RNA sequencing-based screen for reactivation of silenced alleles of autosomal genes

Abstract

In mammalian cells, maternal and paternal alleles usually have similar transcriptional activity. Epigenetic mechanisms such as X-chromosome inactivation (XCI) and imprinting were historically viewed as rare exceptions to this rule. Discovery of autosomal monoallelic expression (MAE) a decade ago revealed an additional allele-specific mode regulating thousands of mammalian genes. Despite MAE prevalence, its mechanistic basis remains unknown. Using an RNA sequencing-based screen for reactivation of silenced alleles, we identified DNA methylation as key mechanism of MAE mitotic maintenance. In contrast with the all-or-nothing allelic choice in XCI, allele-specific expression in MAE loci is tunable, with exact allelic imbalance dependent on the extent of DNA methylation. In a subset of MAE genes, allelic imbalance was insensitive to DNA demethylation, implicating additional mechanisms in MAE maintenance in these loci. Our findings identify a key mechanism of MAE maintenance and provide basis for understanding the biological role of MAE.

Binary outcomes of enhancer activity underlie stable random monoallelic expression

“…Differential DNA methylation of alleles is not broadly associated with all or even most RME genes ( da Rocha and Gendrel, 2019 ; Eckersley-Maslin et al, 2014 ), nor does it appear to be responsible for maintenance of silent alleles in the majority of tested RME genes ( Eckersley-Maslin and Spector, 2014 ; Gupta et al, 2022 ; Marion-Poll et al, 2021 ). Silent Klra and Klrc1 alleles in mouse NK cells were reported to be hypermethylated relative to active alleles ( Rouhi et al, 2007 ; Rouhi et al, 2006 ; Rouhi et al, 2009 ), and our data show that silent Klrk1 alleles in 5′EΔ/5′EΔ NK cells are also hypermethylated.…”

“…The methylation of these promoters, or of Klrk1 promoters as documented here, is not necessarily a sufficient cause of silenced gene expression, however. For example, DNA methylation of promoters is not associated with silent alleles in the case of numerous other RME genes, nor is it necessary for maintaining the silenced state of the large majority of tested RME genes ( da Rocha and Gendrel, 2019 ; Eckersley-Maslin et al, 2014 ; Gupta et al, 2022 ; Marion-Poll et al, 2021 ). In addition, inhibitors of DNA methyltransferases did not result in activation of silenced Klra genes or the Klrc1 gene ( Rouhi et al, 2007 ; Rouhi et al, 2006 ; Rouhi et al, 2009 ).…”

“…Silent Klra and Klrc1 alleles in mouse NK cells were reported to be hypermethylated relative to active alleles ( Rouhi et al, 2007 ; Rouhi et al, 2006 ; Rouhi et al, 2009 ), and our data show that silent Klrk1 alleles in 5′EΔ/5′EΔ NK cells are also hypermethylated. It is doubtful that DNA methylation is sufficient to maintain the silent state, however, since inhibitors of DNA methyltransferases failed to derepress inactive Klra or Klrc1 genes in cell lines ( Rouhi et al, 2007 ; Rouhi et al, 2006 ; Rouhi et al, 2009 ), or other silent RME genes that were hypermethylated ( Eckersley-Maslin and Spector, 2014 ; Gupta et al, 2022 ; Marion-Poll et al, 2021 ). In light of the collective data on DNA methylation and silent alleles in RME we suspect it is not generally causative in maintaining RME.…”

In Vivo Clonal Analysis Reveals Random Monoallelic Expression in Lymphocytes That Traces Back to Hematopoietic Stem Cells

“…To exclude this potential source of discrepancy, we applied the same analytical pipeline to RNA-seq data generated from clonal cells that grew without differentiation. We used the v-Abl pro-B clonal cell lines Abl.1, Abl.2, Abl.3, and Abl.4, which were derived previously from 129S1/SvImJ (129S1) x CAST/EiJ (CAST) F1 female mice ( Zwemer et al, 2012 ), with two replicate RNA-seq libraries prepared and sequenced per sample ( Gupta et al, 2021 ). To control for the possible LOH, exome sequencing data for the Abl.1-4 clones ( Gupta et al, 2021 ) was considered.…”

“…We used the v-Abl pro-B clonal cell lines Abl.1, Abl.2, Abl.3, and Abl.4, which were derived previously from 129S1/SvImJ (129S1) x CAST/EiJ (CAST) F1 female mice ( Zwemer et al, 2012 ), with two replicate RNA-seq libraries prepared and sequenced per sample ( Gupta et al, 2021 ). To control for the possible LOH, exome sequencing data for the Abl.1-4 clones ( Gupta et al, 2021 ) was considered. Genes whose genomic DNA showed total allelic counts of <10 or 0.3<AI>0.7 were excluded from RNA-seq analysis.…”