ئەز   Mevan Fahmi Sami Jacksi

Background: The scaffold protein tyrosine kinase substrate 4 (TKS4) undergoes tyrosine phosphorylation by the epidermal growth factor receptor (EGFR) pathway via Src kinase. The TKS4 deficiency in humans is responsible for the manifestation of a genetic disorder known as Frank–Ter Haar syndrome (FTHS). Based on our earlier investigation, the absence of TKS4 triggers migration, invasion, and epithelial–mesenchymal transition (EMT)-like phenomena while concurrently suppressing cell proliferation in HCT116 colorectal carcinoma cells. This indicates that TKS4 may play a unique role in the progression of cancer. In this study, we demonstrated that the enhancer of zeste homolog 2 (EZH2) and the histone methyltransferase of polycomb repressive complex 2 (PRC2) are involved in the migration, invasion, and EMT-like changes in TKS4-deficient cells (KO). EZH2 is responsible for the maintenance of the trimethylated lysine 27 on histone H3 (H3K27me3). Methods: We performed transcriptome sequencing, chromatin immunoprecipitation, protein and RNA quantitative studies, cell mobility, invasion, and proliferation studies combined with/without the EZH2 activity inhibitor 3-deazanoplanocine (DZNep). Results: We detected an elevation of global H3K27me3 levels in the TKS4 KO cells, which could be reduced with treatment with DZNep, an EZH2 inhibitor. Inhibition of EZH2 activity reversed the phenotypic effects of the knockout of TKS4, reducing the migration speed and wound healing capacity of the cells as well as decreasing the invasion capacity, while the decrease in cell proliferation became stronger. In addition, inhibition of EZH2 activity also reversed most epithelial and mesenchymal markers. We investigated the wider impact of TKS4 deletion on the gene expression profile of colorectal cancer cells using transcriptome sequencing of wild-type and TKS4 knockout cells, particularly before and after treatment with DZNep. Additionally, we observed changes in the expression of several protein-coding genes and long non-coding RNAs that showed a recovery in expression levels following EZH2 inhibition. Conclusions: Our results indicate that the removal of TKS4 causes a notable disruption in the gene expression pattern, leading to the disruption of several signal transduction pathways. Inhibiting the activity of EZH2 can restore most of these transcriptomics and phenotypic effects in colorectal carcinoma cells.

Histone lysine methyltransferases (HKMTs) perform vital roles in cellular life by controlling gene expression programs through the posttranslational modification of histone tails. Since many of them are intimately involved in the development of different diseases, including several cancers, understanding the molecular mechanisms that control their target recognition and activity is vital for the treatment and prevention of such conditions. RNA binding has been shown to be an important regulatory factor in the function of several HKMTs, such as the yeast Set1 and the human Ezh2. Moreover, many HKMTs are capable of RNA binding in the absence of a canonical RNA binding domain. Here, we explored the RNA binding capacity of KMT2D, one of the major H3K4 monomethyl transferases in enhancers, using RNA immunoprecipitation followed by sequencing. We identified a broad range of coding and non-coding RNAs associated with KMT2D and confirmed their binding through RNA immunoprecipitation and quantitative PCR. We also showed that a separated RNA binding region within KMT2D is capable of binding a similar RNA pool, but differences in the binding specificity indicate the existence of other regulatory elements in the sequence of KMT2D. Analysis of the bound mRNAs revealed that KMT2D preferentially binds co-transcriptionally to the mRNAs of the genes under its control, while also interacting with super enhancer- and splicing-related non-coding RNAs. These observations, together with the nuclear colocalization of KMT2D with differentially phosphorylated forms of RNA Polymerase II suggest a so far unexplored role of KMT2D in the RNA processing of the nascent transcripts.

Tks4 is a large scaffold protein in the EGFR signal transduction pathway that is involved in several cellular processes, such as cellular motility, reactive oxygen species-dependent processes, and embryonic development. It is also implicated in a rare developmental disorder, Frank–ter Haar syndrome. Loss of Tks4 resulted in the induction of an EMT-like process, with increased motility and overexpression of EMT markers in colorectal carcinoma cells. In this work, we explored the broader effects of deletion of Tks4 on the gene expression pattern of HCT116 colorectal carcinoma cells by transcriptome sequencing of wild-type and Tks4 knockout (KO) cells. We identified several protein coding genes with altered mRNA levels in the Tks4 KO cell line, as well as a set of long non-coding RNAs, and confirmed these changes with quantitative PCR on a selected set of genes. Our results show a significant perturbation of gene expression upon the deletion of Tks4, suggesting the involvement of different signal transduction pathways over the well-known EGFR signaling.

Long non-coding RNAs (lncRNAs) are found to play crucial roles in several biological processes and have been associated with many complex human diseases including cancers. Several lines of evidences indicate that lncRNAs deregulated in many cancer tissues. In this particular study, differential expression of long intergenic non-coding RNA 663 (LINC00663) was demonstrated in various cancer cell lines and healthy human tissues by using RT-PCR and qPCR methods. While expression level of LINC00663 was most prominent in thyroid gland and uterus, it is least expressed in skeletal muscle tissues. Moreover, LINC00663 was found to be differentially expressed in various cancer cells. Particularly, its expression was highly diminished in DU-145, PC3, HGC-27, CRL-1469, A549, MCF7, and BCPAP cancer cells. Also, LINC00663 expression was most prominent in A172 glioblastoma cells. Additionally, a novel splice variant of LINC00663 RNA was also detected. The sequence and Basic Local Alignment Search Tool (BLAST) analysis results revealed the presence of a novel exonic region between exons 2 and 3. Subsequently, five potential splice variants showing high level of variation have been identified. Secondary structures of these variants with minimum free energy were also demonstrated. Furthermore, putative microRNA (miRNA) binding sites to these variants have been shown. In conclusion, LINC00663 was shown to be differentially expressed in various human tissues and cancer cell lines. Also, LINC00663 undergoes alternative splicing and the novel exonic region alters its secondary structure and its interactions with potential targeting miRNAs. The role of LINC00663 in cancer formation further needs to be investigated with a wide range of studies.

Tyrosine Kinase Substrate 4 (TKS4) is a large scaffold protein in the EGFR signal transduction pathway that is involved in several cellular processes, such as motility, reactive oxygen species-dependent processes, embryonic development and cancer. It is also implicated in a rare developmental disorder, Frank–ter Haar syndrome. Deletion of TKS4 resulted in increased migration, invasion, and EMT-like processes while a decrease in proliferation in HCT116 colorectal carcinoma cells. In this study we demonstrated that enhancer of zeste homolog 2 (EZH2), the histone methyltransferase of the PRC2 is involved in these changes in TKS4 deficient cells. An elevation of local and global H3K27me3 levels were detected in the TKS4 KO cells, and inhibition of EZH2 activity reversed the effects of TKS4 KO, reducing the EMT-like features, migration speed, and invasion capacity of the cells while the decrease in proliferation become stronger. The broader effects of deletion of TKS4 on the gene expression pattern of colorectal carcinoma cells were explored by transcriptome sequencing of WT and TKS4 KO cells before and after treatment with DZNep; the EZH2 inhibitor. Several protein coding genes with altered mRNA levels were identified in the TKS4 KO cell line, as well as a set of long non-coding RNAs, whose expression levels were restored after EZH2 inhibition. In summary, our findings demonstrate a significant perturbation of gene expression upon the deletion of TKS4, suggesting the involvement of different signal transduction pathways over the well-known EGFR signalling.

KMT2D (also known MLL4) is a histone methyltransferase plays a critical role in regulating the epigenetic landscape of cells. Super-enhancers are large clusters of enhancers covering the long region of regulatory DNA and are densely occupied by transcription factors, active histone marks, and co-activators. According to our previous studies, KMT2D is capable of lncRNA binding and most recently KMT2D interacts with various RNA molecules, indicating a potential role in RNA processing

Tks4 is a large scaffold protein in the EGFR signal transduction pathway that is involved in several cellular processes, such as motility, reactive oxygen species-dependent processes, and embryonic development. It is also implicated in a rare developmental disorder, Frank–ter Haar syndrome. Deletion of Tks4 resulted in the increased migration, invasion, and induction of epithelial to mesenchymal transition (EMT)-like processes in HCT116 colorectal carcinoma cells. In this study we demonstrated that the Enhancer of zeste homolog 2 (EZH2), the histone methyltransferase of the Polycomb Repressive complex 2 (PRC2) is involved in these changes in Tks4 deficient cells. We detected an elevation of global H3K27me3 levels in the Tks4 KO cells, and inhibition of EZH2 activity reversed the effects of Tks4 knockout, reducing the EMT-like features, migration speed, and invasion capacity of the cells. Also, we explored the broader effects of deletion of Tks4 on the gene expression pattern of colorectal carcinoma cells by transcriptome sequencing of wild-type and Tks4 knockout (KO) cells before and after treatment with DZNep; the EZH2 inhibitor. We identified several protein coding genes with altered mRNA levels in the Tks4 KO cell line, as well as a set of long non-coding RNAs, whose expression levels were restored after EZH2 inhibition. In summary, our findings demonstrate a significant perturbation of gene expression upon the deletion of Tks4, suggesting the involvement of different signal transduction pathways over the well-known EGFR signaling.

Epithelial-mesenchymal transition (EMT) is a process where the epithelial cells acquire mesenchymal properties by repressing cell–cell junction and losing cell-cell interactions and gaining a more migratory phenotype. The first step in the metastasis formation of cancer is EMT. EMT is involved in different physiological and pathological processes such as embryogenesis, inflammation, wound healing, and cancer metastasis. Various factors have been involved in the regulation of EMT, these include different transcription factors (TFs), several small and long non-coding RNAs, epigenetic modulators and exogenous inducers [1]. Enhancer of zeste homolog 2 (Ezh2) is a histone lysine methyltransferase responsible for the activity of Polycomb Repressive Complex 2 (PRC2). Ezh2 is responsible for the maintenance of the trimethylated lysine 27 on histone H3 (H3K27me3) [2]. Tyrosine kinase substate 4 (Tks4) belongs to the family of scaffold proteins that is tyrosine phosphorylated by EGFR pathway through Src kinase. It is a protein product of SH3PXD2B gene. Tks4 is characterized by a PX domain and four Src homology 3 domains [3]. Tks4 is closely related to Src substrate adaptor proteins that are required for podosome formation, cancer cell invasion and tumor growth [4]. In humans, the Tks4 deficiency is responsible for the development of Frank-ter Haar syndrome. According to our previous study, the absence of Tks4 induces EMT like changes [5]. In this study, we demonstrated that the EZH2 is involved in the EMT like change in Tks4 deficient cells.

Proteins with large intrinsically disordered regions are the second most common drivers of liquid phase separation, and the lysine methyl transferase KMT2D (also known as mixed lineage leukemia 4; MLL4) contains intrinsically disordered regions up to 70% of its full length. KMT2 proteins are responsible for the mono-and di-methylation of histones at enhancers, and tri-methylation of histone H3 lysine 4 (H3K4) at active promoters. According to our previous studies, KMT2D is capable of lncRNA binding, but the molecular background and the physiological relevance of this binding are obscure. As some lncRNAs play their role as molecular scaffolds to enroll chromatin regulators in the formation of membraneless biomolecular condensates and allocate their enzymatic activities to proper locations in the genome to influence the expression of target gene, it is possible that the lncRNA binding of KMT2D is important in its regulation and targeting.

Long noncoding RNA (lncRNA)’lar son yıllarda tüm genom ve transkriptom sekanslama yöntemlerinin gelişmesi ile bilim dünyasında ilgi odağı haline gelmişlerdir. Son yıllarda yapılan çalışmalar ile lncRNA’ların birçok biyolojik fonksiyonu ortaya konmuş ve bu RNA moleküllerinin deregülasyonu kanser başta olmak üzere birçok kompleks hastalık ile ilişkilendirilmiştir. Long intergenic non-protein coding RNA 663 (LINC00663) 19. Kromozomda yerleşiktir ve bu zamana kadar papiller tiroid karsinom haricinde hiçbir patoloji ile ilişkilendirilmemiştir. Bu çalışmada LINC00663’ün farklı insan dokularda ve aynı zamanda farklı kanser hücre hatlarında ekspresyon profilinin belirlenmesi amaçlanmıştır.

Long noncoding RNA (lncRNA)’lar son yıllarda tüm genom ve transkriptom sekanslama yöntemlerinin gelişmesi ile bilim dünyasında ilgi odağı haline gelmişlerdir. Son yıllarda yapılan çalışmalar ile lncRNA’ların birçok biyolojik fonksiyonu ortaya konmuş ve bu RNA moleküllerinin deregülasyonu kanser başta olmak üzere birçok kompleks hastalık ile ilişkilendirilmiştir. Long intergenic non-protein coding RNA 663 (LINC00663) 19. Kromozomda yerleşiktir ve bu zamana kadar papiller tiroid karsinom haricinde hiçbir patoloji ile ilişkilendirilmemiştir. Bu çalışmada LINC00663’ün farklı insan dokularda ve aynı zamanda farklı kanser hücre hatlarında ekspresyon profilinin belirlenmesi amaçlanmıştır.

OBJECTIVES, GOALS, AND EXPECTED OUTCOME The purpose of this workshop is to introduce a new Online Submission and Peer Review System for the Journal of University of Zakho. LENGTH AND PROPOSED DATE The workshop is planned to be a one day long which will take place on Tuesday, 1st of September 2015. It will start at 10:30 am. TARGET AUDIENCE This workshop is addressed to authors, reviewers and journal committee.