Daan Noordermeer

KLF14 acts as a master regulator of gene expression in adipose tissue and variants at the human KLF14 gene show reproducible association with type 2 diabetes and metabolic syndrome. Risk alleles are only pathological when maternally inherited, consistent with the observation that KLF14/Klf14 is a maternally expressed imprinted gene in human and mouse. However, how genomic imprinting is regulated at this important locus is currently unknown. In both species, KLF14/Klf14 is located ∼200 kb away from the paternally expressed imprinted gene MEST/Mest , regulated by a maternal gametic differentially methylated region (gDMR) at its promoter. Although the Klf14 promoter is unmethylated in most tissues, maternally inherited DNA methylation marks are paradoxically required for Klf14 expression. Here, we show that Mest and Klf14 reside within the same topologically associating domain (TAD) in embryonic stem cells (ESCs), defined by biallelic CTCF binding at the boundaries. Using allele-specific 4C-seq, we show that CTCF binding to the unmethylated Mest gDMR generates a paternal allele-specific sub-TAD encompassing Klf14 . CRISPR–Cas9 deletions of the paternal Mest promoter region in ESCs and in vivo in mutant mice result in loss of Mest expression and acquisition of biallelic expression at Klf14 . By analyzing epigenetic marks and chromatin looping in Klf14 -expressing pituitary cells, we identify a putative enhancer element shared by Mest and Klf14 , providing a mechanistic model for the regulation of Klf14 imprinting. This work defines a new role for the Mest maternally methylated gDMR, revealing that it exerts long-range effects via allele-specific modulation of TAD structures and acts as an imprinting control region in the regulation of Klf14 imprinting.

KLF14 acts as a master regulator of gene expression in adipose tissue and variants at the human KLF14 gene show strong and reproducible association with type 2 diabetes and metabolic syndrome. Risk alleles are only pathologic when maternally inherited, consistent with the observation that KLF14/Klf14 is a maternally-expressed imprinted gene in human and mouse. However, how genomic imprinting is regulated at this important locus is currently unknown. In both species, KLF14/Klf14 is located ~200 kb away from the paternally imprinted gene MEST/Mest, which is regulated by a maternal gametic differentially methylated region (gDMR) at its CpG island (CGI) promoter. Although the Klf14 CGI is kept unmethylated in most tissues, maternally-inherited DNA methylation (DNAme) marks are paradoxically required for Klf14 expression in mouse. Here, we show that Mest and Klf14 reside within the same topologically associating domain (TAD) in both mouse embryonic stem cells (ESCs) and differentiated cells, defined by sites of biallelic CTCF binding at the boundaries. Using allele-specific 4C-seq in F1 hybrid ESCs, we show that CTCF binding specifically to the unmethylated Mest gDMR generates a paternal allele-specific sub-TAD encompassing Klf14. We further show through CRISPR-Cas9-mediated mutagenesis, that deletions of the paternal Mest promoter region in ESCs as well as in vivo in mutant mice, results in both loss of Mest expression and acquisition of biallelic expression at Klf14. By analyzing epigenetic marks and chromatin looping in the Klf14-expressing pituitary cell line AtT-20, we identify a putative enhancer element shared by Mest and Klf14, providing a mechanistic model for the regulation of Klf14 imprinting. This work defines a new role for the Mest maternally methylated gDMR, revealing that it exerts long-range effects via allele-specific modulation of TAD structures and consequently act as an imprinting control region (ICR) in the regulation of Klf14 imprinting. Conservation of this TAD organisation at the human locus suggests that a similar regulatory mechanism operates at KLF14.

Centromeres ensure faithful chromosome segregation, yet how new centromeres arise and replace canonical ones remains poorly understood. Here, we investigate a polymorphic centromere repositioning event on the orangutan chromosome 10 using near-telomere-to-telomere assemblies, epigenetic profiling, and population-scale data. We identify striking heterogeneity in canonical centromeres, ranging from large, higher-order repeat α-satellite arrays to short, monomeric α-satellite tracts, alongside the emergence of neocentromeres lacking α-satellite DNA. We show a segmental duplication-mediated deletion of 3.6 Mbp that removed the higher-order repeat array, promoting centromere repositioning and neocentromere formation. Phylogenetic analyses reveal complex evolutionary dynamics, including introgression and incomplete lineage sorting in orangutan lineages. These findings demonstrate that centromere identity can evolve through structural variation and epigenetic reprogramming, highlighting its remarkable plasticity in primate genomes.

Besides genome-wide patterns of replication timing (RT), some genes display allelic replication asynchrony in stem cells, brought about by stochastic events and genetic polymorphisms. Whether epigenetic modifications control asynchronous replication remains unclear. Here, we explore domains controlled by genomic imprinting, where parental DNA methylation imprints mediate allele-specific gene expression. Our genome-wide and locus-specific assays in mono-parental and hybrid mouse ESCs reveal pronounced RT asynchrony—which is parent-of-origin dependent and lost upon neural differentiation—at the Dlk1-Dio3 and Snrpn domains, which both comprise lncRNA polycistrons. Generating a range of mutant lines, we find that asynchronous replication at Dlk1-Dio3 is mediated by differential DNA methylation, and that the lncRNA Meg3 controls early replication across parts of the domain on the maternal chromosome. Moreover, we find no evidence that RT and organisation into TADs are linked in this domain. The combined replication timing, DNA methylation, 3D chromatin structure, and gene expression data highlight how parental methylation imprints and lncRNA expression control replication and can override RT domain organisation.

Animal cells dismantle their nuclear envelope (NE) at the beginning and reconstruct it at the end of mitosis. This process is closely coordinated with spindle pole organization: poles enlarge at mitotic onset and reduce in size as mitosis concludes. The significance of this coordination remains unknown. Here, we demonstrate that Aurora A maintains a pole-localized protein NuMA in a dynamic state during anaphase. Without Aurora A activity, NuMA shifts from a dynamic to a solid state and abnormally accumulates at the poles, causing the segregated chromosome sets to bend around the NuMA-enriched poles. NuMA localization at the poles relies on interactions with dynein/dynactin, its coiled-coil domain, and an intrinsically disordered region (IDR). Mutagenesis experiments revealed that cation- π interactions within IDR are key for NuMA pole localization, while glutamine residues trigger the solid-state transition of NuMA upon Aurora A inhibition. We propose that maintaining the proper material properties of the spindle poles is a key step in choreographing the accurate organization of the nucleus and genome post-mitosis.

The SUMO pathway mainly functions to repress innate immunity in myeloid cells. Inactivating sumoylation triggers a strong, noncanonical, type I interferon (IFN1) response, amplified and coupled with inflammation upon stimulation. These findings transposed to pre-clinical models with the demonstration that sumoylation inhibitors activate antitumor immunity in an IFN1-dependent manner. Yet, how sumoylation represses immune signaling remains largely unknown. Here, we identified MORC3, a negative regulator of IFNB1, as the top SUMO2/3 substrate in myeloid cells. We show that, in monocytes, SUMO functions to repress basal IFNB1 in cis through a single long tandem repeat regulated by MORC3 [MORC3-regulated element (MRE)] that concentrates multiple motifs for the myeloid-enriched PU.1 factor. Inhibiting sumoylation induces a 3D genome reorganization nucleated from the MRE, which acquires both insulator and PU.1-activated enhancer activities, together with loss of H3.3 and H3K9me3 repressive marks and recruitment of PU.1. Paradoxically, MORC3, that interacts with PU.1, is massively recruited, yet unable to repress the MRE. Finally, we show that both sumoylation and MORC3 ATPase cycle are critical for MORC3 repressive activity. Our study thus uncovers an unconventional mechanism in which sumoylation, in concert with MORC3, orchestrates a metastable H3.3/H3K9me3 heterochromatin state on a multi-PU.1 binding platform to prevent an uncontrolled myeloid-specific immune response.

: Animal cells dismantle their nuclear envelope (NE) at the beginning and reconstruct it at the end of mitosis. This process is closely coordinated with spindle pole organization: poles enlarge at mitotic onset and reduce size as mitosis concludes. The significance of this coordination remains unknown. Here, we demonstrate that Aurora A maintains a pole-localized protein NuMA in a dynamic state during anaphase. Without Aurora A, NuMA shifts from a dynamic to a solid phase, abnormally accumulating at the poles, leading to chromosome bending and misshaped nuclei formation around poles. NuMA localization relies on interactions with dynein/dynactin, its coiled-coil domain, and intrinsically disordered region (IDR). Mutagenesis experiments revealed that cation-π interactions within IDR are key for NuMA localization, while glutamine residues trigger its solid-state transition upon Aurora A inhibition. This study emphasizes the role of the physical properties of spindle poles in organizing the nucleus and genome post-mitosis.

Topologically Associating Domains (TADs) are sub-Megabase regions in vertebrate genomes with enriched intra-domain interactions that restrict enhancer-promoter contacts across their boundaries. However, the mechanisms that separate TADs remain incompletely understood. Most boundaries between TADs contain CTCF binding sites (CBSs), which individually contribute to the blocking of Cohesin-mediated loop extrusion. Using genome-wide classification, we show here that TAD boundary width forms a continuum from narrow to highly extended and correlates with CBS distribution, chromatin features, and gene regulatory elements. To investigate how these boundary widths emerge, we modified the Random Cross-Linker (RCL) polymer model to incorporate specific boundary configurations, enabling us to evaluate the differential impact of boundary composition on TAD insulation. Our analysis identifies three generic boundary categories, each influencing TAD insulation differently, with varying local and distal effects on neighboring domains. Notably, we find that increasing boundary width reduces long-range inter-TAD contacts, as confirmed by Hi-C data. While blocking loop extrusion at boundaries indirectly promotes spurious intermingling of neighboring TADs, extended boundaries counteract this effect, emphasizing their role in maintaining genome structure. In conclusion, TAD boundary width not only enhances the efficiency of loop extrusion blocking but may also modulate enhancer-promoter contacts over long distances across TADs boundaries, providing a mechanism for transcriptional regulation. Significance statement: Topologically Associating Domains (TADs) compartmentalize vertebrate genomes to limit cross-domain enhancer-promoter loops. Our study reveals that boundaries between TADs are diverse genomic entities that range from narrow to highly extended. Using an interdisciplinary approach that combines genome-wide data analysis with biophysical polymer modeling, we find that wider TAD boundaries reduce spurious long-range interactions between neighboring domains. Moreover, we reveal how different boundary components can create this difference in insulating capacity. Our identification and characterization of TAD boundary width and composition suggests they have the potential to regulate the formation of enhancer-promoter loops across TAD boundaries at close and long distance.

Currently, routine diagnostics for spinocerebellar ataxia (SCA) look for polyQ repeat expansions and conventional variations affecting the proteins encoded by known SCA genes. However, ~40% of the patients still remain without a genetic diagnosis after routine tests. Increasing evidence suggests that variations in the enhancer regions of genes involved in neurodegenerative disorders can also cause disease. Since the enhancers of SCA genes are not yet known, it remains to be determined whether variations in these regions are a cause of SCA. In this pilot project, we aimed to identify the enhancers of the SCA genes ATXN1, ATXN3, TBP and ITPR1 in the human cerebellum using 4C-seq, publicly available datasets, reciprocal 4C-seq, and luciferase assays. We then screened these enhancers for copy number variants (CNVs) in a cohort of genetically undiagnosed SCA patients. We identified two active enhancers for each of the four SCA genes. CNV analysis did not reveal any CNVs in the enhancers of the four SCA genes in the genetically undiagnosed SCA patients. However, in one patient, we noted a CNV deletion with an unknown clinical significance near one of the ITPR1 enhancers. These results not only reveal elements involved in SCA gene regulation but can also lead to the discovery of novel SCA-causing genetic variants. As enhancer variations are being increasingly recognized as a cause of brain disorders, screening the enhancers of ATXN1, ATXN3, TBP and ITPR1 for variations other than CNVs and identifying and screening enhancers of other SCA genes might elucidate the genetic cause in undiagnosed patients.

During mammalian embryogenesis, both the 5-cytosine DNA methylation (5meC) landscape and three dimensional (3D) chromatin architecture are profoundly remodeled during a process known as ‘epigenetic reprogramming.’ An understudied aspect of epigenetic reprogramming is how the 5meC flux, per se, affects the 3D genome. This is pertinent given the 5meC-sensitivity of DNA binding for a key regulator of chromosome folding: CTCF. We profiled the CTCF binding landscape using a mouse embryonic stem cell (ESC) differentiation protocol that models embryonic 5meC dynamics. Mouse ESCs lacking DNA methylation machinery are able to exit naive pluripotency, thus allowing for dissection of subtle effects of CTCF on gene expression. We performed CTCF HiChIP in both wild-type and mutant conditions to assess gained CTCF–CTCF contacts in the absence of 5meC. We performed H3K27ac HiChIP to determine the impact that ectopic CTCF binding has on cis-regulatory contacts. Using 5meC epigenome editing, we demonstrated that the methyl-mark is able to impair CTCF binding at select loci. Finally, a detailed dissection of the imprinted Zdbf2 locus showed how 5meC-antagonism of CTCF allows for proper gene regulation during differentiation. This work provides a comprehensive overview of how 5meC impacts the 3D genome in a relevant model for early embryonic events.