Mapping of post-translational modifications of spermatid-specific linker histone H1-like protein, HILS1.

In mammalian spermiogenesis, haploid round spermatids undergo dramatic biochemical and morphological changes and transform into motile mature spermatozoa. A majority of the histones are replaced by transition proteins during mid-spermiogenesis and later replaced by protamines, which occupy the sperm chromatin. In mammals, 11 linker histone H1 subtypes have been reported. Among them, H1t, HILS1, and H1T2 are uniquely expressed in testis, with the expression of HILS1 and H1T2 restricted to spermiogenesis. However, there is a lack of knowledge about linker histone role in the nuclear reorganization during mammalian spermiogenesis. Here, we report a method for separation of endogenous HILS1 protein from other rat testis linker histones by reversed-phase high-performance liquid chromatography (RP-HPLC) and identification of 15 novel post-translational modifications of HILS1, which include lysine acetylation and serine/threonine/tyrosine phosphorylation sites. Immunofluorescence studies demonstrate the presence of linker histone HILS1 and HILS1Y78p during different steps of spermiogenesis from early elongating to condensing spermatids.

Identification of genomic targets of transcription factor AEBP1 and its role in survival of glioma cells.

A recent transcriptome analysis of graded patient glioma samples led to identification of AEBP1 as one of the genes upregulated in majority of the primary GBM as against secondary GBM. Aebp1 is a transcriptional repressor that is involved in adipogenesis. It binds to AE-1 element present in the proximal promoter of aP2 gene that codes for fatty acid binding protein (FABP4). A comprehensive study was undertaken to elucidate the role of AEBP1 overexpression in glioblastoma. We employed complementary gene silencing approach to identify the genes that are perturbed in a glioma cell line (U87MG). A total of 734 genes were differentially regulated under these conditions (≥ 1.5-fold, P ≤ 0.05) belonging to different GO categories such as transcription regulation, cell growth, proliferation, differentiation, and apoptosis of which perturbation of 114 genes of these pathways were validated by quantitative real time PCR (qRT-PCR). This approach was subsequently combined with ChIP-chip technique using an Agilent human promoter tiling array to identify genomic binding loci of Aebp1 protein. A subset of these genes identified for Aebp1 occupancy was also validated by ChIP-PCR. Bioinformatics analysis of the promoters identified by ChIP-chip technique revealed a consensus motif GAAAT present in 66% of the identified genes. This consensus motif was experimentally validated by functional promoter assay using luciferase as the reporter gene. Both cellular proliferation and survival were affected in AEBP1-silenced U87MG and U138MG cell lines and a significant percentage of these cells were directed towards apoptosis.

Glioblastoma-specific protein interaction network identifies PP1A and CSK21 as connecting molecules between cell cycle-associated genes.

Glioblastoma (GBM; grade IV astrocytoma) is a very aggressive form of brain cancer with a poor survival and few qualified predictive markers. This study integrates experimentally validated genes that showed specific upregulation in GBM along with their protein-protein interaction information. A system level analysis was used to construct GBM-specific network. Computation of topological parameters of networks showed scale-free pattern and hierarchical organization. From the large network involving 1,447 proteins, we synthesized subnetworks and annotated them with highly enriched biological processes. A careful dissection of the functional modules, important nodes, and their connections identified two novel intermediary molecules CSK21 and protein phosphatase 1 alpha (PP1A) connecting the two subnetworks CDC2-PTEN-TOP2A-CAV1-P53 and CDC2-CAV1-RB-P53-PTEN, respectively. Real-time quantitative reverse transcription-PCR analysis revealed CSK21 to be moderately upregulated and PP1A to be overexpressed by 20-fold in GBM tumor samples. Immunohistochemical staining revealed nuclear expression of PP1A only in GBM samples. Thus, CSK21 and PP1A, whose functions are intimately associated with cell cycle regulation, might play key role in gliomagenesis.

A novel noncoding RNA processed by Drosha is restricted to nucleus in mouse.

Noncoding RNAs constitute a huge repertoire of gene regulatory molecules. Our previous, fine-resolution characterization of a mouse meiotic recombination hotspot from chromosome 8 resulted in identification of 2.4-kb unspliced and polyadenylated noncoding mrhl RNA. The gene is expressed in multiple tissues and is also present in rat but absent in humans. Here we report that the mrhl RNA gets processed to a small 80-nucleotide (nt) RNA species and is mediated by the Drosha complex. We also observe that the 80-nt Drosha product could be processed further to a 22-nt small RNA by Dicer in an in vitro reaction. However, this 22-nt product was not detected in vivo. The 80-nt as well as the 2.4-kb full-length RNA are nuclear-localized, showing distinct punctate nuclear signal. The colocalization of the noncoding RNA with Drosha and Nucleolin suggests the nucleolus as the site of processing of the 2.4-kb primary transcript. Additional foci of the processed 80-nt RNA were also observed outside the nucleolus, suggesting its role in some specific chromatin domain(s). Thus, this study reports a novel noncoding mrhl RNA that is processed and restricted within the cell nucleus.