Dynamic landscape of chromatin accessibility and transcriptomic changes during differentiation of human embryonic stem cells into dopaminergic neurons

César Meléndez-Ramírez,Adolfo López-Ornelas,Ernesto Soto-Reyes,Iván Velasco,Raquel Cuevas-Diaz Duran,Enrique Estudillo,Tonatiuh Barrios-García,Jessica Herrera-Gamboa,Mayela Giacoman-Lozano,Víctor Treviño

doi:10.1038/s41598-021-96263-1

Abstract

Chromatin architecture influences transcription by modulating the physical access of regulatory factors to DNA, playing fundamental roles in cell identity. Studies on dopaminergic differentiation have identified coding genes, but the relationship with non-coding genes or chromatin accessibility remains elusive. Using RNA-Seq and ATAC-Seq we profiled differentially expressed transcripts and open chromatin regions during early dopaminergic neuron differentiation. Hierarchical clustering of differentially expressed genes, resulted in 6 groups with unique characteristics. Surprisingly, the abundance of long non-coding RNAs (lncRNAs) was high in the most downregulated transcripts, and depicted positive correlations with target mRNAs. We observed that open chromatin regions decrease upon differentiation. Enrichment analyses of accessibility depict an association between open chromatin regions and specific functional pathways and gene-sets. A bioinformatic search for motifs allowed us to identify transcription factors and structural nuclear proteins that potentially regulate dopaminergic differentiation. Interestingly, we also found changes in protein and mRNA abundance of the CCCTC-binding factor, CTCF, which participates in genome organization and gene expression. Furthermore, assays demonstrated co-localization of CTCF with Polycomb-repressed chromatin marked by H3K27me3 in pluripotent cells, progressively decreasing in neural precursor cells and differentiated neurons. Our work provides a unique resource of transcription factors and regulatory elements, potentially involved in the acquisition of human dopaminergic neuron cell identity.

Highlights

Chromatin architecture influences transcription by modulating the physical access of regulatory factors to DNA, playing fundamental roles in cell identity
It is important to note that at D14, Tyrosine Hydroxylase (TH) is not expressed; this enzyme is detected by immunocytochemistry in 15% of neurons at D24, reaching 50% of TH + neurons at D28 and this percentage increases to 80% at D4213
We found a higher overlap of assay for transposase-accessible chromatin (ATAC)-Seq D0-specific open chromatin regions (OCRs) with GeneHancer regulatory elements than with D14 and D28 OCRs suggesting that cell type-specific enhancer regions are potentially involved in dopaminergic differentiation

Summary

Introduction

Chromatin architecture influences transcription by modulating the physical access of regulatory factors to DNA, playing fundamental roles in cell identity. DNA is bound to histones forming chromatin, whose structure is dynamic and suffers reversible chemical changes, mainly DNA methylation and histone post-translational modifications These epigenetic modifications have gained increasing interest since they play fundamental roles in modulating gene expression throughout development, differentiation, and in response to environmental cues[1]. Several largescale efforts have characterized transcriptional and epigenetic landscapes of cell lines and tissues[9,10] Even though these studies have identified putative regulatory elements, none of them focused on dopaminergic differentiation. Differentiation of human ESCs to mDA provides an important model for studying the correlation between the dynamic chromatin accessibility landscape and gene expression changes.

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Conclusion