Transcriptional regulation of Ard1b, a testis-predominant isoform of the catalytic subunit of mouse N-alpha terminal acetyltransferase, and the role of protein N-alpha acetylation Investigators: Pang, Fang, Clark, Rennert;in collaboration with Chan, Taft We have cloned a novel Ard1a (Arrest defective 1) gene homolog, known as Ard1b, that demonstrated testis specificity and was highly expressed during male meiosis in the mouse. We have shown Ard1b is functionally equivalent to Ard1a in the reconstitution of N-alpha-acetyltransferase activity in vitro. The testis-specific expression of Ard1b indicates its transcription is suppressed in somatic tissues. We subsequently identified Ard1b transcription is regulated epigenetically by DNA methylation: reactivation of Ard1b transcription occurs after treatment with 5-aza-deoxycytidine in mouse cells that do not express the gene. The two CpG islands located at the 5 end of Ard1b gene are hypermethylated in mouse somatic tissues, but hypomethylated in mouse testicular germ cells. We characterized the promoter region of the Ard1b gene. Reporter assays on different regions upstream of the gene led to identification of two upstream genomic regions that may contain inhibitory and enhancer element(s) for Ard1b transcription. We found the core promoter sequence for Ard1b is localized to a region spanning from -148 to at least +150 base-pairs with respect to the transcriptional start site of the gene, a region which is hypermethylated in cells that do not show Ard1b expression. Within this region, we predicted the presence of binding sequences for several transcription factors. By gene over-expression and knockdown experiments, we confirmed the role of Specificity protein 1 (Sp1) in the activation of Ard1b transcription. Similar experiments are underway to confirm the involvement of the other transcription factors predicted to regulate Ard1b transcription. Next we will examine coordination of DNA methylation and the binding of specific transcription factors in promotion of Ard1b transcription. On the other hand, the availability of Ard1b gene knockout mice enables us to study the role of Ard1b in testicular development as well as the biological significance of protein N-alpha acetylation. With a human cell culture model, we are investigating the difference in biological function between ARD1 and ARD1B, as well as, the global effect of deficiency of protein N-alpha acetylation on cellular function. Transcriptional regulation of Lin28 Investigators: Pang, Cho, Fang, Rennert;in collaboration with Chan Lin28 is a heterochronic gene involved in the temporal control of cell fate determination in C. elegans. It was shown to exert an enhancing effect on the reprogramming of somatic cells to embryonic stem (ES) cell-like state (i.e. iPS cells). In mammals, Lin28 is detected mostly in cells that possess proliferative/renewal capacity (e.g. embryonic stem cells, embryonal carcinoma cells, and mouse type A spermatogonia) and is absent in differentiated cells. The tissue-restricted and developmentally-regulated expression pattern of Lin28 suggests that its expression is subject to temporal and spatial regulation. Our preliminary data showed that Lin28 transcription is not regulated by DNA methylation;we hypothesized Lin28 transcription is primarily activated by the action of specific transcription factors that bind to its promoter. Reporter assays in mouse embryonal carcinoma cells P19 (Lin28-expressing) and mouse embryonic fibroblast cells NIH/3T3 (Lin28-non-expressing), led to localization of the core promoter of Lin28 gene - a region about 400 base-pairs upstream from its start codon. Specific modules of transcription factor binding sites have been predicted within this region, and by gene over-expression studies we have shown that at least Sp1 is able to activate Lin28 transcription in P19 cells. The involvement of other transcription factors in stimulating Lin28 expression, and the mechanism of suppression of Lin28 expression in non-expressing cells, are now under investigation. Effect of vitamin A deficiency on epigenomics of male germ cells: Investigators: Boucheron, Narani, Rennert;in collaboration with Chan The purpose of this project was to study the molecular basis of the effect of vitamin A deficiency (VAD) on spermatogenesis in mice. We first further described the mice model of VAD by performing a time-point study of the VAD diet in mice, which in our knowledge as not been done before, in order to have a better understanding of the chronology of the effect of VAD diet on this animal model. In this purpose, we characterized our experimental model using time-point study of the VAD diet, from 7 weeks up to 28 weeks. For each time point different analyzes allowed us to measure the vitamin A status on the animal: the RBP (Retinol Binding Protein) level in the serum, and the mRNA expression levels of retinoic acid (active metabolite of vitamin A) receptors in the liver, brain and testis. In parallel we used histological techniques to characterize the effect of different time of VAD on the testis of these animals. Our data described the onset of the decrease of the number of differentiated germ cells starting around 16 weeks of diet, coinciding with an the appearance of an increasing number of apoptotic cells. In the second part of our work, we employed expression profiling to study the genetic mechanism of vitamin A deficiency-induced arrest of spermatogonial stem cell differentiation. We isolated enriched population of spermatogonia by STAPUT method (sedimentation velocity at unit gravity, with 2%- 4% BSA gradient). We used a microarray analysis to compare the gene expression levels of these populations. We contrasted gene profiles in cells from controls to mice fed VAD for 18 and 25 weeks, and identified 1900 differentially expressed genes in the 18 weeks VAD group, and 9987 in the 25 weeks VAD group. We then carried out a more comprehensive analysis of the control and VAD 25 week spermatogonia performed bioinformatics analyses to identify clusters of genes and/or signaling pathways known to play a role in the regulation of spermatogenesis that may be affected by VAD. Our initial analyses focused on the retinoid signaling pathways and verify its hypoexpression in spermatogonia of the Vitamin-A-Deficient mice. Our data also highlighted the importance of other pathways in our conditions, NF-κB, thyroid hormones, cell cycle, lipids metabolism, Finally, our results highlight the effects of Vitamin-A on cytoskeleton remodeling and cell adhesion (including cell junctions between germ cells and somatic cells). To conclude, we observed effects of VAD on adult mouse spermatogenesis: arrest of germ cell differentiation, increased germ cells apoptosis, and a general effect on the transcriptome profile of spermatogonia. While these effects on germ cells were sometimes dramatic, our data point toward the importance of the VAD-induced effects on the somatic cells, and their indirect effects on the progression of spermatogenesis. We are studying in parallel the effect of VAD on the Leydig cells, interstitial cells adjacent to the seminiferous tubules, regulating spermatogenesis through the production of testosterone. Our first results show a decrease of the serum concentration of testosterone as an effect of Vitamin-A deficiency. We also performed microarray analyses comparing expression profile of the Leydig cells from VAD and Control animals, using VAD 28 weeks mice and a Control age-paired animals. Our results highlight an effect of VAD on different molecular pathways in these cells, including retinoic acid and thyroid pathways, lipid metabolism, NF-κB, cell adhesion and cell junctions.

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Lee, Tin-Lap; Rennert, Owen M; Chan, Wai-Yee (2012) Revealing the transcriptome landscape of mouse spermatogonial cells by tiling microarray. Methods Mol Biol 825:75-92
Pang, Alan Lap-Yin; Peacock, Stephanie; Johnson, Warren et al. (2009) Cloning, characterization, and expression analysis of the novel acetyltransferase retrogene Ard1b in the mouse. Biol Reprod 81:302-9
Lee, Tin-Lap; Pang, Alan Lap-Yin; Rennert, Owen M et al. (2009) Genomic landscape of developing male germ cells. Birth Defects Res C Embryo Today 87:43-63
Lee, Tin-Lap; Li, Yunmin; Alba, Diana et al. (2009) Developmental staging of male murine embryonic gonad by SAGE analysis. J Genet Genomics 36:215-27
Lee, Tin-Lap; Cheung, Hoi-Hung; Claus, Janek et al. (2009) GermSAGE: a comprehensive SAGE database for transcript discovery on male germ cell development. Nucleic Acids Res 37:D891-7