ALBERT

Description: Abstract 2392 There is increasing recognition of the role of small noncoding RNAs in post-transcriptional regulation of gene expression in diverse tissues of eukaryotic organisms including vertebrates. MicroRNAs (miRNAs) are the best studied amongst these small RNAs and are thought to act by binding to the 3' untranslated regions (3' UTRs) of mature mRNAs in a sequence-specific fashion and preventing the initiation of peptide translation and/ or initiating mRNA degradation. Recent evidence suggests that miRNA-based regulation might involve binding to regions other than 3' UTRs including coding regions. Current approaches to defining miRNA-mRNA interactions are mostly restricted to those based on bio-informatic prediction, protein down-regulation following in-vitro transfection of miRNA precursors and luciferase assays to determine binding to 3' UTRs. None of these methods however show direct interaction between a specific miRNA and its purported target RNA. Bio-informatics-based approaches are also prone to false positive and negative results given the short length of sequence matching, and reliance on heuristics and cross-species conservation. Newer genome-wide approaches like HITS-CLIP (High Throughput Sequencing following Cross Linked Immuno Precipitation, or CLIP-Seq) overcome some of these limitations by directly isolating the miRNA-mRNA interactome bound to argonaute (AGO), a critical component of the rna-induced silencing complex (RISC)1. HITS-CLIP utilizes the ability of ultraviolet (UV) light to cross-link RNAs to proteins in their close proximity. The crosslinked miRNA-mRNA-Ago complexes are then isolated and the RNA reverse transcribed to cDNA libraries and sequenced by next generation sequencing (NGS). Given the widespread role of miRNAs in several vertebrate tissues, we hypothesized that miRNA-regulation of gene expression is operant in the hematopoietic microenvironment (ME) and thus contributes to regulation of hematopoiesis. We hence used HITS-CLIP to analyze the miRNA-mRNA interactome of three key cellular components of the ME: stromal cells, endothelium and macrophages. We have previously reported on the use of the stromal cell lines Hs27a and Hs5 to define specific functional niches within the ME. Hs27a can functionally support primitive hematopoietic stem and progenitor cells (HSPC) in cobblestone areas (CSAs) and express high levels of factors known to support HSPC such as SDF1, Jagged1 and Angiopoietin1. In contrast, Hs5 drives HSPC to mature lineages and secretes high levels of cytokines like IL1, IL6 and GCSF. Human umbilical vein endothelial cells (HUVECs) and MCSF-treated CD14+ cells were utilized for the endothelial and macrophage cultures respectively. The HITS-CLIP datasets from each of these populations were enriched for a putative binding site for miR-9 in the coding region of Matrix Metalloproteinase 2 (MMP2) mRNA. MMP2 belongs to a family of endopeptidases critical in the remodeling of extracellular matrix in several tissues and in the egress/ homing of HSPC to their functional niches in the ME. Functional binding of miR-9 to MMP2 was validated by Western-blotting of stromal cells transfected with miR-9 which revealed 〉 50% reduction of protein levels when compared to control-transfected cells. This was also confirmed by gelatin zymography which showed significantly reduced MMP2 activity in stromal cells transfected with miR-9. Finally, to confirm direct binding of miR-9 to the putative binding region on the MMP2 transcript, we cloned this microRNA responsive region (MRE) downstream of the Renilla luciferase gene and assayed its activity by luciferase assays. MiR-9 transfection down-regulated luciferase activity 〉 50% confirming direct binding to the MRE. Our results show that genome-wide approaches such as HITS-CLIP can be used to define in vivo miRNA-mRNA interactions in the ME and should be considered in studies that define such interactions given the significant false-positive and false negative results associated with approaches based on bio-informatics alone. The approach can also define specific interactions between miRNAs and mRNAs such as MMP2, of relevance to regulation of the hematopoietic ME. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3847 MicroRNAs (miRNAs) are small non-coding RNAs with important roles in the post-transcriptional regulation of up to 30% of all vertebrate genes. Traditional methods to determine miRNA-mRNA interactions have included transcriptional profiling of miRNAs, bio-informatic prediction of miRNA-mRNA binding, analysis of 3` untranslated region (3`UTR) binding of miRNAs and over-expression of miRNAs in relevant cell types. These studies however fall short of demonstrating direct interaction between a miRNA and its target mRNAs. We applied a recently described biochemical technique of high throughput sequencing following cross-linked immune precipitation (HITS-CLIP) to dissecting the miRNA-mRNA interactions in two functionally distinct human marrow stromal cell lines. HITS-CLIP relies on the ability of ultraviolet (UV) radiation to cross-link RNA to proteins they are bound to, followed by immune-precipitation of the RNA-protein complex to isolate the cross-linked RNA and sequencing by high throughput techniques. As miRNA-mRNA interactions occur in close proximity to the argonaute proteins (AGO), an anti-argonaute monoclonal antibody was used to isolate the Ago-miRNA-mRNA complexes. The two stromal cell lines analyzed by HITS-CLIP (designated HS5 and HS27a) were isolated from a normal marrow primary long term culture (LTC), immortalized and extensively characterized for both function and expression profiles (mRNA and miRNA). HS5 was found to secrete growth factors that stimulate proliferation and differentiation of hematopoietic progenitors (G-CSF, IL-6, IL-1α and IL1β), whereas HS27a expresses activities associated with the stem cell niche (CXCL12, Angiopoietin-1, Jag1 etc). In keeping with this, HS5 conditioned media stimulated proliferation and differentiation of isolated CD34+ cells whereas HS27a supported CD34+ cells in an undifferentiated state. Sequence reads from the HITS-CLIP analysis from each of the cell lines were aligned to the human genome using the UCSC genome browser to identify Ago-mRNA and Ago-miRNA binding sites in both the cell lines. Interestingly, corresponding datasets from HS5 and HS27a were similar for the majority of mRNAs and miRNAs, but distinct for those mRNAs (such as Jag1, CXCL12, IL6 and GCSF) and miRNAs (such as miR-886-3p, miR-221, miR-181a and miR-193a) known to be differentially expressed between the two cell lines. We then validated the use of the HITS-CLIP strategy in stromal cells by analyzing one such Ago-mRNA binding site for Jagged1 (Jag1). Jag1 is a ligand for Notch1 and is expressed in those cells that support the hematopoietic stem cell (HSC) niche. The Notch pathway is a highly conserved signaling system critical in regulating several tissue systems including hematopoietic cells. This binding site, 1749 bp downstream of the transcriptional start-site for Jag1 was significantly more enriched in HS5 compared to HS27a. The site was also a predicted binding site for miR-193a, a miRNA over-expressed in HS5 compared to HS27a cells. Over-expression of miR-193a in HS27a cells resulted in the down-regulation of Jag1 protein (as measured by Western blotting). To confirm the direct interaction between Jag1 and miR-193a, we cloned this purported binding site downstream of the luciferase gene and co-transfected the plasmid with miR-193a. Luciferase activity was down-regulated greater than 50% when compared to control transfections suggesting a direct effect of miR-193a on Jag1 transcript. In summary, our data suggest that HITS-CLIP methodology can be used to define in vivo spatial interactions between miRNA and mRNAs in the marrow microenvironment (ME). It can also be used to define miRNA-based regulation of specific genes such as Jag1, which are critical to defining functional niches in the ME. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder with a prevalence ranging from 0.8 to 1.3%. VWD is characterized by incomplete penetrance, variable expressivity with markedly variable VWD antigen levels, even in families with a unifying VWF mutation, suggesting the presence of modifier genes or epigenetic modifications affecting expression of the VWF gene. We had previously diagnosed and characterized an 821-member, multigenerational Amish family in which 121 individuals exhibit a single autosomal-dominant C4120T mutation (R1374C) in the A1 domain of the mature VWF molecule. VWF antigen levels were found to vary significantly, from 9-76 in the individuals exhibiting the C4120T mutation vs 46-483 in the individuals not exhibiting the mutation. To date, very little is known about the epigenetics of VWD and no data exist investigating promoter methylation as a modifier of disease severity. We hypothesized that VWF gene is epigenetically silenced by aberrant DNA methylation resulting in differential expression of VWF protein in patients with VWD. We report our findings suggesting that promoter methylation within the VWF gene may modify VWD severity. Methods: Genomic DNA previously extracted from whole blood using DNeasy kit was CT converted with the use of the EZ DNA Methylation Kit. Primers encompassing a CpG island containing 4 CpG sites in the 5’ region of the VWF gene spanning from 138 to 490 were designed using MethPrimer. The resulting PCR products were gel-extracted and purified, cloned using the TOPO TA Cloning Kit, transformed into competent, Amp resistant E. coli cells and grown on an Ampicillin containing media. This was done in 30 samples, 20 of which contain the disease-characterizing C4120T mutation and 10 which did not. Approximately 20 colonies from each of the 30 samples were randomly chosen and grown in culture media. Plasmid DNA was extracted and sequenced on an ABI-Prism 3100 Genetic Analyzer. The methylation status of 4 CpG dinucleotides of the VWF promoter regions was analyzed using BiQ Analyzer software. Results: We performed ANOVA comparing the high VWF antigen level clones against the low VWF antigen level clones within the C4120T mutation subset and similarly for the controls. Methylation percentage was higher in the low antigen level group at each of the four CpG sites although none reached statistical significance. At two sites, we achieved near statistical significance CpG1 (p=0.100) and CpG2 (p=0.063). Among the control individuals, we did not observe this trend. Methylation was higher in the low antigen group vs the high antigen level group at CpG sites 2 and 3, but lower at CpG sites 1 and 4 with CpG4 bordering on statistical significant (p=0.053). We also performed a Spearman Rank-Order Correlation Coefficient which yielded a near-significant trend between VWF antigen level and methylation percentage within the mutation-bearing, low-expression subset at CpG4 (p=0.064). Discussion: Promoter methylation is known to modify gene expression in numerous disease processes. For the first time, we demonstrate a possible epigenetic effect contributing to the known variance in severity of von Willebrand Disease that is known to correlate with VWF antigen levels. Although our data do not achieve statistical significance, we observed a trend of higher methylation associated with low antigen levels in individuals with the C4120T mutation at two CpG sites. We also observed a near significant association between degree of methylation and VWF antigen levels at 1 CpG site within the mutation-bearing, low antigen level subset. Further studies with larger number of individuals will confirm or refute this observation. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3406 Ionizing radiation causes dose dependent damages in many organs with most pronounced effects on those with high proliferative potential such as the hematopoietic system and gastrointestinal tract. Lympho-hematopoietic failure is often the cause of death following moderate to severe exposures to radiation. Effects of low to moderate (sub-lethal) doses of radiation can be mitigated by cytokines such as granuclocyte colony stimulating factor (GCSF) or Flt-3 ligand; higher doses of radiation require a new hematopoietic system by stem cell transplantation (SCT). As SCT for large numbers of radiation victims is not only costly but impractical following mass casualties (given the need for tissue matching prior to transplantation), there is considerable interest in cellular therapies that can be rapidly expanded, have a long shelf life and do not require tissue matching. Mesenchymal stromal cells (MSC, also referred to as mesenchymal stem cells) have been proposed as one such cellular therapy that could improve survival after moderate to high doses of radiation, but direct evidence for such a clinical benefit is scant in pre-clinical models. To determine if infusion of MSCs following ionizing radiation may rescue hematopoiesis after lethal irradiation in the murine model, we first determined the LD50 (lethal dose 50 or the dose at which 50% of animals survive without specific intervention) in C57/BL6 mice to be between 600 and 700 cGy when a X-Ray irradiator (RS2000) was used as the source of ionizing radiation. We then radiated adult female C57/Bl6 mice (age 6 to 8 weeks) with 700 cGy followed by infusion of either pooled immortalized MSC clones (henceforth referred to as cMSC, 1×106 cells each, n=19) or primary MSC (referred to as pMSC, 1×106 cells each, n=20) by tail vein injection 2 hours later. MSC were syngeneic to the recipients. Control mice received equal volume of PBS (n=21). Survival at the end of 7 weeks after radiation was determined using log-rank test which showed that animals that received either cMSC or pMSCs had significantly improved survival rates (p values of 0.017 and 0.041 respectively) when compared to the control animals that received only PBS (Figure 1). The precise mechanism of action of MSCs after systemic infusion in various tissue injury models is currently undefined. Although trans-differentiation to host tissues has been proposed as one potential mechanism, recent reports have suggested that there is little evidence of persistence of infused MSC after the initial few days in the target tissues making trans-differentiation unlikely. Hence we determined the in vivo distribution kinetics of infused MSC by three techniques: whole-body bioluminescent imaging. (BLI), immune histochemistry (IHC) and quantitative real time polymerase chain reaction (q RT PCR). MSC were labeled with firefly luciferase (ffLuc) using a lentiviral vector and infused to adult female recipients after they were administered 700 cGy radiation (1×106 cells each, n=7). In all mice, strong bioluminescent signals were detected from the chest region at 4 hours after infusion, suggesting an accumulation of infused MSCs in the lungs. The signals rapidly decreased during the first 24 hours, and no bioluminescent signal was detected at 72 hours after infusion. No signals were detectable from other organs (liver, spleen or long bones) at any time point. Ffluc could not be reliably detected in tissues by IHC. Detection of luciferase transcripts in different tissues (lungs, hearts, spleens, livers, guts, muscles, and bone marrow) was performed by quantitative RT PCR at days 1, 4 and 7 (n = 6 each) following infusion. Ffluc transcripts were reliably detected in all animals only in the lungs at 24 hours after MSC infusion, confirming the BLI results. At subsequent time points, ffluc transcripts were detectable at very low levels in a variable proportion of animals from various tissues. In the absence of reliable BLI signals from these extra-pulmonary tissues, we interpret the presence of low levels of ffluc transcripts in these tissues as to have arisen from unviable cells or circulating RNA. Together, these results show that both immortalized MSCs and primary MSCs improve hematopoietic recovery after lethal ionizing radiation, but the infused cells are mostly filtered out by the lungs and their beneficial effect is likely mediated by indirect mechanisms (secondary effector cells in the lungs or secreted cytokines). Disclosures: No relevant conflicts of interest to declare.