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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 autosomal 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.

SUBMITTER: Gupta S

PROVIDER: S-EPMC9210281 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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

Gupta Saumya S Lafontaine Denis L DL Vigneau Sebastien S Mendelevich Asia A Vinogradova Svetlana S Igarashi Kyomi J KJ Bortvin Andrew A Alves-Pereira Clara F CF Nag Anwesha A Gimelbrant Alexander A AA

G3 (Bethesda, Md.) 20220201 2

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 autosomal 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 reac ...[more]

PMID: 35100361

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Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human diseases including Kaposi's sarcoma (KS), a leading cause of cancer in Africa and in patients with AIDS. KS tumor cells harbor KSHV predominantly in a latent form, while typically <5% contain lytic replicating virus. Because both latent and lytic stages likely contribute to cancer initiation and progression, continued dissection of host regulators of this biological switch will provide insights into fundamental pathways controlling the KSHV life cycle and related disease pathogenesis. Several cellular protein kinases have been reported to promote or restrict KSHV reactivation, but our knowledge of these signaling mediators and pathways is incomplete. We employed a polypharmacology-based kinome screen to identifiy specific kinases that regulate KSHV reactivation. Those identified by the screen and validated by knockdown experiments included several kinases that enhance lytic reactivation: ERBB2 (HER2 or neu ), ERBB3 (HER3), ERBB4 (HER4), MKNK2 (MNK2), ITK, TEC, and DSTYK (RIPK5). Conversely, ERBB1 (EGFR1 or HER1), MKNK1 (MNK1) and FRK (PTK5) were found to promote the maintenance of latency. Mechanistic characterization of ERBB2 pro-lytic functions revealed a signaling connection between ERBB2 and the activation of CREB1, a transcription factor that drives KSHV lytic gene expression. These studies provided a proof-of-principle application of a polypharmacology-based kinome screen for the study of KSHV reactivation and enabled the discovery of both kinase inhibitors and specific kinases that regulate the KSHV latent-to-lytic replication switch.Author summaryKaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, a cancer particularly prevalent in Africa. In cancer cells, the virus persists in a quiescent form called latency, in which only a few viral genes are made. Periodically, the virus switches into an active replicative cycle in which most of the viral genes are made and new virus is produced. What controls the switch from latency to active replication is not well understood, but cellular kinases, enzymes that control many cellular processes, have been implicated. Using a cell culture model of KSHV reactivation along with an innovative screening method that probes the effects of many cellular kinases simultaneously, we identified drugs that significantly limit KSHV reactivation, as well as specific kinases that either enhance or restrict KSHV replicative cycle. Among these were the ERBB kinases which are known to regulate growth of cancer cells. Understanding how these and other kinases contribute to the switch leading to production of more infectious virus helps us understand the mediators and mechanisms of KSHV diseases. Additionally, because kinase inhibitors are proving to be effective for treating other diseases including some cancers, identifying ones that restrict KSHV replicative cycle may lead to new approaches to treating KSHV-related diseases.

Dataset Information

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

Publications

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

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