Biology subjects

Sundararajan, K.

Publications and source records attributed to Sundararajan, K..

14 recordsLinked to original sources

bioRxiv2026

Meiotic kinetochore reconstitution identifies chromosome segregation factors

Kinetochores are complex protein assemblies at centromeres that direct and regulate chromosome segregation1. During gamete formation by meiosis, kinetochore specialisations drive two sequential and distinct segregation events to partition half the genome to the offspring2,3. Age-dependent segregation errors in human oocytes are a major cause of reproductive failure and are linked to kinetochore dysfunction4-6, however the molecular composition and function of mammalian meiotic centromeric chromatin and kinetochores remains poorly defined. Here, we reconstitute vertebrate meiosis I and II centromeric chromatin using meiosis I and II endogenous proteins to uncover conserved regulators of chromosome segregation in mammalian meiosis. We find that, in addition to canonical kinetochore proteins, a specific subset of chromatin regulators is enriched on meiotic centromeric chromatin. We discover previously unrecognized kinetochore proteins conserved from frogs to mouse and humans and demonstrate their localisation at mammalian oocyte centromeres. Among these, we show that the newly identified centromeric protein HSPBAP1, is required to maintain chromosome integrity and ensure faithful segregation in both mouse and human oocytes. These findings define the composition of vertebrate meiotic kinetochores and uncover conserved factors that safeguard mammalian meiotic chromosome segregation, a highly error-prone process and major cause of aneuploidy and reproductive failure in humans7.

cell biology↗

bioRxiv2025

Direct In-Sample Sequencing of the 3' Transcriptome Expands the Capabilities of Optical Pooled Screens

We present a platform that directly sequences single guide RNAs and endogenous 3'UTRs in fixed cells while simultaneously measuring protein abundance and cellular morphology. We demonstrate platform capability by performing optical pooled screening of CRISPR-perturbed lung cancer cells. This approach unites direct in-sample RNA sequencing with complementary phenotypic readouts, enabling comprehensive, scalable, and functional genomics analyses within a single experiment.

genomics↗

bioRxiv2025

DiMeLo-cito: a one-tube protocol for mapping protein-DNA interactions reveals CTCF bookmarking in mitosis

Genome regulation relies on complex and dynamic interactions between DNA and proteins. Recently, powerful methods have emerged that leverage third-generation sequencing to map protein-DNA interactions genome-wide. For example, Directed Methylation with Long-read sequencing (DiMeLo-seq) enables mapping of protein-DNA interactions along long, single chromatin fibers, including in highly repetitive genomic regions. However, DiMeLo-seq involves lossy centrifugation-based wash steps that limit its applicability to many sample types. To address this, we developed DiMeLo-cito, a single-tube, wash-free protocol that maximizes the yield and quality of genomic DNA obtained for long-read sequencing. This protocol enables the interrogation of genome-wide protein binding with as few as 100,000 cells and without the requirement of a nuclear envelope, enabling confident measurement of protein-DNA interactions during mitosis. Using this protocol, we detected strong binding of CTCF to mitotic chromosomes in diploid human cells, in contrast with earlier studies in karyotypically unstable cancer cell lines, suggesting that CTCF "bookmarks" specific sites critical for maintaining genome architecture across cell divisions. By expanding the capabilities of DiMeLo-seq to a broader range of sample types, DiMeLo-cito can provide new insights into genome regulation and organization.

genomics↗

bioRxiv2024

Independence of centromeric and pericentromeric chromatin stability on CCAN components

The chromatin of the centromere provides the assembly site for the mitotic kinetochore that couples microtubule attachment and force production to chromosome movement in mitosis. The chromatin of the centromere is specified by nucleosomes containing the histone H3 variant CENP-A. The constitutive centromeric-associated network (CCAN) and kinetochore are assembled on CENP-A chromatin to enable chromosome separation. CENP-A chromatin is surrounded by pericentromeric heterochromatin and bound by the sequence specific binding protein CENP-B. We performed mechanical experiments on mitotic chromosomes while tracking CENP-A and CENP-B to observe the centromeres stiffness and the role of the CCAN. We degraded CENP-C and CENP-N using auxin-inducible degrons, which we verified compromises the CCAN via observation of CENP-T loss. Chromosome stretching revealed that the CENP-A domain does not visibly stretch, even in the absence of CENP-C and/or CENP-N. Pericentromeric chromatin deforms upon force application, stretching approximately 3-fold less than the entire chromosome. CENP-C and/or CENP-N loss has no impact on pericentromere stretching. Chromosome-disconnecting nuclease treatments showed no structural effects on CENP-A. Our experiments show that the core-centromeric chromatin is more resilient and likely mechanically disconnected from the underlying pericentromeric chromatin, while the pericentric chromatin is deformable yet stiffer than the chromosome arms.

cell biology↗

bioRxiv2023

Aberrant phase separation is a common killing strategy of positively charged peptides in biology and human disease

Positively charged repeat peptides are emerging as key players in neurodegenerative diseases. These peptides can perturb diverse cellular pathways but a unifying framework for how such promiscuous toxicity arises has remained elusive. We used mass-spectrometry-based proteomics to define the protein targets of these neurotoxic peptides and found that they all share similar sequence features that drive their aberrant condensation with these positively charged peptides. We trained a machine learning algorithm to detect such sequence features and unexpectedly discovered that this mode of toxicity is not limited to human repeat expansion disorders but has evolved countless times across the tree of life in the form of cationic antimicrobial and venom peptides. We demonstrate that an excess in positive charge is necessary and sufficient for this killer activity, which we name polycation poisoning. These findings reveal an ancient and conserved mechanism and inform ways to leverage its design rules for new generations of bioactive peptides.

cell biology↗

bioRxiv2022

Mapping protein-DNA interactions with DiMeLo-seq

We recently developed Directed Methylation with Long-read sequencing (DiMeLo-seq) to map protein-DNA interactions genome wide. DiMeLo-seq is capable of mapping multiple interaction sites on single DNA molecules, profiling protein binding in the context of endogenous DNA methylation, identifying haplotype specific protein-DNA interactions, and mapping protein-DNA interactions in repetitive regions of the genome that are difficult to study with short-read methods. With DiMeLo-seq, adenines in the vicinity of a protein of interest are methylated in situ by tethering the Hia5 methyltransferase to an antibody using protein A. Protein-DNA interactions are then detected by direct readout of adenine methylation with long-read, single-molecule, DNA sequencing platforms such as Nanopore sequencing. Here, we present a detailed protocol and practical guidance for performing DiMeLo-seq. This protocol can be run on nuclei from fresh, lightly fixed, or frozen cells. The protocol requires 1-2 days for performing in situ targeted methylation, 1-5 days for library preparation depending on desired fragment length, and 1-3 days for Nanopore sequencing depending on desired sequencing depth. The protocol requires basic molecular biology skills and equipment, as well as access to a Nanopore sequencer. We also provide a Python package, dimelo, for analysis of DiMeLo-seq data. Key papersAltemose, N., Maslan, A., Smith, O.K., Sundararajan, K., Brown, R.R., Mishra, R., Detweiler, A.M., Neff, N., Miga, K.H., Straight, A.F. and Streets, A., 2022. DiMeLo-seq: a long-read, single-molecule method for mapping protein-DNA interactions genome wide. Nature Methods, pp.1-13. (https://doi.org/10.1038/s41592-022-01475-6)

genomics↗

bioRxiv2022

EstG is a novel esterase required for cell envelope integrity

Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the -proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell wall-targeting antibiotics, EstG does not demonstrate biochemical activity towards cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which is important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that, instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.

microbiology↗

bioRxiv2021

DiMeLo-seq: a long-read, single-molecule method for mappingprotein-DNA interactions genome-wide

Molecular studies of genome regulation often rely on the ability to map where specific proteins interact with genomic DNA. Existing techniques for mapping protein-DNA interactions genome-wide rely on DNA amplification methods followed by sequencing with short reads, which dissociates joint binding information at neighboring sites, removes endogenous DNA methylation information, and precludes the ability to reliably map interactions in repetitive regions of the genome. To address these limitations, we created a new protein-DNA mapping method, called Directed Methylation with Long-read sequencing (DiMeLo-seq), which methylates DNA near each target proteins DNA binding site in situ, then leverages the ability to distinguish methylated and unmethylated bases on long, native DNA molecules using long-read, single-molecule sequencing technologies. We demonstrate the optimization and utility of this method by mapping the interaction sites of a variety of different proteins and histone modifications across the human genome, achieving a single-molecule binding site resolution of less than 200 bp. Furthermore, we mapped the positions of the centromeric histone H3 variant CENP-A in repetitive regions that are unmappable with short reads, while simultaneously analyzing endogenous CpG methylation and joint binding events on single molecules. DiMeLo-seq is a versatile method that can provide multimodal and truly genome-wide information for investigating protein-DNA interactions.

genomics↗

bioRxiv2021

CENP-N promotes the compaction of centromeric chromatin

The histone variant CENP-A is the epigenetic determinant for the centromere, where it is interspersed with canonical H3 to form a specialized chromatin structure that nucleates the kinetochore. The arrangement of nucleosomes at the centromere into higher order structure is unknown. Here we demonstrate that the CENP-A interacting protein CENP-N promotes the stacking of CENP-A containing mono-nucleosomes and nucleosomal arrays through a previously undefined interaction between the 6 helix of CENP-N with the DNA of a neighboring nucleosome. We describe the cryoEM structures and biophysical characterization of such CENP-N mediated nucleosome stacks and nucleosomal arrays and demonstrate that this interaction is responsible for the formation of densely packed chromatin at the centromere in the cell. Our results provide first evidence that CENP-A, together with CENP-N, promotes specific chromatin higher order structure at the centromere. One-Sentence SummaryThe centromere-associated protein CENP-N promotes centromere-specific nucleosome stacking and higher order structures in vitro and in the cell.

molecular biology↗

bioRxiv2020

Identification and characterization of centromeric sequences in Xenopus laevis

Centromeres play an essential function in cell division by specifying the site of kinetochore formation on each chromosome for mitotic spindle attachment. Centromeres are defined epigenetically by the histone H3 variant CEntromere Protein A (CENP-A). CENP-A nucleosomes maintain the centromere by designating the site for new CENP-A assembly after dilution by replication. Vertebrate centromeres assemble on tandem arrays of repetitive sequences but the function of repeat DNA in centromere formation has been challenging to dissect due to the difficulty in manipulating centromeres in cells. Xenopus laevis egg extracts assemble centromeres in vitro, providing a system for studying centromeric DNA functions. However, centromeric sequences in X. laevis have not been extensively characterized. In this study we combine CENP-A ChIP-seq with a k-mer based analysis approach to identify the X. laevis centromere repeat sequences. By in situ hybridization we show that X. laevis centromeres contain diverse repeat sequences and we map the centromere position on each X. laevis chromosome using the distribution of centromere enriched k-mers. Our identification of X. laevis centromere sequences enables previously unapproachable centromere genomic studies. Our approach should be broadly applicable for the analysis of centromere and other repetitive sequences in any organism.

genomics↗

bioRxiv2019

Determinants of FtsZ C-terminal linker-dependent regulation of cell wall metabolism in Caulobacter crescentus

Bacterial cell division requires assembly of a multi-protein machinery or \"divisome\" that remodels the cell envelope to cause constriction. The cytoskeletal protein FtsZ forms a ring-like scaffold for the divisome at the incipient division site. FtsZ has three major regions - a conserved, polymerizing GTPase domain; a C-terminal conserved (CTC) peptide required for binding membrane-anchoring proteins; and a C-terminal linker (CTL) of poor length and sequence conservation. We previously demonstrated that, in Caulobacter crescentus, the CTL regulates FtsZ polymerization in vitro and cell wall metabolism in vivo. To understand the mechanism of CTL-dependent regulation of cell wall metabolism, here we investigated the impact of the CTL on Z-ring structure in cells and employed genetics to identify molecular determinants of the dominant lethal effects of {Delta}CTL. Deleting the CTL specifically resulted in formation of dense, asymmetric, non-ring FtsZ assemblies in vivo. Moreover, we observed that production of an FtsZ variant with the GTPase domain of Escherichia coli FtsZ fused to the CTC of C. crescentus FtsZ phenocopied the effects of C. crescentus {Delta}CTL, suggesting the CTC mediates signaling to cell wall metabolism. Finally, whereas overproduction of ZapA, FzlC, or FtsEX had slight protective effects against {Delta}CTL, depletion of FtsA partially suppressed the effects of {Delta}CTL. From these results, we propose that the cell wall misregulation downstream of {Delta}CTL results from its aberrant assembly properties and is propagated through the interaction between the CTC of FtsZ and FtsA. Our study provides mechanistic insights into CTL-dependent regulation of cell wall enzymes downstream of FtsZ.\n\nImportanceBacterial cell division is essential and requires the recruitment and regulation of a complex network of proteins needed to initiate and guide constriction and cytokinesis. FtsZ serves as a master regulator for this process, and its function is highly dependent on both its self-assembly into a canonical \"Z-ring\" and interaction with protein binding partners, which results in the activation of enzymes that remodel the cell wall to drive constriction. Using mutants of FtsZ and its binding partners, we have established the role of its C-terminal linker domain in regulating Z-ring organization, as well as the requirement for its C-terminal conserved peptide and interaction with the membrane-anchoring protein FtsA for regulating cell wall remodeling for constriction.

microbiology↗

bioRxiv2018

Agrobacterium tumefaciens divisome proteins regulate the transition from polar growth to cell division

The mechanisms that restrict peptidoglycan biosynthesis to the pole during elongation and re-direct peptidoglycan biosynthesis to mid-cell during cell division in polar-growing Alphaproteobacteria are largely unknown. Here, we demonstrate that although two of the three FtsZ homologs localize to mid-cell, exhibit GTPase activity and form co-polymers, only one, FtsZAT, is required for cell division. We find that FtsZAT is required not only for constriction and cell separation, but also for the termination of polar growth and regulation of peptidoglycan synthesis at mid-cell. Depletion of FtsZ in A. tumefaciens causes a striking phenotype: cells are extensively branched and accumulate growth active poles through tip splitting events. When cell division is blocked at a later stage, polar growth is terminated and ectopic growth poles emerge from mid-cell. Overall, this work suggests that A. tumefaciens FtsZ makes distinct contributions to the regulation of polar growth and cell division.

microbiology↗

bioRxiv2018

Species- and C-terminal linker-dependent variations in the dynamic behavior of FtsZ on membranes in vitro

Bacterial cell division requires the assembly of FtsZ protofilaments into a dynamic structure called the Z-ring. The Z-ring recruits the division machinery and directs local cell wall remodeling for constriction. The organization and dynamics of protofilaments within the Z-ring coordinate local cell wall synthesis during cell constriction, but their regulation is largely unknown. The disordered C-terminal linker (CTL) region of Caulobacter crescentus FtsZ (CcFtsZ) regulates polymer structure and turnover in solution in vitro, and regulates Z-ring structure and activity of cell wall enzymes in vivo. To investigate the contributions of the CTL to the polymerization properties of FtsZ on its physiological platform, the cell membrane, we reconstituted CcFtsZ polymerization on supported lipid bilayers (SLB) and visualized polymer dynamics and structure using total internal reflection fluorescence microscopy. Unlike E. coli FtsZ protofilaments that organized into large, bundled patterns, CcFtsZ protofilaments assembled into small, dynamic clusters on SLBs. Moreover, CcFtsZ lacking its CTL formed large networks of straight filament bundles that underwent slower turnover than the dynamic clusters of wildtype FtsZ. Our in vitro characterization provides novel insights into species- and CTL-dependent differences between FtsZ assembly properties that are relevant to Z-ring assembly and function on membranes in vivo.

microbiology↗

bioRxiv2017

The intrinsically disordered C-terminal linker of FtsZ regulates protofilament dynamics and superstructure in vitro

The bacterial tubulin FtsZ2 polymerizes to form a discontinuous cytokinetic ring that drives bacterial cell division by directing local cell wall synthesis. FtsZ comprises a polymerizing GTPase domain, an intrinsically disordered C-terminal linker (CTL) and a C-terminal conserved -helix (CTC). FtsZ protofilaments align circumferentially in the cell, with the CTC mediating attachment to membrane-associated division proteins. The dynamic turnover and treadmilling of clusters of FtsZ protofilaments guides cell wall synthesis and constriction. The nature and regulation of the interactions that result in the assembly of protofilaments into dynamic clusters is unknown. Here, we describe a role for the CTL of Caulobacter crescentus FtsZ as an intrinsic regulator of lateral interactions between protofilaments in vitro. FtsZ lacking its CTL ({Delta}CTL) shows dramatically increased propensity to form long multifilament bundles compared to wildtype (WT). {Delta}CTL has reduced GTP hydrolysis rate compared to WT. However, reducing protofilament turnover in WT is not sufficient to induce bundling. Surprisingly, binding of the membrane-anchoring protein FzlC disrupts {Delta}CTL bundling in a CTC-dependent manner. Moreover, the CTL affects the ability of FtsZ curving protein FzlA to promote formation of helical bundles. We conclude that the CTL of FtsZ influences polymer structure and dynamics both through intrinsic effects on lateral interactions and turnover and by influencing extrinsic regulation of FtsZ by binding partners. Our characterization of CTL function provides a biochemical handle for understanding the relationship between Z-ring structure and function in bacterial cytokinesis.

microbiology↗