Allele-specific DNA Methylation Research Articles

PARamrfinder: detecting allele-specific DNA methylation on multicore clusters

AbstractThe discovery of Allele-Specific Methylation (ASM) is an important research field in biology as it regulates genomic imprinting, which has been identified as the cause of some genetic diseases. Nevertheless, the high computational cost of the bioinformatic tools developed for this purpose prevents their application to large-scale datasets. Hence, much faster tools are required to further progress in this research field. In this work we present PARamrfinder, a parallel tool that applies a statistical model to identify ASM in data from high-throughput short-read bisulfite sequencing. It is based on the state-of-the-art sequential tool amrfinder, which is able to detect ASM at regional level from Bisulfite Sequencing (BS-Seq) experiments in the absence of Single Nucleotide Polymorphism information. PARamrfinder provides the same Allelically Methylated Regions as amrfinder but at significantly reduced runtime thanks to exploiting the compute capabilities of common multicore CPU clusters and MPI RMA operations to attain an efficient dynamic workload balance. As an example, our tool is up to 567 times faster for real data experiments on a cluster with 8 nodes, each one containing two 16-core processors. The source code of PARamrfinder, as well as a reference manual, is available at https://github.com/UDC-GAC/PARamrfinder.

Allele-specific DNA methylation and gene expression during shoot organogenesis in tissue culture of hybrid poplar.

Plant tissue regeneration is critical for genetic transformation and genome editing techniques. During the regeneration process, changes in epigenetic modifications accompany the cell fate transition. However, how allele-specific DNA methylation in two haplotypes contributes to the transcriptional dynamics during regeneration remains elusive. Here we applied an inter-species hybrid poplar (Populus alba × P. glandulosa cv. 84K) as a system to characterize the DNA methylation landscape during de novo shoot organogenesis at allele level. Both direct and indirect shoot organogenesis showed a reduction in genome-wide DNA methylation. At gene level, non-expressed genes were hypermethylated in comparison with expressed genes. Among the genes exhibiting significant correlations between levels of DNA methylation and gene expression, the expression patterns of 75% of genes were negatively correlated with DNA methylation in the CG context, whereas the correlation patterns in the CHH context were the reverse. The allele-biased DNA methylation was consistent during shoot organogenesis, with fewer than one-thousandth of allele-specific methylation regions shifted. Analysis of allele-specific expression revealed that there were only 1909 genes showing phase-dependent allele-biased expression in the regeneration process, among which the allele pairs with greater differences in transcription factor binding sites at promoter regions exhibited greater differences in allele expression. Our results indicated a relatively independent transcriptional regulation in two subgenomes during shoot organogenesis, which was contributed by cis-acting genomic and epigenomic variations.

Locus-Specific and Stable DNA Demethylation at the H19/IGF2 ICR1 by Epigenome Editing Using a dCas9-SunTag System and the Catalytic Domain of TET1.

DNA methylation is critically involved in the regulation of chromatin states and cell-type-specific gene expression. The exclusive expression of imprinted genes from either the maternal or the paternal allele is regulated by allele-specific DNA methylation at imprinting control regions (ICRs). Aberrant DNA hyper- or hypomethylation at the ICR1 of the H19/IGF2 imprinting locus is characteristic for the imprinting disorders Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS), respectively. In this paper, we performed epigenome editing to induce targeted DNA demethylation at ICR1 in HEK293 cells using dCas9-SunTag and the catalytic domain of TET1. 5-methylcytosine (5mC) levels at the target locus were reduced up to 90% and, 27 days after transient transfection, >60% demethylation was still observed. Consistent with the stable demethylation of CTCF-binding sites within the ICR1, the occupancy of the DNA methylation-sensitive insulator CTCF protein increased by >2-fold throughout the 27 days. Additionally, the H19 expression was increased by 2-fold stably, while IGF2 was repressed though only transiently. Our data illustrate the ability of epigenome editing to implement long-term changes in DNA methylation at imprinting control regions after a single transient treatment, potentially paving the way for therapeutic epigenome editing approaches in the treatment of imprinting disorders.

Paternally imprinted LATE-FLOWERING2 transcription factor contributes to paternal-excess interploidy hybridization barriers in wheat.

Interploidy hybridization between hexaploid and tetraploid genotypes occurred repeatedly during genomic introgression events throughout wheat evolution, and is commonly employed in wheat breeding programs. Hexaploid wheat usually serves as maternal parent because the reciprocal cross generates progeny with severe defects and poor seed germination, but the underlying mechanism is poorly understood. Here, we performed detailed analysis of phenotypic variation in endosperm between two interploidy reciprocal crosses arising from tetraploid (Triticum durum, AABB) and hexaploid wheat (Triticum aestivum, AABBDD). In the paternal- versus the maternal-excess cross, the timing of endosperm cellularization was delayed and starch granule accumulation in the endosperm was repressed, causing reduced germination percentage. The expression profiles of genes involved in nutrient metabolism differed strongly between these endosperm types. Furthermore, expression patterns of parental alleles were dramatically disturbed in interploidy versus intraploidy crosses, leading to increased number of imprinted genes. The endosperm-specific TaLFL2 showed a paternally imprinted expression pattern in interploidy crosses partially due to allele-specific DNA methylation. Paternal TaLFL2 binds to and represses a nutrient accumulation regulator TaNAC019, leading to reduced storage protein and starch accumulation during endosperm development in paternal-excess cross, as confirmed by interploidy crosses between tetraploid wild-type and clustered regularly interspaced palindromic repeats (CRISPR) - CRISPR-associated protein 9 generated hexaploid mutants. These findings reveal a contribution of genomic imprinting to paternal-excess interploidy hybridization barriers during wheat evolution history and explains why experienced breeders preferentially exploit maternal-excess interploidy crosses in wheat breeding programs.

Development of super-specific epigenome editing by targeted allele-specific DNA methylation

BackgroundEpigenome editing refers to the targeted reprogramming of genomic loci using an EpiEditor which may consist of an sgRNA/dCas9 complex that recruits DNMT3A/3L to the target locus. Methylation of the locus can lead to a modulation of gene expression. Allele-specific DNA methylation (ASM) refers to the targeted methylation delivery only to one allele of a locus. In the context of diseases caused by a dominant mutation, the selective DNA methylation of the mutant allele could be used to repress its expression but retain the functionality of the normal gene.ResultsTo set up allele-specific targeted DNA methylation, target regions were selected from hypomethylated CGIs bearing a heterozygous SNP in their promoters in the HEK293 cell line. We aimed at delivering maximum DNA methylation with highest allelic specificity in the targeted regions. Placing SNPs in the PAM or seed regions of the sgRNA, we designed 24 different sgRNAs targeting single alleles in 14 different gene loci. We achieved efficient ASM in multiple cases, such as ISG15, MSH6, GPD1L, MRPL52, PDE8A, NARF, DAP3, and GSPT1, which in best cases led to five to tenfold stronger average DNA methylation at the on-target allele and absolute differences in the DNA methylation gain at on- and off-target alleles of > 50%. In general, loci with the allele discriminatory SNP positioned in the PAM region showed higher success rate of ASM and better specificity. Highest DNA methylation was observed on day 3 after transfection followed by a gradual decline. In selected cases, ASM was stable up to 11 days in HEK293 cells and it led up to a 3.6-fold change in allelic expression ratios.ConclusionsWe successfully delivered ASM at multiple genomic loci with high specificity, efficiency and stability. This form of super-specific epigenome editing could find applications in the treatment of diseases caused by dominant mutations, because it allows silencing of the mutant allele without repression of the expression of the normal allele thereby minimizing potential side-effects of the treatment.

Paternal DNA methylation is remodeled to maternal levels in rice zygote

Epigenetic reprogramming occurs during reproduction to reset the genome for early development. In flowering plants, mechanistic details of parental methylation remodeling in zygote remain elusive. Here we analyze allele-specific DNA methylation in rice hybrid zygotes and during early embryo development and show that paternal DNA methylation is predominantly remodeled to match maternal allelic levels upon fertilization, which persists after the first zygotic division. The DNA methylation remodeling pattern supports the predominantly maternal-biased gene expression during zygotic genome activation (ZGA) in rice. However, parental allelic-specific methylations are reestablished at the globular embryo stage and associate with allelic-specific histone modification patterns in hybrids. These results reveal that paternal DNA methylation is remodeled to match the maternal pattern during zygotic genome reprogramming and suggest existence of a chromatin memory allowing parental allelic-specific methylation to be maintained in the hybrid.

Dynamics of imprinted genes and their epigenetic mechanisms in castor bean seed with persistent endosperm.

Genomic imprinting refers to parent-of-origin-dependent gene expression and primarily occurs in the endosperm of flowering plants, but its functions and epigenetic mechanisms remain to be elucidated in eudicots. Castor bean, a eudicot with large and persistent endosperm, provides an excellent system for studying the imprinting. Here, we identified 131 imprinted genes in developing endosperms and endosperm at seed germination phase of castor bean, involving into the endosperm development, accumulation of storage compounds and specially seed germination. Our results showed that the transcriptional repression of maternal allele of DNA METHYLTRANSFERASE 1 (MET1) may be required for maternal genome demethylation in the endosperm. DNA methylation analysis showed that only a small fraction of imprinted genes was associated with allele-specific DNA methylation, and most of them were closely associated with constitutively unmethylated regions (UMRs), suggesting a limited role for DNA methylation in controlling genomic imprinting. Instead, histone modifications can be asymmetrically deposited in maternal and paternal genomes in a DNA methylation-independent manner to control expression of most imprinted genes. These results expanded our understanding of the occurrence and biological functions of imprinted genes and showed the evolutionary flexibility of the imprinting machinery and mechanisms in plants.

Long-read sequencing reveals the complex structure of extra dic(21;21) chromosome and its biological effects

Complex congenital chromosome abnormalities are rare but often cause severe symptoms. However, the structures and biological impacts of such abnormalities have seldomly been analyzed at the molecular level. Previously, we reported a Japanese female patient with severe developmental defects. The patient had an extra dicentric chromosome 21 (chr21) consisting of two partial chr21 copies fused together within their long arms along with two centromeres and many copy number changes. In this study, we performed whole-genome, transcriptional, and DNA methylation analyses, coupled with novel bioinformatic approaches, to reveal the complex structure of the extra chromosome and its transcriptional and epigenetic changes. Long-read sequencing accurately identified the structures of junctions related to the copy number changes in extra chr21 and suggested the mechanism of the structural changes. Our transcriptome analysis showed the overexpression of genes in extra chr21. Additionally, an allele-specific DNA methylation analysis of the long-read sequencing data suggested that the centromeric region of extra chr21 was hypermethylated, a property associated with the inactivation of one centromere in the extra chromosome. Our comprehensive analysis provides insights into the molecular mechanism underlying the generation of the extra chromosome and its pathogenic roles.

Five nucleotides found in RCTG motifs are essential for post-fertilization methylation imprinting of the H19 ICR in YAC transgenic mice.

Genomic imprinting at the mouse Igf2/H19 locus is controlled by the H19 ICR, within which paternal allele-specific DNA methylation originating in sperm is maintained throughout development in offspring. We previously found that a 2.9 kb transgenic H19 ICR fragment in mice can be methylated de novo after fertilization only when paternally inherited, despite its unmethylated state in sperm. When the 118 bp sequence responsible for this methylation in transgenic mice was deleted from the endogenous H19 ICR, the methylation level of its paternal allele was significantly reduced after fertilization, suggesting the activity involving this 118 bp sequence is required for methylation maintenance at the endogenous locus. Here, we determined protein binding to the 118 bp sequence using an in vitro binding assay and inferred the binding motif to be RCTG by using a series of mutant competitors. Furthermore, we generated H19 ICR transgenic mice with a 5-bp substitution mutation that disrupts the RCTG motifs within the 118 bp sequence, and observed loss of methylation from the paternally inherited transgene. These results indicate that imprinted methylation of the H19 ICR established de novo during the post-fertilization period involves binding of specific factors to distinct sequence motifs within the 118 bp sequence.

Chromosome-level, nanopore-only genome and allele-specific DNA methylation of Pallas's cat, Otocolobus manul.

Pallas's cat, or the manul cat (Otocolobus manul), is a small felid native to the grasslands and steppes of central Asia. Population strongholds in Mongolia and China face growing challenges from climate change, habitat fragmentation, poaching, and other sources. These threats, combined with O. manul's zoo collection popularity and value in evolutionary biology, necessitate improvement of species genomic resources. We used standalone nanopore sequencing to assemble a 2.5 Gb, 61-contig nuclear assembly and 17097 bp mitogenome for O. manul. The primary nuclear assembly had 56× sequencing coverage, a contig N50 of 118 Mb, and a 94.7% BUSCO completeness score for Carnivora-specific genes. High genome collinearity within Felidae permitted alignment-based scaffolding onto the fishing cat (Prionailurus viverrinus) reference genome. Manul contigs spanned all 19 felid chromosomes with an inferred total gap length of less than 400 kilobases. Modified basecalling and variant phasing produced an alternate pseudohaplotype assembly and allele-specific DNA methylation calls; 61 differentially methylated regions were identified between haplotypes. Nearest features included classical imprinted genes, non-coding RNAs, and putative novel imprinted loci. The assembled mitogenome successfully resolved existing discordance between Felinae nuclear and mtDNA phylogenies. All assembly drafts were generated from 158 Gb of sequence using seven minION flow cells.

Genome-Wide DNA Methylation Profile Indicates Potential Epigenetic Regulation of Aging in the Rhesus Macaque Thymus.

We analyzed whole-genome bisulfite sequencing (WGBS) and RNA sequencing data of two young (1 year old) and two adult (9 years old) rhesus macaques (Macaca mulatta) to characterize the genomic DNA methylation profile of the thymus and explore the molecular mechanism of age-related changes in the thymus. Combining the two-omics data, we identified correlations between DNA methylation and gene expression and found that DNA methylation played an essential role in the functional changes of the aging thymus, especially in immunity and coagulation. The hypomethylation levels of C3 and C5AR2 and the hypermethylation level of C7 may lead to the high expressions of these genes in adult rhesus macaque thymuses, thus activating the classical complement pathway and the alternative pathway and enhancing their innate immune function. Adult thymuses had an enhanced coagulation pathway, which may have resulted from the hypomethylation and upregulated expressions of seven coagulation-promoting factor genes (F13A1, CLEC4D, CLEC4E, FCN3, PDGFRA, FGF2 and FGF7) and the hypomethylation and low expression of CPB2 to inhibit the degradation of blood clots. Furthermore, the functional decline in differentiation, activation and maturation of T cells in adult thymuses was also closely related to the changes in methylation levels and gene expression levels of T cell development genes (CD3G, GAD2, ADAMDEC1 and LCK) and the thymogenic hormone gene TMPO. A comparison of the age-related methylated genes among four mammal species revealed that most of the epigenetic clocks were species-specific. Furthermore, based on the genomic landscape of allele-specific DNA methylation, we identified several age-related clustered sequence-dependent allele-specific DNA methylated (cS-ASM) genes. Overall, these DNA methylation patterns may also help to assist with understanding the mechanisms of the aging thymus with the epigenome.

A Role of DNA Methylation within the CYP17A1 Gene in the Association of Genetic and Environmental Risk Factors with Stress-Related Manifestations of Schizophrenia.

As genetic and environmental influences on schizophrenia might converge on DNA methylation (DNAm) within loci which are both associated with the disease and implicated in response to environmental stress, we examined whether DNAm within CYP17A1, a hypothalamus–pituitary–adrenal axis gene which is situated within the schizophrenia risk locus 10q24.32, would mediate genetic and environmental effects on stress-related schizophrenia symptoms. DNAm within an exonic–intronic fragment of CYP17A1 was assessed in the blood of 66 schizophrenia patients and 63 controls using single-molecule real-time bisulfite sequencing. Additionally, the VNTR polymorphism of the AS3MT gene, a plausible causal variant within the 10q24.32 locus, was genotyped in extended patient and control samples (n = 700). The effects of local haplotype, VNTR and a polyenviromic risk score (PERS) on DNAm, episodic verbal memory, executive functions, depression, and suicidality of patients were assessed. Haplotype and PERS differentially influenced DNAm at four variably methylated sites identified within the fragment, with stochastic, additive, and allele-specific effects being found. An allele-specific DNAm at CpG-SNP rs3781286 mediated the relationship between the local haplotype and verbal fluency. Our findings do not confirm that the interrogated DNA fragment is a place where genetic and environmental risk factors converge to influence schizophrenia symptoms through DNAm.

Allele-specific H3K9me3 and DNA methylation co-marked CpG-rich regions serve as potential imprinting control regions in pre-implantation embryo.

Parental DNA methylation and histone modifications undergo distinct global reprogramming in mammalian pre-implantation embryos, but the landscape of epigenetic crosstalk and its effects on embryogenesis are largely unknown. Here we comprehensively analyse the association between DNA methylation and H3K9me3 reprogramming in mouse pre-implantation embryos and reveal that CpG-rich genomic loci with high H3K9me3 signal and DNA methylation level (CHM) are hotspots of DNA methylation maintenance during pre-implantation embryogenesis. We further profile the allele-specific epigenetic map with unprecedented resolution in gynogenetic and androgenetic embryos, respectively, and identify 1,279 allele-specific CHMs, including 19 known imprinting control regions (ICRs). Our study suggests that 22 ICR-like regions (ICRLRs) may regulate allele-specific transcription similarly to known ICRs, and five of them are confirmed to be important for mouse embryo development. Taken together, our study reveals the widespread existence of allele-specific CHMs and largely extends the scope of allele-specific regulation in mammalian pre-implantation embryos.

DNA demethylation affects imprinted gene expression in maize endosperm

BackgroundDNA demethylation occurs in many species and is involved in diverse biological processes. However, the occurrence and role of DNA demethylation in maize remain unknown.ResultsWe analyze loss-of-function mutants of two major genes encoding DNA demethylases. No significant change in DNA methylation has been detected in these mutants. However, we detect increased DNA methylation levels in the mutants around genes and some transposons. The increase in DNA methylation is accompanied by alteration in gene expression, with a tendency to show downregulation, especially for the genes that are preferentially expressed in endosperm. Imprinted expression of both maternally and paternally expressed genes changes in F1 hybrid with the mutant as female and the wild-type as male parental line, but not in the reciprocal hybrid. This alteration in gene expression is accompanied by allele-specific DNA methylation differences, suggesting that removal of DNA methylation of the maternal allele is required for the proper expression of these imprinted genes. Finally, we demonstrate that hypermethylation in the double mutant is associated with reduced binding of transcription factor to its target, and altered gene expression.ConclusionsOur results suggest that active removal of DNA methylation is important for transcription factor binding and proper gene expression in maize endosperm.

Chromosome-scale and haplotype-resolved genome assembly of a tetraploid potato cultivar

Potato is the most widely produced tuber crop worldwide. However, reconstructing the four haplotypes of its autotetraploid genome remained an unsolved challenge. Here, we report the 3.1 Gb haplotype-resolved (at 99.6% precision), chromosome-scale assembly of the potato cultivar ‘Otava’ based on high-quality long reads, single-cell sequencing of 717 pollen genomes and Hi-C data. Unexpectedly, ~50% of the genome was identical-by-descent due to recent inbreeding, which was contrasted by highly abundant structural rearrangements involving ~20% of the genome. Among 38,214 genes, only 54% were present in all four haplotypes with an average of 3.2 copies per gene. Taking the leaf transcriptome as an example, 11% of the genes were differently expressed in at least one haplotype, where 25% of them were likely regulated through allele-specific DNA methylation. Our work sheds light on the recent breeding history of potato, the functional organization of its tetraploid genome and has the potential to strengthen the future of genomics-assisted breeding.

Dual role of allele-specific DNA hypermethylation within the TERT promoter in cancer.

Aberrant activation of telomerase in human cancer is achieved by various alterations within the TERT promoter, including cancer-specific DNA hypermethylation of the TERT hypermethylated oncological region (THOR). However, the impact of allele-specific DNA methylation within the TERT promoter on gene transcription remains incompletely understood. Using allele-specific next-generation sequencing, we screened a large cohort of normal and tumor tissues (n = 652) from 10 cancer types and identified that differential allelic methylation (DAM) of THOR is restricted to cancerous tissue and commonly observed in major cancer types. THOR-DAM was more common in adult cancers, which develop through multiple stages over time, than in childhood brain tumors. Furthermore, THOR-DAM was especially enriched in tumors harboring the activating TERT promoter mutations (TPMs). Functional studies revealed that allele-specific gene expression of TERT requires hypomethylation of the core promoter, both in TPM and TERT WT cancers. However, the expressing allele with hypomethylated core TERT promoter universally exhibits hypermethylation of THOR, while the nonexpressing alleles are either hypermethylated or hypomethylated throughout the promoter. Together, our findings suggest a dual role for allele-specific DNA methylation within the TERT promoter in the regulation of TERT expression in cancer.

A bipartite element with allele-specific functions safeguards DNA methylation imprints at the Dlk1-Dio3 locus.

Loss of imprinting (LOI) results in severe developmental defects, but the mechanisms preventing LOI remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) in pluripotent stem cells. We demonstrate that the IG-DMR consists of two antagonistic elements: a paternally methylated CpG island that prevents recruitment of TET dioxygenases and a maternally unmethylated non-canonical enhancer that ensures expression of the Gtl2 lncRNA by counteracting de novo DNA methyltransferases. Genetic or epigenetic editing of these elements leads to distinct LOI phenotypes with characteristic alternations of allele-specific gene expression, DNA methylation, and 3D chromatin topology. Although repression of the Gtl2 promoter results in dysregulated imprinting, the stability of LOI phenotypes depends on the IG-DMR, suggesting a functional hierarchy. These findings establish the IG-DMR as a bipartite control element that maintains imprinting by allele-specific restriction of the DNA (de)methylation machinery.

ASMdb: a comprehensive database for allele-specific DNA methylation in diverse organisms.

DNA methylation is known to be the most stable epigenetic modification and has been extensively studied in relation to cell differentiation, development, X chromosome inactivation and disease. Allele-specific DNA methylation (ASM) is a well-established mechanism for genomic imprinting and regulates imprinted gene expression. Previous studies have confirmed that certain special regions with ASM are susceptible and closely related to human carcinogenesis and plant development. In addition, recent studies have proven ASM to be an effective tumour marker. However, research on the functions of ASM in diseases and development is still extremely scarce. Here, we collected 4400 BS-Seq datasets and 1598 corresponding RNA-Seq datasets from 47 species, including human and mouse, to establish a comprehensive ASM database. We obtained the data on DNA methylation level, ASM and allele-specific expressed genes (ASEGs) and further analysed the ASM/ASEG distribution patterns of these species. In-depth ASM distribution analysis and differential methylation analysis conducted in nine cancer types showed results consistent with the reported changes in ASM in key tumour genes and revealed several potential ASM tumour-related genes. Finally, integrating these results, we constructed the first well-resourced and comprehensive ASM database for 47 species (ASMdb, www.dna-asmdb.com).

Allele-specific DNA methylation maps in monozygotic twins discordant for psychiatric disorders reveal that disease-associated switching at the EIPR1 regulatory loci modulates neural function.

The non-Mendelian features of phenotypic variations within monozygotic twins are likely complicated by environmental modifiers of genetic effects that have yet to be elucidated. Here, we performed methylome and genome analyses of blood DNA from psychiatric disorder-discordant monozygotic twins to study how allele-specific methylation (ASM) mediates phenotypic variations. We identified that thousands of genetic variants with ASM imbalances exhibit phenotypic variation-associated switching at regulatory loci. These ASMs have plausible causal associations with psychiatric disorders through effects on interactions between transcription factors, DNA methylations, and other epigenomic markers and then contribute to dysregulated gene expression, which eventually increases disease susceptibility. Moreover, we also experimentally validated the model that the rs4854158 alternative C allele at an ASM switching regulatory locus of EIPR1 encoding endosome-associated recycling protein-interacting protein 1, is associated with demethylation and higher RNA expression and shows lower TF binding affinities in unaffected controls. An epigenetic ASM switching induces C allele hypermethylation and then recruits repressive Polycomb repressive complex 2 (PRC2), reinforces trimethylation of lysine 27 on histone 3 and inhibits its transcriptional activity, thus leading to downregulation of EIPR1 in schizophrenia. Moreover, disruption of rs4854158 induces gain of EIPR1 function and promotes neural development and vesicle trafficking. Our study provides a powerful framework for identifying regulatory risk variants and contributes to our understanding of the interplay between genetic and epigenetic variants in mediating psychiatric disorder susceptibility.

Phasing analysis of the transcriptome and epigenome in a rice hybrid reveals the inheritance and difference in DNA methylation and allelic transcription regulation

Hybrids are always a focus of botanical research and have a high practical value in agricultural production. To better understand allele regulation and differences in DNA methylation in hybrids, we developed a phasing pipeline for hybrid rice based on two parental genomes (PP2PG), which is applicable for Iso-Seq, RNA-Seq, and Bisulfite sequencing (BS-Seq). Using PP2PG, we analyzed differences in gene transcription, alternative splicing, and DNA methylation in an allele-specific manner between parents and progeny or different progeny alleles. The phasing of Iso-Seq data provided a great advantage in separating the whole gene structure and producing a significantly higher separation ratio than RNA-Seq. The interaction of hybrid alleles was studied by constructing an allele co-expression network that revealed the dominant allele effect in the network. The expression variation between parents and the parental alleles in progeny showed tissue- or environment-specific patterns, which implied a preference for trans-acting regulation under different conditions. In addition, by comparing allele-specific DNA methylation, we found that CG methylation was more likely to be inherited than CHG and CHH methylation, and its enrichment in genic regions was connected to gene structure. In addition to an effective phasing pipeline, we also identified differentiation in OsWAK38 gene structure that may have led to the expansion of allele functions in hybrids. In summary, we developed a phasing pipeline and provided valuable insights into alternative splicing, interaction networks, trans-acting regulation, and the inheritance of DNA methylation in hybrid rice.