Detection of rare thalassemia mutations using long-read single-molecule real-time sequencing.

Gap- polymerase chain reaction (PCR), reverse dot-blot assay (RDB), real-time PCR based multicolor melting curve analysis (MMCA assay), multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing are conventional methods to diagnose thalassemia but all of them have limitations. In this study, we applied single-molecule real-time (SMRT) sequencing following multiplex long-range PCR to uncover rare mutations in nine patients and their family members. The patients with different results between Gap-PCR and MMCA assay or with phenotype not matching genotype were included. Using SMRT sequencing, we first identified the carriers with αααanti3.7/HKαα, -α762bpα/αα (chr16:172,648-173,409), ααfusion/αQSα (in a trans configuration), two cases with novel gene rearrangements and another case with a novel 341 bp insertion in α-globin gene cluster, respectively. One carrier with --SEA/αααanti4.2, and two carriers with the coexistence of globin variant and an α-globin gene duplication were also found. Most importantly, we could determine two defects in α-globin gene cluster being a cis or trans configuration in a single test. Our results showed that SMRT has great advantages in detection of α-globin gene triplications, rare deletions and determination of a cis or trans configuration. SMRT is a comprehensive and one-step method for thalassemia screening and diagnosis, especially for detection of rare thalassemia mutations.

Publisher Correction: A shared Runx1-bound Zbtb16 enhancer directs innate and innate-like lymphoid lineage development.

In the original PDF version of this Article, which was published on 16 October 2017, the publication date was incorrectly given as 11 October 2017. This has now been corrected in the PDF; the HTML version of the paper was correct from the time of publication.

A shared Runx1-bound Zbtb16 enhancer directs innate and innate-like lymphoid lineage development.

Zbtb16-encoded PLZF is a signature transcription factor (TF) that directs the acquisition of T-helper effector programs during the development of multiple innate lymphocyte lineages, including natural killer T cell, innate lymphoid cell, mucosal-associated invariant T cell and γδ lineages. PLZF is also essential in osteoblast and spermatogonial development. How Zbtb16 itself is regulated in different lineages is incompletely understood. Here, by systematic CRISPR/Cas9-assisted deletions of chromatin accessible regions within the Zbtb16 locus in mouse, we identify a critical enhancer controlling PLZF expression exclusively in innate lymphoid lineages. Multiple sites within this enhancer express canonical motifs for the TF Runx1, which is essential for the development of these lineages. Notably, some regulatory sites control the kinetic rather than the overall level of PLZF expression. Thus, our comprehensive, unbiased analysis of regulatory elements in vivo reveals critical mechanisms of Zbtb16 regulation shared between innate and innate-like lymphoid lineages. Zbtb16-encoded transcription factor PLZF directs the differentiation of multiple innate and innate-like cell lineages, but how Zbtb16 itself is regulated remains unclear. Here the authors show, using CRISPR gene editing, ATAC-seq and ChIP-seq, that specific Runx1-bound enhancer elements critically modulate lineage-dependent expressions of PLZF.

Multiple layers of transcriptional regulation by PLZF in NKT-cell development.

The transcription factor PLZF [promyelocytic leukemia zinc finger, encoded by zinc finger BTB domain containing 16 (Zbtb16)] is induced during the development of innate and innate-like lymphocytes to direct their acquisition of a T-helper effector program, but the molecular mechanisms involved are poorly understood. Using biotinylation-based ChIP-seq and microarray analysis of both natural killer T (NKT) cells and PLZF-transgenic thymocytes, we identified several layers of regulation of the innate-like NKT effector program. First, PLZF bound and regulated genes encoding cytokine receptors as well as homing and adhesion receptors; second, PLZF bound and activated T-helper-specific transcription factor genes that in turn control T-helper-specific programs; finally, PLZF bound and suppressed the transcription of Bach2, a potent general repressor of effector differentiation in naive T cells. These findings reveal the multilayered architecture of the transcriptional program recruited by PLZF and elucidate how a single transcription factor can drive the developmental acquisition of a broad effector program.

Ulcerative Colitis Database: An Integrated Database and Toolkit for Gene Function and Medication Involved in Ulcerative Colitis.

BACKGROUND: Over the last decade, a massive amount of well-annotated genomic data has been accumulated on the pathogenesis and therapies for ulcerative colitis (UC). However, a comprehensive repository is not available yet. METHODS: Ulcerative Colitis Database (UCDB) was constructed using text mining followed by manually curating on the literature to collect the reliable information of UC-related genes, drugs, and susceptibility loci. UC DNA microarray data were collected. R packages were used to implement gene expression analysis toolkit. RESULTS: UCDB includes 4 separate but closely related components: "UC GENE," "UC DRUG," "UC LOCUS," and "UC ANALYSIS." The UC GENE contains comprehensive information for 1151 UC-related genes manually curated from 2919 publications. The UC DRUG includes information for 248 drugs manually curated from 2344 publications. "UC LOCUS" includes 110 UC susceptibility SNP loci, which were collected from 12 Genome-Wide Association Studies. A comprehensive expression quantitative trait loci browser was also implemented. The UC ANALYSIS is an expression analysis toolkit for 37 UC expression array data sets, which contains 1098 samples. The toolkit can be used to do gene expression correlation, clustering, differentially expressed, and Gene Set Enrichment Analysis (GSEA). CONCLUSIONS: UCDB provides a comprehensive collection of well-curated UC-related genes and drugs, and straightforward interfaces for gene expression analyses. UCDB is a useful leading resource for both basic and clinical research and will benefit UC community worldwide. UCDB is freely accessible at http://seiwertlab.uchicago.edu/UCDB.

Expression and regulation of long noncoding RNAs in TLR4 signaling in mouse macrophages.

BACKGROUND: Though long non-coding RNAs (lncRNAs) are emerging as critical regulators of immune responses, whether they are involved in LPS-activated TLR4 signaling pathway and how is their expression regulated in mouse macrophages are still unexplored. RESULTS: By repurposing expression microarray probes, we identified 994 lncRNAs in bone marrow-derived macrophages (BMDMs) and classified them to enhancer-like lncRNAs (elncRNAs) and promoter-associated lncRNAs (plncRNAs) according to chromatin signatures defined by relative levels of H3K4me1 and H3K4me3. Fifteen elncRNAs and 12 plncRNAs are differentially expressed upon LPS stimulation. The expression change of lncRNAs and their neighboring protein-coding genes are significantly correlated. Also, the regulation of both elncRNAs and plncRNAs expression is associated with H3K4me3 and H3K27Ac. Crucially, many identified LPS-regulated lncRNAs, such as lncRNA-Nfkb2 and lncRNA-Rel, locate near to immune response protein-coding genes. The majority of LPS-regulated lncRNAs had at least one binding site among the transcription factors p65, IRF3, JunB and cJun. CONCLUSIONS: We established an integrative microarray analysis pipeline for profiling lncRNAs. Also, our results suggest that lncRNAs can be important regulators of LPS-induced innate immune response in BMDMs.

A negative feedback loop mediated by the Bcl6-cullin 3 complex limits Tfh cell differentiation.

Induction of Bcl6 (B cell lymphoma 6) is essential for T follicular helper (Tfh) cell differentiation of antigen-stimulated CD4(+) T cells. Intriguingly, we found that Bcl6 was also highly and transiently expressed during the CD4(+)CD8(+) (double positive [DP]) stage of T cell development, in association with the E3 ligase cullin 3 (Cul3), a novel binding partner of Bcl6 which ubiquitinates histone proteins. DP stage-specific deletion of the E3 ligase Cul3, or of Bcl6, induced the derepression of the Bcl6 target genes Batf (basic leucine zipper transcription factor, ATF-like) and Bcl6, in part through epigenetic modifications of CD4(+) single-positive thymocytes. Although they maintained an apparently normal phenotype after emigration, they expressed increased amounts of Batf and Bcl6 at basal state and produced explosive and prolonged Tfh responses upon subsequent antigen encounter. Ablation of Cul3 in mature CD4(+) splenocytes also resulted in dramatically exaggerated Tfh responses. Thus, although previous studies have emphasized the essential role of Bcl6 in inducing Tfh responses, our findings reveal that Bcl6-Cul3 complexes also provide essential negative feedback regulation during both thymocyte development and T cell activation to restrain excessive Tfh responses.

TRIM4 modulates type I interferon induction and cellular antiviral response by targeting RIG-I for K63-linked ubiquitination.

RIG-I is a pivotal cytoplasmic sensor that recognizes different species of viral RNAs. This recognition leads to activation of the transcription factors NF-κB and IRF3, which collaborate to induce type I interferons (IFNs) and innate antiviral response. In this study, we identified the TRIM family protein TRIM4 as a positive regulator of RIG-I-mediated IFN induction. Overexpression of TRIM4 potentiated virus-triggered activation of IRF3 and NF-κB, as well as IFN-ß induction, whereas knockdown of TRIM4 had opposite effects. Mechanistically, TRIM4 associates with RIG-I and targets it for K63-linked polyubiquitination. Our findings demonstrate that TRIM4 is an important regulator of the virus-induced IFN induction pathways by mediating RIG-I for K63-linked ubiquitination.

Effect of probiotics on inducing remission and maintaining therapy in ulcerative colitis, Crohn's disease, and pouchitis: meta-analysis of randomized controlled trials.

BACKGROUND: Whether probiotics are beneficial at all stages of treatment in inflammatory bowel disease or superior to placebo remains controversial. METHODS: Two reviewers independently selected randomized controlled trials comparing probiotics with controls in inflammatory bowel disease and extracted data related to remission/response rates, relapse rates, and adverse events. Subanalyses were also performed. RESULTS: Twenty-three randomized controlled trials with a total of 1763 participants met the inclusion criteria. From the meta-analysis, probiotics significantly increase the remission rates in patients with active ulcerative colitis (UC) (P = 0.01, risk ratio [RR] = 1.51). The remission rates were significantly higher in patients with active UC treated with probiotics than placebo (P < 0.0001, RR = 1.80). Unfortunately, subgroup analysis found that only VSL#3 significantly increased the remission rates compared with controls in patients with active UC (P = 0.004, RR = 1.74). Interestingly, VSL#3 (P < 0.00001, RR = 0.18) also significantly reduced the clinical relapse rates for maintaining remission in patients with pouchitis. No significantly different adverse events were detected between probiotics and controls in the treatment of UC (P = 0.94, RR = 0.99) or CD (P = 0.33, RR = 0.87). CONCLUSIONS: Administration of probiotics results in additional benefit in inducing remission of patients with UC. VSL#3 are beneficial for maintaining remission in patients with pouchitis. And, probiotics can provide the similar effect as 5-aminosalicylic acid on maintaining remission of UC, although no additional adverse events presented.

BTB-ZF factors recruit the E3 ligase cullin 3 to regulate lymphoid effector programs.

The differentiation of several T- and B-cell effector programs in the immune system is directed by signature transcription factors that induce rapid epigenetic remodelling. Here we report that promyelocytic leukaemia zinc finger (PLZF), the BTB-zinc finger (BTB-ZF) transcription factor directing the innate-like effector program of natural killer T-cell thymocytes, is prominently associated with cullin 3 (CUL3), an E3 ubiquitin ligase previously shown to use BTB domain-containing proteins as adaptors for substrate binding. PLZF transports CUL3 to the nucleus, where the two proteins are associated within a chromatin-modifying complex. Furthermore, PLZF expression results in selective ubiquitination changes of several components of this complex. CUL3 was also found associated with the BTB-ZF transcription factor BCL6, which directs the germinal-centre B cell and follicular T-helper cell programs. Conditional CUL3 deletion in mice demonstrated an essential role for CUL3 in the development of PLZF- and BCL6-dependent lineages. We conclude that distinct lineage-specific BTB-ZF transcription factors recruit CUL3 to alter the ubiquitination pattern of their associated chromatin-modifying complex. We propose that this new function is essential to direct the differentiation of several T- and B-cell effector programs, and may also be involved in the oncogenic role of PLZF and BCL6 in leukaemias and lymphomas.

Tripartite motif 8 (TRIM8) modulates TNFα- and IL-1ß-triggered NF-κB activation by targeting TAK1 for K63-linked polyubiquitination.

The tripartite motif (TRIM)-containing proteins are a family of proteins that have been known to be involved in divergent biological processes, including important roles in immune responses through regulating various signaling pathways. In this study, we identified a member of the TRIM family, TRIM8, as a positive regulator of tumor necrosis factor-α (TNFα) and interleukin-1ß (IL-1ß)-triggered NF-κB activation. Overexpression of TRIM8 activated NF-κB and potentiated TNFα- and IL-1ß-induced activation of NF-κB, whereas knockdown of TRIM8 had opposite effects. Coimmunoprecipitations indicated that TRIM8 interacted with TGFß activated kinase 1 (TAK1), a serine/threonine kinase essential for TNFα- and IL-ß-induced NF-κB activation. Furthermore, we found that TRIM8 mediated K63-linked polyubiquitination of TAK1 triggered by TNFα and IL-1ß. Our findings demonstrate that TRIM8 serves as a critical regulator of TNFα- and IL-1ß-induced NF-κB activation by mediating K63-linked polyubiquitination of TAK1.

SENP2 negatively regulates cellular antiviral response by deSUMOylating IRF3 and conditioning it for ubiquitination and degradation.

Transcription factor IRF3-mediated type I interferon induction is essential for antiviral innate immunity. We identified the deSUMOylating enzyme Sentrin/SUMO-specific protease  (SENP) 2 as a negative regulator of virus-triggered IFN-ß induction. Overexpression of SENP2 caused IRF3 deSUMOylation, K48-linked ubiquitination, and degradation, whereas depletion of SENP2 had opposite effects. Both the SUMOylation and K48-linked ubiquitination of IRF3 occurred at lysines 70 and 87, and these processes are competitive. The level of virus-triggered IFN-ß was markedly up-regulated and viral replication was reduced in SENP2-deficient cells comparing with wild-type controls. Our findings suggest that SENP2 regulates antiviral innate immunity by deSUMOylating IRF3 and conditioning it for ubiquitination and degradation, and provide an example of cross-talk between the ubiquitin and SUMO pathways in innate immunity.

Virus-triggered ubiquitination of TRAF3/6 by cIAP1/2 is essential for induction of interferon-beta (IFN-beta) and cellular antiviral response.

Viral infection causes activation of transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and cellular antiviral response. Here we show that knockdown of the E3 ubiquitin ligases cIAP1 and cIAP2 markedly inhibited virus-triggered activation of IRF3 and NF-kappaB as well as IFN-beta induction. Knockdown of cIAP1 and cIAP2 also inhibited cytoplasmic dsRNA-triggered cellular antiviral response. Endogenous coimmunoprecipitation experiments indicated that viral infection caused recruitment of cIAP1 and cIAP2 to TRAF3, TRAF6, and VISA. Furthermore, we demonstrated that cIAP1- and cIAP2-mediated virus-triggered ubiquitination of TRAF3 and TRAF6. These findings suggest that virus-triggered ubiquitination of TRAF3 and TRAF6 by cIAP1 and cIAP2 is essential for type I IFN induction and cellular antiviral response.

Regulation of virus-triggered signaling by OTUB1- and OTUB2-mediated deubiquitination of TRAF3 and TRAF6.

Ubiquitination and deubiquitination have emerged as critical post-translational regulatory mechanisms for activation or attenuation of the virus-triggered type I interferon (IFN)(2) induction pathways. In this study, we identified two deubiquitinating enzymes, OTUB1 and OTUB2, as negative regulators of virus-triggered type I IFN induction. Overexpression of OTUB1 and OTUB2 inhibited virus-induced activation of IRF3 and NF-kappaB, transcription of the IFNB1 gene as well as cellular antiviral response, whereas knockdown of OTUB1 and OTUB2 had opposite effects. Coimmunoprecipitations indicated OTUB1 and -2 interacted with TRAF3 and TRAF6, two E3 ubiquitin ligases required for virus-triggered IRF3 and NF-kappaB activation, respectively. Furthermore, we found that OTUB1 and OTUB2 mediated virus-triggered deubiquitination of TRAF3 and -6. These findings suggest that OTUB1 and OTUB2 negatively regulate virus-triggered type I IFN induction and cellular antiviral response by deubiquitinating TRAF3 and -6.

ISG56 is a negative-feedback regulator of virus-triggered signaling and cellular antiviral response.

IFN-stimulated gene 56 (ISG56) is one of the first identified proteins induced by viruses and type I IFNs. In this study, we identified ISG56 as a virus-induced protein associated with MITA, an adapter protein involved in virus-triggered induction of type I IFNs. Overexpression of ISG56 inhibited Sendai virus-triggered activation of IRF3, NF-kappaB, and the IFN-beta promoter, whereas knockdown of ISG56 had opposite effects. Consistently, overexpression of ISG56 reversed cytoplasmic poly(I:C)-induced inhibition of vesicular stomatitis virus (VSV) replication, whereas knockdown of ISG56 inhibited VSV replication. Competitive coimmunoprecipitation experiments indicated that ISG56 disrupted the interactions between MITA and VISA or TBK1, two components in the virus-triggered IFN signaling pathways. These results suggest that ISG56 is a mediator of negative-feedback regulation of virus-triggered induction of type I IFNs and cellular antiviral responses.

The ubiquitin ligase RNF5 regulates antiviral responses by mediating degradation of the adaptor protein MITA.

Viral infection activates transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. MITA (also known as STING) has recently been identified as an adaptor that links virus-sensing receptors to IRF3 activation. Here, we showed that the E3 ubiquitin ligase RNF5 interacted with MITA in a viral-infection-dependent manner. Overexpression of RNF5 inhibited virus-triggered IRF3 activation, IFNB1 expression, and cellular antiviral response, whereas knockdown of RNF5 had opposite effects. RNF5 targeted MITA at Lys150 for ubiquitination and degradation after viral infection. Both MITA and RNF5 were located at the mitochondria and endoplasmic reticulum (ER) and viral infection caused their redistribution to the ER and mitochondria, respectively. We further found that virus-induced ubiquitination and degradation of MITA by RNF5 occurred at the mitochondria. These findings suggest that RNF5 negatively regulates virus-triggered signaling by targeting MITA for ubiquitination and degradation at the mitochondria.

GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth.

Here, we report the identification of GIDE, a mitochondrially located E3 ubiquitin ligase. GIDE contains a C-terminal RING finger domain, which is mostly conserved with those of the IAP family members and is required for GIDE's E3 ligase activity. Overexpression of GIDE induces apoptosis via a pathway involving activation of caspases, since caspase inhibitors, XIAP and an inactive mutant of caspase-9 block GIDE-induced apoptosis. GIDE also activates JNK, and blockage of JNK activation inhibits GIDE-induced release of cytochrome c and Smac as well as apoptosis, suggesting that JNK activation precedes release of cytochrome c and Smac and is required for GIDE-induced apoptosis. These pro-apoptotic properties of GIDE require its E3 ligase activity. When somewhat over- or underexpressed, GIDE slows or accelerates cell growth, respectively. These pro-apoptotic or growth inhibition effects of GIDE may account for its absence in tumor cells.