Biology subjects

Walavalkar, K.

Publications and source records attributed to Walavalkar, K..

7 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↗

bioRxiv2024

Single-cell dynamics of genome-nucleolus interactions captured by nucleolar laser microdissection (NoLMseq)

Gene position in the nuclear space plays an important role in gene regulation. This is exemplified by repressive chromatin domains frequently contacting nuclear lamina or nucleoli. The nucleolus undergoes structural changes in response to various cellular states, potentially impacting genome organization. However, how the 3D-genome organization responds to nucleolar states has remained underinvestigated due to the lack of methods able to identify nucleolar associated domains (NADs) in single cells and under nucleolar stress. To address this, we developed NoLMseq, a method combining laser-capture microdissection and DNA sequencing to map NADs in single cells. NoLMseq identified many unexplored features of chromosome organization around single nucleoli such as NAD heterogeneity among ESCs, culminating in two major populations with distinct chromatin states. NADs prevalently contact nucleoli in a monoallelic manner and allelic nucleolar contact frequency mirrors gene expression and chromatin states. NoLMseq also revealed how chromosomes reorganise around nucleoli under nucleolar stress conditions, highlighting the importance of nucleolus integrity in genome organization and 3D-genome response to nucleolar stress. The results demonstrated that NoLMseq accurately measures chromosome contacts around single healthy and stressed nucleoli and it will be a critical tool to study NADs within biological populations and determine how the 3D-genome responds to nucleolar stress in healthy and disease states.

genomics↗

bioRxiv2022

Cis-regulatory effect of HPV integration is constrained by host chromatin architecture in cervical cancers

Human papillomavirus (HPV) infections are the primary drivers of cervical cancers, and often the HPV DNA gets integrated into the host genome. Although the oncogenic impact of HPV encoded genes (such as E6/E7) is well known, the cis-regulatory effect of integrated HPV DNA on host chromatin structure and gene regulation remains less understood. Here, we investigate the genome-wide patterns of HPV integrations and associated host gene expression changes in the context of chromatin states and topologically associating domains (TADs). We find that HPV integrations are significantly enriched and depleted in active and inactive chromatin regions, respectively. Interestingly, regardless of the chromatin state, the genomic regions flanking HPV integrations showed transcriptional upregulation. Nevertheless, the upregulation (both local and long-range) is mostly confined to the TADs with integration and does not affect the adjacent TADs. Few TADs showed recurrent integrations associated with overexpression of oncogenes within them (such as MYC, PVT1, TP63 and ERBB2), regardless of the proximity. To further understand the long-range effect, we performed HiC and 4C-seq analyses in HeLa and observed chromatin looping interaction between the integrated HPV and MYC/PVT1 regions (situated [~]500 kb apart), leading to allele-specific overexpression of these genes. Again, these chromatin interactions involving integrated HPV are mostly observed within the same TAD. Together, these results suggest the cis-regulatory potential of integrated HPV DNA that drives host gene upregulation at intra-TAD level in cervical cancer. Based on this, we propose HPV integrations can trigger multimodal oncogenic activation to promote cancer progression.

genomics↗

bioRxiv2022

Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of β-hemoglobinopathies

Reactivation of fetal hemoglobin (HbF) is the commonly adapted strategy to ameliorate {beta}-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits the HbF tetramer production affecting the therapeutic benefits. Here, we tested various deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified a 11 kb sequence, encompassing Putative Repressor Region (PRR) to {beta}-globin Exon-1 ({beta}E1), as the core deletion that ablates HbA and exhibit superior production of HbF compared to HPFH or other well-established targets. The PRR-{beta}E1 edited hematopoietic stem and progenitor cells (HSPCs) retained engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice generating HbF+ cells in vivo. Importantly, the editing induces therapeutically relevant levels of HbF to reverse the phenotypes of both sickle cell disease and {beta}-thalassemia major. These results indicate that the PRR-{beta}E1 gene editing in patient HSPCs can potentially lead to superior therapeutic outcomes for {beta}-hemoglobinopathies gene therapy.

biochemistry↗

bioRxiv2021

Active enhancers strengthen TAD insulation by bRNA mediated CTCF enrichment at the TAD boundaries

Vertebrate genomes are partitioned into Topologically Associating Domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at TAD boundaries correlates with better insulation, however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant INK4a/ARF TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancer:promoter interactions and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters we show that active TAD enhancers but not promoters induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs.

genomics↗

bioRxiv2020

Creating Clear and Informative Image-based Figures for Scientific Publications

Scientists routinely use images to display data. Readers often examine figures first; therefore, it is important that figures are accessible to a broad audience. Many resources discuss fraudulent image manipulation and technical specifications for image acquisition; however, data on the legibility and interpretability of images are scarce. We systematically examined these factors in non-blot images published in the top 15 journals in three fields; plant sciences, cell biology and physiology (n=580 papers). Common problems included missing scale bars, misplaced or poorly marked insets, images or labels that were not accessible to colorblind readers, and insufficient explanations of colors, labels, annotations, or the species and tissue or object depicted in the image. Papers that met all good practice criteria examined for all image-based figures were uncommon (physiology 16%, cell biology 12%, plant sciences 2%). We present detailed descriptions and visual examples to help scientists avoid common pitfalls when publishing images. Our recommendations address image magnification, scale information, insets, annotation, and color and may encourage discussion about quality standards for bioimage publishing.

bioinformatics↗

bioRxiv2019

Ligand dependent gene regulation by transient ER clustered enhancers

Unliganded nuclear receptors have been implicated in ligand-dependent gene regulation. However, the underlying mechanisms are not fully understood. Here we demonstrate that unliganded ER binds to specific sites in the genome thereby pre-marking them as future functional enhancers. Upon ligand exposure, ER binds to several EREs relatively proximal to the pre-marked, or persistent, ER-bound sites. Interestingly, the persistent sites interact extensively, via chromatin looping, with the proximal transiently bound sites forming ER clustered enhancers in 3D. CRISPR-based deletion of TFF1 persistent site disrupts the formation of its clustered enhancer resulting in the loss of E2-dependent induced expression of TFF1 and its neighboring genes within the same cluster. The clustered enhancers overlap with nuclear ER puncta that coalesce in a ligand-dependent manner. Furthermore, formation of clustered enhancers, as well as puncta, coincide with the active phase of signaling and their later disappearance results in the loss of gene expression even though persistent sites remain bound by ER. Our results establish the role of persistent unliganded ER binding in priming enhancer clusters in 3D that drive transient, but robust, gene expression in a ligand-dependent fashion.

molecular biology↗