BCL7A-containing SWI/SNF/BAF complexes modulate mitochondrial bioenergetics during neural progenitor differentiation.

Mammalian SWI/SNF/BAF chromatin remodeling complexes influence cell lineage determination. While the contribution of these complexes to neural progenitor cell (NPC) proliferation and differentiation has been reported, little is known about the transcriptional profiles that determine neurogenesis or gliogenesis. Here, we report that BCL7A is a modulator of the SWI/SNF/BAF complex that stimulates the genome-wide occupancy of the ATPase subunit BRG1. We demonstrate that BCL7A is dispensable for SWI/SNF/BAF complex integrity, whereas it is essential to regulate Notch/Wnt pathway signaling and mitochondrial bioenergetics in differentiating NPCs. Pharmacological stimulation of Wnt signaling restores mitochondrial respiration and attenuates the defective neurogenic patterns observed in NPCs lacking BCL7A. Consistently, treatment with an enhancer of mitochondrial biogenesis, pioglitazone, partially restores mitochondrial respiration and stimulates neuronal differentiation of BCL7A-deficient NPCs. Using conditional BCL7A knockout mice, we reveal that BCL7A expression in NPCs and postmitotic neurons is required for neuronal plasticity and supports behavioral and cognitive performance. Together, our findings define the specific contribution of BCL7A-containing SWI/SNF/BAF complexes to mitochondria-driven NPC commitment, thereby providing a better understanding of the cell-intrinsic transcriptional processes that connect metabolism, neuronal morphogenesis, and cognitive flexibility.

Aberrant paracrine signalling for bone remodelling underlies the mutant histone-driven giant cell tumour of bone.

Oncohistones represent compelling evidence for a causative role of epigenetic perturbations in cancer. Giant cell tumours of bone (GCTs) are characterised by a mutated histone H3.3 as the sole genetic driver present in bone-forming osteoprogenitor cells but absent from abnormally large bone-resorbing osteoclasts which represent the hallmark of these neoplasms. While these striking features imply a pathogenic interaction between mesenchymal and myelomonocytic lineages during GCT development, the underlying mechanisms remain unknown. We show that the changes in the transcriptome and epigenome in the mesenchymal cells caused by the H3.3-G34W mutation contribute to increase osteoclast recruitment in part via reduced expression of the TGFß-like soluble factor, SCUBE3. Transcriptional changes in SCUBE3 are associated with altered histone marks and H3.3G34W enrichment at its enhancer regions. In turn, osteoclasts secrete unregulated amounts of SEMA4D which enhances proliferation of mutated osteoprogenitors arresting their maturation. These findings provide a mechanism by which GCTs undergo differentiation in response to denosumab, a drug that depletes the tumour of osteoclasts. In contrast, hTERT alterations, commonly found in malignant GCT, result in the histone-mutated neoplastic cells being independent of osteoclasts for their proliferation, predicting unresponsiveness to denosumab. We provide a mechanism for the initiation of GCT, the basis of which is dysfunctional cross-talk between bone-forming and bone-resorbing cells. The findings highlight the role of tumour/microenvironment bidirectional interactions in tumorigenesis and how this is exploited in the treatment of GCT.

Aberrant chromatin landscape following loss of the H3.3 chaperone Daxx in haematopoietic precursors leads to Pu.1-mediated neutrophilia and inflammation.

Defective silencing of retrotransposable elements has been linked to inflammageing, cancer and autoimmune diseases. However, the underlying mechanisms are only partially understood. Here we implicate the histone H3.3 chaperone Daxx, a retrotransposable element repressor inactivated in myeloid leukaemia and other neoplasms, in protection from inflammatory disease. Loss of Daxx alters the chromatin landscape, H3.3 distribution and histone marks of haematopoietic progenitors, leading to engagement of a Pu.1-dependent transcriptional programme for myelopoiesis at the expense of B-cell differentiation. This causes neutrophilia and inflammation, predisposing mice to develop an autoinflammatory skin disease. While these molecular and phenotypic perturbations are in part reverted in animals lacking both Pu.1 and Daxx, haematopoietic progenitors in these mice show unique chromatin and transcriptome alterations, suggesting an interaction between these two pathways. Overall, our findings implicate retrotransposable element silencing in haematopoiesis and suggest a cross-talk between the H3.3 loading machinery and the pioneer transcription factor Pu.1.

A long hypoxia-inducible factor 3 isoform 2 is a transcription activator that regulates erythropoietin.

Hypoxia-inducible factor (HIF), an αß dimer, is the master regulator of oxygen homeostasis with hundreds of hypoxia-inducible target genes. Three HIF isoforms differing in the oxygen-sensitive α subunit exist in vertebrates. While HIF-1 and HIF-2 are known transcription activators, HIF-3 has been considered a negative regulator of the hypoxia response pathway. However, the human HIF3A mRNA is subject to complex alternative splicing. It was recently shown that the long HIF-3α variants can form αß dimers that possess transactivation capacity. Here, we show that overexpression of the long HIF-3α2 variant induces the expression of a subset of genes, including the erythropoietin (EPO) gene, while simultaneous downregulation of all HIF-3α variants by siRNA targeting a shared HIF3A region leads to downregulation of EPO and additional genes. EPO mRNA and protein levels correlated with HIF3A silencing and HIF-3α2 overexpression. Chromatin immunoprecipitation analyses showed that HIF-3α2 binding associated with canonical hypoxia response elements in the promoter regions of EPO. Luciferase reporter assays showed that the identified HIF-3α2 chromatin-binding regions were sufficient to promote transcription by all three HIF-α isoforms. Based on these data, HIF-3α2 is a transcription activator that directly regulates EPO expression.

Biology and Clinical Implications of the 19q13 Aggressive Prostate Cancer Susceptibility Locus.

Genome-wide association studies (GWAS) have identified rs11672691 at 19q13 associated with aggressive prostate cancer (PCa). Here, we independently confirmed the finding in a cohort of 2,738 PCa patients and discovered the biological mechanism underlying this association. We found an association of the aggressive PCa-associated allele G of rs11672691 with elevated transcript levels of two biologically plausible candidate genes, PCAT19 and CEACAM21, implicated in PCa cell growth and tumor progression. Mechanistically, rs11672691 resides in an enhancer element and alters the binding site of HOXA2, a novel oncogenic transcription factor with prognostic potential in PCa. Remarkably, CRISPR/Cas9-mediated single-nucleotide editing showed the direct effect of rs11672691 on PCAT19 and CEACAM21 expression and PCa cellular aggressive phenotype. Clinical data demonstrated synergistic effects of rs11672691 genotype and PCAT19/CEACAM21 gene expression on PCa prognosis. These results provide a plausible mechanism for rs11672691 associated with aggressive PCa and thus lay the ground work for translating this finding to the clinic.

Rare missense mutations in RECQL and POLG associate with inherited predisposition to breast cancer.

Several known breast cancer susceptibility genes with moderate-to-high risk alleles encode proteins involved in DNA damage response (DDR). As these explain less than half of the hereditary breast cancer cases, additional predisposing alleles are likely to be discovered. Many of the previous studies utilizing massive parallel sequencing have focused on the protein-truncating variants, and the role of rare missense mutations has remained poorly addressed. To identify novel susceptibility factors, we have systematically analyzed the data from our parallel sequencing of 796 DDR genes in 189 Northern Finnish hereditary breast cancer patients for rare missense variants, predicted as deleterious. Thirty-five variants were studied here for the disease association using Finnish breast cancer case (n = 492-2,035) and control (n = 277-1,539) cohorts. As a result, two missense variants in genes involved in DNA replication, RECQL p.I156M and POLG p.L392V, the former involving genomic and the latter mitochondrial DNA replication, showed significant association with risk of breast cancer. Rare RECQL p.I156M allele was observed in breast cancer cases only (6/1,946, 0.3%, p = 0.043), whereas POLG p.L392V was two times more frequent in breast cancer cases (53/2,238, 2.4%) compared to controls (18/1,539, 1.2%, OR = 2.1, 95% CI 1.2-3.5, p = 0.010). Based on the current genetic data, both RECQL p.I156M and POLG p.L392V represent novel breast cancer predisposing alleles.

Whole-genome and Transcriptome Sequencing of Prostate Cancer Identify New Genetic Alterations Driving Disease Progression.

BACKGROUND: Global disparities in prostate cancer (PCa) incidence highlight the urgent need to identify genomic abnormalities in prostate tumors in different ethnic populations including Asian men. OBJECTIVE: To systematically explore the genomic complexity and define disease-driven genetic alterations in PCa. DESIGN, SETTING, AND PARTICIPANTS: The study sequenced whole-genome and transcriptome of tumor-benign paired tissues from 65 treatment-naive Chinese PCa patients. Subsequent targeted deep sequencing of 293 PCa-relevant genes was performed in another cohort of 145 prostate tumors. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The genomic alteration landscape in PCa was analyzed using an integrated computational pipeline. Relationships with PCa progression and survival were analyzed using nonparametric test, log-rank, and multivariable Cox regression analyses. RESULTS AND LIMITATIONS: We demonstrated an association of high frequency of CHD1 deletion with a low rate of TMPRSS2-ERG fusion and relatively high percentage of mutations in androgen receptor upstream activator genes in Chinese patients. We identified five putative clustered deleted tumor suppressor genes and provided experimental and clinical evidence that PCDH9, deleted/loss in approximately 23% of tumors, functions as a novel tumor suppressor gene with prognostic potential in PCa. Furthermore, axon guidance pathway genes were frequently deregulated, including gain/amplification of PLXNA1 gene in approximately 17% of tumors. Functional and clinical data analyses showed that increased expression of PLXNA1 promoted prostate tumor growth and independently predicted prostate tumor biochemical recurrence, metastasis, and poor survival in multi-institutional cohorts of patients with PCa. A limitation of this study is that other genetic alterations were not experimentally investigated. CONCLUSIONS: There are shared and salient genetic characteristics of PCa in Chinese and Caucasian men. Novel genetic alterations in PCDH9 and PLXNA1 were associated with disease progression. PATIENT SUMMARY: We reported the first large-scale and comprehensive genomic data of prostate cancer from Asian population. Identification of these genetic alterations may help advance prostate cancer diagnosis, prognosis, and treatment.

Meta-analysis of gene expression and integrin-associated signaling pathways in papillary renal cell carcinoma subtypes.

Papillary renal cell carcinoma (PRCC) is the second most common renal cell carcinoma (RCC) that can be further subdivided into type 1 (PRCC1) and type 2 (PRCC2) RCCs based on histological and genetic features. PRCC2 is often more aggressive than PRCC1. While integrin-associated protein complexes mediate tumorigenesis and metastases in many types of cancers it is not known whether integrin-mediated signaling impacts PRCC and differs between PRCC1 and PRCC2. In this study, we combined the analysis of five PRCC gene expression datasets derived from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) by using integrative bioinformatics pipelines. We found 1475 differentially expressed genes among which 37 genes were associated with integrin pathways. In comparison with PRCC1, PRCC2 cases showed upregulated expression of α5-integrin (ITGA5) whereas the expression of α6- (ITGA6) and ß8-integrins (ITGB8) was downregulated. Because PRCC2 occurs more frequently in men, the meta-analysis was extended to explore the gender effects. This analysis revealed 8 genes but none of them was related to integrin pathways suggesting that other mechanisms than integrin-mediated signaling underlie the observed gender differences in the pathogenicity of PRCC2.

Membrane-associated collagens with interrupted triple-helices (MACITs): evolution from a bilaterian common ancestor and functional conservation in C. elegans.

BACKGROUND: Collagens provide structural support and guidance cues within the extracellular matrix of metazoans. Mammalian collagens XIII, XXIII and XXV form a unique subgroup of type II transmembrane proteins, each comprising a short N-terminal cytosolic domain, a transmembrane domain and a largely collagenous ectodomain. We name these collagens as MACITs (Membrane-Associated Collagens with Interrupted Triple-helices), and here investigate their evolution and conserved properties. To date, these collagens have been studied only in mammals. Knowledge of the representation of MACITs in other extant metazoans is lacking. This question is of interest for understanding structural/functional relationships in the MACIT family and also for insight into the evolution of MACITs in relation to the secreted, fibrillar collagens that are present throughout the metazoa. RESULTS: MACITs are restricted to bilaterians and are represented in the Ecdysozoa, Hemichordata, Urochordata and Vertebrata (Gnathostomata). They were not identified in available early-diverging metazoans, Lophotrochozoa, Echinodermata, Cephalochordata or Vertebrata (Cyclostomata). Whereas invertebrates encode a single MACIT, collagens XIII/XXIII/XXV of jawed vertebrates are paralogues that originated from the two rounds of en-bloc genome duplication occurring early in vertebrate evolution. MACITs have conserved domain architecture in which a juxta-membrane furin-cleavage site and the C-terminal 34 residues are especially highly conserved, whereas the cytoplasmic domains are weakly conserved. To study protein expression and function in a metazoan with a single MACIT gene, we focused on Caenorhabditis elegans and its col-99 gene. A col-99 cDNA was cloned and expressed as protein in mammalian CHO cells, two antibodies against COL-99 protein were generated, and a col-99-bearing fosmid gene construct col-99::egfp::flag was used to generate transgenic C. elegans lines. The encoded COL-99 polypeptide is 85 kDa in size and forms a trimeric protein. COL-99 is plasma membrane-associated and undergoes furin-dependent ectodomain cleavage and shedding. COL-99 is detected in mouth, pharynx, body wall and the tail, mostly in motor neurons and muscle systems and is enriched at neuromuscular junctions. CONCLUSIONS: Through identification of MACITs in multiple metazoan phyla we developed a model for the evolution of MACITs. The experimental data demonstrate conservation of MACIT molecular and cellular properties and tissue localisations in the invertebrate, C. elegans.

Computational construction of specialized biological networks.

Today we have access to more than 1500 molecular database systems inside the internet. Based on these databases and information systems, computer scientists developed and implemented different methods for the automatic integration and prediction of biological networks. The idea is to use such methods for the automatic prediction and expansion of rudimentary molecular knowledge. However, the inherent data deficiency problem concerning the properties of specialized network hampers the database- and text-mining-based network construction. This paper presents the concept concerning the computational network expansion, namely for the specific biological network-thiol-disulfide redox regulatory network. Besides, a network-contexted document retrieval system (ncDocReSy) is also introduced to assist the network reduction by providing indirectly relevant literature for user's manual curation. NcDocReSy combines literature search with biological network and ranks the retrieved literature according to the network topology. NcDocReSy is implemented as a Cytoscape plugin.

Prediction of thioredoxin and glutaredoxin target proteins by identifying reversibly oxidized cysteinyl residues.

A significant part of cellular proteins undergo reversible thiol-dependent redox transitions which often control or switch protein functions. Thioredoxins and glutaredoxins constitute two key players in this redox regulatory protein network. Both interact with various categories of proteins containing reversibly oxidized cysteinyl residues. The identification of thioredoxin/glutaredoxin target proteins is a critical step in constructing the redox regulatory network of cells or subcellular compartments. Due to the scarcity of thioredoxin/glutaredoxin target protein records in the public database, a tool called Reversibly Oxidized Cysteine Detector (ROCD) is implemented here to identify potential thioredoxin/glutaredoxin target proteins computationally, so that the in silico construction of redox regulatory network may become feasible. ROCD was tested on 46 thioredoxin target proteins in plant mitochondrion, and the recall rate was 66.7% when 50% sequence identity was chosen for structural model selection. ROCD will be used to predict the thioredoxin/glutaredoxin target proteins in human liver mitochondrion for our redox regulatory network construction project. The ROCD will be developed further to provide prediction with more reliability and incorporated into biological network visualization tools as a node prediction component. This work will advance the capability of traditional database- or text mining-based method in the network construction.

Enhanced interferon signaling pathway in oral cancer revealed by quantitative proteome analysis of microdissected specimens using 16O/18O labeling and integrated two-dimensional LC-ESI-MALDI tandem MS.

Oral squamous cell carcinoma (OSCC) remains one of the most common cancers worldwide, and the mortality rate of this disease has increased in recent years. No molecular markers are available to assist with the early detection and therapeutic evaluation of OSCC; thus, identification of differentially expressed proteins may assist with the detection of potential disease markers and shed light on the molecular mechanisms of OSCC pathogenesis. We performed a multidimensional (16)O/(18)O proteomics analysis using an integrated ESI-ion trap and MALDI-TOF/TOF MS system and a computational data analysis pipeline to identify proteins that are differentially expressed in microdissected OSCC tumor cells relative to adjacent non-tumor epithelia. We identified 1233 unique proteins in microdissected oral squamous epithelia obtained from three pairs of OSCC specimens with a false discovery rate of <3%. Among these, 977 proteins were quantified between tumor and non-tumor cells. Our data revealed 80 dysregulated proteins (53 up-regulated and 27 down-regulated) when a 2.5-fold change was used as the threshold. Immunohistochemical staining and Western blot analyses were performed to confirm the overexpression of 12 up-regulated proteins in OSCC tissues. When the biological roles of 80 differentially expressed proteins were assessed via MetaCore analysis, the interferon (IFN) signaling pathway emerged as one of the most significantly altered pathways in OSCC. As many as 20% (10 of 53) of the up-regulated proteins belonged to the IFN-stimulated gene (ISG) family, including ubiquitin cross-reactive protein (UCRP)/ISG15. Using head-and-neck cancer tissue microarrays, we determined that UCRP is overexpressed in the majority of cheek and tongue cancers and in several cases of larynx cancer. In addition, we found that IFN-beta stimulates UCRP expression in oral cancer cells and enhances their motility in vitro. Our findings shed new light on OSCC pathogenesis and provide a basis for the future development of novel biomarkers.

Genomewide analysis of nucleosome density histone acetylation and HDAC function in fission yeast.

We have conducted a genomewide investigation into the enzymatic specificity, expression profiles, and binding locations of four histone deacetylases (HDACs), representing the three different phylogenetic classes in fission yeast (Schizosaccharomyces pombe). By directly comparing nucleosome density, histone acetylation patterns and HDAC binding in both intergenic and coding regions with gene expression profiles, we found that Sir2 (class III) and Hos2 (class I) have a role in preventing histone loss; Clr6 (class I) is the principal enzyme in promoter-localized repression. Hos2 has an unexpected role in promoting high expression of growth-related genes by deacetylating H4K16Ac in their open reading frames. Clr3 (class II) acts cooperatively with Sir2 throughout the genome, including the silent regions: rDNA, centromeres, mat2/3 and telomeres. The most significant acetylation sites are H3K14Ac for Clr3 and H3K9Ac for Sir2 at their genomic targets. Clr3 also affects subtelomeric regions which contain clustered stress- and meiosis-induced genes. Thus, this combined genomic approach has uncovered different roles for fission yeast HDACs at the silent regions in repression and activation of gene expression.