Responsiveness of genes to manipulation of transcription factors in ES cells is associated with histone modifications and tissue specificity.

BACKGROUND: In addition to determining static states of gene expression (high vs. low), it is important to characterize their dynamic status. For example, genes with H3K27me3 chromatin marks are not only suppressed but also poised for activation. However, the responsiveness of genes to perturbations has never been studied systematically. To distinguish gene responses to specific factors from responsiveness in general, it is necessary to analyze gene expression profiles of cells responding to a large variety of disturbances, and such databases did not exist before. RESULTS: We estimated the responsiveness of all genes in mouse ES cells using our recently published database on expression change after controlled induction of 53 transcription factors (TFs) and other genes. Responsive genes (N=4746), which were readily upregulated or downregulated depending on the kind of perturbation, mostly have regulatory functions and a propensity to become tissue-specific upon differentiation. Tissue-specific expression was evaluated on the basis of published (GNF) and our new data for 15 organs and tissues. Non-responsive genes (N=9562), which did not change their expression much following any perturbation, were enriched in housekeeping functions. We found that TF-responsiveness in ES cells is the best predictor known for tissue-specificity in gene expression. Among genes with CpG islands, high responsiveness is associated with H3K27me3 chromatin marks, and low responsiveness is associated with H3K36me3 chromatin, stronger tri-methylation of H3K4, binding of E2F1, and GABP binding motifs in promoters. CONCLUSIONS: We thus propose the responsiveness of expression to perturbations as a new way to define the dynamic status of genes, which brings new insights into mechanisms of regulation of gene expression and tissue specificity.

MicroRNA (miR) 125b regulates cell growth and invasion in pediatric low grade glioma.

Members of the miR-125 family are strongly expressed in several tissues, particularly brain, but may be dysregulated in cancer including adult and pediatric glioma. In this study, miR-125 members were downregulated in pilocytic astrocytoma (PA) as a group compared to non-neoplastic brain in the Agilent platform. In the Nanostring platform, miR-125 members were downregulated primarily in pleomorphic xanthoastrocytomas and gangliogliomas. Using CISH for miR-125b, highest levels of expression were present in grade II tumors (11/33, 33% grade II tumors with 3+ expression compared to 3/70, 4% grade I tumors) (p < 0.001). When focusing on the two histologic subgroups with the largest number of samples, PA and diffuse astrocytoma (DA), the highest expression levels were present in DA, in comparison with the PA group (p = 0.01). Overexpression of miR-125b in pediatric low grade glioma (PLGG) derived cell lines (Res186, Res259, and BT66) resulted in decreased growth and invasion, as well as apoptosis. Additionally, miR-125b overexpression in BT66 resulted in senescence. These findings suggest that miR-125 is frequently underexpressed in PLGG, and overexpression results in a decrease in cell growth and induction of apoptosis, findings that deserve further investigation given its potential as a novel therapeutic strategy for PLGG.

Molecular mechanisms of astrogliosis: new approaches with mouse genetics.

Astrocytes are increasingly being recognized as dynamic participants in many aspects of normal central nervous system function. In disease states, reactive astrocytes undergo complex phenotypic changes, generically referred to as astrogliosis. Unraveling the functions of reactive astrocytes and underlying molecular mechanisms is a difficult problem. The use of genetically modified mice is beginning to yield some answers to long-standing questions in the field. What are the functions of reactive astrocytes? What extracellular factors and intracellular signaling mechanisms are responsible for astrocyte activation in various forms of neural injury? In this review we will highlight studies using astrocyte reporter lines for cellular imaging and lineage tracing, as well as gain- and loss-of-function mutations that have begun to shed light on mechanisms of astrogliosis.

Systematic repression of transcription factors reveals limited patterns of gene expression changes in ES cells.

Networks of transcription factors (TFs) are thought to determine and maintain the identity of cells. Here we systematically repressed each of 100 TFs with shRNA and carried out global gene expression profiling in mouse embryonic stem (ES) cells. Unexpectedly, only the repression of a handful of TFs significantly affected transcriptomes, which changed in two directions/trajectories: one trajectory by the repression of either Pou5f1 or Sox2; the other trajectory by the repression of either Esrrb, Sall4, Nanog, or Tcfap4. The data suggest that the trajectories of gene expression change are already preconfigured by the gene regulatory network and roughly correspond to extraembryonic and embryonic fates of cell differentiation, respectively. These data also indicate the robustness of the pluripotency gene network, as the transient repression of most TFs did not alter the transcriptomes.

Generation of mouse ES cell lines engineered for the forced induction of transcription factors.

Here we report the generation and characterization of 84 mouse ES cell lines with doxycycline-controllable transcription factors (TFs) which, together with the previous 53 lines, cover 7-10% of all TFs encoded in the mouse genome. Global gene expression profiles of all 137 lines after the induction of TFs for 48 hrs can associate each TF with the direction of ES cell differentiation, regulatory pathways, and mouse phenotypes. These cell lines and microarray data provide building blocks for a variety of future biomedical research applications as a community resource.

Uncovering early response of gene regulatory networks in ESCs by systematic induction of transcription factors.

To examine transcription factor (TF) network(s), we created mouse ESC lines, in each of which 1 of 50 TFs tagged with a FLAG moiety is inserted into a ubiquitously controllable tetracycline-repressible locus. Of the 50 TFs, Cdx2 provoked the most extensive transcriptome perturbation in ESCs, followed by Esx1, Sox9, Tcf3, Klf4, and Gata3. ChIP-Seq revealed that CDX2 binds to promoters of upregulated target genes. By contrast, genes downregulated by CDX2 did not show CDX2 binding but were enriched with binding sites for POU5F1, SOX2, and NANOG. Genes with binding sites for these core TFs were also downregulated by the induction of at least 15 other TFs, suggesting a common initial step for ESC differentiation mediated by interference with the binding of core TFs to their target genes. These ESC lines provide a fundamental resource to study biological networks in ESCs and mice.

Development and characterization of a hypomorphic Smith-Lemli-Opitz syndrome mouse model and efficacy of simvastatin therapy.

Smith-Lemli-Opitz syndrome (SLOS) is a genetic syndrome caused by mutations in the 3beta-hydroxysterol Delta(7)-reductase gene (DHCR7). SLOS patients have decreased cholesterol and increased 7-dehydrocholesterol (7-DHC) levels. Dietary cholesterol supplementation improves systemic biochemical abnormalities; however, because of the blood-brain barrier, the central nervous system (CNS) is not treated. Simvastatin therapy has been proposed as a means to treat the CNS. Mice homozygous for a null disruption of Dhcr7, Dhcr7(Delta3-5/Delta3-5), die soon after birth, thus they cannot be used to study postnatal development or therapy. To circumvent this problem, we produced a hypomorphic SLOS mouse model by introducing a mutation corresponding to DHCR7(T93M). Both Dhcr7(T93M/T93M) and Dhcr7(Delta3-5/T93M) mice are viable. Phenotypic findings in Dhcr7(T93M/Delta3-5) mice include CNS ventricular dilatation and two to three syndactyly. Biochemically, both Dhcr7(T93M/T93M) and Dhcr7(T93M/Delta3-5) mice have elevated tissue 7-DHC levels; however, the biochemical defect improved with age. This has not been observed in human patients, and is due to elevated Dhcr7 expression in mouse tissues. Dietary cholesterol therapy improved sterol profiles in peripheral, but not CNS tissues. However, treatment of Dhcr7(T93M/Delta3-5) mice with simvastatin decreased 7-DHC levels in both peripheral and brain tissues. Expression of Dhcr7 increased in Dhcr7(T93M/Delta3-5) tissues after simvastatin therapy, consistent with the hypothesis that simvastatin therapy improves the biochemical phenotype by increasing the expression of a Dhcr7 allele with residual enzymatic activity. We conclude that simvastatin treatment is efficacious in improving the SLOS-associated sterol abnormality found in the brain, and thus has the potential to be an effective therapeutic intervention for behavioral and learning problems associated with SLOS.

3beta-hydroxysterol Delta7-reductase and the Smith-Lemli-Opitz syndrome.

In the final step of cholesterol synthesis, 7-dehydrocholesterol reductase (DHCR7) reduces the double bond at C7-8 of 7-dehydrocholesterol to yield cholesterol. Mutations of DHCR7 cause Smith-Lemli-Opitz syndrome (SLOS). Over 100 different mutations of DHCR7 have been identified in SLOS patients. SLOS is a classical multiple malformation, mental retardation syndrome, and was the first human malformation syndrome shown to result from an inborn error of cholesterol synthesis. This paper reviews the biochemical, molecular, and mutational aspects of DHCR7.