RNAi screening of subtracted transcriptomes reveals tumor suppression by taurine-activated GABAA receptors involved in volume regulation.

To identify coding and non-coding suppressor genes of anchorage-independent proliferation by efficient loss-of-function screening, we have developed a method for enzymatic production of low complexity shRNA libraries from subtracted transcriptomes. We produced and screened two LEGO (Low-complexity by Enrichment for Genes shut Off) shRNA libraries that were enriched for shRNA vectors targeting coding and non-coding polyadenylated transcripts that were reduced in transformed Mouse Embryonic Fibroblasts (MEFs). The LEGO shRNA libraries included ~25 shRNA vectors per transcript which limited off-target artifacts. Our method identified 79 coding and non-coding suppressor transcripts. We found that taurine-responsive GABAA receptor subunits, including GABRA5 and GABRB3, were induced during the arrest of non-transformed anchor-deprived MEFs and prevented anchorless proliferation. We show that taurine activates chloride currents through GABAA receptors on MEFs, causing seclusion of cell volume in large membrane protrusions. Volume seclusion from cells by taurine correlated with reduced proliferation and, conversely, suppression of this pathway allowed anchorage-independent proliferation. In human cholangiocarcinomas, we found that several proteins involved in taurine signaling via GABAA receptors were repressed. Low GABRA5 expression typified hyperproliferative tumors, and loss of taurine signaling correlated with reduced patient survival, suggesting this tumor suppressive mechanism operates in vivo.

Unraveling transcriptome dynamics in human spermatogenesis.

Spermatogenesis is a dynamic developmental process that includes stem cell proliferation and differentiation, meiotic cell divisions and extreme chromatin condensation. Although studied in mice, the molecular control of human spermatogenesis is largely unknown. Here, we developed a protocol that enables next-generation sequencing of RNA obtained from pools of 500 individually laser-capture microdissected cells of specific germ cell subtypes from fixed human testis samples. Transcriptomic analyses of these successive germ cell subtypes reveals dynamic transcription of over 4000 genes during human spermatogenesis. At the same time, many of the genes encoding for well-established meiotic and post-meiotic proteins are already present in the pre-meiotic phase. Furthermore, we found significant cell type-specific expression of post-transcriptional regulators, including expression of 110 RNA-binding proteins and 137 long non-coding RNAs, most of them previously not linked to spermatogenesis. Together, these data suggest that the transcriptome of precursor cells already contains the genes necessary for cellular differentiation and that timely translation controlled by post-transcriptional regulators is crucial for normal development. These established transcriptomes provide a reference catalog for further detailed studies on human spermatogenesis and spermatogenic failure.

The retinoblastoma protein: multitasking to suppress tumorigenesis.

Tumor suppressor activity of the retinoblastoma protein pRB is preserved despite loss of interaction with E2F transcription factors (E2F) or proteins harboring a leucine-x-cysteine-x-glutamic acid motif (LxCxE, where x is any amino acid). This indicates that pRB uses several parallel pathways to suppress tumorigenesis, which may also include E2F- and LxCxE-independent interactions.

RB family tumor suppressor activity may not relate to active silencing of E2F target genes.

The retinoblastoma protein pRB and its two homologs p130 and p107 form the family of pocket proteins and play a major role in cell-cycle regulation and suppression of human and mouse tumorigenesis. Pocket proteins regulate the activity of E2F transcription factors during G1-S transition. Two mechanisms have been described: (i) pocket protein binding blocks the transactivation domain of activator E2Fs, inhibiting E2F-dependent transcription and (ii) E2F-bound pocket proteins can recruit chromatin remodeling proteins containing an LxCxE motif (x encoding any amino acid), resulting in active repression of E2F target genes. To investigate the importance of pRB's LxCxE-interacting motif in cell-cycle control and tumor suppression, we generated mouse embryonic fibroblasts and mice expressing a mutant pRB protein carrying an asparagine for phenylalanine substitution at position 750, abrogating LxCxE binding. Because p130 may compensate for loss of pRB, we studied pRB(N750F) activity in the presence and absence of p130. The pRB-LxCxE interaction was not required for cell-cycle arrest upon mitogen deprivation and cell-cell contact, but did contribute to RAS(V12)- and radiation-induced cell-cycle arrest. Remarkably, the pRB-LxCxE interaction was not required for suppression of in vitro and in vivo transformation, even in the absence of p130. These results indicate that pRB's tumor suppressor activity is not effectuated by active silencing of E2F target genes, but rather by regulation of activator E2Fs or another unidentified mechanism. Furthermore, the in vitro response of pocket protein-perturbed cells to mitogen deprivation and cell-cell contact seems a better predictor of tumor development than the response to ectopic RAS(V12) expression. Cancer Res; 74(18); 5266-76. ©2014 AACR.

Molecular control of rodent spermatogenesis.

Spermatogenesis is a complex developmental process that ultimately generates mature spermatozoa. This process involves a phase of proliferative expansion, meiosis, and cytodifferentiation. Mouse models have been widely used to study spermatogenesis and have revealed many genes and molecular mechanisms that are crucial in this process. Although meiosis is generally considered as the most crucial phase of spermatogenesis, mouse models have shown that pre-meiotic and post-meiotic phases are equally important. Using knowledge generated from mouse models and in vitro studies, the current review provides an overview of the molecular control of rodent spermatogenesis. Finally, we briefly relate this knowledge to fertility problems in humans and discuss implications for future research. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.

Anchorage-independent growth of pocket protein-deficient murine fibroblasts requires bypass of G2 arrest and can be accomplished by expression of TBX2.

Mouse embryonic fibroblasts (MEFs) deficient for pocket proteins (i.e., pRB/p107-, pRB/p130-, or pRB/p107/p130-deficient MEFs) have lost proper G(1) control and are refractory to Ras(V12)-induced senescence. However, pocket protein-deficient MEFs expressing Ras(V12) were unable to exhibit anchorage-independent growth or to form tumors in nude mice. We show that depending on the level of pocket proteins, loss of adhesion induces G(1) and G(2) arrest, which could be alleviated by overexpression of the TBX2 oncogene. TBX2-induced transformation occurred only in the absence of pocket proteins and could be attributed to downregulation of the p53/p21(CIP1) pathway. Our results show that a balance between the pocket protein and p53 pathways determines the level of transformation of MEFs by regulating cyclin-dependent kinase activities. Since transformation of human fibroblasts also requires ablation of both pathways, our results imply that the mechanisms underlying transformation of human and mouse cells are not as different as previously claimed.