Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors

<p>Abstract</p> <p>Background</p> <p>Osteoblast differentiation requires the coordinated stepwise expression of multiple genes. Histone deacetylase inhibitors (HDIs) accelerate the osteoblast differentiation process by blocking the activity of histone deacetylases (HDACs), which alter gene expression by modifying chromatin structure. We previously demonstrated that HDIs and HDAC3 shRNAs accelerate matrix mineralization and the expression of osteoblast maturation genes (e.g. alkaline phosphatase, osteocalcin). Identifying other genes that are differentially regulated by HDIs might identify new pathways that contribute to osteoblast differentiation.</p> <p>Results</p> <p>To identify other osteoblast genes that are altered early by HDIs, we incubated MC3T3-E1 preosteoblasts with HDIs (trichostatin A, MS-275, or valproic acid) for 18 hours in osteogenic conditions. The promotion of osteoblast differentiation by HDIs in this experiment was confirmed by osteogenic assays. Gene expression profiles relative to vehicle-treated cells were assessed by microarray analysis with Affymetrix GeneChip 430 2.0 arrays. The regulation of several genes by HDIs in MC3T3-E1 cells and primary osteoblasts was verified by quantitative real-time PCR. Nine genes were differentially regulated by at least two-fold after exposure to each of the three HDIs and six were verified by PCR in osteoblasts. Four of the verified genes (solute carrier family 9 isoform 3 regulator 1 (Slc9a3r1), sorbitol dehydrogenase 1, a kinase anchor protein, and glutathione S-transferase alpha 4) were induced. Two genes (proteasome subunit, beta type 10 and adaptor-related protein complex AP-4 sigma 1) were suppressed. We also identified eight growth factors and growth factor receptor genes that are significantly altered by each of the HDIs, including Frizzled related proteins 1 and 4, which modulate the Wnt signaling pathway.</p> <p>Conclusion</p> <p>This study identifies osteoblast genes that are regulated early by HDIs and indicates pathways that might promote osteoblast maturation following HDI exposure. One gene whose upregulation following HDI treatment is consistent with this notion is Slc9a3r1. Also known as NHERF1, Slc9a3r1 is required for optimal bone density. Similarly, the regulation of Wnt receptor genes indicates that this crucial pathway in osteoblast development is also affected by HDIs. These data support the hypothesis that HDIs regulate the expression of genes that promote osteoblast differentiation and maturation.</p

Evolution of pathogenicity and sexual reproduction in eight Candida genomes

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.publishe

Differential Expression of Mll-AF9 Up-Regulated Genes Correlates with Enhanced Self-Renewal of Hematopoietic Progenitors.

Abstract In order to understand the pathophysiology of leukemia, we need to study the effects of leukemic oncogenes on the rare hematopoietic stem and progenitor cells. We investigated the self-renewal capabilities of the various hematopoietic cell types derived from Mll-AF9 knock-in mice. We used the murine knock-in model since it offers the advantage of a single copy of the Mll-fusion gene under the control of the endogenous promoter present in every hematopoietic stem/progenitor cell. In methylcellulose cultures, we compared myeloid colony formation of Mll-AF9 cells to wild type progenitor populations over three generations of plating. In the first generation of plating, the Mll-AF9 common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) formed more colonies than the hematopoietic stem cells (HSCs) and common lymphoid progenitors (CLPs). However, at the third generation of plating, colony numbers formed by Mll-AF9 HSCs and CLPs were significantly greater than those formed by CMPs and GMPs. By the third generation only occasional colonies were found in the wild type groups. These results demonstrate that while Mll-AF9 led to an increase in self-renewal of all 4 cell types studied, these effects were more pronounced in HSCs and CLPs. To identify the downstream genes that mediate the growth deregulatory effects of Mll-AF9, we compared gene expression profiles of Mll-AF9 derived cells to their wild type counterparts. To assess gene expression levels, we extracted RNA from wild type and Mll-AF9 HSCs, CLPs, CMPs and GMPs. We then amplified and labeled the RNA for analysis by Affymetrix murine 430 2.0 genome arrays. In an unsupervised analysis, the various Mll-AF9 cells clustered with their corresponding wild type counterparts, indicating that the expression of most genes was not significantly altered by Mll-AF9. To identify the genes that are differentially expressed in the Mll-AF9 derived cells, we performed a two-way ANOVA (with the genotype and cell type as the two variables) allowing for a false discovery rate of 10%. In this analysis, we found that 76 genes were up-regulated in all Mll-AF9 progenitor cells compared to their wild-type counterparts. This list included known targets of Mll-fusion proteins Hoxa5, Hoxa7, Hoxa9 and Hoxa10. Also included were Evi1 and Mef2c, two genes that have been implicated in promoting enhanced self-renewal of murine hematopoietic cells. Importantly, in wild type mice, these 6 genes were expressed at higher levels in HSCs and CLPs compared to CMPs and GMPs (average 3–25 fold). While we observed an average 2–10 fold increase in expression of these genes in all Mll-AF9 cell types compared to their respective wild type controls, the expression level was 3–8 fold higher in Mll-AF9 HSCs and CLPs compared to CMPs and GMPs. Thus, the expression of genes known to be intrinsically related to self-renewal is further enhanced as a result of the Mll-AF9 fusion gene. In conclusion, while activation of the Mll-AF9 genetic program and the resulting enhanced self-renewal occurs in all 4 cell types studied, these effects are greatest in HSCs and CLPs. Thus, HSCs and CLPs are likely to be more efficient than CMPs and GMPs in producing cellular expansion and targets for cooperating mutations resulting in leukemia.</jats:p

Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors-3

<p><b>Copyright information:</b></p><p>Taken from "Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors"</p><p>http://www.biomedcentral.com/1471-2164/8/362</p><p>BMC Genomics 2007;8():362-362.</p><p>Published online 9 Oct 2007</p><p>PMCID:PMC2147034.</p><p></p>ing the indicated HDI or DMSO. mRNAs were isolated at various times over a 6 or 18 hour period and subjected to quantitative real-time PCR with primers for Slc9a3r1 (A and B) or Sdh1 (C and D). Values are relative to those obtained from DMSO-treated samples at each time point and represent the mean of three samples. For (A-D), * denotes a statistically significant change of p < 0.01 and ** denotes p < 0.05 by one-way ANOVA of the HDI-treated sample versus the DMSO-treated sample at that time point. (E) C2C12 cells were cultured in osteogenic medium and 20 nM TSA for the indicated times. Slc9a3r1 protein levels were determined by immunoblotting

Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors-0

<p><b>Copyright information:</b></p><p>Taken from "Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors"</p><p>http://www.biomedcentral.com/1471-2164/8/362</p><p>BMC Genomics 2007;8():362-362.</p><p>Published online 9 Oct 2007</p><p>PMCID:PMC2147034.</p><p></p>days with the HDIs or vehicle added only at day 0. Fold change in ALP activity is shown in relation to values obtained at the start of the culture (day 0). * denotes a statistically significant change of p < 0.01 by one-way ANOVA of the HDI-treated sample versus the DMSO-treated sample at that time point

Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors-5

<p><b>Copyright information:</b></p><p>Taken from "Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors"</p><p>http://www.biomedcentral.com/1471-2164/8/362</p><p>BMC Genomics 2007;8():362-362.</p><p>Published online 9 Oct 2007</p><p>PMCID:PMC2147034.</p><p></p>days with the HDIs or vehicle added only at day 0. Fold change in ALP activity is shown in relation to values obtained at the start of the culture (day 0). * denotes a statistically significant change of p < 0.01 by one-way ANOVA of the HDI-treated sample versus the DMSO-treated sample at that time point

Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors-4

<p><b>Copyright information:</b></p><p>Taken from "Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors"</p><p>http://www.biomedcentral.com/1471-2164/8/362</p><p>BMC Genomics 2007;8():362-362.</p><p>Published online 9 Oct 2007</p><p>PMCID:PMC2147034.</p><p></p>. mRNAs were isolated after 18 hours and subjected to quantitative real-time PCR with primers for Psmb10 (A) or Ap4s1 (B). Values are relative to those obtained from DMSO-treated samples at each time point and represent the mean of three samples. ** denotes p < 0.05 by one-way ANOVA of the HDI-treated sample versus the DMSO-treated sample at that time point

Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors-2

<p><b>Copyright information:</b></p><p>Taken from "Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors"</p><p>http://www.biomedcentral.com/1471-2164/8/362</p><p>BMC Genomics 2007;8():362-362.</p><p>Published online 9 Oct 2007</p><p>PMCID:PMC2147034.</p><p></p>were isolated, reverse transcribed and amplified using semi-quantitative real-time PCR with primers for Slc9a3r1 (A) or Sdh1 (B), Qpct (C) and Psmb10 (D). Comparative threshold values represent the mean of three samples normalized to actin levels

Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors-1

<p><b>Copyright information:</b></p><p>Taken from "Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors"</p><p>http://www.biomedcentral.com/1471-2164/8/362</p><p>BMC Genomics 2007;8():362-362.</p><p>Published online 9 Oct 2007</p><p>PMCID:PMC2147034.</p><p></p>ormatics analysis identified genes that were differentially expressed by more than 1.1 fold in HDI-treated cells relative vehicle (DMSO)-treated cells with a FDR of 0.01 (99% confidence)