Vamsi K. Gangaraju

MicroRNAs: key regulators of stem cells.

The hallmark of a stem cell is its ability to self-renew and to produce numerous differentiated cells. This unique property is controlled by dynamic interplays between extrinsic signalling, epigenetic, transcriptional and post-transcriptional regulations. Recent research indicates that microRNAs (miRNAs) have an important role in regulating stem cell self-renewal and differentiation by repressing the translation of selected mRNAs in stem cells and differentiating daughter cells. Such a role has been shown in embryonic stem cells, germline stem cells and various somatic tissue stem cells. These findings reveal a new dimension of gene regulation in controlling stem cell fate and behaviour.

Drosophila Piwi functions in Hsp90-mediated suppression of phenotypic variation

Canalization, also known as developmental robustness, describes an organisms ability to produce the same phenotype despite genotypic variations and environmental influences. In Drosophila, Hsp90, the trithorax-group proteins and transposon silencing have been previously implicated in canalization. Despite this, the molecular mechanism underlying canalization remains elusive. Here using a Drosophila eye-outgrowth assay sensitized by the dominant Krirregular facets-1(KrIf-1) allele, we show that the Piwi-interacting RNA (piRNA) pathway, but not the short interfering RNA or micro RNA pathway, is involved in canalization. Furthermore, we isolated a protein complex composed of Hsp90, Piwi and Hop, the Hsp70/Hsp90 organizing protein homolog, and we demonstrated the function of this complex in canalization. Our data indicate that Hsp90 and Hop regulate the piRNA pathway through Piwi to mediate canalization. Moreover, they point to epigenetic silencing of the expression of existing genetic variants and the suppression of transposon-induced new genetic variation as two major mechanisms underlying piRNA pathway-mediated canalization.

Yeast Isw1p Forms Two Separable Complexes In Vivo

ABSTRACT There are several classes of ATP-dependent chromatin remodeling complexes, which modulate the structure of chromatin to regulate a variety of cellular processes. The budding yeast, Saccharomyces cerevisiae, encodes two ATPases of the ISWI class, Isw1p and Isw2p. Previously Isw1p was shown to copurify with three other proteins. Here we identify these associated proteins and show that Isw1p forms two separable complexes in vivo (designated Isw1a and Isw1b). Biochemical assays revealed that while both have equivalent nucleosome-stimulated ATPase activities, Isw1a and Isw1b differ in their abilities to bind to DNA and nucleosomal substrates, which possibly accounts for differences in specific activities in nucleosomal spacing and sliding. In vivo, the two Isw1 complexes have overlapping functions in transcriptional regulation of some genes yet distinct functions at others. In addition, these complexes show different contributions to cell growth at elevated temperatures.

Dependency of ISW1a Chromatin Remodeling on Extranucleosomal DNA

ABSTRACT The nucleosome remodeling activity of ISW1a was dependent on whether ISW1a was bound to one or both extranucleosomal DNAs. ISW1a preferentially bound nucleosomes with an optimal length of ∼33 to 35 bp of extranucleosomal DNA at both the entry and exit sites over nucleosomes with extranucleosomal DNA at only one entry or exit site. Nucleosomes with extranucleosomal DNA at one of the entry/exit sites were readily remodeled by ISW1a and stimulated the ATPase activity of ISW1a, while conversely, nucleosomes with extranucleosomal DNA at both entry/exit sites were unable either to stimulate the ATPase activity of ISW1a or to be mobilized. DNA footprinting revealed that a major conformational difference between the nucleosomes was the lack of ISW1a binding to nucleosomal DNA two helical turns from the dyad axis in nucleosomes with extranucleosomal DNA at both entry/exit sites. The Ioc3 subunit of ISW1a was found to be the predominant subunit associated with extranucleosomal DNA when ISW1a is bound either to one or to both extranucleosomal DNAs. These two conformations of the ISW1a-nucleosome complex are suggested to be the molecular basis for the nucleosome spacing activity of ISW1a on nucleosomal arrays. ISW1b, the other isoform of ISW1, does not have the same dependency for extranucleosomal DNA as ISW1a and, likewise, is not able to space nucleosomes.

A regulated PNUTS mRNA to lncRNA splice switch mediates EMT and tumour progression

The contribution of lncRNAs to tumour progression and the regulatory mechanisms driving their expression are areas of intense investigation. Here, we characterize the binding of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) to a nucleic acid structural element located in exon 12 of PNUTS (also known as PPP1R10) pre-RNA that regulates its alternative splicing. HnRNP E1 release from this structural element, following its silencing, nucleocytoplasmic translocation or in response to TGFβ, allows alternative splicing and generates a non-coding isoform of PNUTS. Functionally the lncRNA-PNUTS serves as a competitive sponge for miR-205 during epithelial–mesenchymal transition (EMT). In mesenchymal breast tumour cells and in breast tumour samples, the expression of lncRNA-PNUTS is elevated and correlates with levels of ZEB mRNAs. Thus, PNUTS is a bifunctional RNA encoding both PNUTS mRNA and lncRNA-PNUTS, each eliciting distinct biological functions. While PNUTS mRNA is ubiquitously expressed, lncRNA-PNUTS appears to be tightly regulated dependent on the status of hnRNP E1 and tumour context.

Simultaneous Single-Molecule Detection of Endogenous C-5 DNA Methylation and Chromatin Accessibility Using MAPit

Bisulfite genomic sequencing provides a single-molecule view of cytosine methylation states. After deamination, each cloned molecule contains a record of methylation within its sequence. The full power of this technique is harnessed by treating nuclei with an exogenous DNMT prior to DNA extraction. This exogenous methylation marks regions of accessibility and footprints nucleosomes, as well as other DNA-binding proteins. Thus, each cloned molecule records not only the endogenous methylation present (at CG sites, in mammals), but also the exogenous (GC, when using the Chlorella virus protein M.CviPI). We term this technique MAPit, methylation accessibility protocol for individual templates.

Co-chaperone Hsp70/Hsp90-organizing protein (Hop) is required for transposon silencing and Piwi-interacting RNA (piRNA) biogenesis

Piwi-interacting RNAs (piRNAs) are 26–30-nucleotide germ line-specific small non-coding RNAs that have evolutionarily conserved function in mobile genetic element (transposons) silencing and maintenance of genome integrity. Drosophila Hsp70/90-organizing protein homolog (Hop), a co-chaperone, interacts with piRNA-binding protein Piwi and mediates silencing of phenotypic variations. However, it is not known whether Hop has a direct role in piRNA biogenesis and transposon silencing. Here, we show that knockdown of Hop in the germ line nurse cells (GLKD) of Drosophila ovaries leads to activation of transposons. Hop GLKD females can lay eggs at the same rate as wild-type counterparts, but the eggs do not hatch into larvae. Hop GLKD leads to the accumulation of γ-H2Av foci in the germ line, indicating increased DNA damage in the ovary. We also show that Hop GLKD-induced transposon up-regulation is due to inefficient piRNA biogenesis. Based on these results, we conclude that Hop is a critical component of the piRNA pathway and that it maintains genome integrity by silencing transposons.

Tudor-SN Interacts with Piwi Antagonistically in Regulating Spermatogenesis but Synergistically in Silencing Transposons in Drosophila

Piwi proteins associate with piRNAs and functions in epigenetic programming, post-transcriptional regulation, transposon silencing, and germline development. However, it is not known whether the diverse functions of these proteins are molecularly separable. Here we report that Piwi interacts with Tudor-SN (Tudor staphylococcal nuclease, TSN) antagonistically in regulating spermatogenesis but synergistically in silencing transposons. However, it is not required for piRNA biogenesis. TSN is known to participate in diverse molecular functions such as RNAi, degradation of hyper-edited miRNAs, and spliceosome assembly. We show that TSN colocalizes with Piwi in primordial germ cells (PGCs) and embryonic somatic cells. In adult ovaries and testes, TSN is ubiquitously expressed and enriched in the cytoplasm of both germline and somatic cells. The tsn mutants display a higher mitotic index of spermatogonia, accumulation of spermatocytes, defects in meiotic cytokinesis, a decreased number of spermatids, and eventually reduced male fertility. Germline-specific TSN-expression analysis demonstrates that this function is germline-dependent. Different from other known Piwi interters, TSN represses Piwi expression at both protein and mRNA levels. Furthermore, reducing piwi expression in the germline rescues tsn mutant phenotype in a dosage-dependent manner, demonstrating that Piwi and TSN interact antagonistically in germ cells to regulate spermatogenesis. However, the tsn deficiency has little, if any, impact on piRNA biogenesis but displays a synergistic effect with piwi mutants in transposon de-silencing. Our results reveal the biological function of TSN and its contrasting modes of interaction with Piwi in spermatogenesis, transposon silencing, and piRNA biogenesis.

The Long (lncRNA) and Short (miRNA) of It: TGFβ-Mediated Control of RNA-Binding Proteins and Noncoding RNAs

RNA-binding proteins (RBP) and noncoding RNAs (ncRNA), such as long noncoding RNAs (lncRNA) and microRNAs (miRNA), control co- and posttranscriptional gene regulation (PTR). At the PTR level, RBPs and ncRNAs contribute to pre-mRNA processing, mRNA maturation, transport, localization, turnover, and translation. Deregulation of RBPs and ncRNAs promotes the onset of cancer progression and metastasis. Both RBPs and ncRNAs are altered by signaling cascades to cooperate or compete with each other to bind their nucleic acid targets. Most importantly, transforming growth factor-beta (TGFβ) signaling plays a significant role in controlling gene expression patterns by targeting RBPs and ncRNAs. Because of TGFβ signaling in cancer, RBP-RNA or RNA-RNA interactions are altered and cause enhanced cell growth and tumor cell dissemination. This review focuses on the emerging concepts of TGFβ signaling on posttranscriptional gene regulation and highlights the implications of RBPs and ncRNAs in cancer progression and metastasis. Mol Cancer Res; 16(4); 567–79. ©2018 AACR.

A critical role for nucleoporin 358 (Nup358) in transposon silencing and piRNA biogenesis in Drosophila

Piwi-interacting RNAs (piRNAs) are a class of small noncoding RNAs that bind Piwi proteins to silence transposons and to regulate gene expression. In Drosophila germ cells, the Aubergine (Aub)-Argonaute 3 (Ago3)–dependent ping-pong cycle generates most germline piRNAs. Loading of antisense piRNAs amplified by this cycle enables Piwi to enter the nucleus and silence transposons. Nuclear localization is crucial for Piwi function in transposon silencing, but how this process is regulated remains unknown. It is also not known whether any of the components of the nuclear pore complex (NPC) directly function in the piRNA pathway. Here, we show that nucleoporin 358 (Nup358) and Piwi interact with each other and that a germline knockdown (GLKD) of Nup358 with short hairpin RNA prevents Piwi entry into the nucleus. The Nup358 GLKD also activated transposons, increased genomic instability, and derailed piRNA biogenesis because of a combination of decreased piRNA precursor transcription and a collapse of the ping-pong cycle. Our results point to a critical role for Nup358 in the piRNA pathway, laying the foundation for future studies to fully elucidate the mechanisms by which Nup358 contributes to piRNA biogenesis and transposon silencing.