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Binary outcomes of enhancer activity underlie stable random monoallelic expression

ABSTRACT: We analyzed the natural killer (NK) cell receptor genes as in vivo genetic model to study random monoallelic gene expression (RME). These genes are expressed randomly, and largely monoallelically, in subsets of NK cells to generate a repertoire of specificities. We found that enhancers directly controlled the probability of expression of both RME and related, broadly expressed genes. We present an analysis of the accessibility (ATAC-seq) and both active and repressive histone modifications of elements associated with both expressed and silent alleles of the NK receptor genes in primary cells. The goals of this study were to assess the chromatin state of the silent and active alleles of the monoallelically expressed natural killer (NK) cell receptor genes.

ORGANISM(S): Mus musculus

PROVIDER: GSE181197 | GEO | 2022/04/21

REPOSITORIES: GEO

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Project description:The two copies of a gene are canonically believed to be indistinguishable to the cell and are expressed in the same spatiotemporal manner in the absence of a loss-of-function mutation. The mechanisms by which cells break this symmetry and express a gene from only one of the two alleles of the diploid genome are of great interest. Monoallelic gene expression on the X-chromosome, at imprinted genes, and at olfactory receptor genes has been studied in detail and involves combinations of non-coding RNAs, DNA methylation, and histone modifications. Recently, random monoallelic gene expression (RME), in which a gene can be expressed from either or both alleles in a clonal manner, has been described across many cell types and disease-relevant genes. However little is known about how RME is established and regulated. Here we develop allele-specific ATAC-seq, a rapid and sensitive method for profiling active regulatory DNA allelically and genome-wide, and find that monoallelic chromatin accessibility is extensive, developmentally regulated, and epigenetically inherited. In clonal hybrid F1 murine neural progenitor cells, we find over 1800 monoallelically-accessible DNA elements across autosomes. Randomly monoallelic regulatory elements tend to be promoter-proximal and located at RME genes identified by RNA-seq. Following differentiation, they are highly stable across passages and bookmarked during mitosis. Most RME genes have monoallelic promoters, but surprisingly, the nearby enhancer landscape around RME genes is biallelic. This suggests that RME genes are regulated at the chromatin, not post-transcriptional level, and that highly localized promoter accessibility is the gatekeeper within a permissive regulatory landscape dictating monoallelic vs. biallelic expression. These randomly monoallelic promoters are biallelically accessible in a previous developmental state, indicating that one allele is randomly shut down during NPC specification. Furthermore, the distribution of active alleles for a subset of these randomly monoallelic regulatory elements across clones deviates from a binomial distribution, indicating a highly regulated, non-stochastic mechanism of establishment.

Project description:Natural Killer cells (NK), a major constituent of innate immune system, have the ability to kill the transformed and infected cells without prior sensitization; can be put to immunotherapeutic use against various malignancies. NK cells discriminate between normal cells and transformed cells via a balance of inhibitory and activating signals induced by interactions between NK cell receptors and target cell ligands. Present study investigates whether expansion of NK cells could augment their anti-myeloma (MM) activity. For NK cell expansion, peripheral blood mononuclear cells from healthy donors and myeloma patients were co-cultured with irradiated K562 cells transfected with 4-1BBL and membrane-bound IL15 (K562-mb15-41BBL). A genome-wide profiling approach was performed to identify gene expression signature in expanded NK (ENK) cells and non-expanded NK cells isolated from healthy donors and myeloma patients. A specific set of genes involved in proliferation, migration, adhesion, cytotoxicity, and activation were up regulated post expansion, also confirmed by flow cytometry. Exp-NK cells killed both allogeneic and autologous primary MM cells more avidly than non-exp-NK cells in vitro. Multiple receptors, particularly NKG2D, natural cytotoxicity receptors, and DNAM-1 contributed to target lysis, via a perforin mediated mechanism. In summary, vigorous expansion and high anti-MM activity both in vitro and in vivo provide the rationale for testing exp-NK cells in a clinical trial for high risk MM. Differential gene expression profile in expanded natural killer (ENK) cells and non-expanded natural killer (NK) cells from healthy donors and myeloma patients Eight healthy donor and eight myeloma patients were used in the study. Non-expanded natural killer (NK) cells were isolated from PBMCs of healthy donors and myeloma patients. Expanded natural killer (ENK) cells were generated from same set of samples as mentioned in expansion protocol. All ENK and NK cells were used for gene expression profiling.

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Binary outcomes of enhancer activity underlie stable random monoallelic expression

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