RNA polymerase II-associated proteins reveal pathways affected in VCP-related amyotrophic lateral sclerosis.

Valosin-containing protein (VCP) is a hexameric ATPase associated with diverse cellular activities. Genetic mutations in VCP are associated with several forms of muscular and neuronal degeneration, including amyotrophic lateral sclerosis (ALS). Moreover, VCP mediates UV-induced proteolysis of RNA polymerase II (RNAPII), but little is known about the effects of VCP mutations on the transcriptional machinery. Here, we used silica particle-assisted chromatin enrichment and mass spectrometry to study proteins co-localized with RNAPII in precursor neurons differentiated from VCP-mutant or control induced pluripotent stem cells. Remarkably, we observed diminished RNAPII binding of proteins involved in transcription elongation and mRNA splicing in mutant cells. One of these is SART3, a recycling factor of the splicing machinery, whose knockdown leads to perturbed intron retention in several ALS-associated genes. Additional reduced proteins are RBM45, EIF5A and RNF220, mutations in which are associated with various neurodegenerative disorders and are linked to TDP-43 aggregation. Conversely, we observed increased RNAPII binding of heat shock proteins such as HSPB1. Together, these findings shed light on how transcription and splicing machinery are impaired by VCP mutations, which might contribute to aberrant alternative splicing and proteinopathy in neurodegeneration.

Cell-specific bioorthogonal tagging of glycoproteins.

Altered glycoprotein expression is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function.

Identification of therapeutic targets of the hijacked super-enhancer complex in EVI1-rearranged leukemia.

Deregulation of the EVI1 proto-oncogene by the GATA2 distal hematopoietic enhancer (G2DHE) is a key event in high-risk acute myeloid leukemia carrying 3q21q26 aberrations (3q-AML). Upon chromosomal rearrangement, G2DHE acquires characteristics of a super-enhancer and causes overexpression of EVI1 at 3q26.2. However, the transcription factor (TF) complex of G2DHE remains poorly characterized. The aim of this study was to unravel key components of G2DHE-bound TFs involved in the deregulation of EVI1. We have identified several CEBPA and RUNX1 binding sites to be enriched and critical for G2DHE function in 3q-AML cells. Using ChIP-SICAP (ChIP followed by selective isolation of chromatin-associated proteins), a panel of chromatin interactors of RUNX1 and CEBPA were detected in 3q-AML, including PARP1 and IKZF1. PARP1 inhibition (PARPi) caused a reduction of EVI1 expression and a decrease in EVI1-G2DHE interaction frequency, highlighting the involvement of PARP1 in oncogenic super-enhancer formation. Furthermore, 3q-AML cells were highly sensitive to PARPi and displayed morphological changes with higher rates of differentiation and apoptosis as well as depletion of CD34 + cells. In summary, integrative analysis of the 3q-AML super-enhancer complex identified CEBPA and RUNX1 associated proteins and nominated PARP1 as a potential new therapeutic target in EVI1 + 3q-AML.

Chromatin-Directed Proteomics Identifies ZNF84 as a p53-Independent Regulator of p21 in Genotoxic Stress Response.

The p21WAF1/Cip1 protein, encoded by CDKN1A, plays a vital role in senescence, and its transcriptional control by the tumour suppressor p53 is well-established. However, p21 can also be regulated in a p53-independent manner, by mechanisms that still remain less understood. We aimed to expand the knowledge about p53-independent senescence by looking for novel players involved in CDKN1A regulation. We used a chromatin-directed proteomic approach and identified ZNF84 as a novel regulator of p21 in various p53-deficient cell lines treated with cytostatic dose of doxorubicin. Knock-down of ZNF84, an as-yet un-characterized protein, inhibited p21 gene and protein expression in response to doxorubicin, it attenuated senescence and was associated with enhanced proliferation, indicating that ZNF84-deficiency can favor senescence bypass. ZNF84 deficiency was also associated with transcriptomic changes in genes governing various cancer-relevant processes e.g., mitosis. In cells with ZNF84 knock-down we discovered significantly lower level of H2AX Ser139 phosphorylation (γH2AX), which is triggered by DNA double strand breaks. Intriguingly, we observed a reverse correlation between the level of ZNF84 expression and survival rate of colon cancer patients. In conclusion, ZNF84, whose function was previously not recognized, was identified here as a critical p53-independent regulator of senescence, opening possibilities for its targeting in novel therapies of p53-null cancers.

Protease-resistant streptavidin for interaction proteomics.

Streptavidin-mediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners; however, intense streptavidin-derived peptides impede protein identification by mass spectrometry. Here, we present an approach to chemically modify streptavidin, thus rendering it resistant to proteolysis by trypsin and LysC. This modification results in over 100-fold reduction of streptavidin contamination and in better coverage of proteins interacting with various biotinylated bait molecules (DNA, protein, and lipid) in an overall simplified workflow.

Enrichment of A Rare Subpopulation of miR-302-Expressing Glioma Cells by Serum Deprivation.

OBJECTIVE: MiR-302-367 is a cluster of polycistronic microRNAs that are exclusively expressed in embryonic stem (ES) cells. The miR-302-367 promoter is functional during embryonic development but is turned off in later stages. Motivated by the cancer stem cell hypothesis, we explored the potential expression of miR-302 in brain tumor cell lines. MATERIALS AND METHODS: In the present experimental study, we have tried to expand our knowledge on the expression pattern and functionality of miR302 cluster by quantifying its expression in a series of glioma (A-172, 1321N1, U87MG) and medulloblastoma (DAOY) cell lines. To further assess the functionality of miR-302 in these cell lines, we cloned its promoter core region upstream of the enhanced green fluorescent protein (EGFP) or luciferase encoding genes. RESULTS: Our data demonstrated a very low expression of miR-302 in glioma cell lines, compared with that of embryonal carcinoma cell line NT2 being used as a positive control. The expression of miR-302 promoter-EGFP construct in the aforementioned cell lines demonstrated GFP expression in a rare subpopulation of the cells. Serum deprivation led to the generation of tumorospheres, enrichment of miR-302 positive cells and upregulation of a number of pluripotency genes. CONCLUSION: Taken together, our data suggest that miR-302 could potentially be used as a novel putative cancer stem cell marker to identify and target cancer stem cells within tumor tissues.

Two novel splice variants of SOX2OT, SOX2OT-S1, and SOX2OT-S2 are coupregulated with SOX2 and OCT4 in esophageal squamous cell carcinoma.

Long noncoding RNAs (lncRNAs) have emerged as new regulators of stem cell pluripotency and tumorigenesis. The SOX2 gene, a master regulator of pluripotency, is embedded within the third intron of a lncRNA known as SOX2 overlapping transcript (SOX2OT). SOX2OT has been suspected to participate in regulation of SOX2 expression and/or other related processes; nevertheless, its potential involvement in tumor initiation and/or progression is unclear. Here, we have evaluated a possible correlation between expression patterns of SOX2OT and those of master regulators of pluripotency, SOX2 and OCT4, in esophageal squamous cell carcinoma (ESCC) tissue samples. We have also examined its potential function in the human embryonic carcinoma stem cell line, NTERA2 (NT2), which highly expresses SOX2OT, SOX2, and OCT4. Our data revealed a significant coupregulation of SOX2OT along with SOX2 and OCT4 in tumor samples, compared to the non-tumor tissues obtained from the margin of same tumors. We also identified two novel splice variants of SOX2OT (SOX2OT-S1 and SOX2OT-S2) which coupregulated with SOX2 and OCT4 in ESCCs. Suppressing SOX2OT variants caused a profound alteration in cell cycle distribution, including a 5.9 and 6.9 time increase in sub-G1 phase of cell cycle for SOX2OT-S1 and SOX2OT-S2, respectively. The expression of all variants was significantly diminished, upon the induction of neural differentiation in NT2 cells, suggesting their potential functional links to the undifferentiated state of the cells. Our data suggest a part for SOX2OT spliced variants in tumor initiation and/or progression as well as regulating pluripotent state of stem cells.

A plausible anti-apoptotic role of up-regulated OCT4B1 in bladder tumors.

PURPOSE: To investigate and compare the expression of OCT4B1 between tumor and non-tumor bladder tissues. MATERIALS AND METHODS: We investigated the expression of OCT4B1 in 30 tumor and non-tumor surgical specimens of the bladder, using the TaqMan real-time polymerase chain reaction approach and by carefully designing primers and probes specific for the amplification of the variant. RESULTS: Most tumor and non-tumor samples of the bladder showed OCT4B1 expression, but its expression level was significantly higher in the tumors (P < .002). Moreover, the up-regulation of OCT4B1 was more significant in high-grade tumors compared to the low-grade ones (P < .05). We have also employed the RNA interference strategy to evaluate the functional role of OCT4B1 in a bladder cancer cell line, 5637. Suppression of OCT4B1 caused some changes in cell cycle distribution, and significantly elevated the rate of apoptosis in the cells. CONCLUSION: Our findings suggest that OCT4B1 plays a potential role in tumor initiation and/or progression of the bladder cancer. Additionally, OCT4B1 can be regarded as a new tumor marker for detection, classification, and treatment of the bladder cancer. However, more experimental studies are needed to replicate our findings.

Identification of salt-inducible peptide with putative kinase activity in halophilic bacterium Virgibacillus halodenitrificans.

Strain XII, a moderately halophilic bacterium, expressed a peptide in response to saline media. This peptide was designated as salt-inducible factor (Sif-A). The purpose of this study is to describe Sif-A, which might be involved in the osmoresistance mechanism of strain XII. The complete sequence of sif-A was determined using PCR. sif-A codes for a polypeptide of 20.518 kDa. The polypeptide has a putative signal peptide of 27 amino acids (2.667 kDa) preceding the mature protein (17.869 kDa). Motif analysis of the deduced amino acid sequence indicated that there is a p-loop NTPase domain on the C-terminal of the peptide, which might correlate with its function. The sequence of the 16S rRNA gene was analyzed phylogenetically to classify strain XII. This organism was found to have the closest association with Virgibacillus halodenitrificans, which was proven by its phenotypic characteristics.