BackgroundRNA:DNA hybrids represent a non-canonical nucleic acid structure that has been associated with a range of human diseases and potential transcriptional regulatory functions. Mapping of RNA:DNA hybrids in human cells reveals them to have a number of characteristics that give insights into their functions.ResultsWe find RNA:DNA hybrids to occupy millions of base pairs in the human genome. A directional sequencing approach shows the RNA component of the RNA:DNA hybrid to be purine-rich, indicating a thermodynamic contribution to their in vivo stability. The RNA:DNA hybrids are enriched at loci with decreased DNA methylation and increased DNase hypersensitivity, and within larger domains with characteristics of heterochromatin formation, indicating potential transcriptional regulatory properties. Mass spectrometry studies of chromatin at RNA:DNA hybrids shows the presence of the ILF2 and ILF3 transcription factors, supporting a model of certain transcription factors binding preferentially to the RNA:DNA conformation.ConclusionsOverall, there is little to indicate a dependence for RNA:DNA hybrids forming co-transcriptionally, with results from the ribosomal DNA repeat unit instead supporting the intriguing model of RNA generating these structures intrans. The results of the study indicate heterogeneous functions of these genomic elements and new insights into their formation and stability in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0040-6) contains supplementary material, which is available to authorized users.

A critical step toward understanding autism spectrum disorder (ASD) is to identify both genetic and environmental risk factors. A number of rare copy number variants (CNVs) have emerged as robust genetic risk factors for ASD, but not all CNV carriers exhibit ASD and the severity of ASD symptoms varies among CNV carriers. Although evidence exists that various environmental factors modulate symptomatic severity, the precise mechanisms by which these factors determine the ultimate severity of ASD are still poorly understood. Here, using a mouse heterozygous for Tbx1 (a gene encoded in 22q11.2 CNV), we demonstrate that a genetically-triggered neonatal phenotype in vocalization generates a negative environmental loop in pup-mother social communication. Wild-type pups used individually diverse sequences of simple and complicated call types, but heterozygous pups used individually invariable call sequences with less complicated call types. When played back, representative wild-type call sequences elicited maternal approach, but heterozygous call sequences were ineffective. When the representative wild-type call sequences were randomized, they were ineffective in eliciting vigorous maternal approach behavior. These data demonstrate that an ASD risk gene alters the neonatal call sequence of its carriers and this pup phenotype in turn diminishes maternal care through atypical social communication. Thus, an ASD risk gene induces, through atypical neonatal call sequences, less than optimal maternal care as a negative neonatal environmental factor.

WT1, a critical regulator of kidney development, is a tumor suppressor for nephroblastoma but in some contexts functions as an oncogene. A limited number of direct transcriptional targets of WT1 have been identified to explain its complex roles in tumorigenesis and organogenesis. In this study we performed genome-wide screening for direct WT1 targets, using a combination of ChIP-ChIP and expression arrays. Promoter regions bound by WT1 were highly G-rich and resembled the sites for a number of other widely expressed transcription factors such as SP1, MAZ, and ZNF219. Genes directly regulated by WT1 were implicated in MAPK signaling, axon guidance, and Wnt pathways. Among directly bound and regulated genes by WT1, nine were identified in the Wnt signaling pathway, suggesting that WT1 modulates a subset of Wnt components and responsive genes by direct binding. To prove the biological importance of the interplay between WT1 and Wnt signaling, we showed that WT1 blocked the ability of Wnt8 to induce a secondary body axis during Xenopus embryonic development. WT1 inhibited TCF-mediated transcription activated by Wnt ligand, wild type and mutant, stabilized ␤-catenin by preventing TCF4 loading onto a promoter. This was neither due to direct binding of WT1 to the TCF binding site nor to interaction between WT1 and TCF4, but by competition of WT1 and TCF4 for CBP. WT1 interference with Wnt signaling represents an important mode of its action relevant to the suppression of tumor growth and guidance of development.ChIP-ChIP ͉ microarray ͉ tumor suppressor T he genetic etiology of Wilms tumorigenesis is heterogeneous including loss-of-imprinting of IGF2 (1), deletions and mutations of WT1 (reviewed in ref.2) and the recently identified WTX (3) genes. In addition, somatic mutations in ␤-catenin leading to a stabilized protein are found in 15% of cases and curiously almost all of these mutation cases are found in patients lacking functional WT1 alleles (4-7).The WT1 tumor suppressor gene encodes a zinc finger transcription factor, possibly yielding up to 32 different isoforms (8). The major isoforms differ in the presence or absence of amino acids KTS in the zinc finger region and the presence or absence of a 17-aa stretch in the middle of the protein. The ϪKTS isoforms have been linked to DNA binding-mediated transcriptional control whereas the ϩKTS isoforms have been implicated in RNA processing as well (9). Renal agenesis in the Wt1 knockout mouse and the presence of constitutional mutations in WT1 in a number of renal developmental syndromes indicate a critical role for WT1 in kidney development.

We investigated why some donor-specific antibody-positive patients do not develop antibody-mediated rejection. Of 71 donor-specific antibody-positive patients, 46 had diagnosis of antibody-mediated rejection and 25 had normal biopsies. Fifty donor-specific antibody-negative patients with normal biopsies were used as a control group. A subgroup of 61 patients with available biopsy and 64 with blood samples were analyzed by microarrays. Both donor-specific antibody-positive/antibody-mediated rejection-positive and negative biopsies showed increased expression of gene transcripts associated with cytotoxic T cells, natural killer cells, macrophages, interferon-gamma, and rejection compared to donor-specific antibody-negative biopsies. Regulatory T-cell transcripts were upregulated in donor-specific antibody-positive/antibody-mediated rejection-positive and B-cell transcripts in donor-specific antibody-positive/antibody-mediated rejection-negative biopsies. Whole-blood gene expression analysis showed increased immune activity in only donor-specific antibody-positive/antibody-mediated rejection-positive but not negative patients. During a median follow-up of 36 months, 4 donor-specific antibody-positive/antibody-mediated rejection-negative patients developed antibody-mediated rejection, 12 continued to have donor-specific antibody, but 9 lost their donor-specific antibody. Gene expression profiles did not predict the development of antibody-mediated rejection or the persistence of donor-specific antibody. Thus, donor-specific antibody-positive/antibody-mediated rejection-negative patients had increased rejection-associated gene transcripts in their allografts despite no histologic findings of rejection but not in their blood. This was found in both biopsy and blood samples of donor-specific antibody-positive/antibody-mediated rejection-positive patients.

The diagnostic criteria for antibody-mediated rejection (AMR) are continuously evolving. Here we investigated the clinical and molecular significance of different Banff microvascular inflammation (MVI) scores in transplant kidney biopsies. A total of 356 patients with clinically indicated kidney transplant biopsies were classified into three groups based on MVI scores of 0, 1, 2, or more for Groups 1-3, respectively. Gene expression profiles were assessed using arrays on a representative subset of 93 patients. The incidence of donor-specific anti-HLA antibodies was increased from 25% in Group 1 to 36% in Group 2 and to 54% in Group 3. Acute and chronic AMR were significantly more frequent in Group 3 (15% and 35%) compared with the Group 2 (3% and 15%) and Group 1 (0% and 5%), respectively. Gene expression profiles showed increased interferon-γ and rejection-induced, cytotoxic and regulatory T-cell, natural killer cell-associated and donor-specific antibody (DSA)-selective transcripts in Group 3 compared with Groups 1 and 2. There was no significant difference in gene expression profiles between the Groups 1 and 2. Increased intragraft expression of DSA-selective transcripts was found in the biopsies of C4d- Group 3 patients. Thus, an MVI score of 2 or more was significantly associated with a histological diagnosis of acute and chronic antibody-mediated rejection. Hence, increased intragraft DSA-selective gene transcripts may be used as molecular markers for AMR, especially in C4d- biopsies.

Background: RNA:DNA hybrids represent a non-canonical nucleic acid structure that has been associated with a range of human diseases and potential transcriptional regulatory functions. Mapping of RNA:DNA hybrids in human cells reveals them to have a number of characteristics that give insights into their functions. Results:We find RNA:DNA hybrids to occupy millions of base pairs in the human genome. A directional sequencing approach shows the RNA component of the RNA:DNA hybrid to be purine-rich, indicating a thermodynamic contribution to their in vivo stability. The RNA:DNA hybrids are enriched at loci with decreased DNA methylation and increased DNase hypersensitivity, and within larger domains with characteristics of heterochromatin formation, indicating potential transcriptional regulatory properties. Mass spectrometry studies of chromatin at RNA:DNA hybrids shows the presence of the ILF2 and ILF3 transcription factors, supporting a model of certain transcription factors binding preferentially to the RNA:DNA conformation. Conclusions:Overall, there is little to indicate a dependence for RNA:DNA hybrids forming co-transcriptionally, with results from the ribosomal DNA repeat unit instead supporting the intriguing model of RNA generating these structures in trans. The results of the study indicate heterogeneous functions of these genomic elements and new insights into their formation and stability in vivo.

Purpose Assessing whole-body radiation injury and absorbed dose is essential for remediation efforts following accidental or deliberate exposure in medical, industrial, military, or terrorist incidents. We hypothesize that variations in specific metabolite concentrations extracted from blood plasma would correlate with whole-body radiation injury and dose. Methods and Materials Groups of C57BL/6 mice (n=12 per group) were exposed to 0 Gy, 2 Gy, 4 Gy, 8 Gy, and 10.4 Gy of whole-body γ-radiation. At 24 hours post treatment all animals were euthanized and both plasma and liver biopsies obtained - the latter being used to deconvolve a distinct hepatic radiation injury response within plasma. A semi-quantitative untargeted metabolites/lipid profiling using both GC/MS and LC/MS/MS platforms was performed and identified 354 biochemicals. A second set of C57BL/6 mice (n=6 per group) were used to assess a subset of identified plasma markers beyond 24 hours. Results We identified a cohort of 37 biochemical compounds in plasma that yielded the optimal separation of the irradiated sample groups, with the most correlated metabolites associated with pyrimidine (positively correlated) and tryptophan (negatively correlated) metabolism. The latter were predominantly associated with indole compounds, and there was evidence to indicate that these were also correlated between liver and plasma. No evidence of saturation as a function of dose was observed, as has been noted for studies involving metabolite analysis of urine. Conclusion Plasma profiling of specific metabolites related to the pyrimidine and tryptophan pathways can be used to differentiate whole-body radiation injury and dose response. As the tryptophan associated indole compounds have their origin in the intestinal microbiome and subsequently the liver, these metabolites in particular represent an attractive marker for radiation injury within blood plasma.

Mechanisms of treatment resistance in head and neck squamous cell carcinoma (HNSCC) are not well characterized. In this study, HNSCC tumors from a cohort of prospectively enrolled subjects on an ongoing tissue banking study were divided into those that persisted or recurred locoregionally (n=23) and those that responded without recurrence (n=35). Gene expression was evaluated using llumina HumanHT-12-v3 Expression BeadChip microarrays. Sparse Partial Least Squares – Discriminant Analysis (sPLS-DA) identified 135 genes discriminating treatment-resistant from treatment-sensitive tumors. BCL-xL was identified among 23% of canonical pathways derived from this set of genes using Ingenuity Pathway analysis. The BCL-xL protein was expressed in 8 HNSCC cell lines examined. Cells were treated with the BCL-xL inhibitor, ABT-263 (navitoclax): the average half maximal inhibitory concentration (IC50) was 8.9μM (range 6.6μM – 13.9μM). Combining ABT-263 did not significantly increase responses to 2 Gy radiation or cisplatin in the majority of cell lines. MCL-1, a potential mediator of resistance to ABT-263, was expressed in all cell lines and HNSCC patient tumors, in addition to BCL-xL. Treatment with the MCL-1 inhibitor, A-1210477, in HNSCC cell lines showed an average IC50 of 10.7μM (range, 8.8μM to 12.7μM). Adding A-1210477 to ABT-263 (navitoclax) treatment resulted in an average 7-fold reduction in the required lethal dose of ABT-263 (navitoclax) when measured across all 8 cell lines. Synergistic activity was confirmed in PCI15B, Detroit 562, MDA686LN, and HN30 based on Bliss Independence analysis. This study demonstrates that targeting both BCL-xL and MCL-1 is required to optimally inhibit BCL-family pro-survival molecules in HNSCC, and co-inhibition is synergistic in HNSCC cancer cells.