An integrated approach to characterize transcription factor and microRNA regulatory networks involved in Schwann cell response to peripheral nerve injury

BACKGROUND: The regenerative response of Schwann cells after peripheral nerve injury is a critical process directly related to the pathophysiology of a number of neurodegenerative diseases. This SC injury response is dependent on an intricate gene regulatory program coordinated by a number of transcription factors and microRNAs, but the interactions among them remain largely unknown. Uncovering the transcriptional and post-transcriptional regulatory networks governing the Schwann cell injury response is a key step towards a better understanding of Schwann cell biology and may help develop novel therapies for related diseases. Performing such comprehensive network analysis requires systematic bioinformatics methods to integrate multiple genomic datasets. RESULTS: In this study we present a computational pipeline to infer transcription factor and microRNA regulatory networks. Our approach combined mRNA and microRNA expression profiling data, ChIP-Seq data of transcription factors, and computational transcription factor and microRNA target prediction. Using mRNA and microRNA expression data collected in a Schwann cell injury model, we constructed a regulatory network and studied regulatory pathways involved in Schwann cell response to injury. Furthermore, we analyzed network motifs and obtained insights on cooperative regulation of transcription factors and microRNAs in Schwann cell injury recovery. CONCLUSIONS: This work demonstrates a systematic method for gene regulatory network inference that may be used to gain new information on gene regulation by transcription factors and microRNAs

In Vitro Transformation of Primary Human CD34+ Cells by AML Fusion Oncogenes: Early Gene Expression Profiling Reveals Possible Drug Target in AML

Different fusion oncogenes in acute myeloid leukemia (AML) have distinct clinical and laboratory features suggesting different modes of malignant transformation. Here we compare the in vitro effects of representatives of 4 major groups of AML fusion oncogenes on primary human CD34+ cells. As expected from their clinical similarities, MLL-AF9 and NUP98-HOXA9 had very similar effects in vitro. They both caused erythroid hyperplasia and a clear block in erythroid and myeloid maturation. On the other hand, AML1-ETO and PML-RARA had only modest effects on myeloid and erythroid differentiation. All oncogenes except PML-RARA caused a dramatic increase in long-term proliferation and self-renewal. Gene expression profiling revealed two distinct temporal patterns of gene deregulation. Gene deregulation by MLL-AF9 and NUP98-HOXA9 peaked 3 days after transduction. In contrast, the vast majority of gene deregulation by AML1-ETO and PML-RARA occurred within 6 hours, followed by a dramatic drop in the numbers of deregulated genes. Interestingly, the p53 inhibitor MDM2 was upregulated by AML1-ETO at 6 hours. Nutlin-3, an inhibitor of the interaction between MDM2 and p53, specifically inhibited the proliferation and self-renewal of primary human CD34+ cells transduced with AML1-ETO, suggesting that MDM2 upregulation plays a role in cell transformation by AML1-ETO. These data show that differences among AML fusion oncogenes can be recapitulated in vitro using primary human CD34+ cells and that early gene expression profiling in these cells can reveal potential drug targets in AML

The Origin and Evolution of Mutations in Acute Myeloid Leukemia

SummaryMost mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is “captured” as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse

Differentially expressed microRNAs in chondrocytes from distinct regions of developing human cartilage.

There is compelling in vivo evidence from reports on human genetic mutations and transgenic mice that some microRNAs (miRNAs) play an important functional role in regulating skeletal development and growth. A number of published in vitro studies also point toward a role for miRNAs in controlling chondrocyte gene expression and differentiation. However, information on miRNAs that may regulate a specific phase of chondrocyte differentiation (i.e. production of progenitor, differentiated or hypertrophic chondrocytes) is lacking. To attempt to bridge this knowledge gap, we have investigated miRNA expression patterns in human embryonic cartilage tissue. Specifically, a developmental time point was selected, prior to endochondral ossification in the embryonic limb, to permit analysis of three distinct populations of chondrocytes. The location of chondroprogenitor cells, differentiated chondrocytes and hypertrophic chondrocytes in gestational day 54-56 human embryonic limb tissue sections was confirmed both histologically and by specific collagen expression patterns. Laser capture microdissection was utilized to separate the three chondrocyte populations and a miRNA profiling study was carried out using TaqMan® OpenArray® Human MicroRNA Panels (Applied Biosystems®). Here we report on abundantly expressed miRNAs in human embryonic cartilage tissue and, more importantly, we have identified miRNAs that are significantly differentially expressed between precursor, differentiated and hypertrophic chondrocytes by 2-fold or more. Some of the miRNAs identified in this study have been described in other aspects of cartilage or bone biology, while others have not yet been reported in chondrocytes. Finally, a bioinformatics approach was applied to begin to decipher developmental cellular pathways that may be regulated by groups of differentially expressed miRNAs during distinct stages of chondrogenesis. Data obtained from this work will serve as an important resource of information for the field of cartilage biology and will enhance our understanding of miRNA-driven mechanisms regulating cartilage and endochondral bone development, regeneration and repair

Enriched pathways of predicted genes targeted by differentially expressed miRNAs (DC

<p>Enriched pathways of predicted genes targeted by differentially expressed miRNAs (DC</p

Top 30 most abundantly expressed miRNAs in precursor, differentiated and hypertrophic chondrocytes from gestational day 54–56 human embryonic cartilage tissue.

<p>Highly expressed miRNAs were identified according to their average (2^-ΔCt) values. Delta (Δ) Ct value for each miRNA was calculated by subtracting the Ct value of endogenous control, RNU44, from the Ct value of the specific miRNA. Expression level average (2^−ΔCt) in a region reflects the average of 2^−ΔCt values across all samples in that region.</p

Differentially-expressed miRNAs between differentiated chondrocytes (DC) and hypertrophic chondrocytes (HYP) from gestational day 54–56 human embryonic cartilage tissue.

<p>Fold change (f.c.) expression of miRNAs between cells of DC and HYP regions are shown. The score (d) and q-values for each differentially-expressed miRNA are shown based on SAM analysis (FDR≤5%; n = 8–9).</p

Enriched pathways of predicted genes targeted by differentially expressed miRNAs (PC>DC).

<p>Enriched pathways of predicted genes targeted by differentially expressed miRNAs (PC>DC).</p

Enriched pathways of predicted genes targeted by differentially expressed miRNAs (DC>HYP).

<p>Enriched pathways of predicted genes targeted by differentially expressed miRNAs (DC>HYP).</p

Immunofluorescence staining of different collagen types in a human developing embryonic proximal tibia (gestational day 54).

<p>(<b>A</b>) Localization of the embryonic isoform of type II procollagen (type IIA) in the extracellular matrix (ECM). The anti-IIA antibody recognizes the exon 2-encoded cysteine-rich domain present in the amino propeptide of type IIA procollagen <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075012#pone.0075012-Oganesian1" target="_blank">[43]</a>. These IIA isoforms are generated predominantly by progenitor chondrocytes seen at the periphery and most proximal area of the developing tibia. Some expression of IIA procollagen has been reported in the pre-hypertrophic and hypertrophic region of developing cartilage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075012#pone.0075012-Zhu1" target="_blank">[58]</a> and is also shown here. (<b>B</b>) Type I collagen staining is restricted to the areas of precursor chondrocytes and cells of the perichondrium/periosteum. (<b>C</b>) Type II collagen staining patterns (i.e. the processed triple helical domain of type II collagen) is present throughout the entire developing limb. (<b>D</b>) Collagen X staining is restricted to the ECM containing hypertrophic chondrocytes. Cell nuclei are visualized in blue by DAPI staining. Scale bars = 100 µm. Immunofluorescent images are representative of three independent experiments using gestational day 54 tissue sections from different embryos.</p