Quantifying evolvability in small biological networks

We introduce a quantitative measure of the capacity of a small biological network to evolve. We apply our measure to a stochastic description of the experimental setup of Guet et al. (Science 296:1466, 2002), treating chemical inducers as functional inputs to biochemical networks and the expression of a reporter gene as the functional output. We take an information-theoretic approach, allowing the system to set parameters that optimize signal processing ability, thus enumerating each network's highest-fidelity functions. We find that all networks studied are highly evolvable by our measure, meaning that change in function has little dependence on change in parameters. Moreover, we find that each network's functions are connected by paths in the parameter space along which information is not significantly lowered, meaning a network may continuously change its functionality without losing it along the way. This property further underscores the evolvability of the networks.Comment: 8 pages, 3 figure

<i>Trans</i>-vaccenic acid reprograms CD8⁺ T cells and anti-tumour immunity

Diet-derived nutrients are inextricably linked to human physiology by providing energy and biosynthetic building blocks and by functioning as regulatory molecules. However, the mechanisms by which circulating nutrients in the human body influence specific physiological processes remain largely unknown. Here we use a blood nutrient compound library-based screening approach to demonstrate that dietary trans-vaccenic acid (TVA) directly promotes effector CD8+ T cell function and anti-tumour immunity in vivo. TVA is the predominant form of trans-fatty acids enriched in human milk, but the human body cannot produce TVA endogenously. Circulating TVA in humans is mainly from ruminant-derived foods including beef, lamb and dairy products such as milk and butter, but only around 19% or 12% of dietary TVA is converted to rumenic acid by humans or mice, respectively. Mechanistically, TVA inactivates the cell-surface receptor GPR43, an immunomodulatory G protein-coupled receptor activated by its short-chain fatty acid ligands. TVA thus antagonizes the short-chain fatty acid agonists of GPR43, leading to activation of the cAMP–PKA–CREB axis for enhanced CD8+ T cell function. These findings reveal that diet-derived TVA represents a mechanism for host-extrinsic reprogramming of CD8+ T cells as opposed to the intrahost gut microbiota-derived short-chain fatty acids. TVA thus has translational potential for the treatment of tumours

Protein Arginine Methylation in Candida albicans: Role in Nuclear Transport▿

Protein arginine methylation plays a key role in numerous eukaryotic processes, such as protein transport and signal transduction. In Candida albicans, two candidate protein arginine methyltransferases (PRMTs) have been identified from the genome sequencing project. Based on sequence comparison, C. albicans candidate PRMTs display similarity to Saccharomyces cerevisiae Hmt1 and Rmt2. Here we demonstrate functional homology of Hmt1 between C. albicans and S. cerevisiae: CaHmt1 supports growth of S. cerevisiae strains that require Hmt1, and CaHmt1 methylates Npl3, a major Hmt1 substrate, in S. cerevisiae. In C. albicans strains lacking CaHmt1, asymmetric dimethylarginine and ω-monomethylarginine levels are significantly decreased, indicating that Hmt1 is the major C. albicans type I PRMT1. Given the known effects of type I PRMTs on nuclear transport of RNA-binding proteins, we tested whether Hmt1 affects nuclear transport of a putative Npl3 ortholog in C. albicans. CaNpl3 allows partial growth of S. cerevisiae npl3Δ strains, but its arginine-glycine-rich C terminus can fully substitute for that of ScNpl3 and also directs methylation-sensitive association with ScNpl3. Expression of green fluorescent protein-tagged CaNpl3 proteins in C. albicans strains with and without CaHmt1 provides evidence for CaHmt1 facilitating export of CaNpl3 in this fungus. We have also identified the C. albicans Rmt2, a type IV fungus- and plant-specific PRMT, by amino acid analysis of an rmt2Δ/rmt2Δ strain, as well as biochemical evidence for additional cryptic PRMTs

Regulation of Hematopoietic Stem Cell Quiescence - A Novel Role for p53

Abstract Although the p53 tumor suppressor can elicit cell-cycle arrest or apoptosis in hematopoietic cells upon DNA damage, its function during steady-state hematopoiesis is largely unknown. We demonstrated that the Ets transcription factor MEF/ELF4 regulates both HSC proliferation/self-renewal and quiescence, as Mef null mice exhibit greater numbers of hematopoietic stem cells and the Mef null HSCs are more quiescent than normal. As such, the hematopoietic compartment of Mef null mice shows significant resistance to chemotherapy and radiation (Lacorazza et al., Cancer Cell, 2006). In this study, we have investigated the mechanisms utilized by MEF/ELF4 to regulate the quiescence and self-renewal of hematopoietic stem cells, identifying p53 as a key regulator. We have recently found that Mef null mouse embryonic fibroblasts (mefs) accumulate p53 and undergo premature senescence; MEF appears to surpress the expression of p53 by directly upregulating Mdm2 (G. Sashida et al., submitted). We hypothesized that p53 may play a role in the enhanced stem cell quiescence or the increased HSC frequency seen in Mef null mice. To examine this, we generated p53−/− Mef −/− mice and compared HSC biology in the double knock out mice (p53−/− Mef −/−) vs p53 null mice, Mef null mice and wt mice. Loss of p53 decreased the fraction of Pyronin Ylow Lin-Sca-1+ cells, suggesting fewer quiescent HSCs, and staining of CD34-LSK cells for the proliferation marker Ki67 also showed enhanced HSC proliferation in the absence of p53 (with fewer quiescent cells present). These data suggest that p53 promotes quiescence in HSCs, and in the absence of p53, HSCs more readily enter the cell cycle. When we analyzed the DKO (p53−/− Mef −/−) mice, we observed that the percentage of G0 HSCs returned to normal, indicating that p53 is essential for maintaining the enhanced quiescence of MEF null HSCs. p21 is a major target gene of p53 in cells, and has been shown to play an important role in maintaining HSC quiescence. As expeceted, we found elevated levels of p21 mRNA in MEF null LSK cells and reasoned that p21 may account for their enhanced quiescence. We generated p21 −/− Mef −/− mice, which are viable, born at normal mendelian frequency and appear grossly normal. However, cell cycle analysis of HSCs obtained from p21 −/− Mef −/− mice showed that the enhanced quiescence in MEF null HSCs did not depend on p21, indicating that other p53 target genes play an important role in maintaining stem cell quiescence. We therefore utilized transcript profiling (Microarray studies and quantitative PCR analysis) to identify potential p53-regulated genes that may be differentialy expressed in LSK cells isolated from wild type, p53−/−, Mef −/−, and p53−/− Mef −/− mice. By ChiP and luciferase reporter assays, we show for the first time that Gfi-1 and Necdin are direct transcriptional targets of p53 in HSCs and both Gfi-1 and Necdin regulate the enhanced quiescence exhibited in MEF null HSCs. Taken together, our work defines a novel role for p53 in the maintenance of HSC quiescence. Furthermore, HSC quiescence and self-renewal appear to be mediated by different p53 target genes during steady state hematopoiesis

The p53 tumor suppressor protein is a critical regulator of hematopoietic stem cell behavior

In response to diverse stresses, the tumor suppressor p53 differentially regulates its target genes, variably inducing cell cycle arrest, apoptosis or senescence. Emerging evidence indicates that p53 plays an important role in regulating hematopoietic stem cell (HSC) quiescence, self-renewal, apoptosis and aging. The p53 pathway is activated by DNA damage, defects in ribosome biogenesis, oxidative stress and oncogene induced p19 ARF upregulation. We present an overview of the current state of knowledge about p53 (and its target genes) in regulating HSC behavior, with the hope that understanding the molecular mechanisms that control p53 activity in HSCs and how p53 mutations affect its role in these events may facilitate the development of therapeutic strategies for eliminating leukemia (and cancer) propagating cells

RUNX1/CBFβ Dosage Is Critical for MLL Leukemias Development

Abstract Transcription factors RUNX1/CBFβ play critical roles in hematopoiesis. Both of them are frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. We have previously shown that MLL, RUNX1, and CBFβ interact and form a regulatory complex to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity remains unknown. To determine the impact of MLL fusion protein on RUNX1/CBFβ, we introduced either MLL, MLL-BP (longer N-terminal Flag-tagged MLL construct which contains CXXC domain; 1-1406), or MLL-fusions together with RUNX1, CBFβ, or both RUNX1 and CBFβ into 293T cells. MLL-BP and MLL fusions significantly decreased RUNX1 levels compared with controls (empty vector and MLL). CBFβ protein was mildly decreased by MLL-BP and MLL-fusions when expressed alone. However, when CBFβ was co-expressed with RUNX1, it was significantly decreased compared with controls. The expression levels of RUNX1 and CBFβ proteins in LSK cells from Mll-Af9 knock-in mice were significantly lower than those from wild-type (WT) mice. To confirm these findings in human acute myeloid leukemia (AML), we measured the expression of RUNX1 and CBFβ at both mRNA and protein levels in various leukemia cell lines. The expression levels of RUNX1 and CBFβ proteins were significantly decreased in AML cells with MLL fusion and MLL partial tandem duplication (MLL-PTD) compared with those in AML cells without MLL aberrations. MLL fusions still have CXXC domain. In MLL-PTD, the CXXC domain is duplicated. Our data showed that RUNX1 protein is not only down-regulated by MLL fusion proteins, but also by MLL-BP. Thus, to determine which region is involved in the down-regulation of RUNX1, we introduced a series of MLL deletion mutants into 293T cells and measured RUNX1 protein expression. MLL deletion mutants without CXXC domain had no effect on RUNX1 stability. The construct which contains point mutations in CXXC domain also lacked the ability to reduce RUNX1 expression. Furthermore, overexpression of only CXXC domain and flanking regions could down-regulate RUNX1 protein expression. These results suggest that MLL fusion proteins and the N-terminal MLL portion of MLL fusions down-regulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. To understand the impact of RUNX1/CBFβ down-regulation on hematopoietic stem and progenitor cells (HSPCs), we generated RUNX1+/–/CBFβ+/– mice as a hypomorph model. The percentage of bone marrow (BM) LSK cells from RUNX1+/–/CBFβ+/– mice was significantly increased compared with that from WT mice. Using BM cells from these mice, we performed in vitro CFU assay and in vivo bone marrow transplantation (BMT) assay. BM cells from RUNX1+/–/CBFβ+/– mice provided more colonies in CFU assay compared with those from WT mice. To determine whether restoration of RUNX1 could repress the MLL mediated leukemogenesis, we retrovirally overexpressed WT RUNX1 in BM cells from Mll-Af9 knock-in mice. Using transduced BM cells, we performed in vitro CFU assay and in vivo BMT assay. RUNX1 overexpressed Mll-Af9 (Mll-Af9/RUNX1) cells underwent terminal differentiation after 2 times replating, while control vector transduced Mll-Af9 (Mll-Af9/Control) cells could still be replated more than 4 times. All the recipient mice transplanted with Mll-Af9/Control cells developed AML. In contrast, all the recipient mice transplanted with Mll-Af9/RUNX1 never develop AML. Furthermore, when we treated MLL leukemia cell lines with DOT1L inhibitor (EPZ-5676), RUNX1 protein levels in these MLL leukemia cell lines were significantly increased 48 hours after the treatment in comparing with controls treated with DMSO. However, there was no significant mRNA expression level change of RUNX1within 48 hours. Future studies are needed to fully understand the mechanism of whether this increasing RUNX1 protein level by DOT1L inhibitor is through blocking CXXC domain and flanking regions mediated degradation. In conclusion, MLL aberrations down-regulate RUNX1/CBFβ via their CXXC domain and flanking regions. Down-regulation of RUNX1/CBFβ plays critical role for MLL mediated leukemia development. Targeting RUNX1/CBFβ levels allows us to test novel therapies for MLL leukemias. Disclosures Mulloy: Celgene: Research Funding; Seattle Genetics: Research Funding; Amgen: Research Funding; NovImmune: Research Funding

Figure S7 from <i>NRAS</i> Mutant Dictates AHCYL1-Governed ER Calcium Homeostasis for Melanoma Tumor Growth

Supplementary Figure 7. AHCYL1 deficiency attenuates cell proliferation, decreases ER calcium levels, and activates the UPR.</p

Figure S11 from <i>NRAS</i> Mutant Dictates AHCYL1-Governed ER Calcium Homeostasis for Melanoma Tumor Growth

Supplementary Figure 11. There is positive correlation between ATF2 and AHCYL1 mRNA levels in human cutaneous melanoma patients by TCGA analysis.</p

Figure S3 from <i>NRAS</i> Mutant Dictates AHCYL1-Governed ER Calcium Homeostasis for Melanoma Tumor Growth

Supplementary Figure 3. AHCYL2 does not overexpress and is not critical in human NRAS-mutated melanoma as AHCYL1.</p

Figure S8 from <i>NRAS</i> Mutant Dictates AHCYL1-Governed ER Calcium Homeostasis for Melanoma Tumor Growth

Supplementary Figure 8. AHCYL1 deficiency causes cell growth attenuation, ER calcium decrease, and apoptosis in NRAS-Q61K overexpressed Mel-ST cells.</p