The scaffold attachment factors SAFB1 and SAFB2 are paralogs, which are involved in cell cycle regulation, apoptosis, differentiation, and stress response. They have been shown to function as estrogen receptor co-repressors, and there is evidence for a role in breast tumorigenesis. To identify their endogenous target genes in MCF-7 breast cancer cells, we utilized gene expression array analysis, which was set up in a two-by-four design, with vehicle and estrogen treatment, and control, SAFB1, SAFB2, and SAFB1/SAFB2 siRNA as variables. Using custom chips containing 1.5 kb upstream regulatory region, we identified 541 SAFB1/SAFB2 binding sites in promoters of known genes, with significant enrichment on chromosome 1 and 6. Gene expression analysis revealed that the majority of target genes were induced in the absence of SAFB1 or SAFB2, and less were repressed. In contrast to SAFB2, which shared most of its target genes with SAFB1, SAFB1 had many unique target genes, most of them involved in regulation of the immune system. A subsequent analysis of the estrogen treatment group revealed that twelve percent of estrogen-regulated genes were dependent on SAFB1, with the majority being estrogen-repressed genes. These were primarily genes involved in apoptosis, such as BBC3, NEDD9, and OPG. Thus, this study confirms SAFB1/SAFB2s primary role as co-repressors, and also uncovers a previously unknown role for SAFB1 in regulation of immune genes, and in estrogen-mediated repression of genes.
SAFB1 mediates repression of immune regulators and apoptotic genes in breast cancer cells.
Cell line, TreatmentView Samples
LMP2A of Epstein-Barr virus is a receptor that mimics an activated B cell receptor, BCR. K1 and K15, related receptors of Kaposi sarcoma-associated herpes virus, KSHV, are expressed in virus-associated tumors but their functions are less obvious. We addressed this uncertainty with mutant EBVs encoding the KSHV genes K1 or K15 in lieu of LMP2A and infected primary human B cells with them. K1 and K15 encoded proteins appear to have noncomplementing redundant functions in this model but our findings suggest that both KSHV proteins can replace LMP2As key activities contributing to the survival, activation and proliferation of B cells.
K1 and K15 of Kaposi's Sarcoma-Associated Herpesvirus Are Partial Functional Homologues of Latent Membrane Protein 2A of Epstein-Barr Virus.
Specimen part, SubjectView Samples
Epstein-Barr virus (EBV) has evolved exquisite controls over its host cells, human B lymphocytes, not only directing these cells during latency to proliferate and thereby expand the pool of infected cells, but also to survive and thereby persist for the lifetime of the infected individual. Although these activities ensure the virus is successful, they also make the virus oncogenic, particularly when infected people are immunosuppressed. Here we show, strikingly, that one set of EBV’s miRNAs both sustain BL (Burkitt’s lymphoma) cells in the absence of other viral oncogenes and promote the transformation of primary B lymphocytes. Burkitt’s Lymphoma cells were engineered to lose EBV and found to die by apoptosis and could be rescued by constitutively expressing viral miRNAs in them. Two of these EBV miRNAs were found to target Caspase 3 to inhibit apoptosis at physiological concentrations. Overall design: Examination of RISC associated transcripts under 4 conditions in Sav S1-1 cells
Epstein-Barr virus maintains lymphomas via its miRNAs.
Cell line, Treatment, SubjectView Samples
Long-term pharmacological glucocorticoid therapy causes atrophy and hypofunction of the adrenal cortex. Following glucocorticoids withdrawal, a functional and anatomic regeneration take place, whose cellular and molecular mechanisms are poorly understood
Sonic Hedgehog and WNT Signaling Promote Adrenal Gland Regeneration in Male Mice.
Age, Specimen partView Samples
MicroRNAs (miRNAs) are small, endogenous, non-protein coding RNAs that are an important means of post-transcriptional gene regulation. Deletion of Dicer, a key miRNA processing enzyme, is embryonic lethal in mice, and tissue-specific Dicer deletion results in developmental defects. Using a conditional knockout model, we generated mice lacking Dicer in the adrenal cortex. These Dicer knockout (KO) mice exhibited perinatal mortality and failure of the adrenal cortex during late gestation between embryonic day 16.5 (E16.5) and E18.5. Further study of Dicer KO adrenals demonstrated a significant loss of Sf1 expressing cortical cells that was histologically evident as early as E16.5 coincident with an increase in p21 and cleaved-caspase 3 staining in the cortex. However, peripheral cortical proliferation persisted in KO adrenals as assessed by anti-PCNA staining. To further characterize the embryonic adrenals from Dicer KO mice, we performed microarray analyses for both gene expression and miRNA on purified RNA isolated from control and KO adrenals of E15.5 and E16.5 embryos. Consistent with the absence of Dicer and the associated loss of miRNA-mediated mRNA degradation, we observed an up-regulation of a small subset of adrenal transcripts in Dicer KO mice, most notably the transcripts coded by the genes Nr6a1 and Acvr1c. Indeed, several miRNAs, including let-7, miR-34c, and miR-21 that are predicted to target these genes for degradation, were also markedly down-regulated in Dicer KO adrenals. Together these data suggest a role for miRNA mediated regulation of a subset of genes that are essential for normal adrenal growth and homeostasis.
Dicer deficiency reveals microRNAs predicted to control gene expression in the developing adrenal cortex.
Specimen partView Samples
Vasoregression is a hallmark of vascular eye diseases but the mechanisms involved are still largely unknown. We have recently characterized a rat ciliopathy model which develops primary photoreceptor degeneration and secondary vasoregression. To improve the understanding of secondary vasoregression in retinal neurodegeneration, we used microarray techniques to compare gene expression profiles in this new model before and after retinal vasoregression. Differential gene expression was validated by quantitative RT-PCR, Western blot and immunofluorescence. Of the 374 genes regulated more than twofold, the MHC class II invariant chain CD74 yielded the strongest upregulation, and was allocated to activated microglial cells close to the vessels undergoing vasoregression. Pathway clustering identified genes of the immune system, inflammatory signaling, and components of the complement cascade upregulated during vasoregression. Furthermore, macroglial cells were markedly activated. Together, our data suggest that glial cells involved in retinal immune response participate in the initiation of vasoregression in the retina.
Gene expression profiling of vasoregression in the retina--involvement of microglial cells.
Specimen partView Samples
The SV40 large (LT) and small (st) antigens are produced from a single alternatively spliced pre-mRNA, that when co-expressed, transform a variety of cells in vitro and in vivo. However, 17kT, a relatively uncharacterized third protein that is co-linear with LT for the first 131 amino acids, is also produced from the early viral pre-mRNA by removal of an additional intron from the LT transcript. Here we report a line of transgenic mice expressing a liver-specific dox-inducible viral transcript that fails to yield any detectable LT protein, yet produces abundant 17kT. Comparative analysis of livers of transgenic mice expressing either 17kT or LT demonstrates that while 17kT is a potent stimulator of cell proliferation, it is ineffective at inducing liver tumor development, due in part, to the failure of 17kT to effectively induce the expression of growth regulators and reactivate expression of imprinted and developmentally regulated hepatic genes. These studies highlight key functional differences between LT and 17kT in their ability to transform quiescent primary epithelial cells in vivo, and demonstrate how specific functional domains within LT impact cell-specific gene expression to promote oncogenesis.
Comparative analysis of SV40 17kT and LT function in vivo demonstrates that LT's C-terminus re-programs hepatic gene expression and is necessary for tumorigenesis in the liver.
Sex, Specimen partView Samples