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

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.

Identifying regulators of parental imprinting by CRISPR/Cas9 screening in haploid human embryonic stem cells

In mammals, imprinted genes are regulated by differentially methylated regions (DMRs) that are inherited from germ cells, leading to monoallelic expression in accordance with parent-of-origin. Yet, it is largely unknown how imprinted DMRs are maintained in human embryos despite global DNA demethylation following fertilization. Here, we explored the mechanisms involved in imprinting regulation by employing human parthenogenetic embryonic stem cells (hpESCs), which lack paternal alleles. We show that although global loss of DNA methylation in hpESCs affects most imprinted DMRs, many paternally-expressed genes (PEGs) remain repressed. To search for factors regulating PEGs, we performed a genome-wide CRISPR/Cas9 screen in haploid hpESCs. This revealed ATF7IP as an essential repressor of a set of PEGs, which we further show is also required for silencing sperm-specific genes. Our study reinforces an important role for histone modifications in regulating imprinted genes and suggests a link between parental imprinting and germ cell identity.

Introns control stochastic allele expression bias

Monoallelic expression (MAE) or extreme allele bias can account for incomplete penetrance, missing heritability and non-Mendelian diseases. In cancer, MAE is associated with shorter patient survival times and higher tumor grade. Prior studies showed that stochastic MAE is caused by stochastic epigenetic silencing, in a gene and tissue-specific manner. Here, we used C. elegans to study stochastic MAE in vivo. We found allele bias/MAE to be widespread within C. elegans tissues, presenting as a continuum from fully biallelic to MAE. We discovered that the presence of introns within alleles robustly decreases MAE. We determined that introns control MAE at distinct loci, in distinct cell types, with distinct promoters, and within distinct coding sequences, using a 5’-intron position-dependent mechanism. Bioinformatic analysis showed human intronless genes are significantly enriched for MAE. Our experimental evidence demonstrates a role for introns in regulating MAE, possibly explaining why some mutations within introns result in disease.

Loss of imprinting control of the lncRNA H19-fetal mitogen IGF2 gene cluster in the decidual microenvironment of patients with idiopathic spontaneous miscarriages

Miscarriage, the spontaneous loss of a pregnancy before the fetus achieves viability, is a common complication of pregnancy. Decidualization plays a critical role in the implantation of the embryo. To search for molecular factors underlying miscarriage, we explored the role of long noncoding RNAs (lncRNAs) in the decidual microenvironment, where the molecular crosstalk at the feto–maternal interface occurs. By integrating RNA-seq data from recurrent miscarriage patients and decidualized endometrial stromal cells, we identified H19 , a noncoding RNA that exhibits paternally imprinted monoallelic expression in normal tissues, as the most upregulated lncRNA associated with miscarriage. Aberrant upregulation of H19 lncRNA was observed in decidual tissues derived from patients with spontaneous miscarriage as well as decidualized endometrial stromal cells. The maternally imprinted fetal mitogen Igf2, which is usually reciprocally co-regulated with H19 in the same imprinting cluster, was also upregulated. Notably, both genes underwent loss of imprinting, as H19 and IGF2 were actively transcribed from both parental alleles in decidual tissues. Mechanistically, this loss of imprinting in decidual tissues was associated with the loss of the H3K27m3 suppression marker in the IGF2 promoter, CpG hypomethylation at the central CTCF binding site in the imprinting control center (ICR) that is located between IGF2 and H19 , and the loss of CTCF-mediated intrachromosomal looping. These data provide the first evidence that aberrant control of the ICR epigenotype-intrachromosomal looping- H19/IGF2 imprinting pathway may be a critical epigenetic risk factor in the abnormal decidualization related to miscarriage.

Genome-Wide Analysis of Allele-Specific Expression Genes in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction. Pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL) is the most common pediatric hematological malignancy and it remains an important cause of morbidity and mortality in children. In this study, we performed an allele-specific expression (ASE) analysis of pediatric BCP ALL with the aim to investigate the relationship between cis-regulatory mutations and gene expression patterns. Materials and methods. Twenty-two high hyperdiploid ALL, twenty ETV6/RUNX1-positive ALL, seven TCF3/PBX1-positive ALL and twenty-eight genetically unclassified BCP ALL ("B-other") were subjected to whole genome sequencing, SNP array analysis and RNA sequencing. The binomial test was applied to estimate the allelic bias of heterozygous exonic single nucleotide variants (SNVs) in the RNA sequencing data against the genomic data. Allelic ratios >2 or <0.5, and P values <0.05 were used to identify allele-specific expression protein-coding genes. Results. We identified 12,693 expressed genes, of which 9,672 (76%) had heterozygous exonic SNVs (informative genes), in multiple BCP ALL samples (n>2) in 77 of the investigated samples. Genes with ASE were distributed evenly across the autosomal chromosomes in the different subtypes with a range of 30 - 165 ASE genes per case (median number, 86). We found that 630 (6.5%) genes displayed ASE in multiple BCP ALL samples (n>2), of which only eight autosomal genes had monoallelic expression in more than two investigated samples. This suggests that ASE and monoallelic expression are relatively rare in BCP ALL. Gene enrichment analyses showed that pathways involving negative regulation of natural killer cell-mediated cytotoxicity and cell proliferation were enriched, indicating that ASE events possibly were associated with the cell proliferation and leukemia progression in BCP ALL. Furthermore, the hematopoiesis pathway was also enriched in ASE genes that showed high allelic expression bias (allelic ratios >2.5), suggesting that ASE genes might be associated with leukemia development. Somatic genomic aberrations that could cause ASE were also investigated in this study. All informative cases with TCF3/PBX1 rearrangement (n=4) showed monoallelic expression of the PBX1 gene, likely associated with the PBX1 truncation caused by the fusion. Additionally, CHP1, located in 15q15.1, displayed ASE in one case with an inversion involving that chromosome band, indicating a potential cis-acting element in the inversion region that regulated the CHP1 gene expression. Notably, PAX5 displayed various patterns of ASE in BCP ALL. One of three cases with PAX5/ZCCHC7 gene rearrangements displayed PAX5 ASE while the other two did not, indicating a potential uncovered cis-regulatory element around the PAX5/ZCCHC7 breakpoints. Furthermore, two cases with no PAX5 gene rearrangement displayed monoallelic expression of the PAX5 gene, suggesting that there are additional epigenetic alterations were also involved in the regulation of PAX5 gene expression in BCP ALL. Conclusions. In this study, we have characterized genes displaying ASE in childhood BCP ALL. Our data provide new insight into pathogenesis of BCP ALL and may be used to identify novel targets for treatment. Disclosures No relevant conflicts of interest to declare.

A Comprehensive Characterization of Monoallelic Expression During Hematopoiesis and Leukemogenesis via Single-Cell RNA-Sequencing

Single-cell RNA-sequencing (scRNA-seq) is becoming a powerful tool to investigate monoallelic expression (MAE) in various developmental and pathological processes. However, our knowledge of MAE during hematopoiesis and leukemogenesis is limited. In this study, we conducted a systematic interrogation of MAEs in bone marrow mononuclear cells (BMMCs) at single-cell resolution to construct a MAE atlas of BMMCs. We identified 1,020 constitutive MAEs in BMMCs, which included imprinted genes such as MEG8, NAP1L5, and IRAIN. We classified the BMMCs into six cell types and identified 74 cell type specific MAEs including MTSS1, MOB1A, and TCF12. We further identified 114 random MAEs (rMAEs) at single-cell level, with 78.1% single-allele rMAE and 21.9% biallelic mosaic rMAE. Many MAEs identified in BMMCs have not been reported and are potentially hematopoietic specific, supporting MAEs are functional relevance. Comparison of BMMC samples from a leukemia patient with multiple clinical stages showed the fractions of constitutive MAE were correlated with fractions of leukemia cells in BMMCs. Further separation of the BMMCs into leukemia cells and normal cells showed that leukemia cells have much higher constitutive MAE and rMAEs than normal cells. We identified the leukemia cell-specific MAEs and relapsed leukemia cell-specific MAEs, which were enriched in immune-related functions. These results indicate MAE is prevalent and is an important gene regulation mechanism during hematopoiesis and leukemogenesis. As the first systematical interrogation of constitutive MAEs, cell type specific MAEs, and rMAEs during hematopoiesis and leukemogenesis, the study significantly increased our knowledge about the features and functions of MAEs.

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

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.

Systematic Analysis of Monoallelic Gene Expression and Chromatin Accessibility Across Multiple Tissues in Hybrid Mice

In diploid eukaryotic organisms, both alleles of each autosomal gene are usually assumed to be simultaneously expressed at similar levels. However, some genes can be expressed preferentially or strictly from a single allele, a process known as monoallelic expression. Classic monoallelic expression of X-chromosome-linked genes, olfactory receptor genes and developmentally imprinted genes is the result of epigenetic modifications. Genetic-origin-dependent monoallelic expression, however, is caused by cis-regulatory differences between the alleles. There is a paucity of systematic study to investigate these phenomena across multiple tissues, and the mechanisms underlying such monoallelic expression are not yet fully understood. Here we provide a detailed portrait of monoallelic gene expression across multiple tissues/cell lines in a hybrid mouse cross between the Mus musculus strain C57BL/6J and the Mus spretus strain SPRET/EiJ. We observed pervasive tissue-dependent allele-specific gene expression: in total, 1,839 genes exhibited monoallelic expression in at least one tissue, and 410 genes in at least two tissues. Among these 88 are monoallelic genes with different active alleles between tissues, probably representing genetic-origin-dependent monoallelic expression. We also identified six autosomal monoallelic genes with the active allele being identical in all eight tissues, which are likely novel candidates of imprinted genes. To depict the underlying regulatory mechanisms at the chromatin layer, we performed ATAC-seq in two different cell lines derived from the F1 mouse. Consistent with the global expression pattern, cell-type dependent monoallelic peaks were found, and a higher proportion of C57BL/6J-active peaks were observed in both cell types, implying possible species-specific regulation. Finally, only a small part of monoallelic gene expression could be explained by allelic differences in chromatin organization in promoter regions, suggesting that other distal elements may play important roles in shaping the patterns of allelic gene expression across tissues.

The establishment of variant surface glycoprotein monoallelic expression revealed by single-cell RNA-seq of Trypanosoma brucei in the tsetse fly salivary glands

The long and complex Trypanosoma brucei development in the tsetse fly vector culminates when parasites gain mammalian infectivity in the salivary glands. A key step in this process is the establishment of monoallelic variant surface glycoprotein (VSG) expression and the formation of the VSG coat. The establishment of VSG monoallelic expression is complex and poorly understood, due to the multiple parasite stages present in the salivary glands. Therefore, we sought to further our understanding of this phenomenon by performing single-cell RNA-sequencing (scRNA-seq) on these trypanosome populations. We were able to capture the developmental program of trypanosomes in the salivary glands, identifying populations of epimastigote, gamete, pre-metacyclic and metacyclic cells. Our results show that parasite metabolism is dramatically remodeled during development in the salivary glands, with a shift in transcript abundance from tricarboxylic acid metabolism to glycolytic metabolism. Analysis of VSG gene expression in pre-metacyclic and metacyclic cells revealed a dynamic VSG gene activation program. Strikingly, we found that pre-metacyclic cells contain transcripts from multiple VSG genes, which resolves to singular VSG gene expression in mature metacyclic cells. Single molecule RNA fluorescence in situ hybridisation (smRNA-FISH) of VSG gene expression following in vitro metacyclogenesis confirmed this finding. Our data demonstrate that multiple VSG genes are transcribed before a single gene is chosen. We propose a transcriptional race model governs the initiation of monoallelic expression.

LRFN5 locus structure is influenced by the individual’s sex and associated with autism

BackgroundLRFN5 is a brain-specific gene needed for synaptic development and plasticity. It is the only gene in a large 5.4 Mb topologically associating domain (TAD) on chromosome 14, which we term the LRFN5 locus. This locus is highly conserved, but has extensive copy number variation. MethodsLocus structure was studied by chromatin immunoprecipitation (chIP-onchip) in fibroblasts from individuals with autism and controls, supplemented with a capture-HiC determination of TAD structures in a family trio. LRFN5 expression was studied in foetal brain cell cultures. In addition, locus interaction was studied in four large and independent cohorts by measuring deviations from Hardy-Weinberg equilibrium of a common deletion polymorphism. ResultsWe found that locus structural changes are associated with developmental delay (DD) and autism spectrum disorders (ASD). In a large family, ASD in males segregated with a chromosome 14 haplotype carrying a 172 kb deletion upstream of LRFN5 . In a fibroblast capture-HiC study on an ASD-patient-parent trio, the ASDsusceptible haplotype (in the mother and her autistic son) had a TAD pattern different from both the father and a female control. When the trimethylated histone-3-lysine-9 chromatin (H3K9me3) profiles in fibroblasts from control males (n=6) and females (n=7) were compared, a male-female difference was observed around the LRFN5 gene itself (p<0.01). Intriguingly, in three cohorts of individuals with DD (n=8757), the number of heterozygotes of a common deletion polymorphism upstream of LRFN5 was 20-26% lower than expected from Hardy-Weinberg equilibrium. This indicates early allelic interaction, and the genomic conversions from heterozygosity to wildtype or deletion homozygosity were of equal magnitudes. In a control group of medical students (n=1416), such conversions were three times more common than in the DD-patient cohorts (p=0.00001). Hypothetically, such allelic interaction is needed to establish monoallelic expression, which we found in foetal brain cell cultures. LimitationsThe male-female difference in H3K9me3 profiles was based on fibroblast data from a small number of individuals, and the monoallelic expression data on a single experiment. ConclusionsTaken together, allelic interaction, monoallelic expression and sexdependent differences make the LRFN5 locus attractive for exploring the genetic basis of synaptic memory and high-functioning male autism.