Gametophyte genome activation occurs at pollen mitosis I in maize

Flowering plants alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. One consequence of this life cycle is that plants face substantial selection during the haploid phase (1-3). Pollen actively transcribes its haploid genome (4), providing phenotypic diversity even among pollen grains from a single plant. Currently, the timing that pollen precursors first establish this independence is unclear. Starting with an endowment of transcripts from the diploid parent, when do haploid cells generated by meiosis begin to express genes? Here, we follow the shift to haploid expression in maize pollen using allele-specific RNA-sequencing (RNA-Seq) of single pollen precursors. We observe widespread biallelic expression for 11 days after meiosis, indicating that transcripts synthesized by the diploid sporophyte persist long into the haploid phase. Subsequently, there was a rapid and global conversion to monoallelic expression at pollen mitosis I (PMI), driven by active new transcription from the haploid genome. Genes expressed during the haploid phase showed reduced rates of nonsynonymous relative to synonymous substitutions (dn/ds) if they were expressed after PMI, but not before, consistent with purifying selection acting on the haploid gametophyte. This work establishes the timing with which haploid selection may act in pollen and provides a detailed time-course of gene expression during pollen development.

Somatotroph Tumors and the Epigenetic Status of the GNAS Locus

Background: Forty percent of somatotroph tumors harbor recurrent activating GNAS mutations, historically called the gsp oncogene. In gsp-negative somatotroph tumors, GNAS expression itself is highly variable; those with GNAS overexpression most resemble phenotypically those carrying the gsp oncogene. GNAS is monoallelically expressed in the normal pituitary due to methylation-based imprinting. We hypothesize that changes in GNAS imprinting of gsp-negative tumors affect GNAS expression levels and tumorigenesis. Methods: We characterized the GNAS locus in two independent somatotroph tumor cohorts: one of 23 tumors previously published (PMID: 31883967) and classified by pan-genomic analysis, and a second with 82 tumors. Results: Multi-omics analysis of the first cohort identified a significant difference between gsp-negative and gsp-positive tumors in the methylation index at the known differentially methylated region (DMR) of the GNAS A/B transcript promoter, which was confirmed in the larger series of 82 tumors. GNAS allelic expression was analyzed using a polymorphic Fok1 cleavage site in 32 heterozygous gsp-negative tumors. GNAS expression was significantly reduced in the 14 tumors with relaxed GNAS imprinting and biallelic expression, compared to 18 tumors with monoallelic expression. Tumors with relaxed GNAS imprinting showed significantly lower SSTR2 and AIP expression levels. Conclusion: Altered A/B DMR methylation was found exclusively in gsp-negative somatotroph tumors. 43% of gsp-negative tumors showed GNAS imprinting relaxation, which correlated with lower GNAS, SSTR2 and AIP expression, indicating lower sensitivity to somatostatin analogues and potentially aggressive behavior.

Zfp57 inactivation illustrates the role of ICR methylation in imprinted gene expression during neural differentiation of mouse ESCs

ZFP57 is required to maintain the germline-marked differential methylation at imprinting control regions (ICRs) in mouse embryonic stem cells (ESCs). Although DNA methylation has a key role in genomic imprinting, several imprinted genes are controlled by different mechanisms, and a comprehensive study of the relationship between DMR methylation and imprinted gene expression is lacking. To address the latter issue, we differentiated wild-type and Zfp57-/- hybrid mouse ESCs into neural precursor cells (NPCs) and evaluated allelic expression of imprinted genes. In mutant NPCs, we observed a reduction of allelic bias of all the 32 genes that were imprinted in wild-type cells, demonstrating that ZFP57-dependent methylation is required for maintaining or acquiring imprinted gene expression during differentiation. Analysis of expression levels showed that imprinted genes expressed from the non-methylated chromosome were generally up-regulated, and those expressed from the methylated chromosome were down-regulated in mutant cells. However, expression levels of several imprinted genes acquiring biallelic expression were not affected, suggesting the existence of compensatory mechanisms that control their RNA level. Since neural differentiation was partially impaired in Zfp57-mutant cells, this study also indicates that imprinted genes and/or non-imprinted ZFP57-target genes are required for proper neurogenesis in cultured ESCs.

Placental polyamines regulate acetyl-coA and histone acetylation in a sex-dependent manner

Fetal sex differences play an important role in the pathophysiology of several placenta related pregnancy complications. We previously reported that the maternal circulating level of a polyamine metabolite was altered in a fetal sex-specific manner, and was associated with pre-eclampsia and fetal growth restriction. Here we show that placental polyamine metabolism is altered in these disorders and that polyamines influence widespread changes in gene expression by regulating the availability of acetyl CoA which is necessary for histone acetylation. Sex differences in polyamine metabolism are associated with escape from X chromosome inactivation of the gene encoding the enzyme spermine synthase in female placentas, as evidenced by biallelic expression of the gene in female trophoblasts. Polyamine depletion in primary human trophoblasts impairs glycolysis and mitochondrial metabolism resulting in decreased availability of acetyl-CoA and global histone hypoacetylation, in a sex-dependent manner. Chromatin immunoprecipitation sequencing and RNA sequencing identifies downregulation of progesterone biosynthetic pathways as a key target and polyamine depletion reduced progesterone release in male trophoblasts. Collectively, these findings suggest that polyamines regulate placental endocrine function through metabolic regulation of gene expression, and that sex differences in polyamine metabolism due to XCI escape may buffer the effects of placental dysfunction in pregnancy disorders.

209 Large Offspring Syndrome: Effects of in vitro Production on Embryo Epigenetics and Development

In cattle, the use of assisted reproductive technologies (ART) can result in a congenital overgrowth condition known as large/abnormal offspring syndrome (LOS/AOS). The phenotypic characteristics of LOS include; somatic overgrowth, abdominal wall defects, large organs, breathing difficulties, skeletal defects, hypoglycemia, abnormal placentas, difficulty suckling, and perinatal death. LOS can have detrimental effects on the offspring and dam and also pose managerial and financial challenges to the producer. Research from the Rivera laboratory has demonstrated that LOS is an epigenetic syndrome. As in cattle, ART can promote the development of congenital overgrowth in humans, a condition known as Beckwith Wiedemann Syndrome (BWS). For the past 13 years, the Rivera laboratory has been characterizing LOS and we have shown that LOS and BWS are phenotypically and epigenotypically similar. In our studies, using gestation day ~105 Bos taurus taurus x Bos taurus indicus F1 hybrids, we showed global misregulation of imprinted and non-imprinted transcripts, micro RNAs and global misregulation of DNA methylation. In brief, LOS fetuses displayed variable loss-of-imprinting in kidney, liver, muscle and brain, when compared to controls. Biallelic expression of imprinted genes in LOS was associated with tissue-specific hypomethylation of the normally methylated parental allele. Not only was there loss of allele-specific expression of imprinted genes in LOS, but we also observed differential transcript amounts of these genes between control and overgrown fetuses. In addition, a positive correlation was observed between bodyweight and the number of biallelically expressed imprinted genes in LOS fetuses. From this work, we concluded that LOS is a multi-locus loss-of-imprinting condition. Current work, aims to determine if LOS is identifiable during pregnancy using day 55 fetal ultrasonography and day 55 and 105 maternal blood. In addition, we aim to determine how serum supplementation of culture medium can program preimplantation embryos to develop LOS. Findings will be discussed.

Parental bias in expression and interaction of genes in the equine placenta

Most autosomal genes in the placenta show a biallelic expression pattern. However, some genes exhibit allele-specific transcription depending on the parental origin of the chromosomes on which the copy of the gene resides. Parentally expressed genes are involved in the reciprocal interaction between maternal and paternal genes, coordinating the allocation of resources between fetus and mother. One of the main challenges of studying parental-specific allelic expression (allele-specific expression [ASE]) in the placenta is the maternal cellular remnant at the fetomaternal interface. Horses (Equus caballus) have an epitheliochorial placenta in which both the endometrial epithelium and the epithelium of the chorionic villi are juxtaposed with minimal extension into the uterine mucosa, yet there is no information available on the allelic gene expression of equine chorioallantois (CA). In the current study, we present a dataset of 1,336 genes showing ASE in the equine CA (https://pouya-dini.github.io/equine-gene-db/) along with a workflow for analyzing ASE genes. We further identified 254 potentially imprinted genes among the parentally expressed genes in the equine CA and evaluated the expression pattern of these genes throughout gestation. Our gene ontology analysis implies that maternally expressed genes tend to decrease the length of gestation, while paternally expressed genes extend the length of gestation. This study provides fundamental information regarding parental gene expression during equine pregnancy, a species with a negligible amount of maternal cellular remnant in its placenta. This information will provide the basis for a better understanding of the role of parental gene expression in the placenta during gestation.

Transcriptional bursts explain autosomal random monoallelic expression and affect allelic imbalance

Transcriptional bursts render substantial biological noise in cellular transcriptomes. Here, we investigated the theoretical extent of allelic expression resulting from transcriptional bursting and how it compared to the amount biallelic, monoallelic and allele-biased expression observed in single-cell RNA-sequencing (scRNA-seq) data. We found that transcriptional bursting can explain the allelic expression patterns observed in single cells, including the frequent observations of autosomal monoallelic gene expression. Importantly, we identified that the burst frequency largely determined the fraction of cells with monoallelic expression, whereas the burst size had little effect on monoallelic observations. The high consistency between the bursting model predictions and scRNA-seq observations made it possible to assess the heterogeneity of a group of cells as their deviation in allelic observations from the expected. Finally, both burst frequency and size contributed to allelic imbalance observations and reinforced that studies of allelic imbalance can be confounded from the inherent noise in transcriptional bursting. Altogether, we demonstrate that allele-level transcriptional bursting renders widespread, although predictable, amounts of monoallelic and biallelic expression in single cells and cell populations.

Genome-wide analysis of allele-specific expression of genes in the model diatom Phaeodactylum tricornutum

Recent advances in next generation sequencing technologies have allowed the discovery of widespread autosomal allele-specific expression (aASE) in mammals and plants with potential phenotypic effects. Extensive numbers of genes with allele-specific expression have been described in the diatom Fragilariopsis cylindrus in association with adaptation to external cues, as well as in Fistulifera solaris in the context of natural hybridization. However, the role of aASE and its extent in diatoms remain elusive. In this study, we investigate allele-specific expression in the model diatom Phaeodactylum tricornutum by the re-analysis of previously published whole genome RNA sequencing data and polymorphism calling. We found that 22% of P. tricornutum genes show moderate bias in allelic expression while 1% show nearly complete monoallelic expression. Biallelic expression associates with genes encoding components of protein metabolism while moderately biased genes associate with functions in catabolism and protein transport. We validated candidate genes by pyrosequencing and found that moderate biases in allelic expression were less stable than monoallelically expressed genes that showed consistent bias upon experimental validations at the population level and in subcloning experiments. Our approach provides the basis for the analysis of aASE in P. tricornutum and could be routinely implemented to test for variations in allele expression under different environmental conditions.

Molecular Definition of Pseudohypoparathyroidism Variants

Pseudohypoparathyroidism (PHP) and pseudopseudohypoparathyroidism (PPHP) are caused by mutations and/or epigenetic changes at the complex GNAS locus on chromosome 20q13.3 that undergoes parent-specific methylation changes at several differentially methylated regions (DMRs). GNAS encodes the alpha-subunit of the stimulatory G protein (Gsα) and several splice variants thereof. PHP type Ia (PHP1A) is caused by heterozygous inactivating mutations involving the maternal exons 1-13. Heterozygosity of these maternal GNAS mutations cause PTH-resistant hypocalcemia and hyperphosphatemia because paternal Gsα expression is suppressed in certain organs thus leading to little or no Gsα protein in the proximal renal tubules and other tissues. Besides biochemical abnormalities, PHP1A patients show developmental abnormalities, referred to as Albright’s hereditary osteodystrophy (AHO). Some, but not all of these AHO features are encountered also in patients affected by PPHP, who carry paternal Gsα-specific mutations and typically show no laboratory abnormalities. Autosomal dominant PHP type Ib (AD-PHP1B) is caused by heterozygous maternal deletions within GNAS or STX16, which are associated with loss of methylation at the A/B DMR alone or at all maternally methylated GNAS exons. Loss of methylation of exon A/B and the resulting biallelic expression of A/B transcript reduces Gsα expression thus leading to hormonal resistance. Epigenetic changes at all differentially methylated GNAS regions are also observed in sporadic PHP1B, which is the most frequent PHP1B variant. However, this disease variant remains unresolved at the molecular level, except for rare cases with paternal uniparental isodisomy or heterodisomy of chromosome 20q (patUPD20q).

BIRD: identifying cell doublets via biallelic expression from single cells

ABSTRACT Summary Current technologies for single-cell transcriptomics allow thousands of cells to be analyzed in a single experiment. The increased scale of these methods raises the risk of cell doublets contamination. Available tools and algorithms for identifying doublets and estimating their occurrence in single-cell experimental data focus on doublets of different species, cell types or individuals. In this study, we analyze transcriptomic data from single cells having an identical genetic background. We claim that the ratio of monoallelic to biallelic expression provides a discriminating power toward doublets’ identification. We present a pipeline called BIallelic Ratio for Doublets (BIRD) that relies on heterologous genetic variations, from single-cell RNA sequencing. For each dataset, doublets were artificially created from the actual data and used to train a predictive model. BIRD was applied on Smart-seq data from 163 primary fibroblast single cells. The model achieved 100% accuracy in annotating the randomly simulated doublets. Bonafide doublets were verified based on a biallelic expression signal amongst X-chromosome of female fibroblasts. Data from 10X Genomics microfluidics of human peripheral blood cells achieved in average 83% (±3.7%) accuracy, and an area under the curve of 0.88 (±0.04) for a collection of ∼13 300 single cells. BIRD addresses instances of doublets, which were formed from cell mixtures of identical genetic background and cell identity. Maximal performance is achieved for high-coverage data from Smart-seq. Success in identifying doublets is data specific which varies according to the experimental methodology, genomic diversity between haplotypes, sequence coverage and depth. Supplementary information Supplementary data are available at Bioinformatics online.