxation tests, and stress-strain profiles taken at breaking point, both with and without sodium borohydride reduction. The results from both the MS studies and mechanical testing provide insights into the chemical changes during tendon stretching and directly link these chemical changes to functional collagen properties. [ABSTRACT FROM AUTHOR]

Almost half of all individuals affected by intellectual disability (ID) remain undiagnosed. In the Solve-RD project, exome sequencing (ES) datasets from unresolved individuals with (syndromic) ID (n = 1,472 probands) are systematically reanalyzed, starting from raw sequencing files, followed by genome-wide variant calling and new data interpretation. This strategy led to the identification of a disease-causing de novo missense variant in TUBB3 in a girl with severe developmental delay, secondary microcephaly, brain imaging abnormalities, high hypermetropia, strabismus and short stature. Interestingly, the TUBB3 variant could only be identified through reanalysis of ES data using a genome-wide variant calling approach, despite being located in protein coding sequence. More detailed analysis revealed that the position of the variant within exon 5 of TUBB3 was not targeted by the enrichment kit, although consistent high-quality coverage was obtained at this position, resulting from nearby targets that provide off-target coverage. In the initial analysis, variant calling was restricted to the exon targets ± 200 bases, allowing the variant to escape detection by the variant calling algorithm. This phenomenon may potentially occur more often, as we determined that 36 established ID genes have robust off-target coverage in coding sequence. Moreover, within these regions, for 17 genes (likely) pathogenic variants have been identified before. Therefore, this clinical report highlights that, although compute-intensive, performing genome-wide variant calling instead of target-based calling may lead to the detection of diagnostically relevant variants that would otherwise remain unnoticed. [ABSTRACT FROM AUTHOR]

Collagen is a structural protein whose internal cross-linking critically determines the properties and functions of connective tissue. Knowing how the cross-linking of collagen changes with age is key to understanding why the mechanical properties of tissues change over a lifetime. The current scientific consensus is that collagen cross-linking increases with age and that this increase leads to tendon stiffening. Here, we show that this view should be reconsidered. Using MS-based analyses, we demonstrated that during aging of healthy C57BL/6 mice, the overall levels of collagen cross-linking in tail tendon decreased with age. However, the levels of lysine glycation in collagen, which is not considered a cross-link, increased dramatically with age. We found that in 16-week-old diabetic db/db mice, glycation reaches levels similar to those observed in 98-week-old C57BL/6 mice, while the other cross-links typical of tendon collagen either decreased or remained the same as those observed in 20-week-oldWT mice. These results, combined with findings from mechanical testing of tendons from these mice, indicate that overall collagen cross-linking in mouse tendon decreases with age. Our findings also reveal that lysine glycation appears to be an important factor that contributes to tendon stiffening with age and in diabetes. [ABSTRACT FROM AUTHOR]

Class I phosphoinositide 3-kinases (PI3Ks) are important regulators of neutrophil migration in response to a range of chemoattractants. Their primary lipid products PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 preferentially accumulate near to the leading edge of migrating cells and are thought to act as an important cue organizing molecular and morphological polarization. We have investigated the distribution and accumulation of these lipids independently in mouse neutrophils using eGFP-PH reportersand electron microscopy (EM). We found that authentic mouse neutrophils rapidly polarized their Class I PI3K signalling, as read-out by eGFP-PH reporters, both at the up-gradient leading edge in response to local stimulation with fMLP as well as spontaneously and randomly in response to uniform stimulation. EM studies revealed these events occurred at the plasma membrane, were dominated by accumulation of PtdIns(3,4,5)P 3 , but not PtdIns(3,4)P 2 , and were dependent on PI3Kγ and its upstream activation by both Ras and Gβγs. [ABSTRACT FROM AUTHOR]