Biology subjects

DeMayo, F. J.

Publications and source records attributed to DeMayo, F. J..

18 recordsLinked to original sources

bioRxiv2026

Increased chromatin accessibility following 1α,25-dihydroxyvitamin D3 treatment in human endometrial stromal cells

Vitamin D signaling has recognized roles in female reproductive physiology, but its effects at the chromatin level in endometrial stromal cells are still unclear. Here, we investigated how the active form of vitamin D, 1,25-dihydroxyvitamin D3, or calcitriol, influences the accessible chromatin landscape of human endometrial stromal cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed on T-HESCs treated with either a vehicle or 1,25(OH)2D3. Ligand treatment increased overall chromatin accessibility, shown by higher ATAC-seq signal intensity, while causing only minor changes in the total number of called peaks. Peak annotation revealed that accessible regions were spread across both promoter-proximal and distal genomic areas. Integrating this data with CUT&RUN and RNA sequencing showed that most vitamin D-responsive cistromic modifications and transcripts were linked to nearby open chromatin, though fewer were associated with regions that were significantly differentially accessible. These results suggest that 1,25(OH)2D3-dependent transcription mainly occurs within a permissive, pre-accessible chromatin environment. This study offers new evidence that active vitamin D influences the epigenomic landscape of human endometrial stromal cells, establishing the chromatin-based molecular response to a chemically-defined VDR ligand, 1,25(OH)2D3, relevant to stromal differentiation and preparation for decidualization. HighlightsO_LIFirst evidence suggesting the direct impact of active vitamin D, 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, enhanced the signal intensity of chromatin accessibility in human endometrial stromal cells C_LIO_LIMost accessible chromatin regions were shared between vehicle and ligand-treated human endometrial stromal cells C_LIO_LI1,25(OH)2D3-responsive transcription occurs largely within pre-accessible chromatin in human endometrial stromal cells C_LIO_LIAssay for transposase-accessible chromatin sequencing (ATAC-seq) defines a chromatin-level pharmacologic response to a chemically defined VDR ligand in human endometrial stromal cells C_LI

molecular biology↗

bioRxiv2025

Impact of vitamin D deficiency on defective endometrial decidualization and the repressive role of vitamin D receptor (VDR) in the epigenomic network

Identifying the factors that regulate female reproduction is crucial to understanding how the environment affects female reproductive health. The vitamin D receptor (VDR) and its ligands (primarily 1,25(OH)2D3) have a recognized role in calcium homeostasis; however, their broader impact on female reproduction remains underexplored. We demonstrate that the VDR and its ligands are involved in the hormonal induction of uterine decidualization. Mice fed a vitamin D-deficient diet displayed an impaired hormonally induced decidual response. In a human telomerase reverse transcriptase-immortalized human endometrial stromal cell line (T-HESC), VDR decreased during in vitro decidualization. Small interfering RNA (siRNA) knockdown of VDR in T-HESC enhanced in vitro decidualization, while overexpression of VDR inhibited it. Chromatin accessibility and histone modification analyses revealed that VDR functions as a chromatin regulator, restricting accessibility and repressing transcription in specific genomic regions. Transcriptomic analyses confirmed that VDR broadly modulates gene expression, with most ligand-mediated effects occurring through the VDR. These findings identify VDR as a key regulator of transcriptional and chromatin landscapes in human endometrial stromal cells, offering novel insights into vitamin D signaling in reproduction. This study highlights the potential of targeting vitamin D pathways to treat uterine disorders associated with impaired decidualization and reduced fertility. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/688535v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@6b048org.highwire.dtl.DTLVardef@11f7b4corg.highwire.dtl.DTLVardef@1d7dbfaorg.highwire.dtl.DTLVardef@ab3894_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Deciphering ESR1-driven transcription in human endometrial stromal cells via transcriptome, cistrome, and integration with chromatin landscape

ObjectiveTo investigate ESR1 and estrogen-driven transcription in human endometrial stromal cells. DesignTelomerase-immortalized human endometrial stromal cells were engineered to activate ESR1 using the CRISPR activation system and treated with vehicle or E2. Primary endometrial stromal cells were treated with vehicle or decidualization cocktail. SubjectsBiopsies from two healthy, reproductive-aged volunteers with regular menstrual cycles and no history of gynecological malignancies. Main Outcome MeasuresBulk RNA-sequencing in ESR1-activated and E2-treated cells was compared to identify ligand-independent and -dependent ESR1 actions. Cut&Run was performed in ESR1-activated cells treated with E2 or vehicle to assess the ESR1 cistrome. H3K27ac HiChIP was conducted in primary endometrial stromal cells treated with vehicle or decidualization cocktail to identify hormonally induced chromatin interaction changes. ResultsAmong six tested guide RNAs, the ESR1-3 gRNA induced robust ESR1 activation, which restored E2 responsiveness in THESC. Bulk RNA-seq revealed ESR1-mediated E2-dependent and E2-independent transcriptional programs, regulating pathways involved in inflammation, proliferation, extracellular matrix organization, and cancer. Notably, 72% of differentially expressed genes (DEGs) overlapped with genes active in human endometrial tissue during the proliferative E2 dominant phase, supporting their physiological relevance. Cut&Run-seq identified genome-wide ESR1 binding sites, with most located at distal regulatory elements. To associate distal ESR1 binding sites with genes, we integrated H3K27ac HiChIP chromatin loops in hESC to identify distal ESR1 binding sites that loop to gene promoters. We identified genes regulated by ESR1/E2 through long-range chromatin looping that are involved in stromal cell decidualization, including FOXO1 and IL6R. Additionally, we identified genes implicated in endometrial cancer, including ERRFI1, NRIP1, and EPAS1, suggesting a role for stromal ESR1 driven endometrial pathologies. Functional assays confirmed that ESR1 promotes cell viability and, in the presence of E2, enhances migration. ConclusionsESR1 activation through CRISPR restores E2 responsiveness in endometrial stromal cells. Changes to chromatin architecture support gene expression changes that drive decidualization. Integration of ESR1/E2 transcriptome and cistrome with HiChIP data identifies its role in regulating inflammation, proliferation, and decidualization, as well as its implications in endometrial cancer. This model serves as a powerful tool to study estrogen signaling in endometrial stromal biology and related pathologies.

molecular biology↗

bioRxiv2022

The estrogen receptor α cistrome in human endometrium and epithelial organoids

ContextEndometrial health is impacted by molecular processes that underlie estrogen responses. ObjectiveTo define estrogen regulation of endometrial function by integrating the estrogen receptor alpha (ESR1) cistrome and transcriptome of endometrial biopsies taken from the proliferative and midsecretory phases of the menstrual cycle and hormonally stimulated endometrial epithelial organoids. DesignESR1 ChIPseq and RNAseq were performed on proliferative or mid-secretory endometrial biopsies and on hormone treated organoid cultures. SettingEndometrial samples were obtained from volunteers at outpatient research clinics for ChIPseq and for organoid culture. Patients or Other ParticipantsParticipants were fertile, reproductive aged women with normal cycle length, and without any history of infertility or irregular cycles. In total, 5 new endometrial biopsies obtained from 5 women were used in this study and were analyzed together with previously published cycle stage endometrial RNAseq data. Intervention(s)There were no interventions in this study. Main Outcome Measure(s)The cycle stage specific ESR1 binding sites and gene expression identification of human endometrium and organoid cultures were integrated with changes in gene expression. ResultsGenes with ESR1 binding in whole endometrium were enriched for chromatin modification and regulation of cell proliferation. The distribution of ESR1 binding sites in organoids was more distal to the gene promoter when compared to primary endometrium. Organoid estrogen/ESR1 candidate target genes impacted formation of cellular protrusions, and chromatin modification, ConclusionsAnalysis of the ESR1 cistromes and transcriptomes from endometrium and organoids provides important resources for understanding how estrogen impacts endometrial health and function.

genomics↗

bioRxiv2022

Inserting Cre Recombinase into the Prolactin 8a2 gene for Decidua-Specific recombination in Mice

An estimated 75% of unsuccessful pregnancies are due to implantation failure. Investigating the causes of implantation failure is difficult as decidualization and embryo implantation is a dynamic process. Here we describe a new decidua specific iCre recombinase mouse strain. Utilizing CRISPR/Cas9-based genome editing, a mouse strain was developed that expresses iCre recombinase under the control of the endogenous prolactin family 8, subfamily a, member 2 (Prl8a2) promoter. iCre recombinase activity was examined by crossing with mTmG or Sun1-GFP reporter alleles. iCre activity initiated reporter expression at gestational day 5.5 in the primary decidual zone and continued into mid-gestation (gestational day 9.5), with expression highly concentrated in the anti-mesometrial region. No reporter expression was observed in the ovary, oviduct, pituitary, or skeletal muscle, supporting the tissue specificity of the Prl8a2iCre in the primary decidual zone. This novel iCre line will be a valuable tool for in vivo genetic manipulation and lineage tracing to investigate functions of genetic networks and cellular dynamics associated with decidualization and infertility.

genetics↗

bioRxiv2021

The production of 4,182 mouse lines identifies experimental and biological variables impacting Cas9-mediated mutant mouse line production

The International Mouse Phenotyping Consortium (IMPC) systematically produces and phenotypes mouse lines with presumptive null mutations to provide insight into gene function. The IMPC now uses the programmable RNA-guided nuclease Cas9 for its increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of 3,313 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo gene engineering with Cas9. Mouse line production has two critical steps - generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. A systematic evaluation of the variables impacting success rates identified gene essentiality as the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for using Cas9 to generate alleles in mouse essential genes, many of which are orthologs of genes linked to human disease.

genetics↗

bioRxiv2021

Dependency of LKB1-inactivated lung cancer on aberrant CRTC-CREB activation

Lung cancer with loss-of-function of the LKB1 tumor suppressor is a common aggressive subgroup with no effective therapies. LKB1-deficiency induces constitutive activation of cAMP/CREB-mediated transcription by a family of three CREB-regulated transcription coactivators (CRTC1-3). However, the significance and mechanism of CRTC activation in promoting the aggressive phenotype of LKB1-null cancer remain poorly characterized. Here we observed overlapping CRTC expression patterns and mild growth phenotypes of individual CRTC-knockouts in lung cancer, suggesting functional redundancy of CRTC1-3. We consequently designed a dominant-negative mutant (dnCRTC) to block all three CRTCs to bind and co-activate CREB. Expression of dnCRTC efficiently inhibited the aberrantly activated cAMP/CREB-mediated oncogenic transcriptional program induced by LKB1-deficiency, and specifically blocked the growth of LKB1-inactivated lung cancer. Collectively, this study provides direct proof for an essential role of the CRTC-CREB activation in promoting the malignant phenotypes of LKB1-null lung cancer and proposes the CRTC-CREB interaction interface as a novel therapeutic target.

cancer biology↗

bioRxiv2020

Cell-type specific analysis of physiological action of estrogen in mouse oviducts

One of the endogenous estrogens, 17{beta}-estradiol (E2) is a female steroid hormone secreted from the ovary. It is well established that E2 causes biochemical and histological changes in the uterus. The oviduct response to E2 is virtually unknown in an in vivo environment. In this study, we assessed the effect of E2 on each oviductal cell type, using an ovariectomized-hormone-replacement mouse model, single cell RNA-sequencing (scRNA-seq), in situ hybridization, and cell-type-specific deletion in mice. We found that each cell type in the oviduct responded to E2 distinctively, especially ciliated and secretory epithelial cells. The treatment of exogenous E2 did not drastically alter the transcriptomic profile from that of endogenous E2 produced during estrus. Moreover, we have identified and validated genes of interest in our datasets that may be used as cell- and region-specific markers in the oviduct. Insulin-like growth factor 1 (Igf1) was characterized as an E2-target gene in the mouse oviduct and was also expressed in human Fallopian tubes. Deletion of Igf1 in progesterone receptor (Pgr)-expressing cells resulted in female subfertility, partially due to an embryo developmental defect and embryo retention within the oviduct. In summary, we have shown that oviductal cell types are differentially regulated by E2 and support gene expression changes that are required for normal embryo development and transport in mouse models.

cell biology↗

bioRxiv2020

Identification of a Novel PP2A Regulator, WNK1, as Critical For Uterine Function

WNK1 is an atypical kinase protein ubiquitously expressed in humans and mice. A mutation in its encoding gene causes hypertension in humans which is associated with abnormal ion homeostasis. Our earlier findings demonstrated that WNK1 is critical for in vitro decidualization in human endometrial stromal cells – pointing towards an unrecognized role of WNK1 in female reproduction. Here, we employed a mouse model with conditional WNK1 ablation from the female reproductive tract to define its in vivo role in uterine biology. Loss of WNK1 altered uterine morphology, causing endometrial epithelial hyperplasia, adenomyosis and a delay in embryo implantation, ultimately resulting in compromised fertility. Combining transcriptomic, proteomic and interactomic analyses revealed a novel regulatory pathway whereby WNK1 represses AKT phosphorylation through the phosphatase PP2A in endometrial cells from both humans and mice. We show that WNK1 interacts with PPP2R1A, an isoform of the PP2A scaffold subunit. This interaction stabilizes the PP2A complex, which then dephosphorylates AKT. Therefore, loss of WNK1 reduced PP2A activity, causing AKT hypersignaling. Using FOXO1 as a readout of AKT activity, we demonstrate that there was escalated FOXO1 phosphorylation and nuclear exclusion, leading to a disruption in the regulation of genes that are crucial for embryo implantation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2020

Pten and Dicer1 loss causes poorly-differentiated endometrial adenocarcinoma in mice

Endometrial cancer remains the most common gynecological malignancy in the United States. While the loss of the tumor suppressor, PTEN (phosphatase and tensin homolog), is well studied in endometrial cancer, recent studies suggest that DICER1, the endoribonuclease responsible for miRNA genesis, also plays a significant role in endometrial adenocarcinoma. In an endometrial adenocarcinoma mouse model, which has a conditional uterine deletion of Pten, Dicer1 was also conditionally deleted. Conditional uterine deletion of Dicer1 and Pten resulted in high-penetrance, poorly-differentiated endometrial adenocarcinomas. Poorly-differentiated endometrial adenocarcinomas expressed known markers of clear-cell adenocarcinoma, including Napsin A and HNF1B (hepatocyte nuclear factor 1 homeobox B). Adenocarcinomas were hormone-independent, and treatment with long-term progesterone did not mitigate poorly-differentiated adenocarcinoma, nor did it affect adnexal metastasis. Transcriptomic analyses of uteri or Ishikawa cells with deletion of DICER1 revealed unique transcriptomic profiles and global downregulation of miRNAs. Integration of downregulated miRNAs with upregulated mRNA targets revealed deregulated let-7 and miR-16 target genes, similar to published human DICER1-mutant endometrial cancers from TCGA (The Cancer Genome Atlas). Importantly, these miRNA-target genes, involved in ephrin-receptor and transforming growth factor-beta signaling, represent potential clinical targets for rare, yet deadly, poorly-differentiated endometrial adenocarcinomas in women. This mouse model represents poorly-differentiated endometrial adenocarcinoma and will allow for the discovery of novel mechanisms of hormone-independent endometrial adenocarcinoma from atrophic endometrium. Significance StatementEndometrial cancer is one of the few cancers with an increasing death rate in the United States. The most significant risk factor associated with death is high tumor grade, which occurs most frequently in postmenopausal women, where it develops within an atrophic endometrium. Here, we present a mouse model with conditional deletion of Dicer1, a key enzyme in miRNA genesis, and Pten, a tumor suppressor, that develops poorly-differentiated, steroid hormone-independent, endometrial adenocarcinoma with adnexal metastasis. These high-grade adenocarcinomas develop from an atrophic endometrium and share molecular features with DICER1-mutant human endometrial adenocarcinomas. We anticipate that this preclinical model represents a move toward the discovery of novel mechanisms of hormone-independent development of endometrial adenocarcinoma from atrophic endometrium.

cancer biology↗

bioRxiv2020

Increased FOXL2 Expression Alters Uterine Structures and Functions

Transcription factor FOXL2 exhibits an increase in mRNA levels in eutopic endometrial biopsy in endometriosis patients. While FOXL2 is known of regulating sex differentiation and reproductive function, the impact of elevated FOXL2 expression on uterine physiology remains unknown. To answer this question, we generated mice with over expression of FOXL2 (FOXL2OE) in the female reproductive tract by crossing Foxl2LsL/+ with the Pgrcre model. FOXL2OE uterus showed severe morphological abnormality including abnormal epithelial stratification, blunted adenogenesis, increased endometrial fibrosis and disrupted myometrial morphology. In contrast, increasing FOXL2 levels specifically in uterine epithelium by crossing the Foxl2LsL/+ with the Ltficre mice resulted in the eFOXL2OE mice with uterine epithelial stratification but without defects in endometrial fibrosis and adenogenesis, demonstrating a role of the endometrial stroma in the uterine abnormalities of the FOXL2OE mice. Transcriptomic analysis of 12 weeks old Pgrcre and FOXL2OE uterus at diestrus stage showed a positive correlation of FOXL2OE uterine transcriptome with human endometrium of endometriosis patients. Furthermore, we found FOXL2OE mice were sterile. The infertility was caused in part by a disruption of the hypophyseal ovarian axis resulting in an anovulatory phenotype. The FOXL2OE mice failed to show decidual responses during artificial decidualization in ovariectomized mice which demonstrates the uterine contribution to the infertility phenotype. These data supported that aberrantly increased FOXL2 expressions in the female reproductive tract can disrupt ovarian and uterine functions, particularly, may be involved in the progressions of endometriosis.

developmental biology↗

bioRxiv2019

A resource of targeted mutant mouse lines for 5,061 genes

The International Mouse Phenotyping Consortium reports the generation of new mouse mutant strains for over 5,000 genes from targeted embryonic stem cells on the C57BL/6N genetic background. This includes 2,850 null alleles for which no equivalent mutant mouse line exists, 2,987 novel conditional-ready alleles, and 4,433 novel reporter alleles. This nearly triples the number of genes with reporter alleles and almost doubles the number of conditional alleles available to the scientific community. When combined with more than 30 years of community effort, the total mutant allele mouse resource covers more than half of the genome. The extensively validated collection is archived and distributed through public repositories, facilitating availability to the worldwide biomedical research community, and expanding our understanding of gene function and human disease.

genetics↗

bioRxiv2019

Constitutive Expression of the Progesterone Receptor Isoforms Promotes Hormone-Dependent Development of Ovarian Neoplasms

Abnormal expression of the progesterone receptor (PGR) isoforms, PGRA and PGRB, is often observed in women with reproductive tract cancer. To assess the importance of the PGR isoform ratio in the maintenance of the healthy reproductive tract, mice with Cre recombinase- activated PGRA and PGRB transgenes were bred with the PGRCre mouse model to generate strains expressing either PGRA or PGRB in PGR positive tissues. The PGRB mice developed ovarian neoplasms at 23 weeks of age derived from ovarian luteal cells, while the PGRA expressing mice displayed a reduced frequency of tumor development. Transcriptomic analyses of the ovarian tumors revealed an enhanced AKT pathway signature, which is in agreement with expression changes found in human ovarian adenocarcinoma. Effective treatment with the PGR antagonist RU486 reduced tumor growth and the expression of cell cycle genes. We concluded that tumor growth and proliferation is hormone and PGR isoform dependent. Further analysis of the PGRB cistrome identified binding events of critical mitotic phase entry genes. This work suggests an intriguing mechanism whereby the expression of the PGR isoforms determines in vivo neoplasia through high-jacking of the cell cycle pathway.

cancer biology↗

bioRxiv2019

Dynamic Transcriptome, Accessible Genome and PGR Cistrome Profiles in the Human Myometrium

The myometrium undergoes structural and functional remodeling during pregnancy. We hypothesize that myometrial genomic elements alter correspondingly in preparation for parturition. Human myometrial tissues from nonpregnant (NP) and term pregnant (TP) human subjects were examined by RNAseq, ATACseq and PGR ChIPseq assays to profile transcriptome, assessible genome and PGR occupancy. NP and TP specimens exhibit 2890 differentially expressed genes, reflecting an increase of metabolic, inflammatory and PDGF signaling, among others, in adaptation to pregnancy. At the epigenome level, patterns of accessible genome change between NP and TP myometrium, leading to altered enrichment of binding motifs for hormone and muscle regulators such as the progesterone receptor (PGR), Kruppel-like factors and MEF2A transcription factors. PGR genome occupancy exhibits a significant difference between the two stages of the myometrium, concomitant with distinct transcriptomic profiles including genes such as ENO1, LHDA, and PLCL1 in the glycolytic and calcium signaling pathways. Over-representation of SRF, MYOD and STAT binding motifs in PGR occupying sites further suggests interactions between PGR and major muscle regulators for myometrial gene expression. In conclusion, changes in accessible genome and PGR occupancy are part of the myometrial remodeling process and may serve as mechanisms to formulate the state-specific transcriptome profiles.

genomics↗

bioRxiv2019

Transcriptional and Progesterone Receptor Binding Profiles of the Human Endometrium Reveal Important Pathways and Regulators in the Epithelium During the Window of Implantation

ContextPoor uterine receptivity is one major factor leading to pregnancy loss and infertility. Understanding the molecular events governing successful implantation is hence critical in combating infertility.\n\nObjectiveTo define PGR-regulated molecular mechanisms and epithelial roles in receptivity.\n\nDesignRNA-seq and PGR-ChIP-seq were conducted in parallel to identify PGR-regulated pathways during the WOI in endometrium of fertile women.\n\nSettingEndometrial biopsies from the proliferative and mid-secretory phases were analyzed.\n\nPatients or Other ParticipantsParticipants were fertile, reproductive aged (18-37) women with normal cycle length; and without any history of dysmenorrhea, infertility, or irregular cycles. In total, 42 endometrial biopsies obtained from 42 women were analyzed in this study.\n\nInterventionsThere were no interventions during this study.\n\nMain Outcome MeasuresHere we measured the alterations in gene expression and PGR occupancy in the genome during the WOI, based on the hypothesis that PGR binds uterine chromatin cycle-dependently to regulate genes involved in uterine cell differentiation and function.\n\nResults653 genes were identified with regulated PGR binding and differential expression during the WOI. These were involved in regulating inflammatory response, xenobiotic metabolism, EMT, cell death, interleukin/STAT signaling, estrogen response, and MTORC1 response. Transcriptome of the epithelium identified 3,052 DEGs, of which 658 were uniquely regulated. Transcription factors IRF8 and MEF2C were found to be regulated in the epithelium during the WOI at the protein level, suggesting potentially important functions that are previously unrecognized.\n\nConclusionPGR binds the genomic regions of genes regulating critical processes in uterine receptivity and function.\n\nPrecisUsing a combination of RNA-seq and PGR ChIP-seq, novel signaling pathways and epithelial regulators were identified in the endometrium of fertile women during the window of implantation.

developmental biology↗

bioRxiv2018

Super-enhancer impairment is a link between MLL4 inactivated lung tumors and their vulnerability to glycolysis pathway inhibition

Epigenetic modifiers often harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Here we show that lung-specific loss of the gene encoding the histone methyltransferase MLL4 (alias KMT2D; a COMPASS-like enzyme), which is ranked the most highly inactivated epigenetic modifier in lung cancer, strongly promotes lung adenocarcinoma in mice. Mll4 loss upregulated tumor-promoting programs, including glycolysis. The pharmacological inhibition of glycolysis preferentially impeded tumorigenic growth of human lung cancer cells bearing MLL4-inactivating mutations. Mll4 loss widely impaired epigenomic signals for super-enhancers and enhancers, including the super-enhancer for the circadian rhythm repressor gene Per2, and decreased Per2 expression. Per2 downregulated several glycolytic pathway genes. These findings uncover a distinct tumor-suppressive epigenetic mechanism in which MLL4 enhances Per2-mediated repression of pro-tumorigenic glycolytic genes via super-enhancer activation to suppress lung adenocarcinoma tumorigenesis and also implicate a glycolysis-targeting strategy as a therapeutic intervention for the treatment of MLL4- mutant lung cancer.

cancer biology↗

bioRxiv2018

An ancient fecundability-associated polymorphism creates a new GATA2 binding site in a distal enhancer of HLA-F

Variation in female reproductive traits such as fertility, fecundity, and fecundability are heritable in humans, but identifying and functionally characterizing genetic variants associated with these traits has been challenging. Here we explore the functional significance and evolutionary history of a G/A polymorphism of SNP rs2523393, which we have previously shown is an eQTL for the HLA-F gene and significantly associated with fecundability (time to pregnancy). We replicated the association between rs2523393 genotype and HLA-F expression using GTEx data and demonstrate that HLA-F is up-regulated in the endometrium during the window of implantation and by progesterone in decidual stromal cells. Next, we show that the rs2523393 A allele creates a new GATA2 binding site in a progesterone responsive distal enhancer that loops to the HLA-F promoter. Remarkably, we found that the A allele is derived in the human lineage, that G/A polymorphism arose before the divergence of modern and archaic humans, and is segregating at intermediate to high frequencies across human populations. Remarkably, the derived A is also has been identified in a GWAS as a risk allele for multiple sclerosis. These data suggests that the polymorphism is maintained by antagonistic pleiotropy and a reproduction-health tradeoff in human evolution.

genetics↗

bioRxiv2017

Employing single-stranded DNA donors for the high-throughput production of conditional knockout alleles in mice

The International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene-phenotype associations. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-mediated homology driven repair (HDR) with short and long single-stranded oligodeoxynucleotides (ssODNs and lssODNs). Using pairs of guides and ssODNs donating loxP sites, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 of 30 genes targeted. LoxP sites integrated in cis in at least one F0 for 18 of 23 targeted genes. However, loxP sites were mutagenized in 4 of 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was not influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and a single lssODN to introduce a loxP-flanked exon, conditional allele founders were generated for all 4 genes targeted. Our studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssODNs are amenable to high-throughput production of conditional alleles when they can be employed.

genetics↗