REST reduction is essential for hypoxia-induced neuroendocrine differentiation of prostate cancer cells by activating autophagy signaling.

Prostate cancer (PCa) with neuroendocrine differentiation (NED) is tightly associated with hormone refractory PCa (HRPC), an aggressive form of cancer that is nearly impossible to treat. Determining the mechanism of the development of NED may yield novel therapeutic strategies for HRPC. Here, we first demonstrate that repressor element-1 silencing transcription factor (REST), a transcriptional repressor of neuronal genes that has been implicated in androgen-deprivation and IL-6 induced NED, is essential for hypoxia-induced NED of PCa cells. Bioinformatics analysis of transcriptome profiles of REST knockdown during hypoxia treatment demonstrated that REST is a master regulator of hypoxia-induced genes. Gene set enrichment analysis (GSEA) of hypoxia and REST knockdown co-upregulated genes revealed their correlation with HRPC. Consistently, gene ontology (GO) analysis showed that REST reduction potential associated with hypoxia-induced tumorigenesis, NE development, and AMPK pathway activation. Emerging reports have revealed that AMPK activation is a potential mechanism for hypoxia-induced autophagy. In line with this, we demonstrate that REST knockdown alone is capable of activating AMPK and autophagy activation is essential for hypoxia-induced NED of PCa cells. Here, making using of in vitro cell-based assay for NED, we reveal a new role for the transcriptional repressor REST in hypoxia-induced NED and characterized a sequential molecular mechanism downstream of REST resulting in AMPK phosphorylation and autophagy activation, which may be a common signaling pathway leading to NED of PCa.

K-bZIP Mediated SUMO-2/3 Specific Modification on the KSHV Genome Negatively Regulates Lytic Gene Expression and Viral Reactivation.

SUMOylation is associated with epigenetic regulation of chromatin structure and transcription. Epigenetic modifications of herpesviral genomes accompany the transcriptional switch of latent and lytic genes during the virus life cycle. Here, we report a genome-wide comparison of SUMO paralog modification on the KSHV genome. Using chromatin immunoprecipitation in conjunction with high-throughput sequencing, our study revealed highly distinct landscape changes of SUMO paralog genomic modifications associated with KSHV reactivation. A rapid and widespread deposition of SUMO-2/3, compared with SUMO-1, modification across the KSHV genome upon reactivation was observed. Interestingly, SUMO-2/3 enrichment was inversely correlated with H3K9me3 mark after reactivation, indicating that SUMO-2/3 may be responsible for regulating the expression of viral genes located in low heterochromatin regions during viral reactivation. RNA-sequencing analysis showed that the SUMO-2/3 enrichment pattern positively correlated with KSHV gene expression profiles. Activation of KSHV lytic genes located in regions with high SUMO-2/3 enrichment was enhanced by SUMO-2/3 knockdown. These findings suggest that SUMO-2/3 viral chromatin modification contributes to the diminution of viral gene expression during reactivation. Our previous study identified a SUMO-2/3-specific viral E3 ligase, K-bZIP, suggesting a potential role of this enzyme in regulating SUMO-2/3 enrichment and viral gene repression. Consistent with this prediction, higher K-bZIP binding on SUMO-2/3 enrichment region during reactivation was observed. Moreover, a K-bZIP SUMO E3 ligase dead mutant, K-bZIP-L75A, in the viral context, showed no SUMO-2/3 enrichment on viral chromatin and higher expression of viral genes located in SUMO-2/3 enriched regions during reactivation. Importantly, virus production significantly increased in both SUMO-2/3 knockdown and KSHV K-bZIP-L75A mutant cells. These results indicate that SUMO-2/3 modification of viral chromatin may function to counteract KSHV reactivation. As induction of herpesvirus reactivation may activate cellular antiviral regimes, our results suggest that development of viral SUMO E3 ligase specific inhibitors may be an avenue for anti-virus therapy.

An improved ChIP-seq peak detection system for simultaneously identifying post-translational modified transcription factors by combinatorial fusion, using SUMOylation as an example.

BACKGROUND: Post-translational modification (PTM) of transcriptional factors and chromatin remodelling proteins is recognized as a major mechanism by which transcriptional regulation occurs. Chromatin immunoprecipitation (ChIP) in combination with high-throughput sequencing (ChIP-seq) is being applied as a gold standard when studying the genome-wide binding sites of transcription factor (TFs). This has greatly improved our understanding of protein-DNA interactions on a genomic-wide scale. However, current ChIP-seq peak calling tools are not sufficiently sensitive and are unable to simultaneously identify post-translational modified TFs based on ChIP-seq analysis; this is largely due to the wide-spread presence of multiple modified TFs. Using SUMO-1 modification as an example; we describe here an improved approach that allows the simultaneous identification of the particular genomic binding regions of all TFs with SUMO-1 modification. RESULTS: Traditional peak calling methods are inadequate when identifying multiple TF binding sites that involve long genomic regions and therefore we designed a ChIP-seq processing pipeline for the detection of peaks via a combinatorial fusion method. Then, we annotate the peaks with known transcription factor binding sites (TFBS) using the Transfac Matrix Database (v7.0), which predicts potential SUMOylated TFs. Next, the peak calling result was further analyzed based on the promoter proximity, TFBS annotation, a literature review, and was validated by ChIP-real-time quantitative PCR (qPCR) and ChIP-reChIP real-time qPCR. The results show clearly that SUMOylated TFs are able to be pinpointed using our pipeline. CONCLUSION: A methodology is presented that analyzes SUMO-1 ChIP-seq patterns and predicts related TFs. Our analysis uses three peak calling tools. The fusion of these different tools increases the precision of the peak calling results. TFBS annotation method is able to predict potential SUMOylated TFs. Here, we offer a new approach that enhances ChIP-seq data analysis and allows the identification of multiple SUMOylated TF binding sites simultaneously, which can then be utilized for other functional PTM binding site prediction in future.

Whole-exome sequencing to identify a novel LMNA gene mutation associated with inherited cardiac conduction disease.

BACKGROUND: Inherited cardiac conduction diseases (CCD) are rare but are caused by mutations in a myriad of genes. Recently, whole-exome sequencing has successfully led to the identification of causal mutations for rare monogenic Mendelian diseases. OBJECTIVE: To investigate the genetic background of a family affected by inherited CCD. METHODS AND RESULTS: We used whole-exome sequencing to study a Chinese family with multiple family members affected by CCD. Using the pedigree information, we proposed a heterozygous missense mutation (c.G695T, Gly232Val) in the lamin A/C (LMNA) gene as a candidate mutation for susceptibility to CCD in this family. The mutation is novel and is expected to affect the conformation of the coiled-coil rod domain of LMNA according to a structural model prediction. Its pathogenicity in lamina instability was further verified by expressing the mutation in a cellular model. CONCLUSIONS: Our results suggest that whole-exome sequencing is a feasible approach to identifying the candidate genes underlying inherited conduction diseases.

The chromatin modification by SUMO-2/3 but not SUMO-1 prevents the epigenetic activation of key immune-related genes during Kaposi's sarcoma associated herpesvirus reactivation.

BACKGROUND: SUMOylation, as part of the epigenetic regulation of transcription, has been intensively studied in lower eukaryotes that contain only a single SUMO protein; however, the functions of SUMOylation during mammalian epigenetic transcriptional regulation are largely uncharacterized. Mammals express three major SUMO paralogues: SUMO-1, SUMO-2, and SUMO-3 (normally referred to as SUMO-1 and SUMO-2/3). Herpesviruses, including Kaposi's sarcoma associated herpesvirus (KSHV), seem to have evolved mechanisms that directly or indirectly modulate the SUMO machinery in order to evade host immune surveillance, thus advancing their survival. Interestingly, KSHV encodes a SUMO E3 ligase, K-bZIP, with specificity toward SUMO-2/3 and is an excellent model for investigating the global functional differences between SUMO paralogues. RESULTS: We investigated the effect of experimental herpesvirus reactivation in a KSHV infected B lymphoma cell line on genomic SUMO-1 and SUMO-2/3 binding profiles together with the potential role of chromatin SUMOylation in transcription regulation. This was carried out via high-throughput sequencing analysis. Interestingly, chromatin immunoprecipitation sequencing (ChIP-seq) experiments showed that KSHV reactivation is accompanied by a significant increase in SUMO-2/3 modification around promoter regions, but SUMO-1 enrichment was absent. Expression profiling revealed that the SUMO-2/3 targeted genes are primarily highly transcribed genes that show no expression changes during viral reactivation. Gene ontology analysis further showed that these genes are involved in cellular immune responses and cytokine signaling. High-throughput annotation of SUMO occupancy of transcription factor binding sites (TFBS) pinpointed the presence of three master regulators of immune responses, IRF-1, IRF-2, and IRF-7, as potential SUMO-2/3 targeted transcriptional factors after KSHV reactivation. CONCLUSION: Our study is the first to identify differential genome-wide SUMO modifications between SUMO paralogues during herpesvirus reactivation. Our findings indicate that SUMO-2/3 modification near protein-coding gene promoters occurs in order to maintain host immune-related gene unaltered during viral reactivation.

AVATAR: a database for genome-wide alternative splicing event detection using large scale ESTs and mRNAs.

UNLABELLED: In the past years, identification of alternative splicing (AS) variants has been gaining momentum. We developed AVATAR, a database for documenting AS using 5,469,433 human EST sequences and 26,159 human mRNA sequences. AVATAR contains 12000 alternative splicing sites identified by mapping ESTs and mRNAs with the whole human genome sequence. AVATAR also contains AS information for 6 eukaryotes. We mapped EST alignment information into a graph model where exons and introns are represented with vertices and edges, respectively. AVATAR can be queried using, (1) gene names, (2) number of identified AS events in a gene, (3) minimal number of ESTs supporting a splicing site, etc. as search parameters. The system provides visualized AS information for queried genes. AVAILABILITY: The database is available for free at http://avatar.iecs.fcu.edu.tw/