Project description:Epithelial cells were isolated by FACS from the mammary glands of pubescent (5 week old), estrus adult (10 week old) and diestrus adult (10 week old) female mice. Freshly sorted cells were submitted to a 10X Genomics Chromium System for single cell capture. cDNA synthesis and library preparation was done according to the protocol supplied by the manufacturer. Sequencing was carried out on an Illumina NextSeq500 sequencer using parameters recommended by 10X Genomics.
Project description:RNA Seq data showed 49 differentially altered mRNAs in estrus and diestrus stages of buffalo cell free saliva, that includes 10 mRNAs commonly present in both estrus and diestrus stages, whereas 12 and 27 mRNAs were found to be exclusively present either at the estrus or the diestrus stage respectively. In addition, four mature miRNAs were predicted to be differentially altered in the estrus as compared to the diestrus phase of buffaloes.
Project description:Macrophages have important roles in mammary gland development and tissue homeostasis, but the specific mechanisms that regulate macrophage function need further elucidation. We have identified C/EBPβ as an important macrophage factor that is present in multiple populations of the normal mammary gland. Mammary glands from mice with C/EBPβ-deficient macrophages showed a significant decrease in alveolar budding during the estrus cycle. Deletion of C/EBPβ from macrophages resulted in changes in cytokine production, and an inability of both epithelial cells and macrophages to respond progesterone. RNA sequencing showed significant changes in PR-responsive genes and alterations in the Wnt landscape of epithelial cells in mammary glands that lacked C/EBPbeta in macrophages, which have been shown to mediate stem cell expansion during diestrus. These studies suggest that C/EBPbeta is a critical macrophage factor that facilitates macrophage-epithelial crosstalk during a key stage of mammary gland tissue homeostasis, and have critical implications for how disruption of tissue homeostasis can lead to tumorigenesis.
Project description:Mammary gland development and luminal differentiation occur largely postnatally during puberty and pregnancy. We found that pregnancy had the most significant effects on stem cells, inducing a distinct epigenetic state that remained stable through life. Mammary glands were collected from different reproductive stages to purify different epithelium cell types, which were used for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Hormone dependent uterine gene expression changes that occur during the estrous cycle suggest hormone receptor binding to chromatin may also be dynamic. Therefore, we employed a multi-faceted approach to examine in vivo dynamics of hormone receptor occupancy, chromatin accessibility and chromatin structure by combining RNA-seq, ATAC-seq, HiC-seq and ChIP-seq for estrogen receptor alpha (ERα) and progesterone receptor (PGR). Genome wide, there were extensive estrous cycle dependent changes in ERα and PGR binding as well as chromatin accessibility. There were 4,159 differentially expressed genes between estrus and diestrus. At transcription start sites, accessibility generally correlated with the directionality of gene expression and there was reduced PGR in estrus compared to diestrus but little change in ERα. There were 2,727 enhancers with dynamic accessibility near these genes and 77% of those correlated with directionality of gene expression changes. However, most enhancers were constitutively open (8,694). In both dynamic and constitutively open enhancers, ERα and PgR binding was coordinately lost from diestrus to estrus. Diestrus specific ERα binding and accessible regions were enriched for PGR, FOX, GATA and SOX binding motifs. In contrast, estrus specific ERα binding occurred at transcription factor deserts in relatively closed chromatin while estrus specific accessible regions were highly enriched for ATF, ELF and ELK motifs. Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) revealed many estrous cycle dependent partners of ERα (diestrus, 60; estrus, 24). PGR was found in complex with ERα during diestrus but not estrus supporting coordinated binding of both receptors during diestrus. Two of the cohesin complex proteins, SMC1A and SMC3, were found in complex with ERα during diestrus but not estrus; overlap of SMC1A with ERα confirmed this preferential interaction during diestrus. Additionally, HiC analysis showed more diestrus specific interactions than estrus (476 versus 263) suggesting the SMC1A/ ERα interactions have functional consequences on chromatin structure. Taken together, a complex series of interactions between hormone receptors, chromatin structure and accessibility orchestrate estrous cycle dependent changes in gene expression.
Project description:Hormone dependent uterine gene expression changes that occur during the estrous cycle suggest hormone receptor binding to chromatin may also be dynamic. Therefore, we employed a multi-faceted approach to examine in vivo dynamics of hormone receptor occupancy, chromatin accessibility and chromatin structure by combining RNA-seq, ATAC-seq, HiC-seq and ChIP-seq for estrogen receptor alpha (ERα) and progesterone receptor (PGR). Genome wide, there were extensive estrous cycle dependent changes in ERα and PGR binding as well as chromatin accessibility. There were 4,159 differentially expressed genes between estrus and diestrus. At transcription start sites, accessibility generally correlated with the directionality of gene expression and there was reduced PGR in estrus compared to diestrus but little change in ERα. There were 2,727 enhancers with dynamic accessibility near these genes and 77% of those correlated with directionality of gene expression changes. However, most enhancers were constitutively open (8,694). In both dynamic and constitutively open enhancers, ERα and PgR binding was coordinately lost from diestrus to estrus. Diestrus specific ERα binding and accessible regions were enriched for PGR, FOX, GATA and SOX binding motifs. In contrast, estrus specific ERα binding occurred at transcription factor deserts in relatively closed chromatin while estrus specific accessible regions were highly enriched for ATF, ELF and ELK motifs. Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) revealed many estrous cycle dependent partners of ERα (diestrus, 60; estrus, 24). PGR was found in complex with ERα during diestrus but not estrus supporting coordinated binding of both receptors during diestrus. Two of the cohesin complex proteins, SMC1A and SMC3, were found in complex with ERα during diestrus but not estrus; overlap of SMC1A with ERα confirmed this preferential interaction during diestrus. Additionally, HiC analysis showed more diestrus specific interactions than estrus (476 versus 263) suggesting the SMC1A/ ERα interactions have functional consequences on chromatin structure. Taken together, a complex series of interactions between hormone receptors, chromatin structure and accessibility orchestrate estrous cycle dependent changes in gene expression.
Project description:Hormone dependent uterine gene expression changes that occur during the estrous cycle suggest hormone receptor binding to chromatin may also be dynamic. Therefore, we employed a multi-faceted approach to examine in vivo dynamics of hormone receptor occupancy, chromatin accessibility and chromatin structure by combining RNA-seq, ATAC-seq, HiC-seq and ChIP-seq for estrogen receptor alpha (ERα) and progesterone receptor (PGR). Genome wide, there were extensive estrous cycle dependent changes in ERα and PGR binding as well as chromatin accessibility. There were 4,159 differentially expressed genes between estrus and diestrus. At transcription start sites, accessibility generally correlated with the directionality of gene expression and there was reduced PGR in estrus compared to diestrus but little change in ERα. There were 2,727 enhancers with dynamic accessibility near these genes and 77% of those correlated with directionality of gene expression changes. However, most enhancers were constitutively open (8,694). In both dynamic and constitutively open enhancers, ERα and PgR binding was coordinately lost from diestrus to estrus. Diestrus specific ERα binding and accessible regions were enriched for PGR, FOX, GATA and SOX binding motifs. In contrast, estrus specific ERα binding occurred at transcription factor deserts in relatively closed chromatin while estrus specific accessible regions were highly enriched for ATF, ELF and ELK motifs. Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) revealed many estrous cycle dependent partners of ERα (diestrus, 60; estrus, 24). PGR was found in complex with ERα during diestrus but not estrus supporting coordinated binding of both receptors during diestrus. Two of the cohesin complex proteins, SMC1A and SMC3, were found in complex with ERα during diestrus but not estrus; overlap of SMC1A with ERα confirmed this preferential interaction during diestrus. Additionally, HiC analysis showed more diestrus specific interactions than estrus (476 versus 263) suggesting the SMC1A/ ERα interactions have functional consequences on chromatin structure. Taken together, a complex series of interactions between hormone receptors, chromatin structure and accessibility orchestrate estrous cycle dependent changes in gene expression.
Project description:Hormone dependent uterine gene expression changes that occur during the estrous cycle suggest hormone receptor binding to chromatin may also be dynamic. Therefore, we employed a multi-faceted approach to examine in vivo dynamics of hormone receptor occupancy, chromatin accessibility and chromatin structure by combining RNA-seq, ATAC-seq, HiC-seq and ChIP-seq for estrogen receptor alpha (ERα) and progesterone receptor (PGR). Genome wide, there were extensive estrous cycle dependent changes in ERα and PGR binding as well as chromatin accessibility. There were 4,159 differentially expressed genes between estrus and diestrus. At transcription start sites, accessibility generally correlated with the directionality of gene expression and there was reduced PGR in estrus compared to diestrus but little change in ERα. There were 2,727 enhancers with dynamic accessibility near these genes and 77% of those correlated with directionality of gene expression changes. However, most enhancers were constitutively open (8,694). In both dynamic and constitutively open enhancers, ERα and PgR binding was coordinately lost from diestrus to estrus. Diestrus specific ERα binding and accessible regions were enriched for PGR, FOX, GATA and SOX binding motifs. In contrast, estrus specific ERα binding occurred at transcription factor deserts in relatively closed chromatin while estrus specific accessible regions were highly enriched for ATF, ELF and ELK motifs. Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) revealed many estrous cycle dependent partners of ERα (diestrus, 60; estrus, 24). PGR was found in complex with ERα during diestrus but not estrus supporting coordinated binding of both receptors during diestrus. Two of the cohesin complex proteins, SMC1A and SMC3, were found in complex with ERα during diestrus but not estrus; overlap of SMC1A with ERα confirmed this preferential interaction during diestrus. Additionally, HiC analysis showed more diestrus specific interactions than estrus (476 versus 263) suggesting the SMC1A/ ERα interactions have functional consequences on chromatin structure. Taken together, a complex series of interactions between hormone receptors, chromatin structure and accessibility orchestrate estrous cycle dependent changes in gene expression.
Project description:The emergence of fully antimicrobial resistant Neisseria gonorrhoeae has led global public health agencies to identify a critical need for next generation anti-gonococcal pharmaceuticals. The development and success of these compounds will rely upon valid pre-clinical models of gonorrhoeae infection. We recently developed and reported the first model of upper genital tract gonococcal infection. During initial characterization, we observed significant reproductive cycle-based variation in infection outcome. When uterine infection occurred in the diestrus phase, there was significantly greater pathology than during estrus phase. The aim of this study was to evaluate transcriptional profiles of infected uterine tissue from mice in either estrus or diestrus phase in order to elucidate possible mechanisms for these differences. Genes and biological pathways with phase-independent induction during infection showed a chemokine dominant cytokine response to Neisseria gonorrhoeae. Despite general induction being phase-independent, this common anti-gonococcal response demonstrated greater induction during diestrus phase infection. Greater activity of granulocyte adhesion and diapedesis regulators during diestrus infection, particularly in chemokines and diapedesis regulators, was also shown. In addition to a greater induction of the common anti-gonococcal response, Gene Set Enrichment Analysis (GSEA) identified a diestrus-specific induction of type-1 interferon signaling pathways. This transcriptional analysis of murine uterine gonococcal infection during distinct points in the natural reproductive cycle provided evidence for a common anti-gonococcal response characterized by significant induction of granulocyte chemokine expression and high proinflammatory mediators. The basic biology of this host response to N. gonorrhoeae in estrus and diestrus is similar at the pathway level, but varies drastically in magnitude. Overlaying this, we observed type-1 interferon induction specifically in diestrus infection where greater pathology is observed. This supports recent work suggesting this pathway has a significant, possibly host-detrimental, function in gonococcal infection. Together these findings lay the groundwork for further examination of the role of interferons in gonococcal infection. Additionally, this work enables the implementation of the diestrus uterine infection model using the newly characterized host response as a marker of pathology and its prevention as a correlate of candidate vaccine efficacy and ability to protect against the devastating consequences of N. gonorrhoeae-associated sequelae. Murine microarrays were used to examine transcriptional differences underlying significantly different phenotypes associated with uterine N. gonorrhoeae infection in the estrus versus diestrus phases of the natural reproductive cycle.