Biology subjects

Belmont, A. S.

Publications and source records attributed to Belmont, A. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Rewired mRNA homeostasis enforces cell-size associated senescence

Cellular senescence refers to a permanent state of cell cycle exit and plays a key role in blocking the proliferation of compromised cells. Despite diverse drivers, senescent cells often share common features, one of which is their increased size. Though previously viewed as a passive outcome of cell growth during senescence, recent work shows that excess cell size can actively promote senescence via the p53-p21 pathway, making it both a consequence and a cause of cell cycle arrest. How cells sense their own size and communicate this information to the cell cycle control apparatus remains unresolved. Using genome-wide proximity mapping, we show that excess cell size globally redistributes chromatin between nuclear speckles and the nuclear lamina. These changes correspond with altered gene expression profiles, including the de-repression of typically silenced lamina-associated genes, some of which are involved in the senescence-associated secretory phenotype (SASP). Consistent with altered nuclear speckle function, we find that excess cell size perturbs normal splice isoform usage. In particular, MDM4, a negative regulator of p53 signaling, is differentially spliced when cells become enlarged to yield a non-functional variant, MDM4-S. Re-introducing correctly spliced MDM4 transcripts restores G1 cell cycle progression, thereby reverting senescent phenotypes in enlarged cells. Together, these data indicate that excess cell size is sensed via changes in mRNA homeostasis associated with a reorganized genome, driving both the secretory phenotype and permanent cell cycle exit that are characteristic of cellular senescence.

cell biology↗

bioRxiv2026

HSF1-dependent, long-range directional HSPA1 gene motion to nuclear speckles is coupled to DDX39B condensate dynamics

Approximately 5-10% of the genome, enriched [~]10-fold in the most highly active genes, positions near deterministically within several hundred nm of nuclear speckles (NS). This includes the HSPA1 locus, among the most strongly NS-associated genes. Here we address how HSPA1 endogenous genes and transgenes stably position adjacent to the nuclear speckle periphery, facilitating their gene expression amplification. Live-cell imaging demonstrates a transcription-dependent speckle-anchoring of HSPA1 genes. Removal of this anchoring reveals sustained, long-range, curvilinear oscillations of the HSPA1 genes away from and back to nuclear speckles or between nuclear speckles. Curvilinear transgene movements are dependent on the transcription factor HSF1, largely independent of nuclear actin or cohesin, but unexpectedly spatially and temporally correlated with DDX39B condensate dynamics and dependent on DDX39B protein levels. We propose that stable nuclear speckle association of HSPA1 results from combining metastable speckle anchoring with constitutive, restorative long-range chromosome movements driven by DDX39B condensate dynamics.

cell biology↗

bioRxiv2025

Acidic transcription factors position the genome at nuclear speckles through transcription dependent and independent mechanisms

A small fraction of the genome positions reproducibly near nuclear speckles (NS), increasing expression and/or splicing efficiency of NS-associated genes. How specific genomic regions target to NS remains unclear. Here, we demonstrate that establishment of genome-wide NS-association occurs independent of active transcription. We show that DNA sequences derived from NS-associated regions integrated as transgenes autonomously target to NS. By systematically dissecting one such genomic locus, the COL1A1-SGCA locus, we identified redundant NS-targeting cis regulatory elements, including a [~]600 bp fragment with 17 binding motifs for 8 transcription factors (TFs). Four NS-targeting TFs within this fragment contain acidic activation domains (AADs) that provide both chromatin-context and transcription-dependent NS-targeting, a property that appears common among several other tested AADs. A subset of acidic activator TFs contain an additional, transcription-independent NS-targeting activity. Our findings establish diverse and partially redundant NS-targeting activities, which may facilitate dynamic gene positioning at NS periphery for context-specific transcriptional responses.

cell biology↗

bioRxiv2025

Highly active chromosome regions preferentially associate with two perispeckle networks that partition the interchromatin space

A subset of highly active chromosomal "hot zones" reproducibly positions adjacent to nuclear speckles (NS). Genes within these regions amplify their expression only with NS contact. However, gene expression differences inversely correlate with differences in NS distance, genome-wide. We hypothesized the existence of additional gene expression "niches" away from, but spatially correlated with, NS. Here we report the identification of two dynamic perispeckle patterns of protein concentrations extending outwards from NS and persisting even after NS are eliminated. Highly active chromosome regions which weakly associate with NS instead show close, NS-independent association with these perispeckle patterns. Additionally, transcripts from model intron-containing versus intronless genes associate differentially with these two patterns. While genes within NS-associated genomic regions are predominantly downregulated upon NS depletion, genes associated with perispeckle patterns are biased towards upregulation. We suggest the interchromatin space is partitioned into additional gene expression "niches"- surrounding and extending from NS - that may be involved in mRNA and gene dynamics.

cell biology↗

bioRxiv2025

Nonrandom interchromatin trafficking through dynamic multiphase speckle connections

Nuclear speckles (NS) enhance the expression of NS-associated genes, possibly by elevating local levels of factors involved in multiple steps of gene expression. While dozens of large NS are distributed throughout interchromatin regions, the extent to which NS components dynamically redistribute between NS to adjust to local physiological demands remains unknown. Here we used live-cell imaging of endogenous NS proteins to identify an interchromatin network of connections that functionally link NS throughout the nucleus. Over timescales ranging from tens of seconds to minutes, NS material undergoes bulk transfer through these connections. Multiphase NS-connecting structures form through the dynamic juxtapositions of multiple NS component phases. Each phase exhibits distinct yet recurrent viscoelastic dynamics, but together, they integrate into a more stable, multiphase, NS-connecting structure in an ATP-and transcription-dependent manner. Our findings reveal the existence of a cellular mechanism that facilitates coordinated inter-NS protein trafficking through multiphase connections.

cell biology↗

bioRxiv2024

Chromatin-associated lncRNA-splicing factor condensates regulate hypoxia responsive RNA processing of genes pre-positioned near nuclear speckles

Hypoxia-induced alternative splicing (AS) regulates tumor progression and metastasis. Little is known about how such AS is controlled and whether higher-order genome and nuclear domain (ND) organizations dictate these processes. We observe that hypoxia-responsive alternatively spliced genes position near nuclear speckle (NS), the ND that enhances splicing efficiency. NS-resident MALAT1 long noncoding RNA, induced in response to hypoxia, regulates hypoxia-responsive AS. MALAT1 achieves this by organizing the SR-family of splicing factor, SRSF1, near NS and regulating the binding of SRSF1 to pre-mRNAs. Mechanistically, MALAT1 enhances the recruitment of SRSF1 to elongating RNA polymerase II (pol II) by promoting the formation of phase-separated condensates of SRSF1, which are preferentially recognized by pol II. During hypoxia, MALAT1 regulates spatially organized AS by establishing a threshold SRSF1 concentration near NSs, potentially by forming condensates, critical for pol II-mediated recruitment of SRSF1 to pre-mRNAs.

molecular biology↗

bioRxiv2024

An integrated view of the structure and function of the human 4D nucleome

The dynamic three-dimensional (3D) organization of the human genome (the "4D Nucleome") is closely linked to genome function. Here, we integrate a wide variety of genomic data generated by the 4D Nucleome Project to provide a detailed view of human 3D genome organization in widely used embryonic stem cells (H1-hESCs) and immortalized fibroblasts (HFFc6). We provide extensive benchmarking of 3D genome mapping assays and integrate these diverse datasets to annotate spatial genomic features across scales. The data reveal a rich complexity of chromatin domains and their sub-nuclear positions, and over one hundred thousand structural loops and promoter-enhancer interactions. We developed 3D models of population-based and individual cell-to-cell variation in genome structure, establishing connections between chromosome folding, nuclear organization, chromatin looping, gene transcription, and DNA replication. We demonstrate the use of computational methods to predict genome folding from DNA sequence, uncovering potential effects of genetic variants on genome structure and function. Together, this comprehensive analysis contributes insights into human genome organization and enhances our understanding of connections between the regulation of genome function and 3D genome organization in general.

genomics↗

bioRxiv2024

Beyond A and B Compartments: how major nuclear locales define nuclear genome organization and function

Models of nuclear genome organization often propose a binary division into active versus inactive compartments yet typically overlook nuclear bodies. Here we integrated analysis of sequencing and image-based data to compare genome organization in four human cell types relative to three different nuclear locales: the nuclear lamina, nuclear speckles, and nucleoli. Whereas gene expression correlates mostly with nuclear speckle proximity, DNA replication timing correlates with proximity to multiple nuclear locales. Speckle attachment regions emerge as DNA replication initiation zones whose replication timing and gene composition vary with their attachment frequency. Most facultative LADs retain a partially repressed state as iLADs, despite their positioning in the nuclear interior. Knock out of two lamina proteins, Lamin A and LBR, causes a shift of H3K9me3-enriched LADs from lamina to nucleolus, and a reciprocal relocation of H3K27me3-enriched partially repressed iLADs from nucleolus to lamina. Thus, these partially repressed iLADs appear to compete with LADs for nuclear lamina attachment with consequences for replication timing. The nuclear organization in adherent cells is polarized with nuclear bodies and genomic regions segregating both radially and relative to the equatorial plane. Together, our results underscore the importance of considering genome organization relative to nuclear locales for a more complete understanding of the spatial and functional organization of the human genome.

genomics↗

bioRxiv2023

Nucleolus and centromere TSA-Seq reveals variable localization of heterochromatin in different cell types

Genome differential positioning within interphase nuclei remains poorly explored. We extended and validated TSA-seq to map genomic regions near nucleoli and pericentric heterochromatin in four human cell lines. Our study confirmed that smaller chromosomes localize closer to nucleoli but further deconvolved this by revealing a preference for chromosome arms below 36-46 Mbp in length. We identified two lamina associated domain subsets through their differential nuclear lamina versus nucleolar positioning in different cell lines which showed distinctive patterns of DNA replication timing and gene expression across all cell lines. Unexpectedly, active, nuclear speckle-associated genomic regions were found near typically repressive nuclear compartments, which is attributable to the close proximity of nuclear speckles and nucleoli in some cell types, and association of centromeres with nuclear speckles in hESCs. Our study points to a more complex and variable nuclear genome organization than suggested by current models, as revealed by our TSA-seq methodology.

cell biology↗

bioRxiv2022

Nucleome Browser: An integrative and multimodal data navigation platform for 4D Nucleome

We introduce Nucleome Browser (http://www.nucleome.org), an interactive, multimodal data visualization and exploration platform for 4D Nucleome research. Our tool effectively integrates heterogeneous datasets (e.g., genomics, imaging, 3D genome structure models, and single-cell data) and external data portals by a new adaptive communication mechanism. Nucleome Browser provides a scalable solution for integrating massive amounts of 4D Nucleome data to navigate multiscale nuclear structure and function in a wide range of biological contexts, enabling hypothesis generation and data sharing with the broad community.

bioinformatics↗

bioRxiv2021

Identification of decondensed large-scale chromatin regions by TSA-seq and their localization to a subset of chromatin domain boundaries

Large-scale chromatin compaction is nonuniform across the human genome and correlates with gene expression and genome organization. Current methodologies for assessing large-scale chromatin compaction are indirect and largely based on assays that probe lower levels of chromatin organization, primarily at the level of the nucleosome and/or the local compaction of nearby nucleosomes. These assays assume a one-to-one correlation between local nucleosomal compaction and large-scale compaction of chromosomes that may not exist. Here we describe a method to identify interphase chromosome regions with relatively high levels of large-scale chromatin decondensation using TSA-seq, which produces a signal proportional to microscopic-scale distances relative to a defined nuclear compartment. TSA-seq scores that change rapidly as a function of genomic distance, detected by their higher slope values, identify decondensed large-scale chromatin domains (DLCDs), as then validated by 3D DNA-FISH. DLCDs map near a subset of chromatin domain boundaries, defined by Hi-C, which separate active and repressed chromatin domains and correspond to compartment, subcompartment, and some TAD boundaries. Most DLCDs can also be detected by high slopes of their Hi-C compartment score. In addition to local enrichment in cohesin (RAD21, SMC3) and CTCF, DLCDs show the highest local enrichment to super-enhancers, but are also locally enriched in transcription factors, histone-modifying complexes, chromatin mark readers, and chromatin remodeling complexes. The localization of these DLCDs to a subset of Hi-C chromatin domain boundaries that separate active versus inactive chromatin regions, as measured by two orthogonal genomic methods, suggests a distinct role for DLCDs in genome organization.

genomics↗

bioRxiv2020

SPIN reveals genome-wide landscape of nuclear compartmentalization

Chromosomes segregate differentially relative to distinct subnuclear structures, but this genome-wide compartmentalization, pivotal for modulating genome function, remains poorly understood. New genomic mapping methods can reveal chromosome positioning relative to specific nuclear structures. However, computational methods that integrate their results to identify overall intranuclear chromo-some positioning have not yet been developed. We report SPIN, a new method to identify genome-wide nuclear spatial localization patterns. As a proof-of-principle, we use SPIN to integrate nuclear compartment mapping (TSA-seq and DamID) and chromatin interaction data (Hi-C) from K562 cells to identify 10 spatial compartmentalization states genome-wide relative to nuclear speckles, lamina, and nucleoli. These SPIN states show novel patterns of genome spatial organization and their relation to genome function (transcription and replication timing). Comparisons of SPIN states with Hi-C sub-compartments and lamina-associated domains (LADs) from multiple cell types suggest constitutive compartmentalization patterns. By integrating different readouts of higher-order genome organization, SPIN provides critical insights into nuclear spatial and functional compartmentalization.

bioinformatics↗

bioRxiv2019

TSA-Seq 2.0 reveals both conserved and variable chromosomal distances to nuclear speckles

Genome-wide mapping of chromosomal distances relative to nuclear compartments using TSA-Seq suggests a more deterministic relationship between intranuclear gene position and expression as a function of nuclear speckle distance than radial position. Gene activity increases overall with decreasing distance to nuclear speckles, with active chromosomal regions forming the apex of chromosome loops protruding from the nuclear periphery into the interior. Interestingly, genomic distances to the nearest lamina-associated domain are larger for loop apexes mapping very close to nuclear speckles, suggesting the possibility of genomic "hardwiring" and conservation of speckle-associated regions. To facilitate comparison of genome organization relative to nuclear speckles in human K562, HCT116, HFFc6, and H1 cell lines, here we describe reducing the required cell number 10-20-fold for TSA-Seq by deliberately saturating protein-labeling while preserving distance mapping by the still unsaturated DNA-labeling. Surprisingly, in pair-wise cell line comparisons, only [~]10% of the genome shows a statistically significant shift in relative nuclear speckle distances. These modest shifts in nuclear speckle distance, however, tightly correlate with changes in cell-type specific gene expression. Similarly, half of all loci that contain induced heat-shock protein genes appear pre-positioned close to nuclear speckles, with the remaining showing small shifts towards speckles with transcriptional induction. Speckle association together with chromatin decondensation correlates with expression amplification upon HSPH1 activation. Our results demonstrate a largely "hardwired" genome organization and specific genes moving small mean distances relative to speckles during cell differentiation or physiological transition, suggesting an important role of nuclear speckles in gene expression regulation.

genomics↗

bioRxiv2019

Versatile multi-transgene expression using improved BAC TG-EMBED toolkit, novel BAC episomes, and BAC-MAGIC

Achieving reproducible, stable, and high-level transgene expression in mammalian cells remains problematic. Previously, we attained copy-number-dependent, chromosome-position-independent expression of reporter minigenes by embedding them within a BAC containing the mouse Msh3-Dhfr locus (DHFR BAC). Here we extend this \"BAC TG-EMBED\" approach. First, we report a toolkit of endogenous promoters capable of driving transgene expression over a 0.01-5 fold expression range relative to the CMV promoter, allowing fine-tuning of relative expression levels of multiple reporter genes expressed on a single BAC. Second, we show small variability in both the expression level and long-term expression stability of a reporter gene embedded in BACs containing either transcriptionally active or inactive genomic regions, making choice of BACs more flexible. Third, we describe an intriguing phenomenon in which BAC transgenes are maintained as episomes in a large fraction of stably selected clones. Finally, we demonstrate the utility of BAC TG-EMBED by simultaneously labeling three nuclear compartments in 94% of stable clones using a multi-reporter DHFR BAC, constructed with a combination of synthetic biology and BAC recombineering tools. Our extended BAC TG-EMBED method provides a versatile platform for achieving reproducible, stable simultaneous expression of multiple transgenes maintained either as episomes or stably integrated copies.

bioengineering↗

bioRxiv2019

Transcription amplification by nuclear speckle association

A significant fraction of active chromosome regions and genes reproducibly position near nuclear speckles, but the functional significance of this positioning is unknown. Here we show that Hsp70 BAC transgenes and endogenous genes turn on 2-4 mins after heat shock irrespective of their distance to nuclear speckles. However, we observe 12-56-fold and 3-7-fold higher transcription levels for speckle-associated Hsp70 transgenes and endogenous genes, respectively, after 1-2 hrs heat shock. Several fold higher transcription levels for several genes flanking the Hsp70 locus also correlate with speckle-association at 37 {degrees}C. Live-cell imaging reveals this modulation of Hsp70 transcription temporally correlates with speckle association/disassociation. Our results demonstrate stochastic gene expression dependent on positioning relative to a liquid-droplet nuclear compartment through a \"transcriptional amplification\" mechanism distinct from transcriptional bursting.

cell biology↗

bioRxiv2018

Nuclear speckle fusion via long-range directional motion regulates the number and size of speckles

Although the formation of RNA-protein bodies has been studied intensively, their mobility and how their number and size are regulated are still poorly understood. Here, we show significant increased mobility of nuclear speckles after transcriptional inhibition, including long-range directed motion of one speckle towards another speckle, terminated by speckle fusion, over distances up to 4 um and with velocities between 0.2-1.5 m/min. Frequently, 3 or even 4 speckles follow very similar paths, with new speckles appearing along the path followed by a preceding speckle. Speckle movements and fusion events contribute to fewer but larger speckles after transcriptional inhibition. These speckle movements are not actin-dependent, but occur within chromatin-depleted channels enriched with small granules containing the speckle-marker protein SON. Our observations suggest a mechanism for long-range, directed nuclear speckle movements, contributing to overall regulation of nuclear speckle number and size as well as overall nuclear organization.

cell biology↗

bioRxiv2018

TSA-Seq Mapping of Nuclear Genome Organization

While nuclear compartmentalization is an essential feature of three-dimensional genome organization, no genomic method exists for measuring chromosome distances to defined nuclear structures. Here we describe TSA-Seq, a new mapping method able to estimate mean chromosomal distances from nuclear speckles genome-wide and predict several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple, spatially separated nuclear domains, including two types of transcription \"hot-zones\". Transcription hot-zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, house-keeping genes, genes with low transcriptional pausing, and super-enhancers. Our results demonstrate the capability of TSA-Seq for genome-wide mapping of nuclear structure and suggest a new model for nuclear spatial organization of transcription.

genomics↗

bioRxiv2017

CRISPR/Cas9-mediated Knock-in of an Optimized TetO Repeat for Live Cell Imaging of Endogenous Loci

Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a high-throughput method for tracking any locus of interest is desirable. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions. Compared to alternatives, our method does not require a nearby repetitive sequence and it requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by PCR, was successful at labeling 9 different loci in HCT116 cells with up to 20% efficiency. These loci included both nuclear speckle-associated, euchromatin regions and nuclear lamina-associated, heterochromatin regions. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

cell biology↗