Biology subjects

Kresoja-Rakic, J.

Publications and source records attributed to Kresoja-Rakic, J..

5 recordsLinked to original sources

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

Heterogeneity of RNA editing in mesothelioma and how RNA editing enzyme ADAR2 affects mesothelioma cell growth, response to chemotherapy and tumor microenvironment

We previously observed increased levels of adenosine-deaminase-acting-on-dsRNA (Adar)-dependent RNA editing during mesothelioma development in mice exposed to asbestos. The aim of this study was to characterize and assess the role of ADAR-dependent RNA editing in mesothelioma. Tumors and mesothelioma primary cultures have higher ADAR-mediated RNA editing compared to mesothelial cells. Unsupervised clustering of editing in different genomic regions revealed heterogeneity between tumor samples as well as mesothelioma primary cultures. ADAR2 expression levels are higher in BRCA1-associated protein 1 wild-type tumors, with corresponding changes in RNA editing in transcripts and 3UTR. ADAR2 knockdown and rescue models indicated a role in cell proliferation, altered cell cycle, increased sensitivity to antifolate treatment and type-1 interferon signaling upregulation, leading to changes in the microenvironment in vivo. Our data indicate that RNA editing contributes to mesothelioma heterogeneity and highlights an important role of ADAR2 not only in growth regulation in mesothelioma but also chemotherapy response, in addition to regulating inflammatory response downstream of sensing nucleic acid structures.

cancer biology↗

bioRxiv2021

Heterogeneity and genomic loci of ubiquitous Cre reporter transgenes in zebrafish

The most-common strategy for zebrafish Cre/lox-mediated lineage labeling experiments combines ubiquitously expressed, lox-based Switch reporter transgenes with tissue-specific Cre or 4-OH-Tamoxifen-inducible CreERT2 driver lines. Although numerous Cre driver lines have been produced, only a few broadly expressed Switch reporters exist in zebrafish and their generation by random transgene integration has been challenging due to position-effect sensitivity of the lox-flanked recombination cassettes. Here, we compare commonly used Switch reporter lines for their recombination efficiency and reporter expression pattern during zebrafish development. Using different experimental setups, we show that ubi:Switch and hsp70l:Switch outperform current generations of two additional Switch reporters due to favorable transgene integration sites. Our comparisons also document preferential Cre-dependent recombination of ubi:Switch and hsp70l:Switch in distinct zebrafish tissues at early developmental stages. To investigate what genomic features may influence Cre accessibility and lox recombination efficiency in highly functional Switch lines, we mapped these transgenes and charted chromatin dynamics at their integration sites. Our data documents the heterogeneity among lox-based Switch transgenes towards informing suitable transgene selection for lineage labeling experiments. Our work further proposes that ubi:Switch and hsp70l:Switch define genomic integration sites suitable for universal transgene or switch reporter knock-in in zebrafish.

genetics↗

bioRxiv2020

Genome-wide maps of nucleolus interactions reveal distinct layers of repressive chromatin domains

Eukaryotic chromosomes are folded into hierarchical domains, enabling the organization of the genome into functional compartments. Nuclear periphery and nucleolus are two nuclear landmarks thought to contribute to repressive chromosome architecture. However, while the role of nuclear lamina (NL) in genome organization has been well documented, the function of the nucleolus remains under-investigated due to the lack of methods for genome-wide maps of nucleolar associated domains (NADs). Here we established a method based on a Dam-fused engineered nucleolar histone H2B that marks DNA contacting the nucleolus. NAD-maps of ESCs and neural progenitors revealed layers of genome compartmentalization with distinct, repressive chromatin states based on the interaction with the nucleolus, NL, or both. NADs showed higher H3K9me2 and lower H3K27me3 content than regions exclusively interacting with NL. Upon ESC differentiation, chromosomes around the nucleolus acquire a more compact, rigid architecture whereas NADs specific for ESCs decrease their interaction strength within the repressive B-compartment strength, unlocking neural genes from repressive nuclear environment. The methodologies here developed will make possible to include the contribution of the nucleolus in future studies investigating the relationship between nuclear space and genome function.

genomics↗

bioRxiv2020

Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma

The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration, and their dysregulation can result in congenital anomalies of the viscera, ventral wall defects, and mesothelioma tumors. Nonetheless, the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here, we combine genetic lineage tracing, in toto live imaging, and single-cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm (LPM). Our single-cell analysis uncovers a post-gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos, we chart the origin of mesothelial progenitors to the lateral-most, hand2-expressing LPM and confirm evolutionary conservation in mouse. Our time-lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation, documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally, hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium, we find de novo expression of LPM-associated transcription factors, and in particular of Hand2, indicating the re-initiation of a developmental transcriptional program in mesothelioma. Taken together, our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2, contributing to our understanding of mesothelial pathologies and mesothelioma.

developmental biology↗