The transcription factor BCL-6 controls early development of innate-like T cells.

Innate T cells, including invariant natural killer T (iNKT) and mucosal-associated innate T (MAIT) cells, are a heterogeneous T lymphocyte population with effector properties preprogrammed during their thymic differentiation. How this program is initiated is currently unclear. Here, we show that the transcription factor BCL-6 was transiently expressed in iNKT cells upon exit from positive selection and was required for their proper development beyond stage 0. Notably, development of MAIT cells was also impaired in the absence of Bcl6. BCL-6-deficient iNKT cells had reduced expression of genes that were associated with the innate T cell lineage, including Zbtb16, which encodes PLZF, and PLZF-targeted genes. BCL-6 contributed to a chromatin accessibility landscape that was permissive for the expression of development-related genes and inhibitory for genes associated with naive T cell programs. Our results revealed new functions for BCL-6 and illuminated how this transcription factor controls early iNKT cell development.

Diabetes risk gene and Wnt effector Tcf7l2/TCF4 controls hepatic response to perinatal and adult metabolic demand.

Most studies on TCF7L2 SNP variants in the pathogenesis of type 2 diabetes (T2D) focus on a role of the encoded transcription factor TCF4 in ß cells. Here, a mouse genetics approach shows that removal of TCF4 from ß cells does not affect their function, whereas manipulating TCF4 levels in the liver has major effects on metabolism. In Tcf7l2(-/-) mice, the immediate postnatal surge in liver metabolism does not occur. Consequently, pups die due to hypoglycemia. By combining chromatin immunoprecipitation with gene expression profiling, we identify a TCF4-controlled metabolic gene program that is acutely activated in the postnatal liver. In concordance, adult liver-specific Tcf7l2 knockout mice show reduced hepatic glucose production during fasting and display improved glucose homeostasis when maintained on high-fat diet. Furthermore, liver-specific TCF4 overexpression increases hepatic glucose production. These observations imply that TCF4 directly activates metabolic genes and that inhibition of Wnt signaling may be beneficial in metabolic disease.

Transcription factor achaete scute-like 2 controls intestinal stem cell fate.

The small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. We recently demonstrated the existence of a long-lived pool of cycling stem cells defined by Lgr5 expression and intermingled with post-mitotic Paneth cells at crypt bottoms. We have now determined a gene signature for these Lgr5 stem cells. One of the genes within this stem cell signature is the Wnt target Achaete scute-like 2 (Ascl2). Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and ectopic crypts on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to disappearance of the Lgr5 stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate.

Catchet-MS identifies IKZF1-targeting thalidomide analogues as novel HIV-1 latency reversal agents.

A major pharmacological strategy toward HIV cure aims to reverse latency in infected cells as a first step leading to their elimination. While the unbiased identification of molecular targets physically associated with the latent HIV-1 provirus would be highly valuable to unravel the molecular determinants of HIV-1 transcriptional repression and latency reversal, due to technical limitations, this has been challenging. Here we use a dCas9 targeted chromatin and histone enrichment strategy coupled to mass spectrometry (Catchet-MS) to probe the differential protein composition of the latent and activated HIV-1 5'LTR. Catchet-MS identified known and novel latent 5'LTR-associated host factors. Among these, IKZF1 is a novel HIV-1 transcriptional repressor, required for Polycomb Repressive Complex 2 recruitment to the LTR. We find the clinically advanced thalidomide analogue iberdomide, and the FDA approved analogues lenalidomide and pomalidomide, to be novel LRAs. We demonstrate that, by targeting IKZF1 for degradation, these compounds reverse HIV-1 latency in CD4+ T-cells isolated from virally suppressed people living with HIV-1 and that they are able to synergize with other known LRAs.

Distinct transcriptional profile of blood mononuclear cells in Behçet's disease: insights into the central role of neutrophil chemotaxis.

OBJECTIVES: Both innate and adaptive immune responses are reportedly increased in Behçet's disease (BD), a chronic, relapsing systemic vasculitis lying at the intersection between autoinflammation and autoimmunity. To further study pathophysiologic molecular mechanisms operating in BD, we searched for transcriptome-wide changes in blood mononuclear cells from these patients. METHODS: We performed 3' mRNA next-generation sequencing-based genome-wide transcriptional profiling followed by analysis of differential expression signatures, Kyoto Encyclopedia of Genes and Genomes pathways, GO biological processes and transcription factor signatures. RESULTS: Differential expression analysis clustered the transcriptomes of 13 patients and one healthy subject separately from those of 10 healthy age/gender-matched controls and one patient. Among the total of 17 591 expressed protein-coding genes, 209 and 31 genes were significantly upregulated and downregulated, respectively, in BD vs controls by at least 2-fold. The most upregulated genes comprised an abundance of CC- and CXC-chemokines. Remarkably, the 5 out of top 10 upregulated biological processes involved leucocyte recruitment to peripheral tissues, especially for neutrophils. Moreover, NF-kB, TNF and IL-1 signalling pathways were prominently enhanced in BD, while transcription factor activity analysis suggested that the NF-kB p65/RELA subunit action underlies the observed differences in the BD transcriptome. CONCLUSION: This RNA-sequencing analysis in peripheral blood mononuclear cells derived from patients with BD does not support a major pathogenetic role for adaptive immunity-driven mechanisms, but clearly points to the action of aberrant innate immune responses with a central role played by upregulated neutrophil chemotaxis.

Correction: The Leukemia-Associated Mllt10/Af10-Dot1l Are Tcf4/ß-Catenin Coactivators Essential for Intestinal Homeostasis.

[This corrects the article DOI: 10.1371/journal.pbio.1000539.].

Spontaneous development of hepatocellular carcinoma with cancer stem cell properties in PR-SET7-deficient livers.

PR-SET7-mediated histone 4 lysine 20 methylation has been implicated in mitotic condensation, DNA damage response and replication licensing. Here, we show that PR-SET7 function in the liver is pivotal for maintaining genome integrity. Hepatocyte-specific deletion of PR-SET7 in mouse embryos resulted in G2 phase arrest followed by massive cell death and defect in liver organogenesis. Inactivation at postnatal stages caused cell duplication-dependent hepatocyte necrosis, accompanied by inflammation, fibrosis and compensatory growth induction of neighboring hepatocytes and resident ductal progenitor cells. Prolonged necrotic regenerative cycles coupled with oncogenic STAT3 activation led to the spontaneous development of hepatic tumors composed of cells with cancer stem cell characteristics. These include a capacity to self-renew in culture or in xenografts and the ability to differentiate to phenotypically distinct hepatic cells. Hepatocellular carcinoma in PR-SET7-deficient mice displays a cancer stem cell gene signature specified by the co-expression of ductal progenitor markers and oncofetal genes.

Smyd3-associated regulatory pathways in cancer.

SMYD3 is a member of the SET and MYND-domain family of methyl-transferases, the increased expression of which correlates with poor prognosis in various types of cancer. In liver and colon tumors, SMYD3 is localized in the nucleus, where it interacts with RNA Pol II and H3K4me3 and functions as a selective transcriptional amplifier of oncogenes and genes that control cell proliferation and metastatic spread. Smyd3 expression has a high discriminative power for the characterization of liver tumors and positively correlates with poor prognosis. In lung and pancreatic cancer, SMYD3 acts in the cytoplasm, potentiating oncogenic Ras/ERK signaling through the methylation of the MAP3K2 kinase and the subsequent release from its inhibitor. A clinico-pathological analysis of lung cancer patients uncovers prognostic significance of SMYD3 only for first progression survival. However, stratification of patients according to their smoking history significantly expands the prognostic value of SMYD3 to overall survival and other features, suggesting that smoking-related effects saturate the clinical analysis and mask the function of SMYD3 as an oncogenic potentiator.

Wnt-induced transcriptional activation is exclusively mediated by TCF/LEF.

Active canonical Wnt signaling results in recruitment of ß-catenin to DNA by TCF/LEF family members, leading to transcriptional activation of TCF target genes. However, additional transcription factors have been suggested to recruit ß-catenin and tether it to DNA. Here, we describe the genome-wide pattern of ß-catenin DNA binding in murine intestinal epithelium, Wnt-responsive colorectal cancer (CRC) cells and HEK293 embryonic kidney cells. We identify two classes of ß-catenin binding sites. The first class represents the majority of the DNA-bound ß-catenin and co-localizes with TCF4, the prominent TCF/LEF family member in these cells. The second class consists of ß-catenin binding sites that co-localize with a minimal amount of TCF4. The latter consists of lower affinity ß-catenin binding events, does not drive transcription and often does not contain a consensus TCF binding motif. Surprisingly, a dominant-negative form of TCF4 abrogates the ß-catenin/DNA interaction of both classes of binding sites, implying that the second class comprises low affinity TCF-DNA complexes. Our results indicate that ß-catenin is tethered to chromatin overwhelmingly through the TCF/LEF transcription factors in these three systems.

Systematic integration of RNA-Seq statistical algorithms for accurate detection of differential gene expression patterns.

RNA-Seq is gradually becoming the standard tool for transcriptomic expression studies in biological research. Although considerable progress has been recorded in the development of statistical algorithms for the detection of differentially expressed genes using RNA-Seq data, the list of detected genes can differ significantly between algorithms. We present a new method (PANDORA) that combines multiple algorithms toward a summarized result, more efficiently reflecting true experimental outcomes. This is achieved through the systematic combination of several analysis algorithms, by weighting their outcomes according to their performance with realistically simulated data sets generated from real data. Results supported by the analysis of both simulated and real data from different organisms as well as correlation with PolII occupancy demonstrate that PANDORA improves the detection of differential expression. It accomplishes this by optimizing the tradeoff between standard performance measurements, such as precision and sensitivity.

Hypoxia inducible NOD2 interacts with 3-O-sulfogalactoceramide and regulates vesicular homeostasis.

BACKGROUND: Oxygen sensing in mammalian cells is a conserved signaling pathway regulated by hypoxia inducible factor type 1 (HIF-1). Inadequate oxygen supply (hypoxia) is common to many pathological disorders where autophagy plays an import role. The aim of this study was the identification and characterization of novel HIF-1 target genes that promote autophagy during hypoxia. METHODS: Whole genome Chromatin Immune Precipitation from hypoxic HeLa cells was used to identify novel HIF-1 target genes. Hypoxia induced expression and transcription regulation was studied in wild type and HIF-deficient cells. siRNA silencing of candidate genes was used to establish their role during autophagy. Recombinant protein was used for screening immobilized glycosylated lipids to identify potential ligands. RESULTS: We identified the Nucleotide Oligomerization Domain 2 (NOD2/CARD15) as a novel HIF-1 target and 3-O-sulfo-galactoceramide (sulfatide) and Mycobacterium sp. specific sulfolipid-1 as the first NOD2 ligands that both compete for binding to NOD2. Loss of NOD2 function impaired autophagy upstream of the autophagy inhibitor chloroquine by reducing the number of acidic vesicles. Inhibition of sulfatide synthesis elicited defects in autophagy similar to the NOD2 loss of function but did not influence NOD2-mediated NF-kB signaling. CONCLUSIONS: Our findings suggest that the interaction of NOD2 with sulfatide may mediate the balance between autophagy and inflammation in hypoxic cells. GENERAL SIGNIFICANCE: These findings may lead to a better understanding of complex inflammatory pathologies like Crohn's disease and tuberculosis where both NOD2 and hypoxia are implicated.

Integrated genome-wide analysis of transcription factor occupancy, RNA polymerase II binding and steady-state RNA levels identify differentially regulated functional gene classes.

Routine methods for assaying steady-state mRNA levels such as RNA-seq and micro-arrays are commonly used as readouts to study the role of transcription factors (TFs) in gene expression regulation. However, cellular RNA levels do not solely depend on activity of TFs and subsequent transcription by RNA polymerase II (Pol II), but are also affected by RNA turnover rate. Here, we demonstrate that integrated analysis of genome-wide TF occupancy, Pol II binding and steady-state RNA levels provide important insights in gene regulatory mechanisms. Pol II occupancy, as detected by Pol II ChIP-seq, was found to correlate better with TF occupancy compared to steady-state RNA levels and is thus a more precise readout for the primary transcriptional mechanisms that are triggered by signal transduction. Furthermore, analysis of differential Pol II occupancy and RNA-seq levels identified genes with high Pol II occupancy and relatively low RNA levels and vice versa. These categories are strongly enriched for genes from different functional classes. Our results demonstrate a complementary value in Pol II chip-seq and RNA-seq approaches for better understanding of gene expression regulation.

Fingerprinting Breast Milk; insights into Milk Exosomics.

Breast milk, often referred to as "liquid gold," is a complex biofluid that provides essential nutrients, immune factors, and developmental cues for newborns. Recent advancements in the field of exosome research have shed light on the critical role of exosomes in breast milk. Exosomes are nanosized vesicles that carry bioactive molecules, including proteins, lipids, nucleic acids, and miRNAs. These tiny messengers play a vital role in intercellular communication and are now being recognized as key players in infant health and development. This paper explores the emerging field of milk exosomics, emphasizing the potential of exosome fingerprinting to uncover valuable insights into the composition and function of breast milk. By deciphering the exosomal cargo, we can gain a deeper understanding of how breast milk influences neonatal health and may even pave the way for personalized nutrition strategies.

The leukemia-associated Mllt10/Af10-Dot1l are Tcf4/ß-catenin coactivators essential for intestinal homeostasis.

Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/ß-catenin complexes. In colorectal cancer, activating mutations in Wnt pathway components cause inappropriate activation of TCF4/ß-catenin-driven transcription. Despite the passage of a decade after the discovery of TCF4 and ß-catenin as the molecular effectors of the Wnt signal, few transcriptional activators essential and unique to the regulation of this transcription program have been found. Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l as Tcf4/ß-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a ß-catenin-dependent manner, resulting in H3K79 methylation over their coding regions in vivo in proliferative crypts of mouse small intestine in colorectal cancer and Wnt-inducible HEK293T cells. Depletion of MLLT10/AF10 in colorectal cancer and Wnt-inducible HEK293T cells followed by expression array analysis identifies MLLT10/AF10 and DOT1L as essential activators to a large extent dedicated to Wnt target gene regulation. In contrast, previously published ß-catenin coactivators p300 and BRG1 displayed a more pleiotropic target gene expression profile controlling Wnt and other pathways. tcf4, mllt10/af10, and dot1l are co-expressed in Wnt-driven tissues in zebrafish and essential for Wnt-reporter activity. Intestinal differentiation defects in apc-mutant zebrafish can be rescued by depletion of Mllt10 and Dot1l, establishing these genes as activators downstream of Apc in Wnt target gene activation in vivo. Morpholino-depletion of mllt10/af10-dot1l in zebrafish results in defects in intestinal homeostasis and a significant reduction in the in vivo expression of direct Wnt target genes and in the number of proliferative intestinal epithelial cells. We conclude that Mllt10/Af10-Dot1l are essential, largely dedicated activators of Wnt-dependent transcription, critical for maintenance of intestinal proliferation and homeostasis. The methyltransferase DOT1L may present an attractive candidate for drug targeting in colorectal cancer.

TCF4 and CDX2, major transcription factors for intestinal function, converge on the same cis-regulatory regions.

Surprisingly few pathways signal between cells, raising questions about mechanisms for tissue-specific responses. In particular, Wnt ligands signal in many mammalian tissues, including the intestinal epithelium, where constitutive signaling causes cancer. Genome-wide analysis of DNA cis-regulatory regions bound by the intestine-restricted transcription factor CDX2 in colonic cells uncovered highly significant overrepresentation of sequences that bind TCF4, a transcriptional effector of intestinal Wnt signaling. Chromatin immunoprecipitation confirmed TCF4 occupancy at most such sites and co-occupancy of CDX2 and TCF4 across short distances. A region spanning the single nucleotide polymorphism rs6983267, which lies within a MYC enhancer and confers colorectal cancer risk in humans, represented one of many co-occupied sites. Co-occupancy correlated with intestine-specific gene expression and CDX2 loss reduced TCF4 binding. These results implicate CDX2 in directing TCF4 binding in intestinal cells. Co-occupancy of regulatory regions by signal-effector and tissue-restricted transcription factors may represent a general mechanism for ubiquitous signaling pathways to achieve tissue-specific outcomes.

SRC and TKS5 mediated podosome formation in fibroblasts promotes extracellular matrix invasion and pulmonary fibrosis.

The activation and accumulation of lung fibroblasts resulting in aberrant deposition of extracellular matrix components, is a pathogenic hallmark of Idiopathic Pulmonary Fibrosis, a lethal and incurable disease. In this report, increased expression of TKS5, a scaffold protein essential for the formation of podosomes, was detected in the lung tissue of Idiopathic Pulmonary Fibrosis patients and bleomycin-treated mice. Τhe profibrotic milieu is found to induce TKS5 expression and the formation of prominent podosome rosettes in lung fibroblasts, that are retained ex vivo, culminating in increased extracellular matrix invasion. Tks5+/- mice are found resistant to bleomycin-induced pulmonary fibrosis, largely attributed to diminished podosome formation in fibroblasts and decreased extracellular matrix invasion. As computationally predicted, inhibition of src kinase is shown to potently attenuate podosome formation in lung fibroblasts and extracellular matrix invasion, and bleomycin-induced pulmonary fibrosis, suggesting pharmacological targeting of podosomes as a very promising therapeutic option in pulmonary fibrosis.

Wnt signalling induces maturation of Paneth cells in intestinal crypts.

Wnt signalling, which is transduced through beta-catenin/TCF4, maintains the undifferentiated state of intestinal crypt progenitor cells. Mutational activation of the pathway initiates the adenomacarcinoma sequence. Whereas all other differentiated epithelial cells migrate from the crypt onto the villus, Paneth cells home towards the source of Wnt signals--that is, the crypt bottom. Here, we show that expression of a Paneth gene programme is critically dependent on TCF4 in embryonic intestine. Moreover, conditional deletion of the Wnt receptor Frizzled-5 abrogates expression of these genes in Paneth cells in the adult intestine. Conversely, adenomas in Apc-mutant mice and colorectal cancers in humans inappropriately express these Paneth-cell genes. These observations imply that Wnt signals in the crypt can separately drive a stem-cell/progenitor gene programme and a Paneth-cell maturation programme. In intestinal cancer, both gene programmes are activated simultaneously.

Interactive Analysis, Exploration, and Visualization of RNA-Seq Data with SeqCVIBE.

The rise of modern gene expression profiling techniques, such as RNA-Seq, has generated a wealth of high-quality datasets spanning all fields of current biological research. The large data sets and the continually expanding applications for which they can be mined, such as the investigation of alternative splicing and others, have created novel challenges for data management, exploration, analysis, and visualization. Although a large variety of RNA-Seq data analysis software packages has emerged, both open-source and commercial, most fail to simultaneously address the above challenges, while they lack obvious functionalities, such as estimating RNA abundance over non-annotated genomic regions of interest in real time. We have developed SeqCVIBE, an R Shiny web application for the interactive exploration, analysis, visualization, and genome browsing of large RNA-Seq datasets. SeqCVIBE allows for multiple on-the-fly visualizations and calculations, such as differential expression analysis, averaging genomic signals over specific regions of the genome, and calculating RNA abundances over custom, potentially non-annotated regions, such as novel long non-coding RNAs. In addition, SeqCVIBE comprises a database for pre-analyzed data, where users can navigate and explore results, as well as perform a variety of basic on-the-fly analyses and export the outcomes. Finally, we demonstrate the value of SeqCVIBE in the elucidation of the interplay of a novel lincRNA, WiNTRLINC1, and Wnt signaling in colon cancer.

Bee foraging preferences, microbiota and pathogens revealed by direct shotgun metagenomics of honey.

Honeybees (Apis mellifera) continue to succumb to human and environmental pressures despite their crucial role in providing essential ecosystem services. Owing to their foraging and honey production activities, honeybees form complex relationships with species across all domains, such as plants, viruses, bacteria and other hive pests, making honey a valuable biomonitoring tool for assessing their ecological niche. Thus, the application of honey shotgun metagenomics (SM) has paved the way for a detailed description of the species honeybees interact with. Nevertheless, SM bioinformatics tools and DNA extraction methods rely on resources not necessarily optimized for honey. In this study, we compared five widely used taxonomic classifiers using simulated species communities commonly found in honey. We found that Kraken 2 with a threshold of 0.5 performs best in assessing species distribution. We also optimized a simple NaOH-based honey DNA extraction methodology (Direct-SM), which profiled species seasonal variability similarly to an established column-based DNA extraction approach (SM). Both approaches produce results consistent with melissopalinology analysis describing the botanical landscape surrounding the apiary. Interestingly, we detected a strong stability of the bacteria constituting the core and noncore gut microbiome across seasons, pointing to the potential utility of honey for noninvasive assessment of bee microbiota. Finally, the Direct-SM approach to detect Varroa correlates well with the biomonitoring of mite infestation observed in hives. These observations suggest that Direct-SM methodology has the potential to comprehensively describe honeybee ecological niches and can be tested as a building block for large-scale studies to assess bee health in the field.

Obesity Reshapes the Microbial Population Structure along the Gut-Liver-Lung Axis in Mice.

The microbiome is emerging as a major player in tissue homeostasis in health and disease. Gut microbiome dysbiosis correlates with several autoimmune and metabolic diseases, while high-fat diets and ensuing obesity are known to affect the complexity and diversity of the microbiome, thus modulating pathophysiology. Moreover, the existence of a gut-liver microbial axis has been proposed, which may extend to the lung. In this context, we systematically compared the microbiomes of the gut, liver, and lung of mice fed a high-fat diet to those of littermates fed a matched control diet. We carried out deep sequencing of seven hypervariable regions of the 16S rRNA microbial gene to examine microbial diversity in the tissues of interest. Comparison of the local microbiomes indicated that lung tissue has the least diverse microbiome under healthy conditions, while microbial diversity in the healthy liver clustered closer to the gut. Obesity increased microbial complexity in all three tissues, with lung microbial diversity being the most modified. Obesity promoted the expansion of Firmicutes along the gut-liver-lung axis, highlighting staphylococcus as a possible pathologic link between obesity and systemic pathophysiology, especially in the lungs.