Biology subjects

Benoukraf, T.

Publications and source records attributed to Benoukraf, T..

8 recordsLinked to original sources

bioRxiv2024

ColocZStats: A Z-Stack Signal Colocalization Extension Tool for 3D Slicer

Confocal microscopy has evolved as a widely adopted imaging technique in molecular biology and is frequently utilized to achieve accurate subcellular localization of proteins. Applying colocalization analysis on image z-stacks obtained from confocal fluorescence microscopes is a dependable method to reveal the association between different molecules. In addition, despite the established advantages and growing adoption of 3D visualization software in various microscopy research domains, there has been a scarcity of systems supporting colocalization analysis within a user-specified region of interest (ROI). In this context, several broadly employed biological image visualization platforms were meticulously explored in this study to comprehend the current landscape. It has been observed that while these applications can generate three-dimensional (3D) reconstructions for the z-stacks and in some cases transfer them into an immersive Virtual Reality (VR) scene, there is still a lack of support for performing quantitative colocalization analysis on such images based on a user-defined ROI and thresholding levels. To address these issues, an extension called ColocZStats has been developed for 3D Slicer, a widely used free and open-source software package for image analysis and scientific visualization. With a custom-designed user-friendly interface, ColocZStats allows investigators to conduct intensity thresholding and ROI selection on imported 3D image stacks. It can deliver several essential colocalization metrics for structures of interest and produce reports in the form of diagrams and spreadsheets.

bioinformatics↗

bioRxiv2023

Increased spontaneous Ca2+ activity in Cardiac Purkinje cells after myocardial infarction; A consequence of a dramatic shift of SERCA isoforms as potential adaptation to acute ischemia?

BackgroundStudies of Purkinje cells (Pcells) from canine hearts have suggested an increase of Ca2+-release by the sarcoplasmic reticulum (SR) but also reported a potential augmentation of SR-Ca2+-uptake after MI. Abnormal increase of SR-Ca2+-uptake in heart cells is novel and contrasts with the reduction of this function in cells of failing heart. Our study examined the origin of this increased SR-Ca2+-uptake by considering a change in SR-Ca2+ pump (SERCA2) expression in Purkinje fibers (PFs) post MI. MethodsPcells were isolated from canine hearts 48Hrs post MI. Intracellular Ca2+-activity was captured by confocal microscopy. Purkinje-typical Ca2+ events were analyzed to probe the regional Ca2+-dynamics within Pcells. A Purkinje-specific numerical model assisted in the interpretation of Ca2+-anomalies detected in Pcells Ca2+-transients. SR-Ca2+-uptake system was studied by immunofluorescence in Pcells from canine, ovine and human hearts post MI. SERCA protein and gene expressions in PFs and myocardium were measured by Western Blots and RT-qPCR in a classical porcine model of MI. Results48Hrs after MI, Pcells showed 60% increase in spark-rate and 37% acceleration of Ca2+ wave decay. In the model of normal wave, 35% increase of Ca2+-uptake rate reproduced the actual post-MI wave alterations. In apparent contrast with increased Ca2+-uptake rate, SERCA2 protein expression was reduced in canine, sheep, and human Pcells after MI. In pig MI model, the protein level of cardiac-specific SERCA2-splicing variant SERCA2a was reduced by 52% in the whole infarcted ventricle whereas the "non-cardiac" SERCA2b level was increased by 120%. In the infarcted regions, PFs showed 30% downregulation of SERCA2a gene expression and 630% upregulation of SERCA2b. ConclusionOur results confirm that elevated spontaneous Ca2+-activity in post-MI PFs is due to increased SR-Ca2+-uptake within Pcells. Data suggest that a replacement of "cardiac" SERCA2a by the "non-cardiac" SERCA2b sub-isoform in cardiac cells in response to ischemia is implicated in this alteration.

cell biology↗

bioRxiv2021

Formation and recycling of an active epigenetic mark mediated by cell cycle-specific RNAs

The mechanisms by which epigenetic modifications are established in gene regulatory regions of active genes remain poorly understood. The data presented show that the establishment and recycling of a major epigenetic mark, the acetylated form of the replacement histone H2A.Z, is regulated by cell cycle-specific long noncoding RNAs encoded in regions adjacent to the promoters of active genes. These transcripts, termed SPEARs (S Phase EArly RNAs), are induced in early S phase: their expression precedes that of the downstream genes on which they exert their regulatory action. SPEARs drive the modification and deposition of the acetylated form of histone H2A.Z by bringing together the replacement histone and the histone acetyl transferase TIP60. This widespread bimodal pathway constitutes a novel RNA-mediated mechanism for the establishment of epigenetic marks and cell-specific epigenetic profiles, thereby providing a unifying explanation for the accuracy and persistence of epigenetic marks on chromatin.

genetics↗

bioRxiv2020

Targeted intragenic demethylation initiates chromatin rewiring for gene activation

Aberrant DNA methylation in the region surrounding the transcription start site is a hallmark of gene silencing in cancer. Currently approved demethylating agents lack specificity and exhibit high toxicity. Herein we show, using the p16 gene as an example, that targeted demethylation of the promoter-exon 1-intron 1 (PrExI) region initiates an epigenetic wave of local chromatin remodeling and distal long-range interactions, culminating in gene-locus specific activation. Through development of CRISPR-DiR (DNMT1-interacting RNA), in which ad hoc edited guides block methyltransferase activity in a locus-specific fashion, we demonstrate that demethylation is coupled to epigenetic and topological changes. These results suggest the existence of a specialized "demethylation firing center (DFC)" which can be switched on by an adaptable and selective RNA-mediated approach for locus-specific transcriptional activation. One Sentence SummaryLocus demethylation via CRISPR-DiR reshapes chromatin structure and specifically reactivates its cognate gene.

genetics↗

bioRxiv2020

TIP60 acetylates H2AZ and regulates doxorubicin-induced DNA damage sensitivity through RAD51 transcription

TIP60, a lysine acetyltransferase and H2AZ, a histone H2A variant are involved in transcription and DNA repair. Recent studies suggest that H2AZ acetylation is dependent on TIP60. Here, we show that TIP60 acetylates both isoforms of H2AZ in vitro and in cells. Utilizing ChIP-seq and RNA-seq to identify the genes regulated by TIP60-dependent acetylation of H2AZ, we find that TIP60-dependent acetylation of H2AZ correlates with the expression of genes involved in DNA damage repair, amongst several other pathways. In line with this, TIP60-depleted cells exhibit increased sensitivity to the DNA damage-inducing drug doxorubicin. Restoring the expression level of RAD51, one of the genes involved in the DNA damage repair pathway, partially rescues the doxorubicin sensitivity due to TIP60 depletion. Overall, our study uncovers a role for TIP60 in regulating doxorubicin-induced DNA damage sensitivity in a manner dependent on RAD51 transcription.

molecular biology↗

bioRxiv2020

Three-dimensional Genome Organization Maps in Normal Haematopoietic Stem Cells and Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is a highly lethal blood cancer arising due to aberrant differentiation of haematopoietic stem cells. Here we obtained 3D genome organization maps by Hi-C in the CD34+ haematopoietic stem cells from three healthy individuals and eight individuals with AML, and found that AML have increased loops to oncogenes compared with normal CD34+ cells. The MEIS1 oncogenic transcription factor is regulated by a Frequently Interacting Region (FIRE). This FIRE is only present in normal bone marrow samples, and four of eight AML sample. FIRE presence is associated with MEIS1 expression. CRISPR excision of a FIRE boundary led to loss of MEIS1 and reduced cell growth. Moreover, MEIS1 can bind to the promoter of HOXA9, and HOXA9 shows gain of Acute Myeloid Leukemia-specific super-enhancers that loop to the HOXA9 promoter. SignificanceWe found that Acute Myeloid Leukemias have more chromatin loops to oncogenes compared with normal blood stem cells. We identified heterogeneity in chromatin interactions at oncogenes, and heterogeneity in super-enhancers that loop to oncogenes, as two key epigenetic mechanisms that underlie MEIS1 and HOXA9 oncogene expression respectively.

cancer biology↗

bioRxiv2019

NanoVar: Accurate Characterization of Patients’ Genomic Structural Variants Using Low-Depth Nanopore Sequencing

Despite the increasing relevance of structural variants (SV) in the development of many human diseases, progress in novel pathological SV discovery remains impeded, partly due to the challenges of accurate and routine SV characterization in patients. The recent advent of third-generation sequencing (3GS) technologies brings promise for better characterization of genomic aberrations by virtue of having longer reads. However, the applications of 3GS are restricted by their high sequencing error rates and low sequencing throughput. To overcome these limitations, we present NanoVar, an accurate, rapid and low-depth (4X) 3GS SV caller utilizing long-reads generated by Oxford Nanopore Technologies. NanoVar employs split-reads and hard-clipped reads for SV detection and utilizes a neural network classifier for true SV enrichment. In simulated data, NanoVar demonstrated the highest SV detection accuracy (F1 score = 0.91) amongst other long-read SV callers using 12 gigabases (4X) of sequencing data. In patient samples, besides the detection of genomic aberrations, NanoVar also uncovered many normal alternative sequences or alleles which were present in healthy individuals. The low sequencing depth requirements of NanoVar enable the use of Nanopore sequencing for accurate SV characterization at a lower sequencing cost, an approach compatible with clinical studies and large-scale SV-association research.

bioinformatics↗

bioRxiv2019

ZBTB33 (Kaiso) methylated binding sites are associated with primed heterochromatin

BackgroundZBTB33, also known as Kaiso, is a member of the zinc finger and BTB/POZ family. In contrast to many transcription factors, ZBTB33 has the ability to bind both a sequence-specific consensus and methylated DNA. Although these dual binding preferences enable ZBTB33 to function as an active as well as repressive regulator of gene expression, little is known about the underlining molecular mechanisms.\n\nResultsIn this study, we aimed to investigate the role of ZBTB33 as a methylated DNA binding factor. We took advantage of the latest releases of the ENCODE sequencing datasets, including ZBTB33 ChIP- seq, whole genome bisulfite sequencing (WGBS), histone mark ChIP-seq and sequencing assays determining the chromatin states, to characterize the chromatin landscapes surrounding methylated ZBTB33 binding sites. Interestingly, our integrative analyses demonstrated that the majority of methylated ZBTB33 binding sites were located within condensed chromatin, which are inaccessible to DNase I and Tn5 transposase. Moreover, these sites were carrying a newly revealed histone post-translational modification signature, with significant enrichment of mono-methylation at lysine 4 of histone 3 (H3K4me1) and a complete absence of other active or expected repressive histone marks.\n\nConclusionsOverall, our analyses revealed that ZBTB33 has the unique ability to bind methylated DNA across heterochromatin in a transition state, suggesting a potential role for ZBTB33 in heterochromatin priming.

genomics↗