Inference of putative cell-type-specific imprinted regulatory elements and genes during human neuronal differentiation

Abstract

Genomic imprinting results in gene expression bias caused by parental chromosome of origin and occurs in genes with important roles during human brain development. However, the cell-type and temporal specificity of imprinting during human neurogenesis is generally unknown. By detecting within-donor allelic biases in chromatin accessibility and gene expression that are unrelated to cross-donor genotype, we inferred imprinting in both primary human neural progenitor cells and their differentiated neuronal progeny from up to 85 donors. We identified 43/20 putatively imprinted regulatory elements (IREs) in neurons/progenitors, and 133/79 putatively imprinted genes in neurons/progenitors. Although 10 IREs and 42 genes were shared between neurons and progenitors, most putative imprinting was only detected within specific cell types. In addition to well-known imprinted genes and their promoters, we inferred novel putative IREs and imprinted genes. Consistent with both DNA methylation-based and H3K27me3-based regulation of imprinted expression, some putative IREs also overlapped with differentially methylated or histone-marked regions. Finally, we identified a progenitor-specific putatively imprinted gene overlapping with copy number variation that is associated with uniparental disomy-like phenotypes. Our results can therefore be useful in interpreting the function of variants identified in future parent-of-origin association studies.

Figure 1

Identification of putatively imprinted genes and REs. (A) Schematic cartoon of experimental design and methods. Imprinted genes and REs can be detected using parental genotype data. In this study, we do not know parent-of-origin information, and we infer imprinted genes and REs using high dispersion at heterozygous SNPs. (B) Comparison of putatively imprinted genes and REs in neurons, progenitors and known imprinted genes/ICEs (left). Overlap between putatively imprinted genes and imprinted promoters in neurons or progenitors (right). (C) Ideogram of putative neuron/progenitor imprinted genes and REs on the human genome. The names of known imprinted genes and regions are bolded.

Figure 2

Putatively imprinted genes and REs at the AS/PWS locus. (A) Coverage plot of ATAC-seq in neurons and progenitors at the AS/PWS locus and SNPs in imprinted REs and genes. (B) Allelic ATAC-seq counts for selected heterozygous SNPs in putatively imprinted REs in (A) showing a characteristic imprinting pattern. (C) Allelic RNA-seq counts for selected SNPs in putatively imprinted genes in (A) also showing a characteristic imprinting pattern.

Figure 3

Epigenetic modifications and transcription factor binding at putatively imprinted REs. (A) GC content of putatively imprinted and biallelically accessible REs in neurons (left) and progenitors (right). (B) Overlap of putatively imprinted and biallelically accessible REs with human CpG islands in neurons (left) and progenitors (right). (C) Putative imprinted genes in progenitors and neurons overlapped with oocyte hyperDMR-related genes and oocyte hyperDMR-unrelated genes (25). (D) Putative nIREs and pIREs overlap with human morula H3K27me3 ChIP-seq peaks, which exhibit a strong bias toward the maternal allele (25). (E) Enriched TF motifs from JASPAR 2016 vertebrates core dataset within putatively imprinted REs as compared with biallelically accessible REs.

Figure 4

Putative imprinted REs and genes indicating isoform-specific imprinting in neurons. (A) Coverage plot of ATAC-seq in neurons and progenitors at GET1 locus. (B) Allelic ATAC-seq counts for the SNPs in the putatively imprinted RE in (A). (C) Methylation ratio in human fetal brain and 12-year-old brain (88) showing overlap with the ICE. (D) Coverage plot of ATAC-seq in neurons and progenitors at the ZNF331 locus. Boundaries of ZNF331 isoforms showed neuron-specific putatively imprinted expression patterns are indicated by blue arrows. Boundaries of ZNF331 imprinted isoforms in LCL are indicated by red arrows. (E) Allelic ATAC/RNA-seq counts for SNPs in the putatively imprinted RE and gene in (D).

Figure 5

Progenitor-specific DLK1 putative imprinting at Kagami Ogata syndrome paternal UPD locus. (A) Coverage plot of ATAC-seq in neurons and progenitors at Kagami Ogata syndrome paternal UPD locus. DLK1 showed putative progenitor-specific imprinted expression overlapping the copy number variation. (B) Copy number variation in the 14q32 imprinted gene cluster related to Temple syndrome and Kagami Ogata syndrome. (C) Allelic RNA-seq counts for the SNPs in MEG3 (rs10147988) and MEG8 (rs12879413) in neurons. (D) Allelic RNA-seq counts for the SNPs in MEG3 (rs10147988), MEG8 (rs12879413) and DLK1 in progenitors (rs1802710).