Functional dissection of inherited non-coding variation influencing multiple myeloma risk.

Thousands of non-coding variants have been associated with increased risk of human diseases, yet the causal variants and their mechanisms-of-action remain obscure. In an integrative study combining massively parallel reporter assays (MPRA), expression analyses (eQTL, meQTL, PCHiC) and chromatin accessibility analyses in primary cells (caQTL), we investigate 1,039 variants associated with multiple myeloma (MM). We demonstrate that MM susceptibility is mediated by gene-regulatory changes in plasma cells and B-cells, and identify putative causal variants at six risk loci (SMARCD3, WAC, ELL2, CDCA7L, CEP120, and PREX1). Notably, three of these variants co-localize with significant plasma cell caQTLs, signaling the presence of causal activity at these precise genomic positions in an endogenous chromosomal context in vivo. Our results provide a systematic functional dissection of risk loci for a hematologic malignancy.

Analysis of Trans-Ancestral SLE Risk Loci Identifies Unique Biologic Networks and Drug Targets in African and European Ancestries.

Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disorder with a prominent genetic component. Individuals of African ancestry (AA) experience the disease more severely and with an increased co-morbidity burden compared to European ancestry (EA) populations. We hypothesize that the disparities in disease prevalence, activity, and response to standard medications between AA and EA populations is partially conferred by genomic influences on biological pathways. To address this, we applied a comprehensive approach to identify all genes predicted from SNP-associated risk loci detected with the Immunochip. By combining genes predicted via eQTL analysis, as well as those predicted from base-pair changes in intergenic enhancer sites, coding-region variants, and SNP-gene proximity, we were able to identify 1,731 potential ancestry-specific and trans-ancestry genetic drivers of SLE. Gene associations were linked to upstream and downstream regulators using connectivity mapping, and predicted biological pathways were mined for candidate drug targets. Examination of trans-ancestral pathways reflect the well-defined role for interferons in SLE and revealed pathways associated with tissue repair and remodeling. EA-dominant genetic drivers were more often associated with innate immune and myeloid cell function pathways, whereas AA-dominant pathways mirror clinical findings in AA subjects, suggesting disease progression is driven by aberrant B cell activity accompanied by ER stress and metabolic dysfunction. Finally, potential ancestry-specific and non-specific drug candidates were identified. The integration of all SLE SNP-predicted genes into functional pathways revealed critical molecular pathways representative of each population, underscoring the influence of ancestry on disease mechanism and also providing key insight for therapeutic selection.

Epigenetic therapy induces transcription of inverted SINEs and ADAR1 dependency.

Cancer therapies that target epigenetic repressors can mediate their effects by activating retroelements within the human genome. Retroelement transcripts can form double-stranded RNA (dsRNA) that activates the MDA5 pattern recognition receptor1-6. This state of viral mimicry leads to loss of cancer cell fitness and stimulates innate and adaptive immune responses7,8. However, the clinical efficacy of epigenetic therapies has been limited. To find targets that would synergize with the viral mimicry response, we sought to identify the immunogenic retroelements that are activated by epigenetic therapies. Here we show that intronic and intergenic SINE elements, specifically inverted-repeat Alus, are the major source of drug-induced immunogenic dsRNA. These inverted-repeat Alus are frequently located downstream of 'orphan' CpG islands9. In mammals, the ADAR1 enzyme targets and destabilizes inverted-repeat Alu dsRNA10, which prevents activation of the MDA5 receptor11. We found that ADAR1 establishes a negative-feedback loop, restricting the viral mimicry response to epigenetic therapy. Depletion of ADAR1 in patient-derived cancer cells potentiates the efficacy of epigenetic therapy, restraining tumour growth and reducing cancer initiation. Therefore, epigenetic therapies trigger viral mimicry by inducing a subset of inverted-repeats Alus, leading to an ADAR1 dependency. Our findings suggest that combining epigenetic therapies with ADAR1 inhibitors represents a promising strategy for cancer treatment.

A small intergenic region of lp17 is required for evasion of adaptive immunity and induction of pathology by the Lyme disease spirochete.

The causative agent of Lyme disease, Borrelia burgdorferi, harbours a single linear chromosome and upwards of 23 linear and circular plasmids. Only a minority of these plasmids, including linear plasmid 17, are maintained with near-absolute fidelity during extended in vitro passage, and characterisation of any putative virulence determinants they encode has only recently begun. In this work, a mutant lacking a ~4.7 kb fragment of lp17 was studied. Colonisation of murine tissues by this lp17 mutant was significantly impaired, as was the ability to induce carditis and arthritis. The deficiency in tissue colonisation was alleviated in severe combined immunodeficient (SCID) mice, implicating a role for this plasmid region in adaptive immune evasion. Through genetic complementation, the mutant phenotype could be fully attributed to a 317 bp intergenic region that corresponds to the discontinued bbd07 ORF and upstream sequence. The intergenic region was found to be transcriptionally active, and mutant spirochetes lacking this region exhibited an overall difference in the antigenic profile during infection of an immunocompetent murine host. Overall, this study is the first to provide evidence for the involvement of lp17 in colonisation of joint and heart tissues, along with the associated pathologies caused by the Lyme disease spirochete.

Impact of human sepsis on CCCTC-binding factor associated monocyte transcriptional response of Major Histocompatibility Complex II components.

BACKGROUND: Antigen presentation on monocyte surface to T-cells by Major Histocompatibility Complex, Class II (MHC-II) molecules is fundamental for pathogen recognition and efficient host response. Accordingly, loss of Major Histocompatibility Complex, Class II, DR (HLA-DR) surface expression indicates impaired monocyte functionality in patients suffering from sepsis-induced immunosuppression. Besides the impact of Class II Major Histocompatibility Complex Transactivator (CIITA) on MHC-II gene expression, X box-like (XL) sequences have been proposed as further regulatory elements. These elements are bound by the DNA-binding protein CCCTC-Binding Factor (CTCF), a superordinate modulator of gene transcription. Here, we hypothesized a differential interaction of CTCF with the MHC-II locus contributing to an altered monocyte response in immunocompromised septic patients. METHODS: We collected blood from six patients diagnosed with sepsis and six healthy controls. Flow cytometric analysis was used to identify sepsis-induced immune suppression, while inflammatory cytokine levels in blood were determined via ELISA. Isolation of CD14++ CD16-monocytes was followed by (i) RNA extraction for gene expression analysis and (ii) chromatin immunoprecipitation to assess the distribution of CTCF and chromatin modifications in selected MHC-II regions. RESULTS: Compared to healthy controls, CD14++ CD16-monocytes from septic patients with immune suppression displayed an increased binding of CTCF within the MHC-II locus combined with decreased transcription of CIITA gene. In detail, enhanced CTCF enrichment was detected on the intergenic sequence XL9 separating two subregions coding for MHC-II genes. Depending on the relative localisation to XL9, gene expression of both regions was differentially affected in patients with sepsis. CONCLUSION: Our experiments demonstrate for the first time that differential CTCF binding at XL9 is accompanied by uncoupled MHC-II expression as well as transcriptional and epigenetic alterations of the MHC-II regulator CIITA in septic patients. Overall, our findings indicate a sepsis-induced enhancer blockade mediated by variation of CTCF at the intergenic sequence XL9 in altered monocytes during immunosuppression.

Clustered intergenic region sequences as predictors of factor H Binding Protein expression patterns and for assessing Neisseria meningitidis strain coverage by meningococcal vaccines.

Factor H binding protein (fHbp) is a major protective antigen in 4C-MenB (Bexsero®) and Trumenba®, two serogroup B meningococcal vaccines, wherein expression level is a determinant of protection. Examination of promoter-containing intergenic region (IGR) sequences indicated that nine fHbp IGR alleles covered 92% of 1,032 invasive meningococcal strains with variant 1 fHbp alleles. Relative expression values for fHbp were determined for 79 meningococcal isolates covering ten IGR alleles by quantitative reverse transcriptase polymerase chain reaction (qRT PCR). Derivation of expression clusters of IGR sequences by linear regression identified five expression clusters with five nucleotides and one insertion showing statistically associations with differences in expression level. Sequence analysis of 273 isolates examined by the Meningococcal Antigen Typing Scheme, a sandwich ELISA, found that coverage depended on the IGR expression cluster and vaccine peptide homology combination. Specific fHbp peptide-IGR expression cluster combinations were designated as 'at risk' for coverage by 4C-MenB and were detected in multiple invasive meningococcal disease cases confirmed by PCR alone and occurring in partially-vaccinated infants. We conclude that sequence-based analysis of IGR sequences is informative for assessing protein expression and has utility for culture-independent assessments of strain coverage by protein-based vaccines.

Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response.

CRISPR loci and their associated (Cas) proteins encode a prokaryotic immune system that protects against viruses and plasmids. Upon infection, a low fraction of cells acquire short DNA sequences from the invader. These sequences (spacers) are integrated in between the repeats of the CRISPR locus and immunize the host against the matching invader. Spacers specify the targets of the CRISPR immune response through transcription into short RNA guides that direct Cas nucleases to the invading DNA molecules. Here we performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection. We identified a mutation, I473F, that increases the rate of spacer acquisition by more than two orders of magnitude. Our results highlight the role of Cas9 during CRISPR immunization and provide a useful tool to study this rare process and develop it as a biotechnological application.

The cytokines IL-21 and GM-CSF have opposing regulatory roles in the apoptosis of conventional dendritic cells.

Interleukin-21 (IL-21) has broad actions on T and B cells, but its actions in innate immunity are poorly understood. Here we show that IL-21 induced apoptosis of conventional dendritic cells (cDCs) via STAT3 and Bim, and this was inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). ChIP-Seq analysis revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3. Expression of IL-21 in vivo decreased cDC numbers, and this was prevented by GM-CSF. Moreover, repetitive α-galactosylceramide injection of mice induced IL-21 but decreased GM-CSF production by natural killer T (NKT) cells, correlating with decreased cDC numbers. Furthermore, adoptive transfer of wild-type CD4+ T cells caused more severe colitis with increased DCs and interferon-γ (IFN-γ)-producing CD4+ T cells in Il21r(-/-)Rag2(-/-) mice (which lack T cells and have IL-21-unresponsive DCs) than in Rag2(-/-) mice. Thus, IL-21 and GM-CSF exhibit cross-regulatory actions on gene regulation and apoptosis, regulating cDC numbers and thereby the magnitude of the immune response.

Basal chromatin modification at the IL-4 gene in helper T cells.

Chromatin immunoprecipitations in naive CD4, but not CD8, T cells, demonstrated association of the IL-4 promoter with acetylated histone. Histone modifications and rapid IL-4 transcription were absent in conserved noncoding sequence 1 (CNS-1)(-/-) cells lacking an 8-kb-distant enhancer in the IL-4/IL-13 intergenic region, but also in CD4(-/-) and Itk(-/-) cells, which have similar Th2 deficiencies. Histones associated with the IL-13 promoter were not similarly acetylated in naive T cells, but became acetylated in differentiated Th2 cells. Conversely, Th1 differentiation induced histone methylation at the type 2 cytokine locus. Like CD4(-/-) and Itk(-/-) mice, CNS-1(-/-) BALB/c mice were highly resistant to the Th2-inducing protozoan, Leishmania major. CNS-1 deficiency led to failure of IL-4 gene repositioning to heterochromatin after Th1 polarization, possibly related to the presence of reiterative Ikaros binding sites in the intergenic element. Hyperacetylation of nonexpressed genes may serve to mark lineage-specific loci for rapid expression and further modification.

Hepatocyte NF-1 and STAT6 cooperate with additional DNA-binding factors to activate transcription of the human polymeric Ig receptor gene in response to IL-4.

Secretory IgA and IgM, which protect the mucosal surfaces, are generated by selective transport of locally produced polymeric (p)Igs through the epithelial barrier by the pIgR. The expression of this receptor, and hence the generation of secretory Igs, is modulated by numerous extracellular factors. We have previously identified a STAT6 site in intron 1 of the human pIgR gene that is required for the slow and de novo protein synthesis-dependent IL-4-mediated transcriptional activation of the gene. In this study, we show that this intronic IL-4-responsive enhancer is confined to a 250-bp region that is highly conserved in the murine pIgR gene. The enhancer was dependent on the cooperation between the STAT6 site and at least four additional DNA elements. EMSA experiments demonstrated binding by hepatocyte NF-1 to one of these DNA elements. Extensive overlap in the tissue distribution of hepatocyte NF-1 and pIgR suggests that this transcription factor contributes to tissue-specific pIgR expression. Changing the helical phase between the STAT6 site and downstream DNA elements greatly reduced the strength of the IL-4 response, suggesting that the precise organization of this enhancer is important for its proper function. Thus, several transcription factors cooperate in this enhanceosome to mediate IL-4 responsiveness in HT-29 epithelial cells.

Immunity as a function of the unicellular state: implications of emerging genomic data.

Instead of being greeted as supporting the growing corpus of immunological theory, recent advances in the bioinformatic analysis of genomes have often surprised the discoverers and failed to attract the attention of immunologists. In fact, the view that multicellular immune systems are adaptations of already highly evolved unicellular immune systems that are capable of self/not-self discrimination can assist our comprehension of phenomena, such as 'junk' DNA, genetic polymorphism and the ubiquity of repetitive elements. For instance, the 'hidden transcriptome', revealed by run-on transcription of genes or repetitive elements, contains a diverse repertoire of RNA 'immune receptors' with the potential to form double-stranded RNA with viral RNA 'antigens', thus triggering intracellular alarms.

Defined flanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string DNA vaccine.

Loss of immunogenic epitopes by tumors has urged the development of vaccines against multiple epitopes. Recombinant DNA technologies have opened the possibility to develop multiepitope vaccines in a relatively rapid and efficient way. We have constructed four naked DNA-based multiepitope vaccines, containing CTL, Th cell, and B cell epitopes of the human papillomavirus type 16. Here we show that gene gun-mediated vaccination with an epitope-based DNA vaccine protects 100% of the vaccinated mice against a lethal tumor challenge. The addition of spacers between the epitopes was crucial for the epitope-induced tumor protection, as the same DNA construct without spacers was significantly less effective and only protected 50% of the mice. When tested for therapeutic potential, only the epitope construct with defined spacers significantly reduced the size of established tumors, but failed to induce tumor regression. Only after targeting the vaccine-encoded protein to the protein degradation pathway by linking it to ubiquitin, the vaccine-induced T cell-mediated eradication of 100% of 7-day established tumors in mice. The finding that defined flanking sequences around epitopes and protein targeting dramatically increased the efficacy of epitope string DNA vaccines against established tumors will be of importance for the further development of multiepitope DNA vaccines toward clinical application.