Genome Wide Analysis of Hepatic Gene Silencing in Mammalian Cell Variants

Using microarray data, a genome wide analysis of hepatic gene-silencing in four hepatoma variant cell lines was carried out. The purpose of this analysis was to identify candidate genes that may be able to restore liver function in non-functional cell variants, ultimately giving insight into the mechanisms behind global hepatic gene expression. Based on a selection scheme allowing only for genes activated or repressed at least 5 fold in 2 out of the 4 variant cell lines, 225 genes were found to be repressed while a total of 76 genes were found to be activated. Of the repressed genes identified, fourteen candidate genes were chosen based on known function as transcription factors or involvement in signal transduction pathways. One gene of particular interest, ONECUT1 (or HNF6), was analyzed for the capability to restore liver function in one of the cell variant lines. This was done by stable transfection of ONECUT1 into the H11-variant cell line, followed by qPCR to monitor liver gene reactivation. Measurement for liver reactivation was assessed by comparing gene expression values of known liver-specific genes (such as albumin) of parental hepatoma cells to those of variant cell lines. Data indicates that ONECUT1 is not able to directly restore liver function in the variant cell lines. However, the thorough analysis of hepatic gene silencing paves the way for genetic rescue experiments designed to identify genes involved with hepatic function and the genetic programs responsible for establishing and maintaining liver specific gene expression

The mirn23a and mirn23b microrna clusters are necessary for proper hematopoietic progenitor cell production and differentiation

Mice deficient for microRNA (miRNA) cluster mirn23a exhibit increased B lymphopoiesis at the expense of myelopoiesis, whereas hematopoietic stem and progenitor cell (HSPC) populations are unchanged. Mammals possess a paralogous mirn23b gene that can give rise to three mature miRNAs (miR-23b, miR-24-1, and miR-27b) that have identical seed/mRNA-targeting sequences to their mirn23a counterparts. To assess whether compound deletion of mirn23a and mirn23b exacerbates the hematopoietic phenotype observed in mirn23a−/− mice, we generated a compound mirn23a−/−mirn23bfl/fl:Mx1-Cre conditional knockout mouse and assayed hematopoietic development after excision of mirn23b. Loss of both genes in adult bone marrow further skewed HSPC differentiation toward B cells at the expense of myeloid cells, demonstrating a dosage-dependent effect on regulating cell differentiation. Strikingly, double-knockout (DKO) mice had decreased bone marrow cellularity with significantly decreased hematopoietic stem cell and HSPC populations, a phenotype not observed in mice deficient for mirn23a alone. Competitive transplantation assays showed decreased contribution of mirn23a−/−mirn23b−/− HSPCs to hematopoietic lineages at 6 and 12 weeks after transplantation. Defects in the proliferation of mirn23a−/−b−/− HSPCs was not observed; however, DKO cells were more apoptotic compared with both wild-type and mirn23a−/− cells. Together, our data show that complete loss of mirn23a/mirn23b miRNAs results in decreased blood production and affects lineage output in a concentration-dependent manner

Arid3b Is Critical for B Lymphocyte Development

Arid3a and Arid3b belong to a subfamily of ARID (AT-rich interaction domain) transcription factors. The Arid family is involved in regulating chromatin accessibility, proliferation, and differentiation. Arid3a and Arid3b are closely related and share a unique REKLES domain that mediates their homo- and hetero-multimerization. Arid3a was originally isolated as a B cell transcription factor binding to the AT rich matrix attachment regions (MARS) of the immunoglobulin heavy chain intronic enhancer. Deletion of Arid3a results in a highly penetrant embryonic lethality with severe defects in erythropoiesis and hematopoietic stem cells (HSCs). The few surviving Arid3a-/- (<1%) animals have decreased HSCs and early progenitors in the bone marrow, but all mature lineages are normally represented in the bone marrow and periphery except for B cells. Arid3b-/- animals die around E7.5 precluding examination of hematopoietic development. So it is unclear whether the phenotype of Arid3a loss on hematopoiesis is dependent or independent of Arid3b. In this study we circumvented this limitation by also examining hematopoiesis in mice with a conditional allele of Arid3b. Bone marrow lacking Arid3b shows decreased common lymphoid progenitors (CLPs) and downstream B cell populations while the T cell and myeloid lineages are unchanged, reminiscent of the adult hematopoietic defect in Arid3a mice. Unlike Arid3a-/- mice, HSC populations are unperturbed in Arid3b-/- mice. This study demonstrates that HSC development is independent of Arid3b, whereas B cell development requires both Arid3a and Arid3b transcription factors

Genome-wide analysis of hepatic gene silencing in hepatoma cell variants

AbstractGenome-wide gene expression profiling was carried out on rat hepatoma cells and compared to profiles of hepatoma “variant” cell lines derived via a stringent selection protocol that enriches for rare cells (<1 in 100,000 cells) that fail to drive liver function. Results show 132 genes that are strongly (>5-fold) repressed in each of the four variant cell lines tested. An additional 68 genes were repressed in 3 of 4 variant cell lines. Importantly, several of the repressed genes are members of transcriptional activation pathways, suggesting that they may contribute to maintaining the hepatic phenotype. Ectopic expression of the HNF1A gene in a variant cell line resulted in activation of 56 genes, 37 of which were included in the repressed data set. These data suggest that a high level of reprogramming occurs when hepatoma cells convert to a non-differentiated phenotype, a process that can be partially reversed by the introduction of transcription factors

CD133 Promotes Adhesion to the Ovarian Cancer Metastatic Niche

Cancer stem cells (CSCs) are an attractive therapeutic target due to their predicted role in both metastasis and chemoresistance. One of the most commonly agreed on markers for ovarian CSCs is the cell surface protein CD133. CD133+ ovarian CSCs have increased tumorigenicity, resistance to chemotherapy, and increased metastasis. Therefore, we were interested in defining how CD133 is regulated and whether it has a role in tumor metastasis. Previously we found that overexpression of the transcription factor, ARID3B , increased the expression of PROM1 (CD133 gene) in ovarian cancer cells in vitro and in xenograft tumors. We report that ARID3B directly regulates PROM1 expression. Importantly, in a xenograft mouse model of ovarian cancer, knockdown of PROM1 in cells expressing exogenous ARID3B resulted in increased survival time compared with cells expressing ARID3B and a control short hairpin RNA. This indicated that ARID3B regulation of PROM1 is critical for tumor growth. Moreover, we hypothesized that CD133 may affect metastatic spread. Given that the peritoneal mesothelium is a major site of ovarian cancer metastasis, we explored the role of PROM1 in mesothelial attachment. PROM1 expression increased adhesion to mesothelium in vitro and ex vivo. Collectively, our work demonstrates that ARID3B regulates PROM1 adhesion to the ovarian cancer metastatic niche

CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

CUX1 is a homeodomain-containing transcription factor that is essential for the development and differentiation of multiple tissues. CUX1 is recurrently mutated or deleted in cancer, particularly in myeloid malignancies. However, the mechanism by which CUX1 regulates gene expression and differentiation remains poorly understood, creating a barrier to understanding the tumor-suppressive functions of CUX1. Here, we demonstrate that CUX1 directs the BAF chromatin remodeling complex to DNA to increase chromatin accessibility in hematopoietic cells. CUX1 preferentially regulates lineage-specific enhancers, and CUX1 target genes are predictive of cell fate in vivo. These data indicate that CUX1 regulates hematopoietic lineage commitment and homeostasis via pioneer factor activity, and CUX1 deficiency disrupts these processes in stem and progenitor cells, facilitating transformation

Supplementary_Material – Supplemental material for CD133 Promotes Adhesion to the Ovarian Cancer Metastatic Niche

<p>Supplemental material, Supplementary_Material for CD133 Promotes Adhesion to the Ovarian Cancer Metastatic Niche by Lynn Roy, Alexander Bobbs, Rachel Sattler, Jeffrey L Kurkewich, Paige B Dausinas, Prakash Nallathamby and Karen D Cowden Dahl in Cancer Growth and Metastasis</p

The miR-23a~27a~24-2 microRNA cluster buffers transcription and signaling pathways during hematopoiesis

<div><p>MicroRNA cluster <i>mirn23a</i> has previously been shown to promote myeloid development at the expense of lymphoid development in overexpression and knockout mouse models. This polarization is observed early in hematopoietic development, with an increase in common lymphoid progenitors (CLPs) and a decrease in all myeloid progenitor subsets in adult bone marrow. The pool size of multipotential progenitors (MPPs) is unchanged; however, in this report we observe by flow cytometry that polarized subsets of MPPs are changed in the absence of <i>mirn23a</i>. Additionally, in vitro culture of MPPs and sorted MPP transplants showed that these cells have decreased myeloid and increased lymphoid potential in vitro and in vivo. We investigated the mechanism by which <i>mirn23a</i> regulates hematopoietic differentiation and observed that <i>mirn23a</i> promotes myeloid development of hematopoietic progenitors through regulation of hematopoietic transcription factors and signaling pathways. Early transcription factors that direct the commitment of MPPs to CLPs (Ikzf1, Runx1, Satb1, Bach1 and Bach2) are increased in the absence of <i>mirn23a</i> miRNAs as well as factors that commit the CLP to the B cell lineage (FoxO1, Ebf1, and Pax5). Mirn23a appears to buffer transcription factor levels so that they do not stochastically reach a threshold level to direct differentiation. Intriguingly, <i>mirn23a</i> also inversely regulates the PI3 kinase (PI3K)/Akt and BMP/Smad signaling pathways. Pharmacological inhibitor studies, coupled with dominant active/dominant negative biochemical experiments, show that both signaling pathways are critical to <i>mirn23a</i>’s regulation of hematopoietic differentiation. Lastly, consistent with <i>mirn23a</i> being a physiological inhibitor of B cell development, we observed that the essential B cell transcription factor EBF1 represses expression of <i>mirn23</i>a. In summary, our data demonstrates that <i>mirn23a</i> regulates a complex array of transcription and signaling pathways to modulate adult hematopoiesis.</p></div

Arid3b^-/- B cells produce normal serum IgG and have no defect in response to LPS during ex vivo culture.

<p><b>A)</b> Peripheral blood was collected from control (N = 4) and <i>Arid3b</i>^<i>-/-</i> (N = 5) mice via cheek bleed and analyzed for total serum IgG levels. No difference was observed between control and <i>Arid3b</i>^<i>-/-</i> mice. <b>B)</b> Splenic B cells were isolated from control (N = 3) or <i>Arid3b</i>^<i>-/-</i> (N = 3) cohorts by negative selection of CD43 (Ly-48). Cells were stained with CFSE and cultured in LPS for 72 hours before analyzing by flow cytometry. Representative plots are shown. P values determined by unpaired students t-test. <b>C)</b> No difference was observed in the percentage of proliferating cells between control and <i>Arid3b</i>^<i>-/-</i> mice. <b>D)</b> Cultures were also analyzed for their ability to differentiate into plasma cells by cell surface markers B220 and CD138. <b>E)</b> No difference was observed in the percent of short lived plasma cells between control and <i>Arid3b</i>^<i>-/-</i> cultures.</p

Splenic B and T cell populations are unchanged in ARID3B null mice.

<p>Spleens were harvested from <i>Arid3b</i>^<i>fl/fl</i>: <i>Mx1-Cre</i> (denoted Arid3b^-/-) mice for analysis of B cell and T cell populations by flow cytometry, 12 weeks after final pIpC injection. <b>A)</b> B220+ B cells were analyzed in control (N = 11) and <i>Arid3b</i>^<i>-/-</i> (N = 10) mice. No statistically significant differences were observed in B220+ splenic B cell populations. <b>B)</b> Splenic follicular zone, marginal zone, and transitional zone B cells were evaluated using cell surface markers CD21 and IgM. Representative plots are shown. Control (N = 5) and <i>Arid3b</i>^<i>-/-</i> (N = 7) mice were analyzed. <b>C)</b> <i>Arid3b</i>^<i>-/-</i> mice had significantly decreased T1-T2 cell population with significantly increased follicular zone population. No difference was observed in marginal zone B cells. <b>D)</b> T cell populations were analyzed in spleens of control (N = 6) and Arid3b^-/- (N = 9) mice.<b>E)</b> No significant differences were observed in the CD4+ T cell populations. <b>F)</b> No significant differences were observed in CD8+ T cell populations. <b>G)</b> Control (N = 7) and <i>Arid3b</i>^<i>-/-</i> (N = 7) peritoneal cells were collected by peritoneal lavage and analyzed for cell surface expression of CD5 and B220 to identify B1 and B2 B cell subsets. <b>H)</b> A modest but significant increase was observed in the B1a population with a concomitant decrease in the B1b population, while the B2 population was unchanged. P values determined by unpaired students t-test.</p