Stage-specific Gene Expression Research Articles

Association of microRNAs with Argonaute proteins in the malaria mosquito Anopheles gambiae after blood ingestion

Drastic changes in gene expression occur after adult female mosquitoes take a blood meal and use the nutrients for egg maturation. A growing body of evidence indicates that microRNAs (miRNAs) contribute to this tightly controlled tissue- and stage-specific gene expression. To investigate the role of miRNAs, we monitored miRNA expression in the mosquito Anopheles gambiae during the 72-h period immediately after blood feeding. We also measured the association of miRNAs with Argonaute 1 (Ago1) and Argonaute 2 (Ago2) to assess the functional status of individual miRNA species. Overall, 173 mature miRNAs were precipitated with Ago1 and Ago2, including 12 new miRNAs, the orthologs of which are found thus far only in other Anopheles species. Ago1 is the predominant carrier of miRNAs in Anopheles gambiae. The abundance and Ago loading of most of the mature miRNAs were relatively stable after blood ingestion. However, miRNAs of the miR-309/286/2944 cluster were considerably upregulated after blood feeding. Injection of the specific antagomir for miR-309 resulted in smaller developing oocytes and ultimately fewer eggs. In addition, the Ago association of some miRNAs was not proportional to their cellular abundance, suggesting that integration of miRNAs into the Ago complexes is regulated by additional mechanisms.

Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression.

ABSTRACTAneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania, a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro-cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro, aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness.

Nascent RNA sequencing reveals mechanisms of gene regulation in the human malaria parasite Plasmodium falciparum.

Gene expression in Plasmodium falciparum is tightly regulated to ensure successful propagation of the parasite throughout its complex life cycle. The earliest transcriptomics studies in P. falciparum suggested a cascade of transcriptional activity over the course of the 48-hour intraerythrocytic developmental cycle (IDC); however, the just-in-time transcriptional model has recently been challenged by findings that show the importance of post-transcriptional regulation. To further explore the role of transcriptional regulation, we performed the first genome-wide nascent RNA profiling in P. falciparum. Our findings indicate that the majority of genes are transcribed simultaneously during the trophozoite stage of the IDC and that only a small subset of genes is subject to differential transcriptional timing. RNA polymerase II is engaged with promoter regions prior to this transcriptional burst, suggesting that Pol II pausing plays a dominant role in gene regulation. In addition, we found that the overall transcriptional program during gametocyte differentiation is surprisingly similar to the IDC, with the exception of relatively small subsets of genes. Results from this study suggest that further characterization of the molecular players that regulate stage-specific gene expression and Pol II pausing will contribute to our continuous search for novel antimalarial drug targets.

Global analysis of canola genes targeted by SHORT HYPOCOTYL UNDER BLUE 1 during endosperm and embryo development

Seed development in dicots includes early endosperm proliferation followed by growth of the embryo to replace the endosperm. Endosperm proliferation in dicots not only provides nutrient supplies for subsequent embryo development but also enforces a space limitation, influencing final seed size. Overexpression of Arabidopsis SHORT HYPOCOTYL UNDER BLUE1::uidA (SHB1:uidA) in canola produces large seeds. We performed global analysis of the canola genes that were expressed and influenced by SHB1 during early endosperm proliferation at 8days after pollination (DAP) and late embryo development at 13 DAP. Overexpression of SHB1 altered the expression of 973 genes at 8 DAP and 1035 genes at 13 DAP. We also surveyed the global SHB1 association sites, and merging of these sites with the RNA sequencing data identified a set of canola genes targeted by SHB1. The 8-DAP list includes positive and negative genes that influence endosperm proliferation and are homologous to Arabidopsis MINI3, IKU2, SHB1, AGL62, FIE and AP2. We revealed a major role for SHB1 in canola endosperm development based on the dynamics of SHB1-altered gene expression, the magnitude of SHB1 chromatin immunoprecipitation enrichment and the over-representation of eight regulatory genes for endosperm development. Our studies focus on an important agronomic trait in a major crop for global agriculture. The datasets on stage-specific and SHB1-induced gene expression and genes targeted by SHB1 also provide a useful resource in the field of endosperm development and seed size engineering. Our practices in an allotetraploid species will impact similar studies in other crop species.

Analysis of the whole developmental period reveals stage-specific gene expression profiles of the Indianmeal moth, Plodia interpunctella

The Indianmeal moth, Plodia interpunctella, is a global pest of stored and processed food products. Although its biology and management have been widely studied, there is limited information on the roles of specific genes during development at the molecular level. To get better understanding of physiological roles of each gene underlying in insect development, it is necessary to determine the precise and comprehensive profiles of each gene throughout development. Here, we determined the whole developmental expression patterns of 13 genes related to growth, stress, metabolism, and host defense by using quantitative real-time RT-PCR. Our results showed that the expression profiles of the 13 genes were unique throughout the development of P. interpunctella. Expression levels of stress-associated genes such as heat-shock protein genes (hsps) and an antioxidant gene, thioredoxin peroxidase (Tpx), were highest in both egg and adult stages. Expression levels of both ecdysone receptor (EcR) and ultraspiracle (USP) were differentially the highest in the pupal and wandering phase of the fifth instar larvae, respectively. Expression levels of immune-related genes such as hemolin and β-1,3 glucan recognition protein (βgrp) were the highest during the wandering larval and pupal stages, while those of prophenoloxidase (ProPO), superoxide dismutase (SOD), and two storage protein genes (SP1 and SP2) were the highest during the wandering larval stage. This study provides comprehensive insights for understanding the functional roles of genes especially hsps and Tpx, which are stage-specifically regulated during the development of P. interpunctella.

Nuclear Compartmentalization Contributes to Stage-Specific Gene Expression Control in Trypanosoma cruzi.

In the protozoan parasite Trypanosoma cruzi, as in other trypanosomatids, transcription of protein coding genes occurs in a constitutive fashion, producing large polycistronic transcription units. These units are composed of non-functionally related genes which are pervasively processed to yield each mRNA. Therefore, post-transcriptional processes are crucial to regulate gene expression. Considering that nuclear compartmentalization could contribute to gene expression regulation, we comparatively studied the nuclear, cytoplasmic and whole cell transcriptomes of the non-infective epimastigote stage of T. cruzi, using RNA-Seq. We found that the cytoplasmic transcriptome tightly correlates with the whole cell transcriptome and both equally correlate with the proteome. Nonetheless, 1,200 transcripts showed differential abundance between the nuclear and cytoplasmic fractions. For the genes with transcript content augmented in the nucleus, significant structural and compositional differences were found. The analysis of the reported epimastigote translatome and proteome, revealed scarce ribosome footprints and encoded proteins for them. Ontology analyses unveiled that many of these genes are distinctive of other parasite life-cycle stages. Finally, the relocalization of transcript abundance in the metacyclic trypomastigote infective stage was confirmed for specific genes. While gene expression is strongly dependent on transcript steady-state level, we here highlight the importance of the distribution of transcripts abundance between compartments in T. cruzi. Particularly, we show that nuclear compartmentation is playing an active role in the developmental stage determination preventing off-stage expression.

Genetic analysis of Ikaros target genes and tumor suppressor function in BCR-ABL1+ pre-B ALL.

Inactivation of the tumor suppressor gene encoding the transcriptional regulator Ikaros (IKZF1) is a hallmark of BCR-ABL1+ precursor B cell acute lymphoblastic leukemia (pre-B ALL). However, the mechanisms by which Ikaros functions as a tumor suppressor in pre-B ALL remain poorly understood. Here, we analyzed a mouse model of BCR-ABL1+ pre-B ALL together with a new model of inducible expression of wild-type Ikaros in IKZF1 mutant human BCR-ABL1+ pre-B ALL. We performed integrated genome-wide chromatin and expression analyses and identified Ikaros target genes in mouse and human BCR-ABL1+ pre-B ALL, revealing novel conserved gene pathways associated with Ikaros tumor suppressor function. Notably, genetic depletion of different Ikaros targets, including CTNND1 and the early hematopoietic cell surface marker CD34, resulted in reduced leukemic growth. Our results suggest that Ikaros mediates tumor suppressor function by enforcing proper developmental stage-specific expression of multiple genes through chromatin compaction at its target genes.

The bxdPRE/TRE element terminates passing through transcription.

The regulation of PRE/TRE activity is required for appropriate tissue and stage-specific gene expression. However, the molecular principles of PRE/TRE activity control remain unknown. Here we show that PRE/TRE element from Ubx regulatory region efficiently terminates passing through transcription.

Organ- and Growing Stage-Specific Expression of Solanesol Biosynthesis Genes in Nicotiana tabacum Reveals Their Association with Solanesol Content.

Solanesol is a noncyclic terpene alcohol that is composed of nine isoprene units and mainly accumulates in solanaceous plants, especially tobacco (Nicotiana tabacum L.). In the present study, RNA-seq analyses of tobacco leaves, stems, and roots were used to identify putative solanesol biosynthesis genes. Six 1-deoxy-d-xylulose 5-phosphate synthase (DXS), two 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), two 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD), four 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (IspE), two 2-C-methyl-d-erythritol 2,4-cyclo-diphosphate synthase (IspF), four 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), two 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IspH), six isopentenyl diphosphate isomerase (IPI), and two solanesyl diphosphate synthase (SPS) candidate genes were identified in the solanesol biosynthetic pathway. Furthermore, the two N. tabacum SPS proteins (NtSPS1 and NtSPS2), which possessed two conserved aspartate-rich DDxxD domains, were highly homologous with SPS enzymes from other solanaceous plant species. In addition, the solanesol contents of three organs and of leaves from four growing stages of tobacco plants corresponded with the distribution of chlorophyll. Our findings provide a comprehensive evaluation of the correlation between the expression of different biosynthesis genes and the accumulation of solanesol, thus providing valuable insight into the regulation of solanesol biosynthesis in tobacco.

Enhancers and their dynamics during hematopoietic differentiation and emerging strategies for therapeutic action.

Cellular differentiation requires precisely regulated tissue-specific and developmental stage-specific gene expression patterns. Numerous studies have highlighted the predictive power of enhancers on lineage-specific gene expression programs and have started to unravel their mechanisms of action. We review here the dynamics of the enhancer landscape during hematopoietic differentiation and how enhancers function in the context of the 3D organization of the genome. We further discuss the involvement of aberrant enhancer activity in human diseases and emerging strategies aiming at controlling enhancer activity and chromosome topology for therapeutic purposes.

GExplore 1.4: An expanded web interface for queries on Caenorhabditis elegans protein and gene function.

Genetic high-throughput experiments often result in hundreds or thousands of genes satisfying certain experimental conditions. Grouping and prioritizing a large number of genes for further analysis can be a time-consuming challenge. In 2009 we developed a web-based user interface, GExplore, to assist with large-scale data-mining related to gene function in Caenorhabditis elegans. The underlying database contained information about Caenorhabditis elegans genes and proteins including domain organization of the proteins, phenotypic descriptions, expression data and Gene Ontology Consortium annotations. These data enable users to quickly obtain an overview of biological and biochemical functions of a large number of genes at once. Since its inception the underlying database has been updated and expanded significantly. Here we describe the current version of GExplore 1.4, documenting the changes since the original release. GExplore 1.4 now contains information about the domain organization of the proteomes of 9 nematode species, can display the location of Caenorhabditis elegans mutations with respect to the domain organization of the proteins, and includes stage-specific RNAseq gene expression data generated by the modENCODE project. The underlying database has been reorganized to facilitate independent updates of the different parts of the database and to allow the addition of novel data sets in the future. The web interface is available under http://genome.sfu.ca/gexplore.

Epigenetic regulation of ganglioside expression in neural stem cells and neuronal cells.

The structural diversity and localization of cell surface glycosphingolipids (GSLs), including gangliosides, in glycolipid-enriched microdomains (GEMs, also known as lipid rafts) render them ideally suited to play important roles in mediating intercellular recognition, interactions, adhesion, receptor function, and signaling. Gangliosides, sialic acid-containing GSLs, are most abundant in the nerve tissues. The quantity and expression pattern of gangliosides in brain change drastically throughout development and these changes are mainly regulated through stage-specific expression of glycosyltransferase genes. We previously demonstrated for the first time that efficient histone acetylation of the glycosyltransferase genes in mouse brain contributes to the developmental alteration of ganglioside expression. We further demonstrated that acetylation of histones H3 and H4 on the N-acetylgalactosaminyltransferase I (GalNAcT, GA2/GM2/GD2/GT2-synthase; B4galnt1) gene promoter resulted in recruitment of trans-activation factors. In addition, we showed that epigenetic activation of the GalNAcT gene was detected and accompanied by an apparent induction of neuronal differentiation of neural stem cells (NSCs) responding to an exogenous supplement of ganglioside GM1. Most recently, we found that nuclear GM1 binds with acetylated histones on the promoters of the GalNAcT as well as on the NeuroD1 genes in differentiated neurons. Here, we will introduce epigenetic regulation of ganglioside synthase genes in neural development and neuronal differentiation of NSCs.

Quantifying promoter activity during the developmental cycle of Chlamydia trachomatis.

Chlamydia trachomatis is an important human pathogen that undergoes a characteristic development cycle correlating with stage-specific gene expression profiles. Taking advantage of recent developments in the genetic transformation in C. trachomatis, we constructed a versatile green fluorescent protein (GFP) reporter system to study the development-dependent function of C. trachomatis promoters in an attempt to elucidate the mechanism that controls C. trachomatis adaptability. We validated the use of the GFP reporter system by visualizing the activity of an early euo gene promoter. Additionally, we uncovered a new ompA promoter, which we named P3, utilizing the GFP reporter system combined with 5′ rapid amplification of cDNA ends (RACE), in vitro transcription assays, real-time quantitative RT-PCR (RT-qPCR), and flow cytometry. Mutagenesis of the P3 region verifies that P3 is a new class of C. trachomatis σ66-dependent promoter, which requires an extended −10 TGn motif for transcription. These results corroborate complex developmentally controlled ompA expression in C. trachomatis. The exploitation of genetically labeled C. trachomatis organisms with P3-driven GFP allows for the observation of changes in ompA expression in response to developmental signals. The results of this study could be used to complement previous findings and to advance understanding of C. trachomatis genetic expression.

Dynamic Competing Histone H4 K5K8 Acetylation and Butyrylation Are Hallmarks of Highly Active Gene Promoters

SummaryRecently discovered histone lysine acylation marks increase the functional diversity of nucleosomes well beyond acetylation. Here, we focus on histone butyrylation in the context of sperm cell differentiation. Specifically, we investigate the butyrylation of histone H4 lysine 5 and 8 at gene promoters where acetylation guides the binding of Brdt, a bromodomain-containing protein, thereby mediating stage-specific gene expression programs and post-meiotic chromatin reorganization. Genome-wide mapping data show that highly active Brdt-bound gene promoters systematically harbor competing histone acetylation and butyrylation marks at H4 K5 and H4 K8. Despite acting as a direct stimulator of transcription, histone butyrylation competes with acetylation, especially at H4 K5, to prevent Brdt binding. Additionally, H4 K5K8 butyrylation also marks retarded histone removal during late spermatogenesis. Hence, alternating H4 acetylation and butyrylation, while sustaining direct gene activation and dynamic bromodomain binding, could impact the final male epigenome features.

Identification of extracellular vesicle-borne periostin as a feature of muscle-invasive bladder cancer.

Muscle-invasive bladder cancer (MIBC) is an aggressive malignancy with high mortality, and heterogeneity in MIBC results in variable clinical outcomes, posing challenges for clinical management. Extracellular vesicles (EVs) derived from MIBC have been shown to promote cancer progression. EVs derived from bladder cell lines were subjected to proteomic analysis, and periostin was chosen for further characterization due to its stage-specific gene expression profile. Knockdown of periostin by RNA interference reduces invasiveness in vitro and produces a rounder morphology. Importantly, treating low grade BC cells with periostin-rich EVs promotes cell aggressiveness and activates ERK oncogenic signals, and periostin suppression reverses these effects. These data suggest that MIBC might transfer periostin in an EV-mediated paracrine manner to promote the disease. To determine the potential of periostin as a bladder cancer indicator, patient urinary EVs were examined and found to have markedly higher levels of periostin than controls. In addition, immunohistochemical staining of a bladder cancer tissue microarray revealed that the presence of periostin in MIBC cells is correlated with worse prognosis. In conclusion, periostin is a component of bladder cancer cells associated with poor clinical outcome, and EVs can transfer oncogenic molecules such as periostin to affect the tumor environment and promote cancer progression.

Genetic Tools to Study T Cell Development.

Genetics tools, and especially the ability to enforce, by transgenesis, or disrupt, by homologous recombination, gene expression in a cell-specific manner, have revolutionized the study of immunology and propelled the laboratory mouse as the main model to study immune responses. Perhaps more than any other aspect of immunology, the study of T cell development has benefited from these technologies. This brief chapter summarizes genetic tools specific to T cell development studies, focusing on mouse strains with lineage- and stage-specific expression of the Cre recombinase, or expressing unique antigen receptor specificities. It ends with a broader discussion of strategies to enforce ectopic lineage and stage-specific gene expression.

Tissue- and stage-specific Wnt target gene expression is controlled subsequent to β-catenin recruitment to cis-regulatory modules.

Key signalling pathways, such as canonical Wnt/β-catenin signalling, operate repeatedly to regulate tissue- and stage-specific transcriptional responses during development. Although recruitment of nuclear β-catenin to target genomic loci serves as the hallmark of canonical Wnt signalling, mechanisms controlling stage- or tissue-specific transcriptional responses remain elusive. Here, a direct comparison of genome-wide occupancy of β-catenin with a stage-matched Wnt-regulated transcriptome reveals that only a subset of β-catenin-bound genomic loci are transcriptionally regulated by Wnt signalling. We demonstrate that Wnt signalling regulates β-catenin binding to Wnt target genes not only when they are transcriptionally regulated, but also in contexts in which their transcription remains unaffected. The transcriptional response to Wnt signalling depends on additional mechanisms, such as BMP or FGF signalling for the particular genes we investigated, which do not influence β-catenin recruitment. Our findings suggest a more general paradigm for Wnt-regulated transcriptional mechanisms, which is relevant for tissue-specific functions of Wnt/β-catenin signalling in embryonic development but also for stem cell-mediated homeostasis and cancer. Chromatin association of β-catenin, even to functional Wnt-response elements, can no longer be considered a proxy for identifying transcriptionally Wnt-regulated genes. Context-dependent mechanisms are crucial for transcriptional activation of Wnt/β-catenin target genes subsequent to β-catenin recruitment. Our conclusions therefore also imply that Wnt-regulated β-catenin binding in one context can mark Wnt-regulated transcriptional target genes for different contexts.

Reproductive/Developmental Abnormalities Induced by Epigenetic Aberrations and Possible Environmental Causes.

Foetal environmental factors, including maternal nutrition, hormonal disturbance, and chemical exposure, affect foetal growth and can cause birth defects. Recent studies have shown the link of poor foetal growth with increased risks of coronary heart disease, type II diabetes, kidney disease, and brain disorders in adulthood. Epigenetic modifications, such as DNA methylation and histone modifications, are involved in tissue- and developmental stage-specific gene expression and silencing, and they can be transmitted stably through mitotic cell division, thereby inducing long-term changes in gene regulation. Developmental programming during the foetal period, therefore, could affect adult health through epigenetic mechanisms. In fact, many studies using animal models have demonstrated that nutrient manipulation during pregnancy induces epigenetic alterations at specific loci or globally in the offspring. In this review, we summarize our findings that elucidate the effects of in utero environments on the human placental epigenome.

A Library of Sickle Cell Anemia Induced Pluripotent Stem Cells of Diverse Haplotypes and Ethnicities

The mutation causing sickle cell anemia (rs334, GAG-GTG, glu6val) had several independent origins in Africa, the Middle East and India and spread throughout parts of the world by wars, slave trading and population migrations. The genetic background upon which the HbS mutation occurred, or the β-globin gene (HBB) haplotype, is associated with differences in the phenotype of this disease and the ability of affected individuals to synthesize fetal hemoglobin (HbF). The main modifier of the disease phenotype is the level of HbF in the blood of affected individuals. HbF inhibits the polymerization of HbS, the proximate cause of disease pathophysiology. As part of the NHLBI NextGen consortium (U01HL107443) we established a library of induced pluripotent stem cells (iPSC) from patients with sickle cell anemia of diverse HBB haplotypes and HbF phenotypes. The purpose of establishing this library was to allow genetic studies of globin gene expression during the erythroid differentiation of iPSC of diverse genotypes. During these studies we have implemented an efficient and highly reproducible platform for the production of large numbers of sickle cell anemia-specific iPSC, derived and characterized a novel in vitro system for the production of an unlimited supply of erythroid lineage cells from the directed differentiation of normal and disease-specific iPSC and used this system to recapitulate erythroid-lineage ontogeny in vitro with the sequential development of primitive and definitive erythropoiesis, accompanied by the appropriate expression of stage-specific globin genes. We have recently finished whole genome DNA and RNA sequencing analysis in some of these lines aimed at identifying developmental gene expression profile differences between erythroid precursors that produce primarily HbF and those that produce primarily HbA or HbS as part of our search for novel HbF genetic modifiers associated with markedly elevated HbF levels found in sickle cell anemia patients naturally, or in response to hydroxyurea treatment. Furthermore, our labs are also focusing on using a CRISPR-based gene editing platform to study the effect of novel HbF genetic modifiers and explore globin switching. Cell lines established are shown in the table.Table 1Number of subjects recruited to date98Number of subjects with iPSC lines established56Average number of iPSC lines per subject3 (total of 158 lines generated)Quality control status of iPSC linesAll lines are expanded and banked, mycoplasma free, express pluripotency markersSubjects with target cells differentiated (erythrocytes)25Samples have been collected on African American patients with sickle cell anemia with diverse HBB haplotypes, predominantly homozygotes and compound heterozygotes for the Benin and Bantu haplotypes, Saudi Arabian patients with the Arab-Indian haplotype and the Saudi Benin haplotype that is characterized by HbF levels about twice as high as in African Benin haplotype patients and from Brazilian patients who are predominantly homozygotes for the Bantu haplotype that typically is associated with the lowest HbF of all HBB haplotypes. This iPSC-based library and the data associated with it represents a valuable readily available resource for the sickle cell research community and all the generated lines will be available for distribution early in 2016 through WiCell. DisclosuresNo relevant conflicts of interest to declare.

Analysis of Stage-Specific Gene Expression Profiles in the Uterine Endometrium during Pregnancy in Pigs.

The uterine endometrium plays a critical role in regulating the estrous cycle and the establishment and maintenance of pregnancy in mammalian species. Many studies have investigated the expression and function of genes in the uterine endometrium, but the global expression pattern of genes and relationships among genes differentially expressed in the uterine endometrium during gestation in pigs remain unclear. Thus, this study investigated global gene expression profiles using microarray in pigs. Diverse transcriptome analyses including clustering, network, and differentially expressed gene (DEG) analyses were performed to detect endometrial gene expression changes during the different gestation stages. In total, 6,991 genes were found to be differentially expressed by comparing genes expressed on day (D) 12 of pregnancy with those on D15, D30, D60, D90 and D114 of pregnancy, and clustering analysis of detected DEGs distinguished 8 clusters. Furthermore, several pregnancy-related hub genes such as ALPPL2, RANBP17, NF1B, SPP1, and CST6 were discovered through network analysis. Finally, detected hub genes were technically validated by quantitative RT-PCR. These results suggest the complex network characteristics involved in uterine endometrial gene expression during pregnancy and indicate that diverse patterns of stage-specific gene expression and network connections may play a critical role in endometrial remodeling and in placental and fetal development to establish and maintenance of pregnancy in pigs.