Studying a new species using high-throughput sequencing requires a high-quality reference genome. However, assembling chromosome length sequences remains challenging. Recent advances in chromatin conformation capture (Hi-C) have provided a new approach to scaffolding genome assemblies, and the last ten years have seen a proliferation of such methods. However, to our knowledge no comprehensive benchmarking of Hi-C scaffolders has been conducted to date. Through a literature review we identify the most popular Hi-C scaffolders - Lachesis, HiRise, 3d-dna, SALSA, and AllHiC. We test their ability to scaffold four well studied genomes - S. cerevisiae, L. tarentolae, A. thaliana, and H. sapiens. Scaffolders are tasked with both scaffolding fragmented versions of the reference genome as well as de novo assemblies derived from long read datasets. We find that all scaffolders can exceed 80% accuracy under ideal circumstances but that their performance quickly deteriorates under more challenging conditions. Surprisingly, many scaffolders also show poor performance on the best assemblies, where contigs are near chromosome length. Overall, we find that HiRise and Lachesis offer the best performance on average across all conditions. We compare the performance of five Hi-C scaffolders using multiple reference species under both ideal and real-life conditions, thereby illuminating their strengths and weaknesses.

Apios americana and Apios priceana are tuber-forming legumes native to North America with ecological value and agricultural potential. The lack of genomic resources has limited comparative studies and crop improvement for these species. Here, we report high-quality, haplotype-resolved, chromosome-level genome assemblies for both the species. The assemblies were generated from high-fidelity long-read data, and the primary assemblies were scaffolded using Omni-C chromatin conformation maps. The genome sizes of the primary haplotype assemblies were 1.53 Gb for A. americana and 1.85 Gb for A. priceana, each represented by 11 pseudochromosomes. The BUSCO completeness scores ranged from 98.6% to 99.0%. Approximately 26,000 predicted genes (30,000-33,000 predicted mRNA transcripts) were identified per haplotype in A. americana and A. priceana, respectively. Repeat annotation revealed that over 80% of both genomes consist of interspersed repetitive elements, with the most abundant being long terminal repeat (LTR) retrotransposons. These genomic resources will support trait mapping and structural variation analyses in Apios, and more broadly, comparative genomics within the legume family.

Aspergillus flavus, along with its notorious secondary metabolite aflatoxin B1 (AFB1), seriously endangers human health. Histone methyltransferase complex COMPASS (complex of proteins associated with Set1) plays a crucial role in regulating aflatoxin biosynthesis and virulence of A. flavus, but the underlying mechanism is unclear. Here, we find that Bre2, the key subunit of COMPASS, regulates AFB1 biosynthesis, fungal morphogenesis, and virulence through modulation of H3K4 methylation. ChIP-seq and biochemical analyses reveal that chromatin remodeling factor (CRF) Arp9 is directly targeted by Bre2, and Arp9 exerts bio-functions through interacting with the other CRFs such as RSC8, Arp7, and Sth1. ATAC-seq results indicate that Arp9 contributes to fungal pathogenicity by modulating chromatin conformation of genes that are involved in secondary metabolism, morphogenesis, and virulence. The study reveals an epigenetic signaling pathway mediated by chromatin remodeler Arp9 and provides a potential strategy for the control of pathogenic fungi and mycotoxins.

The three-dimensional (3D) organization of cis-regulatory elements (CREs) is critical in transcription control. However, capturing transcriptome, epigenome and 3D genome from the same single cells remains challenging. Here we present scHiCAR (single-cell Hi-C with assay for transposase-accessible chromatin and RNA sequencing), a plate-based combinatorial barcoding method that simultaneously profiles mRNA, open chromatin and chromosome conformation capture from the same cells. Compared to existing single-cell 3D genome methods, scHiCAR more efficiently enriches long-range cis-interactions anchored at candidate CREs (cCREs). Applied to 1.62 million mouse brain cells and complemented with a deep-learning-based loop caller, scHiCAR accurately defines cell-type-specific transcriptomes, accessible cCREs and 5-kb-resolution enhancer-promoter pairs across 22 brain cell types. scHiCAR also performs robustly in challenging tissues such as skeletal muscle, enabling trimodal single-cell-level analysis of gene regulation dynamics during muscle stem cell regeneration. By providing a scalable and cost-effective system for single-cell trimodal analysis of gene-regulatory landscapes in complex tissues, scHiCAR reveals gene-locus-specific regulatory roles of 3D genome reorganization in transcriptional control.

Abstract Background: Despite an undoubted benefit of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) in estrogen receptor positive (ER+) breast cancer (BC), resistance eventually occurs. DNA-Sequencing studies identified genomic alterations that did not fully recapitulate the resistance landscape. Therefore, we explored whether changes in three-dimensional (3-D) chromatin landscape, potentially induced by single-nucleotide variants (SNVs) of non-coding regulatory regions, are involved in resistance to CDK4/6i. Methods: We generated ER+/HER2- palbociclib-resistant T47D and MCF7 BC cells (T47D-PR and MCF7-PR), by exposing parental cells to increasing doses of the drug until resistance occurred. In resistant and parental cells, we performed: 1) High-throughput Chromosome Conformation Capture (Hi-C), to investigate 3D chromatin remodelling associated with drug resistance; 2) Whole Genome Sequencing (WGS), to detect SNVs, insertions or deletions (InDels, <50 bp) acquired in PR cells; 3) ATAC-Seq to identify transcriptional factors (TFs) potentially leading to drug resistance and 4) RNA-Seq for gene expression. Results: Hi-C identified 2,189 differential interactions (loops) between T47D and T47D-PR cells (FDR < 0.05). In detail, T47D-PR cells showed 844 loops gained and 1,345 loops lost. Similarly, we found 896 gained and 1,086 lost interactions in MCF7-PR cells. We intersected coordinates of differential loops with list of candidate enhancers and promoters from cis-Regulatory Elements by ENCODE. We found that ∼80% of all differential interactions in PR cells were enhancers, suggesting their involvement in transcriptional reprogramming. Consistently, analysis of integrated RNA-Seq and Hi-C data revealed that gained/lost loops were associated with changes in expression of their target genes in PR vs. parental cells. By WGS, in T47D-PR cells, we found 5,465 acquired SNVs and 5,248 InDels. Notably, 61.7% and 29.4% SNVs occurred at intergenic and intron genomic regions, respectively. Next, 2,695 (51.3%) and 2,137 (40.7%) InDels occurred at intergenic and intron regions, respectively. In MCF7-PR cells, we found 7,386 acquired SNVs and 5,206 InDels. More in detail, 4,602 (62.3%) and 2,102 (28.5%) SNVs occurred at intergenic and intron regions. Finally, 2,629 (50.5%) and 2,165 (41.6%) InDels occurred at intergenic and intron loci, respectively. We are investigating enrichment of acquired genomic alterations in anchors of loops, compared to the rest of the genome, in PR cells, thus influencing the 3D chromatin conformation. ATAC-Seq revealed 5,223 (LogFC <-1) and 4,361 (LogFC >1) newly closed and open regions in T47D-PR vs. T47D cells, respectively. Next, in MCF7-PR vs. MCF7 cells, we found 948 and 1,625 newly closed and open regions, respectively. De novo HOMER motif analysis of newly open regions in T47D-PR and MCF7-PR cells, revealed enrichment for binding sites of FOS family members (such as Fos, Fosl1, Fosl2) and AP-1 transcription factor subunits among the top enriched motifs in both PR cells. We are testing whether therapeutic inhibition of AP-1 transcriptional complex might overcome resistance to CDK4/6i. Instead, motif analysis of newly closed regions revealed that FOXA1 was the top enriched motif in PR cells. Consistently, intersection of ATAC-Seq data with those from publicly available ChIP-seq datasets (Toolkit for Cistrome Data Browser) revealed enrichment for ERα binding sites and other ERα-interacting TFs, such as FOXA1, GATA3, and GREB1. Indeed, PR cells exhibited reduced sensitivity to fulvestrant and faster estrogen-independent growth, compared to parental cells. Conclusions: SNVs and chromatin remodeling are involved in resistance to CDK4/6i. Results from our study may help to identify novel therapeutic vulnerabilities in ER+ BC. Citation Format: F. Pepe, F. Messina, F. Napolitano, G. Nassa, A. Salvati, D. Memoli, C. Fierro, D. Esposito, S. Belli, C. Ascione, G. Attanasio, A. Vallefuoco, A. Benish, L. Formisano, R. Bianco, A. Servetto. Chromatin remodelling is involved in resistance to CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PD3-04.

This study aims to investigate structural and expression-level alterations in the histone demethylase KDM4C gene in patients with rheumatoid arthritis (RA) and elucidate its role in the disease's epigenetic basis. KDM4C sequencing was performed in RA patients, and identified variants were mapped to functional domains, including the JmjN and PHD-type2 regions. KDM4C expression was assessed by quantitative PCR in RA and control groups. Molecular findings were analyzed alongside patients' clinical characteristics and treatment histories. A rare missense mutation (c.79C > T, R27W) located in the JmjN domain was detected in a single patient with RA. In addition, a synonymous variant was identified in the PHD-type2 domain. The R27W variant is predicted by in silico analyses to impair protein homodimerization, while the synonymous change has been hypothesized to influence translational efficiency. Neither variant has previously been linked to RA. KDM4C mRNA expression was significantly reduced in patients with RA. This reduction was particularly evident in individuals with high CRP/ESR levels, positive RF and anti-CCP status, and treatment resistance. Taken together, the molecular and clinical findings suggest a potential functional alteration of KDM4C activity. Specifically, KDM4C may have a reduced capacity to remove repressive methylation marks on histone H3, particularly H3K9me3. These findings suggest that KDM4C dysregulation may promote a closed chromatin conformation in immune cells. This state may lead to silencing of genes involved in immune regulation and increased expression of inflammatory genes, thereby contributing to the pathogenesis of RA. KDM4C appears to be an important epigenetic regulator in RA pathogenesis. The coexistence of structural variants and decreased expression underscores its potential as a diagnostic biomarker and therapeutic target. To our knowledge, this is the first study providing integrated clinical and genetic evidence linking KDM4C dysregulation to RA.

Chromatin interactions establish spatial proximity between distant regulatory elements and their target genes, significantly influencing gene expression, and phenotypic traits. In this study, we present a plant chromatin interaction prediction model called PlantCTCIP based on Convolutional Neural Networks and Transformer. PlantCTCIP demonstrated superior performance compared to the conventional models. Specifically, PlantCTCIP improved the average AUC of chromatin interaction predictions by 14.56% across the four species in PPI (proximal promoter interaction) mode. Similarly, PlantCTCIP improved the average AUC of chromatin interaction predictions by 9.6% in the PDI (distal promoter interaction) mode. We constructed genome-wide chromatin interaction maps for four plants (maize, rice, cotton and wheat) through PlantCTCIP, further used the Hi-C experiment to validate correctness of the predicted PPIs and PDIs. Some key motifs that influence chromatin interactions are identified, and they are significantly enriched in expression quantitative trait loci (eQTLs) and open chromatin regions. We also analysed the enrichment and species specificity of the transcription factors (TF) and synergistic network of TFs that affect PPIs and PDIs of four crops. Using cloned genes (ZmRAVL1, ZmRPG, ZmRap2.7 and GaFZ) of maize and cotton as examples, PlantCTCIP can assist in identifying target genes regulated by distal elements and mining functional sites combined with chromatin conformation capture (3C) experiments. This research helps to analyse the regulatory mechanism of gene expression and provides novel perspectives for intelligent design breeding of diverse crops. PlantCTCIP is available at http://www.plantctcip.com.

Cheirotonus jansoni is a nationally protected and ornamental beetle species endemic to southern China, northern Burma, and Vietnam. Due to habitat degradation and illegal collection, its populations have declined significantly, prompting urgent conservation efforts. Here, we present a high-quality, chromosome-level genome assembly for C. jansoni to support future ecological and evolutionary studies. Male adults were collected from Qingliangfeng national nature reserve, and high molecular weight DNA was extracted for sequencing. The genome was assembled using PacBio long reads and scaffolded with Hi-C chromatin conformation data. The final assembly spans approximately 620.03 Mb, with a scaffold N50 of 101.60 Mb and 98.9% of sequences anchored to 10 chromosomes. Benchmarking with BUSCO revealed a 93.6% completeness rate, indicating high assembly quality. This genomic resource provides a valuable foundation for studying the genetic basis of adaptation, population decline, and conservation planning in C. jansoni and related species.

The casuarina moth, Lymantria xylina, is a serious pest threatening subtropical regions through severe defoliation and strong invasive potential. Despite its economic impact and high invasion risk, a high-quality reference genome remains lacking. To bridge this knowledge gap, we generated a chromosome-level genome assembly for L. xylina combining Illumina short-reads, Oxford Nanopore long-reads, and high-throughput chromatin conformation capture (Hi-C) scaffolding data. Following long-reads based assembly and Hi-C scaffolding, the final genome assembly totals 977.74 Mb, with 930.50 Mb (95.17%) of sequences anchored onto 31 pseudo-chromosomes, achieving a scaffold N50 of 34.15 Mb. The genome assembly, featuring fully assembled telomeres on all 31 pseudo-chromosomes, demonstrates 94.5% Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness and high accuracy with consensus quality value of 31.72. Repetitive elements constitute 77.18% of the genome, and 18,484 protein-coding genes were predicted, with 95.21% functionally annotated. This high-quality genome assembly provides a critical foundation for elucidating interaction mechanisms with host plants and natural enemies (nucleopolyhedrovirus, Beauveria bassiana), for developing enhanced pest management and control strategies.

Epigenetic modifications regulate chromatin conformation and transcription factor accessibility to regulatory regions of the genome, controlling gene expression without altering the DNA sequence itself, and being stably transmitted throughout cell divisions. One of the most well studied epigenetic marks is DNA methylation, which controls the monoallelic, parental-origin dependent expression of imprinted genes. Paternal imprinting marks are established in the male germ line, so that mature gametes - the spermatozoa - transmit correct imprints to the future embryo. Anomalies in the establishment and/or maintenance of imprinting marks can interfere with embryonic and placental development and/or result in the birth of children affected by imprinting syndromes, such as Silver-Russell (SRS) and Beckwith-Wiedemann (BWS). Here, we review the literature on the observations of imprinting errors in the male gamete, in the context of disturbances in spermatogenesis resulting in male infertility, focusing on the observations described by our group and others. We provide a clinical perspective on the implementation of sperm methylation analysis as a tool to improve diagnostic and therapeutic strategies in Assisted Reproduction Technologies (ART) and highlight the importance of understanding the molecular mechanisms underlying spermatogenic defects and male infertility.

The basal ganglia regulate motor, cognitive, and affective behaviors, and their dysfunction underlies diverse neurological and psychiatric disorders. Comprehensive, accessible multi-omics resources are needed to understand the regulatory mechanisms governing basal ganglia cell types. Here we present an open, interactive web-based platform for exploring single-cell multi-omics datasets from basal ganglia, generated using 10X Multiome, snm3C-seq, and Paired-Tag technologies from the BICAN (NIH BRAIN Initiative Cell Atlas Network) consortium. The platform is available at https://basalganglia.epigenomes.net/ and enables integrated visualization of gene expression, chromatin accessibility, DNA methylation, histone modifications, and chromatin conformation across cell types and human, macaque, marmoset, and mouse species, with direct genome browser support and comparative epigenomic functionality. Representative analyses demonstrate cell-type-specific regulatory landscapes, conserved and species-specific regulatory elements, and links between epigenomic regulation and transcription. This resource provides a scalable, community-oriented foundation for advancing basal ganglia biology and interpreting regulatory mechanisms relevant to brain function and disease.

Heavy ion beam irradiation-induced mutational profiles in Saccharomyces cerevisiae and their dependencies on dose and intracellular state inform an enhanced microbial breeding strategy.

SummaryThe human basal ganglia (BG), subcortical nuclei fundamental to motor regulation and cognitive modulation, is constructed from neurons produced during gestation in the adjacent ganglionic eminences (GEs). GEs are transient structures in the ventral prenatal brain that also generate GABAergic inhibitory neurons which migrate to destinations in the BG, cortex and other destinations. This study aims to elucidate the epigenomic and 3D-genomic dynamics involved in the specification and maturation of GEs and GE-derived neurons, using single-nucleus methyl-3C sequencing (snm3C-seq), highly-multiplexed spatial transcriptomics, and chromatin+RNA single-molecule imaging. Our multi-modal data support a heterogeneous temporal progression across GE subregions, with the lateral GE (LGE) showing declining neurogenic activity in mid-gestation and caudal GE (CGE) exhibiting ongoing developmental progression through infancy. We identified regulatory programs that specify subtypes of BG principal cells, medium spiny neurons (MSN), via synchronized maturation of the 3D-epigenome. In infant brains, we found a transient short-range enriched (SE) chromatin conformation during the transition between oligodendrocyte progenitors (OPCs) and oligodendrocytes (ODCs), and a temporary shift toward Long-range Enriched (LE) chromatin conformation in projection neurons, extending previous works showing the differentiation of neurons and glial cells is associated with permanent SE and LE conformation, respectively. Lastly, we found that gene regulatory regions active in MSNs were enriched in loci associated with genetic risk for neuropsychiatric disease. Our study delineates the highly complex, lineage-specific 3D genomic dynamics in ventral progenitors and basal ganglia populations of the perinatal human brain.

Onthophagus binodis is a coprophagous scarab beetle native to southern Africa. This species and many others in the tribe Onthophagini have been introduced to farms across multiple continents in the context of cattle pasture management efforts. The ecosystem services provided by this species, along with the amenability of comparative developmental and evolutionary studies in this clade, contribute to its role as an emerging insect model system. Here, we present sex-specific chromosomal-level genome assemblies for O. binodis generated from a combination of PacBio long reads and HiC chromatin conformation sequencing. The completeness of the 950.5 Mb female assembly and the 880.5 Mb male assembly is indicated by a contig length N50 of at least 58.6 Mb. BUSCO single-copy and duplicated completeness scores were 99.0% and 0.9% for the female assembly and 97.4% and 2.1% for the male assembly. Gene modeling identified at least 15,403 gene models in each genome with an average transcript length of 1.6 kb. Comparative analyses with other Onthophagini genomes indicated a dramatic expansion of repetitive sequences, which now comprise over 75% of this species' genome and have driven the expansion of overall genome size to nearly twice that of close relatives. We combined the best-assembled chromosome-scale scaffolds from each sex to generate a hybrid reference assembly for this species. Comparative genomic analyses show that the nine autosomes and the X chromosome identified here in O. binodis are likely conserved throughout Onthophagini. Our sex-specific sequencing approach allowed us to identify putative Y chromosome sequences in the male assembly via coverage mapping and k-mer abundance comparisons. These genomes will be of great value to the scientific community as resources for studying insect genome evolution, development, and ecology.

Fontitrygon garouaensis (Smooth Freshwater Stingray) is an evolutionarily distinct and globally endangered species and is currently the only stingray known to be strictly adapted to African freshwater systems. Lack of a reference genome has limited studies of its adaptive evolution and genomic architecture. Here, we present the first chromosome-level genome assembly of F. garouaensis using a hybrid approach that integrates PacBio HiFi long reads, Illumina short reads, and Hi-C chromatin conformation capture. The assembly spans 4.19 Gb with 41 anchored chromosomes, a scaffold N50 of 86.49 Mb, and a contig N50 of 16.58 Mb. Overall, 84.07% of the genome was assigned to chromosomes, with 65.27% repetitive elements. BUSCO analysis showed 93.3% completeness, confirming a highly contiguous genome. We annotated 29,804 protein-coding genes, with 98.28% functionally annotated. Repetitive elements comprised 65.27% of the genome, including lineage-specific expansions of DNA transposons (7.13%) and LTRs (19.31%). This genomic resource establishes a foundation for systematic, evolutionary, and conservation research on this threatened species.

Abstract Although next-generation sequencing has emerged as a powerful tool for diagnosing rare diseases (RD), many cases of inherited metabolic diseases (IMD) remain unsolved, hindering the diagnosis, clinical and therapeutic management of the patients. The primary aim of this study is to address the most elusive cases by applying long-read sequencing (LRS) targeted to the gene of interest on seven patients ( FARS2 , GYS2 , PEX1 , SLC2A1 , AGL , ACAT1 , and ACADM ), identifying six novel pathogenic variants including two intronic variants, a structural variant and three transposable elements (TE) insertions. In addition, we have demonstrated the effect on splicing of an exonic variant previously reported as missense. Functional genetic tests specific for the expected effect of each variant of uncertain significance were designed, such as minigenes analysis or chromatin conformation capture assay. From the TE insertions, two were located in the genomic region of GYS2 or PEX1 , causing a reduction in their mRNA expression. The third was located 7.6 kb downstream of SLC2A1 ; it alters the interaction between the SLC2A1 promoter and its distal regulatory element via the establishment of a loop with the 3’ border of the native topologically associating domain. This study shows that the combination of LRS and functional genetic assays confers a powerful approach for expanding the mutational spectrum of IMD, adding data to improve the diagnosis of this large group of RD.

Chromatin conformation is thought to be critical for enhancer function, but its dynamic, nanoscale organization is difficult to measure directly. Here we introduce PLOTTED (Probabilistic Localization of Oligopaint Tagged Target Element Distances), an integrated imaging and computational framework that infers chromatin architecture from targeted high-resolution imaging of cis-regulatory modules (CRMs). PLOTTED generates spatial distance distributions between DNA loci, enabling quantitative modeling of chromatin configurations across developmental time, spatial axes, and genotypes. Applying PLOTTED to the brinker locus in Drosophila embryos, we measured distances among three CRMs and used chromatin geometry as a proxy for regulatory activity. In wild type, CRM configurations shift dynamically at nuclear cycle 13, whereas these changes are delayed in mutants and vary along the dorsal-ventral and anterior-posterior axes. Importantly, these conformational changes correlate with altered gene expression. Together, our findings position PLOTTED as a probabilistic, single-locus framework for interpreting chromatin architecture in development and disease.

Structural variants (SVs) that disrupt topologically associating domains can cause disease by rewiring enhancer-promoter interactions. Duplications involving GPR101 are known to cause X-linked acrogigantism (X-LAG) through ectopic GPR101 expression, but not all of these duplications are pathogenic. This presents a diagnostic challenge, especially in the prenatal setting. We evaluated POSTRE, a tool that predicts the regulatory impact of SVs, to distinguish pathogenic from benign GPR101 duplications. We analyzed seven non-pathogenic duplications and 27 known X-LAG-associated duplications. To enable predictions in an X-LAG-relevant tissue, enhancer maps built using H3K27ac ChIP-seq, ATAC-seq, and RNA-seq data derived from human anterior pituitary samples (NIH research protocol 97-CH-0076, Clinicaltrials.gov Identifier NCT00001595, submitted on 11 March 1999) were integrated into POSTRE. POSTRE correctly classified all 34 duplications as benign or pathogenic. In addition, one X-LAG case with mild clinical features (i.e. severe growth hormone hypersecretion without pituitary tumorigenesis) was found to include only 2/5 VGLL1 enhancers, whereas all typical X-LAG cases had ≥4 enhancers duplicated. This suggests that partial enhancer hijacking at VGLL1 could explain the different clinical features in this individual. These findings support the utility of POSTRE to support diagnostic pipelines when interpreting SVs affecting chromatin architecture in pituitary disease and highlight its potential to reduce uncertainty in genetic counseling without requiring chromatin conformation capture assays.

Cyperus difformis is a globally problematic weed in rice fields, posing a significant threat to rice yield. While chemical herbicides are commonly used for its control, the species often escapes management due to its rapid evolution of resistance to widely used herbicides. To better understand the mechanisms underlying herbicide resistance, insights into the genetics and genomics of C. difformis are essential. In this study, we present a telomere-to-telomere genome assembly of C. difformis, generated by combining PacBio HiFi, Oxford Nanopore Technologies (ONT), MGI short reads and high-throughput chromatin conformation capture (Hi-C) technologies. The assembled genome spans 226 Mb with a scaffold N50 of 13.08 Mb. Utilizing Hi-C interaction data, 97.24% of the contigs were anchored to 18 chromosomes, with 35 telomeres successfully defined. Further analysis identified 75.73 Mb of repetitive sequences and 21,069 protein-coding genes, of which 91.8% (19,347 genes) were functionally annotated. This high-quality genome provides a valuable resource for studies in population genetics, phylogeny, comparative genomics, adaptive evolution, and functional genomics of C. difformis.

Plasmodium falciparum is responsible for what appears to be a never-ending public health issue in the developing world. With repeated infections, a gradual semi-immunity to severe malaria can be acquired but this is disrupted when women become pregnant as the parasite cytoadheres in the placenta to prevent splenic clearance. This change in tissue tropism is due to specific transcription of the antigenically variable adhesin VAR2CSA. To better understand the molecular mechanisms activating var2csa and antigenic variation overall, we used a combination of phenotypic and systems biology assays. We first established phenotypically homogenous populations of VAR2CSA expressing and placenta binding parasites that were shown to exclusively transcribe var2csa while all other var genes remained silenced. We also confirmed that the transcriptional activation was strongly associated with distinct depletion of repressive H3K9me3 marks. Further, we used chromatin conformation capture as a high-resolution approach to determine interchromosomal interactions and established that transcriptional activation is linked to a small yet significant repositioning of var2csa relative to heterochromatic telomeric clusters. Lastly, we demonstrated that occupancy of 5-methylcytosine was present in all var genes but independent of transcriptional repression and switching. All together, these findings provide insights at high resolution into the potential role of 5-methylcytosine in P. falciparum and increase our understanding of the mechanisms regulating antigenic variation at the epigenetics and chromatin structure level.