Biallelic Expression Research Articles

Targeted Long-Read Bisulfite Sequencing Identifies Differences in the TERT Promoter Methylation Profiles between TERT Wild-Type and TERT Mutant Cancer Cells.

Simple SummaryTERT promoter methylation is enriched in cancers lacking TERT genetic alterations (wild-type cancers), but its functional impact on TERT transcription remains elusive. We developed a long-read bisulfite-sequencing platform to characterize the TERT promoter methylation profile at a single-molecule level. In wild-type cancer cell lines, both epialleles were hypermethylated symmetrically on the TERT distal promoter. In the core and proximal promoter, by contrast, the transcribed epialleles were significantly more hypomethylated than the silent epialleles. Decitabine-therapy reduced the core and proximal (not the distal) promoter methylation and reactivated the silent allele. We showed that TERT allele-specific expression is amenable to in vitro epigenetic manipulation in wild-type cancers.Background: TERT promoter methylation, located several hundred base pairs upstream of the transcriptional start site, is cancer specific and correlates with increased TERT mRNA expression and poorer patient outcome. Promoter methylation, however, is not mutually exclusive to TERT activating genetic alterations, as predicted for functionally redundant mechanisms. To annotate the altered patterns of TERT promoter methylation and their relationship with gene expression, we applied a Pacific Biosciences-based, long-read, bisulfite-sequencing technology and compared the differences in the methylation marks between wild-type and mutant cancers in an allele-specific manner. Results: We cataloged TERT genetic alterations (i.e., promoter point mutations or structural variations), allele-specific promoter methylation patterns, and allele-specific expression levels in a cohort of 54 cancer cell lines. In heterozygous mutant cell lines, the mutant alleles were significantly less methylated than their silent, mutation-free alleles (p < 0.05). In wild-type cell lines, by contrast, both epialleles were equally methylated to high levels at the TERT distal promoter, but differentially methylated in the proximal regions. ChIP analysis showed that epialleles with the hypomethylated proximal and core promoter were enriched in the active histone mark H3K4me2/3, whereas epialleles that were methylated in those regions were enriched in the repressive histone mark H3K27me3. Decitabine therapy induced biallelic expression in the wild-type cancer cells, whereas the mutant cell lines were unaffected. Conclusions: Long-read bisulfite sequencing analysis revealed differences in the methylation profiles and responses to demethylating agents between TERT wild-type and genetically altered cancer cell lines. The causal relation between TERT promoter methylation and gene expression remains to be established.

Multi-omics analysis reveals multiple mechanisms causing Prader-Willi like syndrome in a family with a X;15 translocation.

Prader-Willi syndrome (PWS; MIM# 176270) is a neurodevelopmental disorder caused by the loss of expression of paternally imprinted genes within the PWS region located on 15q11.2. It is usually caused by either maternal uniparental disomy of chromosome 15 (UPD15) or 15q11.2 recurrent deletion(s). Here, we report a healthy carrier of a balanced X;15 translocation and her two daughters, both with the karyotype 45,X,der(X)t(X;15)(p22;q11.2),-15. Both daughters display symptoms consistent with haploinsufficiency of the SHOX gene and PWS. We explored the architecture of the derivative chromosomes and investigated effects on gene expression in patient-derived neural cells. First, a multiplex ligation-dependent probe amplification methylation assay was used to determine the methylation status of the PWS-region revealing maternal UPD15 in daughter 2, explaining her clinical symptoms. Next, short read whole genome sequencing and 10X genomics linked read sequencing was used to pinpoint the exact breakpoints of the translocation. Finally, we performed transcriptome sequencing on neuroepithelial stemcells from the mother and from daughter 1 and observed biallelic expression of genes in the PWS region (including SNRPN) in daughter 1. In summary, our multi-omics analysis highlights two different PWS mechanisms in one family and provide an example of how structural variation can affect imprinting through long-range interactions.

Abstract 5725: HOXA9 demonstrates allelic imbalance in KMT2A-rearranged infant acute lymphoblastic leukemia

Abstract Background: KMT2A-rearrangement is a characteristic finding in approximately 70% of infant acute lymphoblastic leukemia (ALL) cases. We previously utilized a computational screen to find that KMT2A-r infant ALL demonstrated somatic allele specific expression for 431 genes when compared to non-KMT2A-r samples (Abstract Number 3649, AACR 2019). The gene that exhibited the most differential allele specific expression (ASE) pattern was HOXA9. Here, we follow up that analysis with manual examination of additional infant ALL samples. Methods: We performed whole genome sequencing (WGS) and RNA sequencing (RNAseq) on peripheral blood or bone marrow samples from 48 infants with ALL (33 KMT2A-r cases and 15 non-KMT2A-r cases) treated on Children’s Oncology Group protocol AALL0631, at diagnosis (DX), at remission (MD), and at relapse (RL), if applicable. WGS data at MD was phased using a 1000 Genomes reference panel. HOXA9 expression was quantified in transcripts per million (TPM). Biallelic expression was manually calculated on the phased genome for samples with at least 1 germline single nucleotide polymorphism (SNP) within HOXA9 and at least 5 aligned RNAseq reads over the HOXA9 transcript. ASE was defined as minor allele fraction (MAF; allele fraction for the less prevalent allele) of ≤ 0.35. Whole genome bisulfite sequencing data and sequencing-derived copy number data were also analyzed to investigate possible explanations for HOXA9 ASE. Results: We identified diagnostic samples from 21 patients, 9 KMT2A-AFF1, 9 KMT2A-MLLT1, and 3 non-KMT2A-r, that passed manual filtering. Data included 5 patients with paired DX and RL samples. Of the 15 KMT2A-r samples unable to be analyzed for ASE, 13 lacked a SNP for phasing and 2 had &amp;lt; 5 reads. For the 12 non-KMT2A-r samples unanalyzable for ASE, 6 lacked a SNP and 6 had &amp;lt; 5 reads. Average HOXA9 expression was 15.17 TPM (range 0.01 to 44.34) in KMT2A-r samples and 2.91 TPM (range 0 to 29.29) in non-KMT2A-r samples. ASE of HOXA9 (MAF ≤ 0.35) was observed in 14 samples, 72% of KMT2A-r (13 of 18 samples) and 33% of non-KMT2A-r (1 of 3 samples). RL samples were found to have HOXA9 ASE in all cases with ASE of HOXA9 in the DX sample. Of the 18 KMT2A-r samples, 6 of 9 with KMT2A-AFF1 and 7 of 9 with KMT2A-MLLT1 demonstrated HOXA9 ASE. Frequency of HOXA9 ASE at diagnosis was similar between patients known to later relapse versus those known to remain in remission. Allele specific methylation, mono-allelic deletion, or copy-neutral loss of heterozygosity (cn-LOH) for HOXA9 were not observed in KMT2A-r nor non-KMT2A-r samples. Conclusion: Our expanded cohort supports the ASE of HOXA9 in KMT2A-r infant ALL. This phenomenon did not appear to segregate by KMT2A-r partner gene, nor could it be explained by allele specific methylation or copy number variation (e.g., deletion of one HOXA9 allele or cn-LOH). Overall, further investigation into a causal mechanism is needed. Citation Format: Sarah E. Mc Dermott, Byunggil Yoo, Warren A. Cheung, Emily Farrow, Bing Ge, Margaret Gibson, Patrick A. Brown, Erin Guest, Tomi Pastinen, Midhat S. Farooqi. HOXA9 demonstrates allelic imbalance in KMT2A-rearranged infant acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5725.

Placental sex-dependent spermine synthesis regulates trophoblast gene expression through acetyl-coA metabolism and histone acetylation

Placental function and dysfunction differ by sex but the mechanisms are unknown. Here we show that sex differences in polyamine metabolism are associated with escape from X chromosome inactivation of the gene encoding spermine synthase (SMS). Female placental trophoblasts demonstrate biallelic SMS expression, associated with increased SMS mRNA and enzyme activity. Polyamine depletion in primary trophoblasts reduced glycolysis and oxidative phosphorylation resulting in decreased acetyl-coA availability and global histone hypoacetylation in a sex-dependent manner. Chromatin-immunoprecipitation sequencing and RNA-sequencing identifies progesterone biosynthesis as a target of polyamine regulated gene expression, and polyamine depletion reduced progesterone release in male trophoblasts. The effects of polyamine depletion can be attributed to spermine as SMS-silencing recapitulated the effects on energy metabolism, histone acetylation, and progesterone release. In summary, spermine metabolism alters trophoblast gene expression through acetyl-coA biosynthesis and histone acetylation, and SMS escape from X inactivation explains some features of human placental sex differences.

Retinoic acid signaling acts as a rheostat to balance Treg function.

Regulatory T cells (Tregs) promote immune homeostasis by maintaining self-tolerance and regulating inflammatory responses. Under certain inflammatory conditions, Tregs can lose their lineage stability and function. Previous studies have reported that ex vivo exposure to retinoic acid (RA) enhances Treg function and stability. However, it is unknown how RA receptor signaling in Tregs influences these processes in vivo. Herein, we employed mouse models in which RA signaling is silenced by the expression of the dominant negative receptor (DN) RARα in all T cells. Despite the fact that DNRARα conventional T cells are hypofunctional, Tregs had increased CD25 expression, STAT5 pathway activation, mTORC1 signaling and supersuppressor function. Furthermore, DNRARα Tregs had increased inhibitory molecule expression, amino acid transporter expression, and metabolic fitness and decreased antiapoptotic proteins. Supersuppressor function was observed when wild-type mice were treated with a pharmacologic pan-RAR antagonist. Unexpectedly, Treg-specific expression of DNRARα resulted in distinct phenotypes, such that a single allele of DNRARα in Tregs heightened their suppressive function, and biallelic expression led to loss of suppression and autoimmunity. The loss of Treg function was not cell intrinsic, as Tregs that developed in a noninflammatory milieu in chimeric mice reconstituted with DNRARα and wild-type bone marrow maintained the enhanced suppressive capacity. Fate mapping suggested that maintaining Treg stability in an inflammatory milieu requires RA signaling. Our findings indicate that RA signaling acts as a rheostat to balance Treg function in inflammatory and noninflammatory conditions in a dose-dependent manner.

Single Cell RNA-Seq Analysis of Human Red Cells.

Human red blood cells (RBCs), or erythrocytes, are the most abundant blood cells responsible for gas exchange. RBC diseases affect hundreds of millions of people and impose enormous financial and personal burdens. One well-recognized, but poorly understood feature of RBC populations within the same individual are their phenotypic heterogeneity. The granular characterization of phenotypic RBC variation in normative and disease states may allow us to identify the genetic determinants of red cell diseases and reveal novel therapeutic approaches for their treatment. Previously, we discovered diverse RNA transcripts in RBCs that has allowed us to dissect the phenotypic heterogeneity and malaria resistance of sickle red cells. However, these analyses failed to capture the heterogeneity found in RBC sub-populations. To overcome this limitation, we have performed single cell RNA-Seq to analyze the transcriptional heterogeneity of RBCs from three adult healthy donors which have been stored in the blood bank conditions and assayed at day 1 and day 15. The expression pattern clearly separated RBCs into seven distinct clusters that include one RBC cluster that expresses HBG2 and a small population of RBCs that express fetal hemoglobin (HbF) that we annotated as F cells. Almost all HBG2-expessing cells also express HBB, suggesting bi-allelic expression in single RBC from the HBG2/HBB loci, and we annotated another cluster as reticulocytes based on canonical gene expression. Additional RBC clusters were also annotated based on the enriched expression of NIX, ACVR2B and HEMGN, previously shown to be involved in erythropoiesis. Finally, we found the storage of RBC was associated with an increase in the ACVR2B and F-cell clusters. Collectively, these data indicate the power of single RBC RNA-Seq to capture and discover known and unexpected heterogeneity of RBC population.

Dental pulp stem cells as a promising model to study imprinting diseases

Parental imprinting is an epigenetic process leading to monoallelic expression of certain genes depending on their parental origin. Imprinting diseases are characterized by growth and metabolic issues starting from birth to adulthood. They are mainly due to methylation defects in imprinting control region that drive the abnormal expression of imprinted genes. We currently lack relevant animal or cellular models to unravel the pathophysiology of growth failure in these diseases. We aimed to characterize the methylation of imprinting regions in dental pulp stem cells and during their differentiation in osteogenic cells (involved in growth regulation) to assess the interest of this cells in modeling imprinting diseases. We collected dental pulp stem cells from five controls and four patients (three with Silver-Russell syndrome and one with Beckwith-Wiedemann syndrome). Methylation analysis of imprinting control regions involved in these syndromes showed a normal profile in controls and the imprinting defect in patients. These results were maintained in dental pulp stem cells cultured under osteogenic conditions. Furthermore, we confirmed the same pattern in six other loci involved in imprinting diseases in humans. We also confirmed monoallelic expression of H19 (an imprinted gene) in controls and its biallelic expression in one patient. Extensive imprinting control regions methylation analysis shows the strong potential of dental pulp stem cells in modeling imprinting diseases, in which imprinting regions are preserved in culture and during osteogenic differentiation. This will allow to perform in vitro functional and therapeutic tests in cells derived from dental pulp stem cells and generate other cell-types.

BrewerIX enables allelic expression analysis of imprinted and X-linked genes from bulk and single-cell transcriptomes

Genomic imprinting and X chromosome inactivation (XCI) are two prototypical epigenetic mechanisms whereby a set of genes is expressed mono-allelically in order to fine-tune their expression levels. Defects in genomic imprinting have been observed in several neurodevelopmental disorders, in a wide range of tumours and in induced pluripotent stem cells (iPSCs). Single Nucleotide Variants (SNVs) are readily detectable by RNA-sequencing allowing the determination of whether imprinted or X-linked genes are aberrantly expressed from both alleles, although standardised analysis methods are still missing. We have developed a tool, named BrewerIX, that provides comprehensive information about the allelic expression of a large, manually-curated set of imprinted and X-linked genes. BrewerIX does not require programming skills, runs on a standard personal computer, and can analyze both bulk and single-cell transcriptomes of human and mouse cells directly from raw sequencing data. BrewerIX confirmed previous observations regarding the bi-allelic expression of some imprinted genes in naive pluripotent cells and extended them to preimplantation embryos. BrewerIX also identified misregulated imprinted genes in breast cancer cells and in human organoids and identified genes escaping XCI in human somatic cells. We believe BrewerIX will be useful for the study of genomic imprinting and XCI during development and reprogramming, and for detecting aberrations in cancer, iPSCs and organoids. Due to its ease of use to non-computational biologists, its implementation could become standard practice during sample assessment, thus raising the robustness and reproducibility of future studies.

Gametophyte genome activation occurs at pollen mitosis I in maize.

Flowering plants alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. Pollen actively transcribes its haploid genome, providing phenotypic diversity even among pollen grains from a single plant. In this study, we used allele-specific RNA sequencing of single pollen precursors to follow the shift to haploid expression in maize pollen. We observed 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, driven by active new transcription from the haploid genome. Genes showed evidence of increased purifying selection if they were expressed after (but not before) pollen mitosis I. This work establishes the timing during which haploid selection may act in pollen.

Nemo-Dependent, ATM-Mediated Signals from RAG DNA Breaks at Igk Feedback Inhibit V κ Recombination to Enforce Igκ Allelic Exclusion.

Monoallelic AgR gene expression underlies specific adaptive immune responses. AgR allelic exclusion is achieved by sequential initiation of V(D)J recombination between alleles and resultant protein from one allele signaling to prevent recombination of the other. The ATM kinase, a regulator of the DNA double-strand break (DSB) response, helps enforce allelic exclusion through undetermined mechanisms. ATM promotes repair of RAG1/RAG2 (RAG) endonuclease-induced DSBs and transduces signals from RAG DSBs during Igk gene rearrangement on one allele to transiently inhibit RAG1 protein expression, Igk accessibility, and RAG cleavage of the other allele. Yet, the relative contributions of ATM functions in DSB repair versus signaling to enforce AgR allelic exclusion remain undetermined. In this study, we demonstrate that inactivation in mouse pre-B cells of the NF-κB essential modulator (Nemo) protein, an effector of ATM signaling, diminishes RAG DSB-triggered repression of Rag1/Rag2 transcription and Igk accessibility but does not result in aberrant repair of RAG DSBs like ATM inactivation. We show that Nemo deficiency increases simultaneous biallelic Igk cleavage in pre-B cells and raises the frequency of B cells expressing Igκ proteins from both alleles. In contrast, the incidence of biallelic Igκ expression is not elevated by inactivation of the SpiC transcriptional repressor, which is induced by RAG DSBs in an ATM-dependent manner and suppresses Igk accessibility. Thus, we conclude that Nemo-dependent, ATM-mediated DNA damage signals enforce Igκ allelic exclusion by orchestrating transient repression of RAG expression and feedback inhibition of additional Igk rearrangements in response to RAG cleavage on one Igk allele.

Potential immunological effects of gender-affirming hormone therapy in transgender people - an unexplored area of research.

There are well-described sex-based differences in how the immune system operates. In particular, cisgender (cis) females have a more easily activated immune system; associated with an increased prevalence of autoimmune diseases and adverse events following vaccinations. Conversely, cis males have a higher threshold for immune activation, and are more prone to certain infectious diseases, such as coronavirus disease (COVID-19). Oestrogen and testosterone have immune-modulatory properties, and it is likely that these contribute to the sexual dimorphism of the immune system. There are also important immune-related genes located on the X chromosome, such as toll-like receptor (TLR) 7/8; and the mosaic bi-allelic expression of such genes may contribute to the state of immune hyperactivation in cis females. The scientific literature strongly suggests that sex-based differences in the functioning of the immune system are related to both X-linked genes and immune modulation by sex hormones. However, it is currently not clear how this impacts transgender (trans) people receiving gender-affirming hormonal therapy. Moreover, it is estimated that in Australia, at least 2.3% of adolescents identify as trans and/or gender diverse, and referrals to specialist gender-affirming care are increasing each year. Despite the improving social awareness of trans people, they remain chronically underrepresented in the scientific literature. In addition, a small number of case studies describe new onset autoimmune disorders in adult trans females following oestrogen use. However, there is currently minimal long-term research with an immunological focus on trans people. Therefore, to ensure the positive health outcomes of trans people, it is crucial that the role of sex hormones in immune modulation is investigated further.

Genomic imprinting of the IGF2R/AIR locus is conserved between bovines and mice

Genomic imprinting is an epigenetic phenomenon that leads to genes monoallelically expressed in a parent-of-origin-specific manner and plays an important role in the embryonic development and postnatal growth of mammals. Imprinted genes usually occur in clusters in a chromosomal region and are regulated by a cis-acting imprinting control region that involves differential DNA methylation modification. Igf2r, Slc22a2 and Slc22a3 are three maternally expressed genes on mouse chromosome 17. The paternally expressed long noncoding RNA (lncRNA) Air and the nonimprinted gene Slc22a1 are also located in the imprinted region. Comparative characterization of imprinted clusters between species is useful for us to understand the biological significance and epigenetic regulating mechanism of genomic imprinting. The aim of this study was to analyze the allelic expression pattern of AIR and SLC22A1-3 genes in cattle and to determine the role of DNA methylation in regulating gene expression. Allelic expression analysis was performed in bovine adult tissues and term placenta using an SNP-based approach. We found that IGF2R, AIR and SLC22A3 were monoallelically expressed in all detected bovine somatic tissues, including heart, liver, spleen, lung, kidney, muscle, fat and brain. In bovine placenta, IGF2R and SLC22A3 are maternally expressed; however, the AIR gene is paternally expressed. Tissue-specific monoallelic expression of SLC22A2 is detected in bovines, with monoallelic expression in the spleen and brain but biallelic expression in kidney tissues. SLC22A1 is only detected in bovine liver and kidney tissues and is biallelicly expressed, which is consistent with the imprint expression in mice. To determine the possible role of DNA methylation in regulating the monoallelic/imprinted expression of bovine IGF2R, AIR, SLC22A2, and SLC22A3 genes, we analyzed the DNA methylation status of CpG islands in the first exon of SLC22A2, the promoter region of SLC22A3 and region 2 in the second intron of the IGF2R gene by bisulfite sequencing. Two differentially methylated regions (DMRs) were detected in the first exon of bovine SLC22A3 and the common regions of IGF2R and AIR. This suggests that DNA methylation is involved in the regulation of monoallelic/imprinted expression of IGF2R, AIR and SLC22A3 genes in cattle.

Epigenetic Dysregulation of KCNK9 Imprinting and Triple-Negative Breast Cancer.

Simple SummaryGenomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. Here, we showed that KCNK9 is imprinted in breast tissue and identified the differentially methylated region (DMR) controlling its imprint status. Hypomethylation at the DMR, coupled with biallelic expression of KCNK9, occurred in 63% of triple-negative breast cancers (TNBC). The association between hypomethylation and TNBC status was highly significant in African-Americans (p = 0.006), but not in Caucasians (p = 0.70). The high frequency of KCNK9 DMR hypomethylation in TNBC and non-cancerous breast tissue from high-risk women provides evidence that hypomethylation of the KNCK9 DMR/TASK3 overexpression may provide a new target for prevention of TNBC.Genomic imprinting is an inherited form of parent-of-origin specific epigenetic gene regulation that is dysregulated by poor prenatal nutrition and environmental toxins. KCNK9 encodes for TASK3, a pH-regulated potassium channel membrane protein that is overexpressed in 40% of breast cancer. However, KCNK9 gene amplification accounts for increased expression in <10% of these breast cancers. Here, we showed that KCNK9 is imprinted in breast tissue and identified a differentially methylated region (DMR) controlling its imprint status. Hypomethylation at the DMR, coupled with biallelic expression of KCNK9, occurred in 63% of triple-negative breast cancers (TNBC). The association between hypomethylation and TNBC status was highly significant in African-Americans (p = 0.006), but not in Caucasians (p = 0.70). KCNK9 hypomethylation was also found in non-cancerous tissue from 77% of women at high-risk of developing breast cancer. Functional studies demonstrated that the KCNK9 gene product, TASK3, regulates mitochondrial membrane potential and apoptosis-sensitivity. In TNBC cells and non-cancerous mammary epithelial cells from high-risk women, hypomethylation of the KCNK9 DMR predicts for increased TASK3 expression and mitochondrial membrane potential (p < 0.001). This is the first identification of the KCNK9 DMR in mammary epithelial cells and demonstration that its hypomethylation in breast cancer is associated with increases in both mitochondrial membrane potential and apoptosis resistance. The high frequency of hypomethylation of the KCNK9 DMR in TNBC and non-cancerous breast tissue from high-risk women provides evidence that hypomethylation of the KNCK9 DMR/TASK3 overexpression may serve as a marker of risk and a target for prevention of TNBC, particularly in African American women.

Functional organization of the maternal and paternal human 4D Nucleome

SummaryEvery human somatic cell inherits a maternal and a paternal genome, which work together to give rise to cellular phenotypes. However, the allele-specific relationship between gene expression and genome structure through the cell cycle is largely unknown. By integrating haplotype-resolved genome-wide chromosome conformation capture, mature and nascent mRNA, and protein binding data from a B lymphoblastoid cell line, we investigate this relationship both globally and locally. We introduce the maternal and paternal 4D Nucleome, enabling detailed analysis of the mechanisms and dynamics of genome structure and gene function for diploid organisms. Our analyses find significant coordination between allelic expression biases and local genome conformation, and notably absent expression bias in universally essential cell cycle and glycolysis genes. We propose a model in which coordinated biallelic expression reflects prioritized preservation of essential gene sets.

Why Females Do Better: The X Chromosomal TLR7 Gene-Dose Effect in COVID-19.

A male sex bias has emerged in the COVID-19 pandemic, fitting to the sex-biased pattern in other viral infections. Males are 2.84 times more often admitted to the ICU and mortality is 1.39 times higher as a result of COVID-19. Various factors play a role in this, and novel studies suggest that the gene-dose of Toll-Like Receptor (TLR) 7 could contribute to the sex-skewed severity. TLR7 is one of the crucial pattern recognition receptors for SARS-CoV-2 ssRNA and the gene-dose effect is caused by X chromosome inactivation (XCI) escape. Female immune cells with TLR7 XCI escape have biallelic TLR7 expression and produce more type 1 interferon (IFN) upon TLR7 stimulation. In COVID-19, TLR7 in plasmacytoid dendritic cells is one of the pattern recognition receptors responsible for IFN production and a delayed IFN response has been associated with immunopathogenesis and mortality. Here, we provide a hypothesis that females may be protected to some extend against severe COVID-19, due to the biallelic TLR7 expression, allowing them to mount a stronger and more protective IFN response early after infection. Studies exploring COVID-19 treatment via the TLR7-mediated IFN pathway should consider this sex difference. Various factors such as age, sex hormones and escape modulation remain to be investigated concerning the TLR7 gene-dose effect.

The Role of Long Non-coding RNAs in Human Imprinting Disorders: Prospective Therapeutic Targets.

Genomic imprinting is a term used for an intergenerational epigenetic inheritance and involves a subset of genes expressed in a parent-of-origin-dependent way. Imprinted genes are expressed preferentially from either the paternally or maternally inherited allele. Long non-coding RNAs play essential roles in regulating this allele-specific expression. In several well-studied imprinting clusters, long non-coding RNAs have been found to be essential in regulating temporal- and spatial-specific establishment and maintenance of imprinting patterns. Furthermore, recent insights into the epigenetic pathological mechanisms underlying human genomic imprinting disorders suggest that allele-specific expressed imprinted long non-coding RNAs serve as an upstream regulator of the expression of other protein-coding or non-coding imprinted genes in the same cluster. Aberrantly expressed long non-coding RNAs result in bi-allelic expression or silencing of neighboring imprinted genes. Here, we review the emerging roles of long non-coding RNAs in regulating the expression of imprinted genes, especially in human imprinting disorders, and discuss three strategies targeting the central long non-coding RNA UBE3A-ATS for the purpose of developing therapies for the imprinting disorders Prader–Willi syndrome and Angelman syndrome. In summary, a better understanding of long non-coding RNA-related mechanisms is key to the development of potential therapeutic targets for human imprinting disorders.

Large-scale analysis of imprinting in naive human pluripotent stem cells reveals recurrent aberrations and a potential link to FGF signaling.

SummaryGenomic imprinting is a parent-of-origin dependent monoallelic expression of genes. Previous studies showed that conversion of primed human pluripotent stem cells (hPSCs) into naive pluripotency is accompanied by genome-wide loss of methylation that includes imprinted loci. However, the extent of aberrant biallelic expression of imprinted genes is still unknown. Here, we analyze loss of imprinting (LOI) in a large cohort of both bulk and single-cell RNA sequencing samples of naive and primed hPSCs. We show that naive hPSCs exhibit high levels of non-random LOI, with bias toward paternally methylated imprinting control regions. Importantly, we show that different protocols used for the primed to naive conversion led to different extents of LOI, tightly correlated to FGF signaling. This analysis sheds light on the process of LOI occurring during the conversion to naive pluripotency and highlights the importance of these events when modeling disease and development or when utilizing the cells for therapy.

Somatotroph Tumors and the Epigenetic Status of the GNAS Locus.

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. 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. 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. 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.

C-Terminal BRE Overexpression in 11q23-Rearranged and t(8;16) Acute Myeloid Leukemia Is Caused By Intragenic Transcription Initiation

Abstract TextOverexpression of the BRE (Brain and Reproductive organ-Expressed ) gene defines a distinct pediatric and adult acute myeloid leukemia (AML) subgroup (Balgobind et al, Leukemia 2010, and Noordermeer et al, Blood 2011 and 2012). BRE overexpression specifically associates with 11q23/MLL/KMT2A-rearranged AML, occurring in 70% and 50% of 11q23 childhood and adult AML cases, respectively. In adult 11q23-rearranged AML, BRE overexpression significantly associates with younger age. BRE functions in BRCA1-A (Breast Cancer 1) complex mediated DNA damage repair, BRISC (BRCC3 isopeptidase complex) complex mediated mitotic spindle assembly and in protection from apoptosis and senescence. Another constituent of the BRCA1-A/BRISC complexes is BRCC3 which is recurrently mutated in AML, especially in cases with a TET2 mutation (Huang et al., Haematologica, 2015). BRE is relatively highly expressed in blood cells in general but its exact function in hematopoiesis is unknown.By performing genome-wide ChIP-sequencing analyses on primary KMT2A-MLLT3 patient material (n=9) we identified a promoter enriched for active chromatin marks (H3K4me3/H3K27Ac) in BRE intron 4, exclusively in AML cases with BRE overexpression. Both 5' RACE end RNA-sequencing identified strong biallelic expression of a previously unknown C-terminal BRE transcript. This transcript starts near the intragenic promoter, with BRE intron 4 sequences spliced to exon 5 and downstream sequences. RNA-seq and qRT-PCR showed that the downstream transcript is expressed 5-8-fold higher compared to the full-length transcript, explaining BRE overexpression. Of note, two start codons in exon 5 of the downstream BRE transcript may cause the formation of N-terminally truncated BRE isoforms.By performing a qRT-PCR that specifically recognizes the new BRE transcript on samples from a large cohort of pediatric and adult AML (n=195) we observed that it was highly expressed in 60% of 11q23/ KMT2A -rearranged (n=58) and 70% of t(8;16)/ KAT6A-CREBBP cases (n=20), while it was virtually absent from other AML subsets (n=101) and normal hematopoietic tissues (n=14). In gene reporter assays, the AML-specific fusion protein KMT2A-MLLT3 transactivated the intragenic BRE promoter. Further epigenome analyses revealed 97 additional intragenic promoter marks frequently bound by KMT2A in AML with C-terminal BRE overexpression. The bound genes may be part of a context-dependent KMT2A-MLLT3-driven oncogenic program, because they were higher expressed in this AML subtype compared to other cytogenetically-defined AML groups (p<0.01). C-terminal BRE might be a contributor to this oncogenic program because in a case with relapsed AML, we observed an ins(11;2) fusing CHORD1 in frame to BRE exon 5. The CHORDC1 fusion to BRE exon 5 is remarkable because the alternate BRE transcript starts with exon 1b also fused to exon 5. This means that if the CHORDC1-BRE and alternate BRE transcripts would be translated, they would contain a similar C-terminal part of the BRE protein. The CHORDC1 disruption may also contribute to AML pathogenesis because it was recently shown that heterozygous Chord1 (a.k.a. Morgana) inactivation in mice results in the spontaneous development of atypical chronic myeloid leukemia (Di Savino, Blood, 2015). We conclude that abnormal expression of C-terminal alternate BRE sequences specific for AML is caused by distinct, non-random events; by a chromosomal abnormality and by intragenic transcription initiation in 11q23/ KMT2A -rearranged and t(8;16)/ KAT6A-CREBBP AML. These findings warrant further studies to determine whether leukemia-specific alternate BRE contributes to disease pathogenesis and how abnormal intragenic transcription activation in AML is brought about. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.