BORIS/CTCFL epigenetically reprograms clustered CTCF binding sites into alternative transcriptional start sites.

BACKGROUND: Pervasive usage of alternative promoters leads to the deregulation of gene expression in carcinogenesis and may drive the emergence of new genes in spermatogenesis. However, little is known regarding the mechanisms underpinning the activation of alternative promoters. RESULTS: Here we describe how alternative cancer-testis-specific transcription is activated. We show that intergenic and intronic CTCF binding sites, which are transcriptionally inert in normal somatic cells, could be epigenetically reprogrammed into active de novo promoters in germ and cancer cells. BORIS/CTCFL, the testis-specific paralog of the ubiquitously expressed CTCF, triggers the epigenetic reprogramming of CTCF sites into units of active transcription. BORIS binding initiates the recruitment of the chromatin remodeling factor, SRCAP, followed by the replacement of H2A histone with H2A.Z, resulting in a more relaxed chromatin state in the nucleosomes flanking the CTCF binding sites. The relaxation of chromatin around CTCF binding sites facilitates the recruitment of multiple additional transcription factors, thereby activating transcription from a given binding site. We demonstrate that the epigenetically reprogrammed CTCF binding sites can drive the expression of cancer-testis genes, long noncoding RNAs, retro-pseudogenes, and dormant transposable elements. CONCLUSIONS: Thus, BORIS functions as a transcription factor that epigenetically reprograms clustered CTCF binding sites into transcriptional start sites, promoting transcription from alternative promoters in both germ cells and cancer cells.

CTCF mediates chromatin looping via N-terminal domain-dependent cohesin retention.

The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin complex function together to shape chromatin architecture in mammalian cells, but the molecular details of this process remain unclear. Here, we demonstrate that a 79-aa region within the CTCF N terminus is essential for cohesin positioning at CTCF binding sites and chromatin loop formation. However, the N terminus of CTCF fused to artificial zinc fingers was not sufficient to redirect cohesin to non-CTCF binding sites, indicating a lack of an autonomously functioning domain in CTCF responsible for cohesin positioning. BORIS (CTCFL), a germline-specific paralog of CTCF, was unable to anchor cohesin to CTCF DNA binding sites. Furthermore, CTCF-BORIS chimeric constructs provided evidence that, besides the N terminus of CTCF, the first two CTCF zinc fingers, and likely the 3D geometry of CTCF-DNA complexes, are also involved in cohesin retention. Based on this knowledge, we were able to convert BORIS into CTCF with respect to cohesin positioning, thus providing additional molecular details of the ability of CTCF to retain cohesin. Taken together, our data provide insight into the process by which DNA-bound CTCF constrains cohesin movement to shape spatiotemporal genome organization.

Identification and validation of salivary proteomic signatures for non-invasive detection of ovarian cancer.

Ovarian cancer (OC) is one of the most lethal cancers among all gynecological malignancies. An effective and non-invasive screening approach is needed urgently to reduce high mortality rate. The purpose of this study was to identify the salivary protein signatures (SPS) for non-invasive detection of ovarian cancer. Differentially expressed SPS were identified by fluorescence-based 2D-DIGE coupled with MALDI/TOF-MS. The expression levels of three differential proteins (Lipocalin-2, indoleamine-2, 3-dioxygenase1 (IDO1) and S100A8) were validated using western blotting and ELISA. Immunohistochemistry and qRT-PCR were performed in an independent cohort of ovarian tumor tissues. 25 over expressed and 19 under expressed (p<0.05) proteins between healthy controls and cancer patients were identified. Lipocalin-2, IDO1 and S100A8 were selected for initial verification and successfully verified by immunoassay. Diagnostic potential of the candidate biomarkers was evaluated by ROC analysis. The selected biomarkers were further validated by immunohistochemistry in an independent cohort of ovarian tissues. The global expression of selected targets was also analyzed by microarray and validated using qRT-PCR to strengthen our hypothesis. Tumor secreted proteins identified by 'dual-omics' strategy, whose concentration are significantly high in ovarian cancer patients have obvious potential to be used as screening biomarker after large scale validation.

Comparative proteomic analysis of Taurine, Indicine, and crossbred (Bos taurus × Bos indicus) bull spermatozoa for identification of proteins related to sperm malfunctions and subfertility in crossbred bulls.

Subfertility is one of the most common problems observed among Taurine × Indicine crossbred bulls in tropical countries; however, the etiology remain unknown in most of the cases. In present study, we compared the proteomic profile of spermatozoa from crossbred bulls (Bos taurus × Bos indicus) against their purebred parent lines (Holstein Friesian [Taurine] and Tharparkar [Indicine]) to find out alteration in expressions of proteins, if any. The proteomic profiles of freshly ejaculated spermatozoa from these breeds were compared by two-dimensional difference gel electrophoresis, and differentially expressed proteins were identified through mass spectrometry. It was observed that compared to Holstein Friesian, nine proteins were underexpressed and eight proteins were overexpressed (P < 0.05) in the spermatozoa of crossbred bulls. Similarly, four proteins were overexpressed and four proteins were underexpressed (P < 0.05) in the spermatozoa of crossbred bulls compared to Tharparkar bulls. In concurrent three breed comparison, 14 proteins were found to be differentially expressed (P < 0.05) between these breeds. From the findings of the study, it is apparent that the expression levels of several functionally significant proteins are either upregulated or downregulated in spermatozoa of crossbred bulls, which might be related to high incidence of subfertility in these bulls.

Proteomic analysis of mature Lagenaria siceraria seed.

Lagenaria siceraria (bottle gourd) class belongs to Magnoliopsida family curcurbitaceae that is a traditionally used medicinal plant. Fruit of this plant are widely used as a therapeutic vegetable in various diseases, all over the Asia and Africa. Various parts of this plant like fruit, seed, leaf and root are used as alternative medicine. In the present study, primarily, we have focused on proteomic analysis of L. siceraria seed using phenol extraction method for protein isolation. Twenty-four colloidal coomassie blue stained protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) after resolving on two-dimensional gel electrophoresis. Out of 24 identified protein spots, four were grouped as unidentified proteins which clearly suggest that less work has been done in the direction of plant seed proteomics. These proteins have been found to implicate in various functions such as biosynthesis of plant cell wall polysaccharides and glycoproteins, serine/threonine kinase activity, plant disease resistance and transferase activity against insects by means of insecticidal and larval growth inhibitory, anti-HIV, antihelmintic and antimicrobial properties. By Blast2GO annotation analysis, amongst the identified proteins of L. siceraria, molecular function for majority of proteins has indispensable role in catalytic activity, few in binding activity and antioxidant activity; it is mostly distributed in cell, organelle, membrane and macromolecular complex. Most of them involved in biological process such as metabolic process, cellular process, response to stimulus, single organism process, signalling, biological recognition, cellular component organization or biogenesis and localization.

Identification of putative fertility markers in seminal plasma of crossbred bulls through differential proteomics.

Sub-fertility is a major problem in crossbred bulls leading to disintegration of breeding systems and huge economic loss. Identification of some potential biomarkers to determine the latent fertility of bulls accurately has long been the interest of researchers. In this study, we analyzed the proteome of seminal plasma (SP) from bulls with varying fertility to identify the fertility-associated proteins. The proteomic profile of high- and low-fertile bulls was compared by two-dimensional difference gel electrophoresis and differentially expressed proteins were identified through matrix-assisted laser desorption/ionization-time of flight/mass spectrometry. Out of the 18 differentially expressed proteins (P < 0.05), 9 were overexpressed in SP of high-fertile bulls and 9 were overexpressed in SP of low-fertile bulls. The differential expressions ranged from 1.5- to 5.5-fold between the two groups, where protection of telomeres-1 protein (POT1) was highly overexpressed (2.9-fold) in high-fertile group and prostaglandin E2 receptor EP3 (PTGER3) was highly abundant (5.5-fold) in low-fertile group. The protein interaction network was elucidated using STRING software tool, and the functional bioinformatics analysis was done using Blast2Go software. Most of the differentially expressed proteins were found to be involved in cellular processes and biological regulation with binding and catalytic function. It is inferred that the expression of certain proteins in the SP varied with bull fertility, and concurrent appraisal of their expression along with other fertility assays may help in determining bull fertility.