The function of Structural maintenance of chromosome flexible domain containing 1 (Smchd1) was examined during mouse preimplantation development using an siRNA knockdown approach. Transient SMCHD1 deficiency during the period between fertilization and morula/early blastocyst stage compromised embryo viability and resulted in reduced cell number, reduced embryo diameter, and reduced nuclear volumes at the morula stage. RNAseq analysis of Smchd1 knockdown morulae revealed aberrant increases in expression of mRNAs related to the trophoblast lineage, indicating SMCHD1 inhibits trophoblast lineage gene expression and promotes inner cell mass formation. siRNA knockdown also reduced expression of cell proliferation genes, including S-phase kinase-associated protein 2 (Skp2). Smchd1 expression was elevated in Caudal type homeobox transcription factor 2 (Cdx2)-/- blastocysts, indicating enriched expression, and further indicating a role in inner cell mass development. These results indicate that Smchd1 plays dual roles in the preimplantation embryo, promoting a lineage-appropriate pattern of gene expression supporting inner cell mass formation, whilst controlling lineage formation and gene expression in the trophectoderm. Overall design: Effects of SMCHD1 siRNA knockdown were tested in mouse embryos
Novel key roles for structural maintenance of chromosome flexible domain containing 1 (Smchd1) during preimplantation mouse development.
Treatment, SubjectView Samples
Nod2 has been extensively characterized as a bacterial sensor that induces an antimicrobial and inflammatory gene expression program. Therefore, it is unclear why Nod2 mutations that disrupt bacterial recognition are paradoxically among the highest risk factors for Crohns disease, which involves an exaggerated immune response directed at intestinal bacteria. Previous studies from our lab have shown that mice deficient in Atg16L1, another Crohns disease susceptibility gene, develop abnormalities in Paneth cells, specialized epithelial cells in the small intestine involved in antimicrobial responses.
Bacterial sensor Nod2 prevents inflammation of the small intestine by restricting the expansion of the commensal Bacteroides vulgatus.
Age, Specimen partView Samples
To characterize the transcriptional program that governs terminal granulocytic differentation in vivo, we performed comprehensive microarray analysis of human bone marrow population highly enriched for promyelocytes, myelocytes / metamyelocytes and neotrophils.
Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum.
Specimen partView Samples
Internal tandem duplication (ITD) of the fms-related tyrosine kinase-3 (FLT3) gene occurs in 30% acute myeloid leukemias (AML) and confers a poor prognosis. Thirteen relapsed or chemo-refractory FLT3-ITD+ AML patients were treated with sorafenib (200-400 mg twice daily). Twelve patients showed clearance or near clearance of bone marrow (BM) myeloblasts after 27 (range 2184) days with evidence of differentiation of leukemia cells. The sorafenib response was lost in most patients after 72 (range 54287) days but the FLT3 and downstream effectors remained suppressed. Four pairs patients (before sorafenib treatment and after sorafenib relapse), total eight samples from four patients at the two time-points were subjected to microarray analysis. Gene expression profiling showed that leukemia cells which have become sorafenib resistant expressed a number of genes including ALDH1A1, JAK3 and MMP15, whose functions were unknown in AML. NOD/SCID mice transplanted with leukemia cells from patients before and during sorafenib resistance recapitulated the clinical results. Both ITD and tyrosine kinase domain (TKD) mutations at D835 were identified in leukemia initiating cells (LIC) from samples before sorafenib treatment. LIC bearing the D835 mutant have expanded during sorafenib treatment and dominated during the subsequent clinical resistance. These results suggested that sorafenib have selected more aggressive sorafenib-resistant subclones carrying both FLT3-ITD and D835 mutations and might provide important leads to further improvement of treatment outcome with FLT3 inhibitors.
Sorafenib treatment of FLT3-ITD(+) acute myeloid leukemia: favorable initial outcome and mechanisms of subsequent nonresponsiveness associated with the emergence of a D835 mutation.
Specimen partView Samples
Low-grade serous ovarian carcinoma is believed to arise from serous borderline ovarian tumors, yet the progression from serous borderline tumors to low-grade serous ovarian carcinoma remains poorly understood. The purpose of this study was to identify differentially expressed genes between the two groups. Expression profiles were generated from 6 human ovarian surface epithelia (HOSE), 8 serous borderline ovarian tumors (SBOT), 13 low-grade serous ovarian carcinomas (LG), and 22 high-grade serous ovarian carcinomas (HG). The anterior gradient homolog 3 (AGR3) gene was found to be highly upregulated in serous borderline ovarian tumors; this finding was validated by real-time quantitative RT-PCR, Western blotting, and immunohistochemistry. Anti-AGR3 immunohistochemistry was performed on an additional 56 LG and 103 HG tissues and the results were correlated with clinical data. Expression profiling determined that 1254 genes were differentially expressed (P < 0.005) between SBOT, LG and HG tumors. Serous borderline ovarian tumors exhibited robust positive staining for AGR3, with a lower percentage of tumor cells stained in LG and HG. Immunofluorescence staining indicated that AGR3 expression was limited to ciliated cells. Tumor samples with a high percentage (>10%) of AGR3 positively stained tumor cells were associated with improved longer median survival in both the LG (P = 0.013) and HG (P = 0.008) serous ovarian carcinoma groups. The progression of serous borderline ovarian tumors to low-grade serous ovarian carcinoma may involve the de-differentiation of ciliated cells. AGR3 could serve as a prognostic marker for survival in patients with low-grade and high-grade serous ovarian carcinomas.
The anterior gradient homolog 3 (AGR3) gene is associated with differentiation and survival in ovarian cancer.
Specimen partView Samples
Land plants can reproduce sexually by developing an embryo from a fertilized egg cell. However, embryos can also be formed from other cell types in many plant species. A key question is thus how embryo identity in plants is controlled, and how this process is modified during non-zygotic embryogenesis. The Arabidopsis zygote divides to produce an embryonic lineage and an extra-embryonic suspensor. Yet, normally quiescent suspensor cells can develop a second embryo when the initial embryo is damaged, or when response to the signaling molecule auxin is locally blocked. Here we have used auxin-dependent suspensor embryogenesis as a model to determine transcriptome changes during embryonic reprogramming. We find that reprogramming is complex and accompanied by large transcriptomic changes prior to anatomic changes. This analysis revealed a strong enrichment for genes encoding components of auxin homeostasis and response among misregulated genes. Strikingly, deregulation among multiple auxin-related gene families converged upon re-establishment of cellular auxin levels or response. This suggests a remarkable degree of feedback regulation to create resilience in auxin response during embryo development. Starting from the transcriptome of auxin-deregulated embryos, we identify an auxin-dependent bHLH transcription factor network that mediates the activity of this hormone in suppressing embryo development from the suspensor.
A Robust Auxin Response Network Controls Embryo and Suspensor Development through a Basic Helix Loop Helix Transcriptional Module.
Specimen partView Samples
RUNX1 is a frequent target of translocations in acute myeloid leukemia whereby its DNA binding domain fuses to different epigenetic regulators. To assess how different RUNX1 fusion proteins interact with the epigenome we compared the global binding patterns and the chromatin landscape of t(8;21) and t(3;21) AML which express RUNX1-ETO and RUNX1-EVI-1, respectively. We found that differential prognosis for these types of AML is reflected in fundamental differences in gene expression, chromatin landscape, binding patterns of the fusion proteins and other transcription factors as identified by genome-wide digital footprinting in patients. As previously shown for RUNX1-ETO, knockdown of RUNX1-EVI-1 expression initiates differentiation of t(3;21) cells which is associated with up-regulation of genes vital for myeloid differentiation, including C/EBPa. Furthermore, by expressing either dominant-negative C/EBP or an inducible C/EBPa construct in t(3;21) cells we show that C/EBPa is necessary and sufficient for the differentiation response of these cells to RUNX1-EVI-1 knockdown. Overall design: RNA-seq expreiments have been used to study the chromatin landscape of t(8;21) and t(3;21) AML
RUNX1-ETO and RUNX1-EVI1 Differentially Reprogram the Chromatin Landscape in t(8;21) and t(3;21) AML.
Specimen part, SubjectView Samples
B7x (B7-H4 or B7S1) is the seventh member of the B7 family and the in vivo function remains largely unknown. Despite new genetic data linking the B7x gene with autoimmune diseases, how exactly it contributes to peripheral tolerance and autoimmunity is unclear. Here we showed that B7x protein was not detected on antigen-presenting cells or T cells in both human and mice, which is unique in the B7 family. As B7x protein is expressed in some peripheral cells such as pancreatic b cells, we utilized a CD8 T cell-mediated diabetes model (AI4ab) in which CD8 T cells recognize an endogenous self-antigen, and found that mice lacking B7x developed more severe diabetes than control AI4ab mice. Conversely, mice overexpressing B7x in the b cells (Rip-B7xAI4ab) were diabetes free. Furthermore, adoptive transfer of effector AI4ab CD8 T cells induced diabetes in control mice, but not in Rip-B7xAI4ab mice. Mechanistic studies revealed that pathogenic effector CD8 T cells were capable of migrating to the pancreas but failed to robustly destroy tissue when encountering local B7x in Rip-B7xAI4ab mice. Although AI4ab CD8 T cells in Rip-B7xAI4ab mice and AI4ab mice showed similar cytotoxic function, cell death, and global gene expression profiles, these cells had greater proliferation in AI4ab mice than in RIP-B7xAI4ab mice. These results suggest that B7x in nonlymphoid organs prevents peripheral autoimmunity partially through inhibiting proliferation of tissue-specific CD8 T cells and that local overexpression of B7x on pancreatic b cells is sufficient to abolish CD8 T cell-induced diabetes.
B7x in the periphery abrogates pancreas-specific damage mediated by self-reactive CD8 T cells.
Specimen part, DiseaseView Samples