Biology subjects

Kaplan, N.

Publications and source records attributed to Kaplan, N..

15 recordsLinked to original sources

bioRxiv2026

Micro-C-TALE: BAC-based targeted high-resolution measurement of 3D chromatin interactions

Chromosome conformation capture methods have greatly advanced our understanding of genome organization, but obtaining high-resolution interaction maps of selected genomic regions remains costly due to the sequencing requirements. Here, we introduce Micro-C-TALE, a new chromosome conformation capture method that combines Micro-C, which uses micrococcal nuclease to improve the resolution and sensitivity of chromatin interaction measurements, and C-TALE, which uses probes generated from bacterial artificial chromosomes (BACs) for targeted enrichment. This combination enables the generation of high-resolution interaction maps without requiring custom synthesis of capture oligonucleotides. We applied Micro-C-TALE to a 214 kb region in human HAP1 cells, obtaining detailed interaction maps with approximately 5-6 million sequenced read pairs. Compared with published genome-wide Micro-C data, Micro-C-TALE yielded a higher amount of informative interactions within the targeted region despite ~550-fold lower sequencing depth. To evaluate the sensitivity of the method to fine-scale changes in genome organization, we introduced a heterozygous CTCF binding site into the targeted region using CRISPR-Cas9. Comparing Micro-C-TALE to the restriction enzyme-based C-TALE, we found Micro-C-TALE yielded a ~5-fold higher fraction of informative reads and detected novel CTCF-mediated point interactions associated with the inserted CTCF site, which were barely discernible using C-TALE. Our results demonstrate that Micro-C-TALE provides a flexible and cost-effective approach for targeted high-resolution measurement of chromatin interactions, facilitating the investigation of fine-scale genome organization and its response to genetic perturbations.

genomics↗

bioRxiv2026

Angiotensin (1-7)-based Proteomimetic Polymers as Topicals for the Remediation and Repair of the Cornea following Injury

The cornea is the outermost layer of the eye, most vulnerable to injury including from chemical exposure, either environmental, industrial or chemical weapons. For example, exposure of the eye to sulfur mustard (SM) can cause mustard gas keratopathy, resulting in devastating consequences for vision. Available treatments for corneal mustard keratopathy are either ineffective, or if used for a prolonged time, have deleterious side effects. Here, we aimed to harness the anti-inflammatory and anti-angiogenic peptide angiotensin-(1-7), known as Ang-(1-7), that is derived from the renin-angiotensin system (RAS). We designed and developed a protein-like polymer (PLP) displaying a brush of norleucine modified Ang (1-7) peptides (NLE Ang (1-7) PLP), which is a more potent derivative of Ang-(1-7), for administration as an eye drop. In a mouse model of nitrogen mustard (NM)-induced corneal injury, topical treatment and intraperitoneal (i.p.) injection of NLE Ang (1-7) PLP persistently minimized: (i) corneal haze; (ii) edema; (iii), expression of pro-inflammatory and pro-angiogenic genes; and (iv) neovascularization and conjunctivalization as evidenced after a prolonged treatment and recovery period. Collectively, our observations have demonstrated the efficacy of this novel polymer proteomimetic based eye drop treatment in reducing chemical agent-induced corneal injury as well as indicating its potential application in other ocular surface diseases associated with inflammation and angiogenesis.

bioengineering↗

bioRxiv2026

Microbiome-mediated Biotransformation of Human Lactoferrin (effera(R)) Enhances Epithelial Barrier Function in an Ex Vivo Adult Model

Lactoferrin is a multifunctional iron-binding glycoprotein that supports intestinal barrier function, immune regulation, and a favorable gut microbial environment. However, the contribution of gut microbial biotransformation to its gastrointestinal activity remains poorly understood. We investigated whether effera(R), a precision fermentation-derived recombinant human lactoferrin, supports intestinal barrier function through microbiome-mediated mechanisms. effera(R) underwent simulated upper gastrointestinal digestion followed by ex vivo colonic fermentation using the validated SIFR(R) technology pipeline, which employs bioreactors that are inoculated with fecal microbiota from six healthy adult donors. Microbial activity was evaluated by measuring short-chain fatty acid (SCFA) production, bacterial cell density, and microbiome composition. effera(R) produced dose-dependent increases in the production of SCFAs and bacterial cell density demonstrating enhanced microbial metabolic activity. These metabolic changes were accompanied by shifts in key microbial groups within Bacillota_A and Bacteroidota. Intact effera(R) and cell-free post-colonic fermentation-derived products were evaluated in a Caco-2/THP-1 epithelial-immune co-culture model under basal and lipopolysaccharide-challenged conditions. Whereas intact protein did not significantly improve epithelial barrier integrity, effera(R)s post-colonic fermentation-derived products significantly enhanced transepithelial electrical resistance (TEER) under basal conditions and produced an even stronger barrier-protective response following LPS challenge. Across matched doses, effera(R) consistently generated greater TEER responses than bovine lactoferrin. Improved barrier function was accompanied by increased expression of tight-junction-associated targets ZO-1 and occludin and reduced secretion of CXCL-10 and IL-8. Together, these findings demonstrate that microbial biotransformation enhances the biological activity of effera(R), linking increased microbial metabolism with improved epithelial barrier integrity and modulation of inflammatory signaling. This integrated study provides a strong mechanistic foundation for the use of human lactoferrin in adult gut-health applications and offers valuable guidance for future adult clinical studies and infant-relevant investigations.

microbiology↗

bioRxiv2026

CTCF-mediated cis-regulatory chromatin insulation enforces a central B-cell tolerance checkpoint

The generation of a diverse and self-tolerant B cell repertoire is essential for adaptive immunity and is achieved through V(D)J recombination. In mice, Ig{kappa} is the dominant light chain, whereas Ig{lambda} rearrangement typically occurs in response to nonproductive or autoreactive Ig{kappa} recombination, a process termed receptor editing. Recombination at the RS element deletes the Ig{kappa} constant exon, silencing the locus and enabling Ig{lambda} expression. However, the epigenetic regulatory framework that orchestrates and governs receptor editing remains poorly defined. Here, we identify a CTCF-binding insulator element (CBE) within the 3' Ig{kappa} super-enhancer (3'-SE{kappa}) that regulates receptor editing and directs the {kappa}-to-{lambda} switch required for Ig{lambda} B-cell development. Mechanistically, loss of this CBE activates an insulated enhancer within the 3'-SE{kappa}, causing aberrant V{kappa} rearrangements and altered chromatin interactions through disrupted loop extrusion dynamics. Notably, loss of this CBE in mice leads to increased autoantibody production by ten weeks of age, demonstrating that CBE-mediated chromatin architecture shapes B cell fate by constraining autoreactive potential. Collectively, our findings define a novel CTCF-dependent cis-regulatory insulation checkpoint that connects chromatin loop extrusion to antigen-driven receptor editing, thereby enforcing B-cell tolerance.

immunology↗

bioRxiv2024

Fast Low-Input Efficient Hi-C

We developed FLIE (Fast Low-Input Efficient) Hi-C, a highly optimized and simplified version of Hi-C. Our method shortens the duration of Hi-C from five days to two days and decreases key reagent amounts 5-20 fold, while maintaining experimental controls and data quality. Importantly, the method also reduces the required input material by 100-1000 fold, as we demonstrate by generating detailed high complexity interaction maps from low-input sorted mouse frozen neuron nuclei and from 5,000-10,000 human Hap1 cells.

genomics↗

bioRxiv2024

Large-scale manipulation of radial positioning does not affect most aspects of genome organization

Although the spatial organization of the genome is closely linked to biological function, genome structure is highly stochastic. Within this heterogeneity, specific function-associated structural elements must be maintained, even when the nucleus is deformed due to physiological physical constraints. The radial positioning of genomic loci - their distance from the nuclear periphery - has long been considered an important feature of genome organization which is correlated with both structural elements and genomic activity. In the current study, we developed an experimental system for manipulating the radial positioning of the genome, by expressing the sperm-specific protein Prm1 in somatic cells. By microscopy, we observe that initial Prm1 nuclear foci develop within 72 hours into a large Prm1 focus occupying most of the nuclear interior, while the entire genome is driven towards the nuclear periphery, resulting in a 3-5 fold reduction in the volume that the genome occupies. Noting that this system enables isolation of a pure population of cells with reorganized nuclei, we then used Hi-C to study the effects of this perturbation. Remarkably, we find that interaction patterns are largely robust to this major nuclear reorganization, with minor changes which mostly reflect a strengthening of heterochromatin self-interactions. Our experimental system provides means for manipulating nuclear organization in a reproducible manner, potentially allowing to examine radial positioning decoupled from other features of genome organization. Highlighting the complementary nature of microscopic and genomic methods, our work further suggests a remarkable resilience of genome structure such that large-scale nuclear changes, including chromosome compression and changes in radial positioning, can occur without extensive alteration of functional genome organization.

genomics↗

bioRxiv2024

Cell cycle-driven transcriptome maturation confers multilineage competence to cardiopharyngeal progenitors

During development, stem and progenitor cells divide and transition through germ layer- and lineage-specific multipotent states to generate the diverse cell types that compose an animal. Defined changes in biomolecular composition underlie the progressive loss of potency and acquisition of lineage-specific characteristics. For example, multipotent cardiopharyngeal progenitors display multilineage transcriptional priming, whereby both the cardiac and pharyngeal muscle programs are partially active and coexist in the same progenitor cells, while their daughter cells engage in a cardiac or pharyngeal muscle differentiation path only after cell division. Here, using the tunicate Ciona, we studied the acquisition of multilineage competence and the coupling between fate decisions and cell cycle progression. We showed that multipotent cardiopharyngeal progenitors acquire the competence to produce distinct Tbx1/10 (+) and (-) daughter cells shortly before mitosis, which is necessary for Tbx1/10 activation. By combining transgene-based sample barcoding with single cell RNA-seq (scRNA-seq), we uncovered transcriptome-wide dynamics in migrating cardiopharyngeal progenitors as cells progress through G1, S and G2 phases. We termed this process "transcriptome maturation", and identified candidate "mature genes", including the Rho GAP-coding gene Depdc1, which peak in late G2. Functional assays indicated that transcriptome maturation fosters cardiopharyngeal competence, in part through multilineage priming and proper oriented and asymmetric division that influences subsequent fate decisions, illustrating the concept of "behavioral competence". Both classic feedforward circuits and coupling with cell cycle progression drive transcriptome maturation, uncovering distinct levels of coupling between cell cycle progression and fateful molecular transitions. We propose that coupling competence and fate decision with the G2 and G1 phases, respectively, ensures the timely deployment of lineage-specific programs.

developmental biology↗

bioRxiv2023

CTCF-mediated 3D chromatin predetermines the gene expression program in the male germline

Spermatogenesis is a unidirectional differentiation process that generates haploid sperm, but how the gene expression program that directs this process is established is largely unknown. Here we determine the high-resolution 3D chromatin architecture of male germ cells during spermatogenesis and show that CTCF-mediated 3D chromatin predetermines the gene expression program required for spermatogenesis. In undifferentiated spermatogonia, CTCF-mediated chromatin contacts on autosomes pre-establish meiosis-specific super-enhancers (SE). These meiotic SE recruit the master transcription factor A-MYB in meiotic spermatocytes, which strengthens their 3D contacts and instructs a burst of meiotic gene expression. We also find that at the mitosis-to-meiosis transition, the germline-specific Polycomb protein SCML2 resolves chromatin loops that are specific to mitotic spermatogonia. Moreover, SCML2 and A-MYB establish the unique 3D chromatin organization of sex chromosomes during meiotic sex chromosome inactivation. We propose that CTCF-mediated 3D chromatin organization enforces epigenetic priming that directs unidirectional differentiation, thereby determining the cellular identity of the male germline.

developmental biology↗

bioRxiv2023

A genome-wide nucleosome-resolution map of promoter-centered interactions in human cells corroborates the enhancer-promoter looping model

The enhancer-promoter looping model, in which enhancers activate their target genes via physical contact, has long dominated the field of gene regulation. However, the ubiquity of this model has been questioned due to evidence of alternative mechanisms and the lack of its systematic validation, primarily owing to the absence of suitable experimental techniques. In this study, we present a new MNase-based proximity ligation method called MChIP-C, allowing for the measurement of protein-mediated chromatin interactions at single-nucleosome resolution on a genome-wide scale. By applying MChIP-C to study H3K4me3 promoter-centered interactions in K562 cells, we found that it had greatly improved resolution and sensitivity compared to restriction endonuclease-based C-methods. This allowed us to identify EP300 histone acetyltransferase and the SWI/SNF remodeling complex as potential candidates for establishing and/or maintaining enhancer-promoter interactions. Finally, leveraging data from published CRISPRi screens, we found that most functionally-verified enhancers do physically interact with their cognate promoters, supporting the enhancer-promoter looping model.

genomics↗

bioRxiv2022

Hypothesis-driven probabilistic modelling enables a principled perspective of genomic compartments

The Hi-C method has revolutionized the study of genome organization, yet interpretation of Hi-C interaction frequency maps remains a major challenge. Genomic compartments are a checkered Hi-C interaction pattern suggested to represent the partitioning of the genome into two self-interacting states associated with active and inactive chromatin. Based on a few elementary mechanistic assumptions, we derive a generative probabilistic model of genomic compartments, called deGeco. Testing our model, we find it can explain observed Hi-C interaction maps in a highly robust manner, allowing accurate inference of interaction probability maps from extremely sparse data without any training of parameters. Taking advantage of the interpretability of the model parameters, we then test hypotheses regarding the nature of genomic compartments. We find clear evidence of multiple states, and that these states self-interact with different affinities. We also find that the interaction rules of chromatin states differ considerably within and between chromosomes. Inspecting the molecular underpinnings of a four-state model, we show that a simple classifier can use histone marks to predict the underlying states with 87% accuracy. Finally, we observe instances of mixed-state loci and analyze these loci in single-cell Hi-C maps, finding that mixing of states occurs mainly at the population level.

genomics↗

bioRxiv2022

Recruitment of transcriptional effectors by Cas9 creates cis regulatory elements and demonstrates distance-dependent transcriptional regulation

It is essential to regulate the expression of genes, such as those encoding the proteins of the cardiac sarcomere. This regulation is often mediated by cis regulatory elements termed enhancers and repressors that recruit transcription factors to gene-distal sites. However, the relationship between transcription factors recruitment to gene-distant sites and the regulation of gene expression is not fully understood. Specifically, it is unclear if such recruitment to any genomic site is sufficient to form an enhancer or repressor at the site, and what is the relationship between the cis regulatory elements position and its ability to control the transcription of distant genes. Using dead Cas9 to recruit either viral or endogenous transcription factor activation domains, we demonstrate that targeting naive genomic sites lacking open chromatin or active enhancer marks is sufficient to alter the chromatin signature of the target site, the distant gene promoter, and significantly induce the distant gene expression, even across chromatin insulating loci. The magnitude of induction is affected by the distance between the activation site and the cognate gene in a non-linear manner. Dead Cas9 mediated recruitment of repression domains behave similarly to activation in that targeting of non-regulatory regions could repress gene expression with a nonlinear distance dependence and across chromatin insulating loci. These findings expand the models of enhancer generation and function by showing that an arbitrary genomic site can become a regulatory element and interact epigenetically and transcriptionally with a distant promoter. They also provide new fundamental insights into the rules governing gene expression.

genomics↗

bioRxiv2020

KMD clustering: Robust generic clustering of biological data

The noisy and high-dimensional nature of biological data has spawned advanced clustering algorithms that are tailored for specific biological datatypes. However, the performance of such methods varies greatly between datasets and they require post hoc tuning of cryptic hyperparameters. We present k minimal distance (KMD) clustering, a general-purpose method based on a generalization of single and average linkage hierarchical clustering. We introduce a generalized silhouette-like function to eliminate the cryptic hyperparameter k, and use sampling to enable application to million-object datasets. Rigorous comparisons to general and specialized clustering methods on simulated, mass cytometry and scRNA-seq datasets show consistent high performance of KMD clustering across all datasets.

bioinformatics↗

bioRxiv2020

Asymmetric second generation genomic incompatibility in interspecific crosses between Ciona robusta and Ciona intestinalis

Reproductive isolation is central to speciation, but interspecific crosses between two closely related species can produce viable and fertile hybrids. Two different species in the tunicate genus Ciona, Ciona robusta and Ciona intestinalis can produce hybrids. However, wild sympatric populations display limited gene flow, suggesting the existence of obstacles to interspecific reproduction that remain unknown. Here, we took advantage of a closed inland culture system to cross C. robusta with C. intestinalis and established F1 and F2 hybrids. We monitored post-embryonic development, survival, and sexual maturation to further probe the physiological mechanisms underlying reproductive isolation. Partial viability of first and second generation hybrids indicated that both pre- and postzygotic mechanisms contributed to genomic incompatibilities in hybrids. Asymmetrical second generation inviability and infertility suggested that interspecific genomic incompatibilities involved interactions between the maternal, zygotic and mitochondrial genomes during development. This study paves the way to quantitative genetic approaches to study the mechanisms underlying genomic incompatibilities and other complex traits in the genome-enabled Ciona model.

evolutionary biology↗

bioRxiv2018

Three invariant Hi-C interaction patterns: applications to genome assembly

Assembly of reference-quality genomes from next-generation sequencing data is a key challenge in genomics. Recently, we and others have shown that Hi-C data can be used to address several outstanding challenges in the field of genome assembly. This principle has since been developed in academia and industry, and has been used in the assembly of several major genomes. In this paper, we explore the central principles underlying Hi-C-based assembly approaches, by quantitatively defining and characterizing three invariant Hi-C interaction patterns on which these approaches can build: Intrachromosomal interaction enrichment, distance-dependent interaction decay and local interaction smoothness. Specifically, we evaluate to what degree each invariant pattern holds on a single locus level in different species, cell types and Hi-C map resolutions. We find that these patterns are generally consistent across species and cell types but are affected by sequencing depth, and that matrix balancing improves consistency of loci with all three invariant patterns. Finally, we overview current Hi-C-based assembly approaches in light of these invariant patterns and demonstrate how local interaction smoothness can be used to easily detect scaffolding errors in extremely sparse Hi-C maps. We suggest that simultaneously considering all three invariant patterns may lead to better Hi-C-based genome assembly methods.

genomics↗

bioRxiv2017

Study of the mitotic chromatin shows involvement of histone modifications in bookmarking and reveals nucleosome deposition patterns

Mitosis encompasses key molecular changes including chromatin condensation, nuclear envelope breakdown, and reduced transcription levels. Immediately after mitosis, the interphase chromatin structure is reestablished and transcription resumes. The reestablishment of the interphase chromatin is probably achieved by bookmarking, i.e., the retention of at least partial information during mitosis. Yet, while recent studies demonstrate that chromatin accessibility is generally preserved during mitosis and is only locally modulated, the exact details of the bookmarking process and its components are still unclear. To gain a deeper understanding of the mitotic bookmarking process, we merged proteomics, immunofluorescence, and ChIP-seq approaches to study the mitotic and interphase genomic organization of human cells. We focused on key histone modifications and employed HeLa-S3 cells as a model system. Generally, we observed a global concordance between the genomic organization of histone modifications in interphase and mitosis, yet the abundance of the two types of modifications we investigated was different. Whereas histone methylation patterns remain highly similar, histone acetylation patterns show a general reduction while maintaining their genomic organization. These results demonstrate that the epigenomic landscape can serve as a major component of the mitotic bookmarking process. Next, to further investigate mitosis-associated chromatin changes, we followed up on previous studies that showed that nucleosome depleted regions (NDRs) become occupied by a nucleosome during mitosis. Surprisingly, we observed that the nucleosome introduced into the NDR during mitosis encompasses a distinctive set of histone modifications, differentiating it from the surrounding nucleosomes. We show that the nucleosomes near the NDR appear to both shift into the NDR during mitosis and adopt a unique modification pattern. HDAC inhibition by the small molecule TSA reverts this pattern. These results provide evidence for a mitotic deposition and change in the modifications of the nucleosomes surrounding the NDR. Altogether, by merging multiple approaches, our study provides evidence to support a model where mitotic bookmarking is achieved by histone modifications and uncovers new insights into the deposition of nucleosomes during mitosis.

genomics↗