Epigenetics and genetics of hematopoietic stem cells heterogeneity

Abstract In diploid eukaryotic organisms, most genes are expressed biallelically. However, there are exceptions where the expression occurs in a monoallelic pattern that results from a differential allele-specific transcription based on the different epigenetic marking of the two alleles. At the level of cells, there are three classes of monoallelic expression regulated by epigenetic mechanisms: parent-of-origin imprinting, X chromosome inactivation (XCI), and random autosomal monoallelic expression (RMAE). Biased repopulations obtained from single-cell transplantation assays revealed that the pool of hematopoietic stem cells (HSCs) is heterogeneous, reflecting the epigenetic differences of individual cells. According to a model in which the allele-specific expression patterns are established during differentiation in embryonic stem cells and are stably propagated through cell divisions, it is assumed that HSCs carry genes (and alleles) with these stable epigenetic marks. Therefore, the analysis of epigenetic states in the stem cell population at the clonal level is necessary to understand its heterogeneity and diversity. Here we evaluated for the first time the persistence of allele-specific epigenetic states in the hematopoietic system in vivo using allelic imbalance as a readout. We created a monoclonal hematopoietic system in mice by single HSC transplantation and then analyzed the emerging lymphoid progeny using a genome-wide transcriptomics approach. We revealed that in the single-HSC derived hematopoietic cells, XCI is stably maintained through extensive proliferation and differentiation, whereas the vast majority of autosomal genes lack the stable clonal patterns of random monoallelic expression. This finding shows that the recurrent parallels between XCI and RMAE are misleading, suggesting that different mechanisms underlie these two classes of monoallelic expression. Additionally, we show that this in vivo clonal approach, which is free of genetic manipulation, can replace the artificial strategies that have been used to study tissue-specific XCI. Finally, stable allele-specific expression patterns were found in a rare number of genes (14 genes, <0.2%) in the progeny of a single HSC, indicating that these patterns were already present in the original HSC used for transplantation. However, the number of genes with stable monoallelic expression in cells that underwent differentiation steps is much lower than the numbers previously reported in studies using clonal cell lines in vitro without extensive differentiation (~2–15%). To reconcile these observations, we propose that most allele-specific expression patterns in autosomal genes are metastable and can be erased and reestablished at different differentiation stages.Resumo Nos organismos eucarióticos diplóides, a maioria dos genes são expressos bialelicamente. No entanto, existem excepções em que, ao nível das células, a expressão ocorre num padrão monoalélico que resulta de uma transcrição diferencial dos alelos de base epigenética. Existem três classes de expressão monoalélica regulada por mecanismos epigenéticos: imprinting de origem parental, inactivação do cromossoma X (XCI*) e expressão aleatória monoalélica autossómica (RMAE). Populações enviesadas obtidas a partir de ensaios de transplante de uma única célula revelaram que o conjunto de células estaminais hematopoiéticas (HSCs) é heterogéneo, reflectindo as diferenças epigenéticas de células individuais. Segundo um modelo em que os padrões da expressão específica de alelos são estabelecidos durante a diferenciação de células estaminais embrionárias e são propagados depois de forma estável através de divisões celulares, as HSCs carregam genes (e alelos) com marcas epigenéticas estáveis. A análise a nível clonal dos estados epigenéticos das células estaminais é necessária para entender a sua heterogeneidade e diversidade. Nesta tese, avaliamos pela primeira vez a persistência de estados epigenéticos entre os alelos no sistema hematopoiético in vivo usando o desequilíbrio da expressão alélica como ferramenta de leitura. O trabalho baseou-se na criação de um sistema hematopoiético monoclonal em ratinho por transplante de uma única HSC e no subsequente estudo da progenia linfóide emergente por análise transcriptómica de todo o genoma. Nas células hematopoiéticas resultantes de uma única HSC, verificámos que a XCI é mantida de forma estável após extensa proliferação e diferenciação, enquanto a vasta maioria dos genes autossómicos não estão sob RMAE. Assim, os paralelismos recorrentes na literatura entre XCI e RMAE são enganosos, porque estes dois fenómenos não têm a mesma estabilidade e serão regulados por diferentes mecanismos. Além disso, demonstramos que esta abordagem clonal com base num sistema sem manipulação genética pode ser uma estratégia para estudar a XCI específica de tecidos in vivo. Por fim, um padrão de RMAE foi encontrado num número raro de genes (14 genes, <0,2% do total) em células linfóides resultantes de uma única HSC, indicando que esses padrões já estavam presentes na HSC original usada no transplante. No entanto, o número de genes com RMAE em células que passaram por etapas de diferenciação é muito menor do que o número relatado anteriormente em estudos usando linhagens celulares clonais in vitro sem diferenciação extensa (~2–15%). Para conciliar estas observações, propomos que a maioria dos padrões de RMAE são meta-estáveis, isto é, passíveis de eliminação e restauração em diferentes estados de diferenciação

X-Chromosome Inactivation and Autosomal Random Monoallelic Expression as “Faux Amis”

Funding: The work of NK and VMB was funded by iNOVA4Health – UIDB/Multi/04462/2020 and UIDP/Multi/04462/2020, a program financially supported by Fundação para a Ciência e Tecnologia (FCT)/Ministério da Educação e Ciência through national funds, and the FCT grant PTDC/BEX-BCM/5900/2014. CFA-P has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 752806. A-VG was supported by Fundação para a Ciência e Tecnologia (FCT), Portugal, through an assistant research contract (CEECIND/02085/2018) and the project grant PTDC/MEDOUT/4301/2020 IC&DT.X-chromosome inactivation (XCI) and random monoallelic expression of autosomal genes (RMAE) are two paradigms of gene expression regulation where, at the single cell level, genes can be expressed from either the maternal or paternal alleles. X-chromosome inactivation takes place in female marsupial and placental mammals, while RMAE has been described in mammals and also other species. Although the outcome of both processes results in random monoallelic expression and mosaicism at the cellular level, there are many important differences. We provide here a brief sketch of the history behind the discovery of XCI and RMAE. Moreover, we review some of the distinctive features of these two phenomena, with respect to when in development they are established, their roles in dosage compensation and cellular phenotypic diversity, and the molecular mechanisms underlying their initiation and stability.publishersversionpublishe

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

Funding Information: This work has received funding from the FCT (Fundação para a Ciência e a Tecnologia) under grants PTDC/BEX-BCM/5900/2014 and IF/ 1823 01721/2014/CP1252/CT0005, and European Union’s Horizon 2020 research and innovation programme under grant agreement No. 752806. NK received a fellowship (PD/BD/114164/2016) from FCT. Publisher Copyright: Copyright © 2022 Kubasova, Alves-Pereira, Gupta, Vinogradova, Gimelbrant and Barreto.Evaluating the epigenetic landscape in the stem cell compartment at the single-cell level is essential to assess the cells’ heterogeneity and predict their fate. Here, using a genome-wide transcriptomics approach in vivo, we evaluated the allelic expression imbalance in the progeny of single hematopoietic cells (HSCs) as a read-out of epigenetic marking. After 4 months of extensive proliferation and differentiation, we found that X-chromosome inactivation (XCI) is tightly maintained in all single-HSC derived hematopoietic cells. In contrast, the vast majority of the autosomal genes did not show clonal patterns of random monoallelic expression (RME). However, a persistent allele-specific autosomal transcription in HSCs and their progeny was found in a rare number of cases, none of which has been previously reported. These data show that: 1) XCI and RME in the autosomal chromosomes are driven by different mechanisms; 2) the previously reported high frequency of genes under RME in clones expanded in vitro (up to 15%) is not found in clones undergoing multiple differentiation steps in vivo; 3) prior to differentiation, HSCs have stable patterns of autosomal RME. We propose that most RME patterns in autosomal chromosomes are erased and established de novo during cell lineage differentiation.publishersversionpublishe