Chromatin Interaction Changes during the iPSC-NPC Model to Facilitate the Study of Biologically Significant Genes Involved in Differentiation

Abstract

Given the difficulties of obtaining diseased cells, differentiation of neurons from patient-specific human induced pluripotent stem cells (iPSCs) with neural progenitor cells (NPCs) as intermediate precursors is of great interest. While cellular and transcriptomic changes during the differentiation process have been tracked, little attention has been given to examining spatial re-organization, which has been revealed to control gene regulation in various cells. To address the regulatory mechanism by 3D chromatin structure during neuronal differentiation, we examined the changes that take place during differentiation process using two cell types that are highly valued in the study of neurodegenerative disease - iPSCs and NPCs. In our study, we used Hi-C, a derivative of chromosome conformation capture that enables unbiased, genome-wide analysis of interaction frequencies in chromatin. We showed that while topologically associated domains remained mostly the same during differentiation, the presence of differential interacting regions in both cell types suggested that spatial organization affects gene regulation of both pluripotency maintenance and neuroectodermal differentiation. Moreover, closer analysis of promoter–promoter pairs suggested that cell fate specification is under the control of cis-regulatory elements. Our results are thus a resourceful addition in benchmarking differentiation protocols and also provide a greater appreciation of NPCs, the common precursors from which required neurons for applications in neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, schizophrenia and spinal cord injuries are utilized.

RFX4 is an intrinsic factor for neuronal differentiation through induction of proneural genes POU3F2 and NEUROD1

“…However, few studies have investigated the mechanism of action of endogenous induction of the expression of these factors in cells. In our previous studies, we examined changes in gene expression and various epigenetic states, such as histone modification, chromatin accessibility, and chromatin interactions, during the differentiation of iPSCs into NPCs [29,30]. We reanalyzed our data to identify the intrinsic factors that determine the differentiation of stem cells into neuronal cells by integrate them in more analytical methods.…”

Rare Does Not Mean Worthless: How Rare Diseases Have Shaped Neurodevelopment Research in the NGS Era

“…This technique has been successfully applied, for example, to monitor the changes in the 3D chromatin-structure that can take place during the in vitro process that differentiates iPSCs to NPCs both in mice and humans. The presence of differentially interacting regions in both cell types has been found, thus suggesting that spatial organization affects the gene regulation of both pluripotency maintenance and neuroectodermal differentiation as well as the transition from precursors to terminally differentiated neurons [ 78 , 79 ]. Chromosomal conformation changes during in vitro differentiation from NPCs to neurons or astrocytes revealed that the majority of these conformational plasticity changes (i) occur in genes that regulate neuronal connectivity and chromatin remodeling, (ii) are cell specific, and (iii) include interactions that are anchored in common variant sequences that confer a heritable risk for schizophrenia in neurons [ 80 ].…”