Biology subjects

Chhatriwala, M.

Publications and source records attributed to Chhatriwala, M..

4 recordsLinked to original sources

bioRxiv2024

NGN3 oscillatory expression controls the timing of human pancreatic endocrine differentiation

Understanding protein expression dynamics is crucial for the mechanistic understanding of cell differentiation. We investigate the dynamics of NGN3, a transcription factor critical for pancreatic endocrine development, including their function and decoding mechanisms. A knock-in endogenous reporter shows that the expression of NGN3 protein oscillates with a 13-hour periodicity in human iPS-derived endocrine progenitors and is switched off as cells differentiate to {beta}-like and -like cells. Increasing the stability of NGN3 protein results in one broad peak of expression instead of oscillations, with a larger peak to trough fold-change. This leads to precocious endocrine differentiation to both {beta}-like and -like cells and precocious expression of key NGN3 target genes. Single-cell analysis of dynamics, mathematical modelling and bioinformatics suggest that decoding of NGN3 oscillations occurs by fold-change detection via an incoherent feedforward motif that explains both normal and precocious differentiation. Our findings suggest that oscillatory NGN3 dynamics control the timing of differentiation, but not fate specification.

developmental biology↗

bioRxiv2022

Generation of functional hepatocytes by forward programming with nuclear receptors

Production of large quantities of hepatocytes remains a major challenge for a number of clinical applications in the biomedical field. Directed differentiation of human pluripotent stem cells (hPSC) into hepatocyte-like cells (HLCs) provides an advantageous solution and a number of protocols have been developed for this purpose. However, these methods usually follow different steps of liver development in vitro which is time consuming and requires complex culture conditions. In addition, HLCs lack the full repertoire of functionalities characterising primary hepatocytes. Here, we explore the interest of forward programming to generate hepatocytes from hPSCs and to bypass these limitations. This approach relies on the overexpression of 3 hepatocyte nuclear factors (HNF1A, HNF6 and FOXA3) in combination with different nuclear receptors expressed in the adult liver using the OPTi-OX platform. Forward programming allows for the rapid production of hepatocytes (FoP-Heps) with functional characteristics using a simplified process. We also uncovered that the overexpression of nuclear receptors such as RORc can enhance specific functionalities of FoP-Heps thereby validating its role in lipid/glucose metabolism. Together, our results show that forward programming could offer a versatile alternative to direct differentiation for generating hepatocytes in vitro.

developmental biology↗

bioRxiv2019

Single-cell RNA-seq of differentiating iPS cells reveals dynamic genetic effects on gene expression

Recent developments in stem cell biology have enabled the study of cell fate decisions in early human development that are impossible to study in vivo. However, understanding how development varies across individuals and, in particular, the influence of common genetic variants during this process has not been characterised. Here, we exploit human iPS cell lines from 125 donors, a pooled experimental design, and single-cell RNA-sequencing to study population variation of endoderm differentiation. We identify molecular markers that are predictive of differentiation efficiency, and utilise heterogeneity in the genetic background across individuals to map hundreds of expression quantitative trait loci that influence expression dynamically during differentiation and across cellular contexts.

genomics↗

bioRxiv2018

Combined single cell profiling of expression and DNA methylation reveals splicing regulation and heterogeneity

BackgroundAlternative splicing is a key regulatory mechanism in eukaryotic cells and increases the effective number of functionally distinct gene products. Using bulk RNA sequencing, splicing variation has been studied across human tissues and in genetically diverse populations. This has identified disease-relevant splicing events, as well as associations between splicing and genomic variations, including sequence composition and conservation. However, variability in splicing between single cells from the same tissue or cell type and its determinants remain poorly understood.\n\nResultsWe applied parallel DNA methylation and transcriptome sequencing to differentiating human induced pluripotent stem cells to characterize splicing variation (exon skipping) and its determinants. Our results shows that variation in single-cell splicing can be accurately predicted based on local sequence composition and genomic features. We observe moderate but consistent contributions from local DNA methylation profiles to splicing variation across cells. A combined model that is built based on sequence as well as DNA methylation information accurately predicts different splicing modes of individual cassette exons (AUC=0.85). These categories include the conventional inclusion and exclusion patterns, but also more subtle modes of cell-to-cell variation in splicing. Finally, we identified and characterized associations between DNA methylation and splicing changes during cell differentiation.\n\nConclusionsOur study yields new insights into alternative splicing at the single-cell level and reveals a previously underappreciated link between DNA methylation variation and splicing.

bioinformatics↗