Ovol1 represses its own transcription by competing with transcription activator c-Myb and by recruiting histone deacetylase activity

Ovol1 belongs to a family of evolutionarily conserved zinc finger proteins that act downstream of key developmental signaling pathways such as Wnt and TGF-β/BMP. It plays important roles in epithelial and germ cell development, particularly by repressing c-Myc and Id2 genes and modulating the balance between proliferation and differentiation of progenitor cells. In this study, we show that Ovol1 negatively regulates its own expression by binding to and repressing the activity of its promoter. We further demonstrate that Ovol1 uses both passive and active repression mechanisms to auto-repress: (1) it antagonizes transcriptional activation of c-Myb, a known positive regulator of proliferation, by competing for DNA binding; (2) it recruits histone deacetylase activity to the promoter via an N-terminal SNAG repressor domain. At Ovol1 cognate sites in the endogenous Ovol1 promoter, c-Myb binding correlates with increased histone acetylation, whereas the expression of Ovol1 correlates with a displacement of c-Myb from the DNA and decreased histone acetylation. Collectively, our data suggest that Ovol1 restricts its own expression by counteracting c-Myb activation and histone acetylation of the Ovol1 promoter

Two-hybrid analysis of Ty3 capsid subdomain interactions

<p>Abstract</p> <p>Background</p> <p>The yeast retrotransposon Ty3 forms stable virus-like particles. Gag3, the major structural protein, is composed of capsid, spacer and nucleocapsid domains. The capsid domain of Gag3 was previously modeled as a structure similar to retrovirus capsid.</p> <p>Findings</p> <p>Two-hybrid analysis was used to understand the interactions that contribute to particle assembly. Gag3 interacted with itself as predicted based on its role as the major structural protein. The N-terminal subdomain (NTD) of the capsid was able to interact with itself and with the C-terminal subdomain (CTD) of the capsid, but interacted less well with intact Gag3. Mutations previously shown to block particle assembly disrupted Gag3 interactions more than subdomain interactions.</p> <p>Conclusions</p> <p>The findings that the NTD interacts with itself and with the CTD are consistent with previous modeling and a role similar to that of the capsid in retrovirus particle structure. These results are consistent with a model in which the Gag3-Gag3 interactions that initiate assembly differ from the subdomain interactions that potentially underlie particle stability.</p

Host support of Ty3 retrotransposition in Saccharomyces cerevisiae

Ty3 is a Saccharomyces cereviae LTR retrotransposon. The structure of Ty3 is similar to that of a simple retrovirus. It is 5.4 kb in length and encodes overlapping GAG3 and POL3 reading frames flanked by 340 bp long terminal repeats. Expression of Ty3 results in production of Gag3 and Gag3-Pol3 polyproteins which assemble together with genomic RNA into in association with P-body proteins. VLPs are also associated with these clusters. The nucleo-capsid domain of Ty3 Gag3 is required in trans for recruitment of Ty3 RNA into P bodies. The untranslated regions of Ty3 RNA are sufficient in cis for recruitment of RNA to P bodies, but the GAG3-POL3 coding domain of the RNA can also confer association with P body proteins. In contrast, only the untranslated sequences confer packaging of a mini-Ty3 transcript. Upon assembly, Gag3 is processed into capsid, spacer, and nucleocapsid. Gag3-Pol3 is processed into those proteins and protease, junction, reverse transcriptase, and integrase. We propose that P-body proteins promote Ty3 VLP assembly and a mass spectrometry approach is being taken to further define the components of these dynamic complexes. However, in spite of genetic evidence that P-body proteins play a positive role in Ty3 production, these intracellular foci may also act as host traps to down-regulate transposition. Ty3-P body clusters become perinuclear over time and are physically associated with nuclear pores. A specific class of FG nucleoporins are required for Ty3 nuclear entry

Ovol1 regulates the growth arrest of embryonic epidermal progenitor cells and represses c-myc transcription

Transcriptional control plays a key role in regulating epidermal proliferation and differentiation. Although ample information has been obtained on how epidermal homeostasis is controlled in adult skin, less is known about the control of proliferation/differentiation of epidermal stem/progenitor cells in the developing embryo. Ovol1, encoding a zinc finger protein homologous to Drosophila melanogaster Ovo, is expressed in embryonic epidermal progenitor cells that are transiting from proliferation to terminal differentiation. In this study, we demonstrate a function for Ovol1 in interfollicular epidermal development. In its absence, developing epidermis fails to properly restrict the proliferative potential of progenitor cells, and cultured keratinocytes fail to efficiently undergo growth arrest in response to extrinsic growth-inhibitory signals. We present molecular evidence that c-myc expression is up-regulated in Ovol1-deficient suprabasal cells and that Ovol1 represses c-myc transcription by directly binding to its promoter. Collectively, our findings indicate that Ovol1 is required for proliferation exit of committed epidermal progenitor cells and identify c-myc as an Ovol1 target

Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress

Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages

Pygo2 expands mammary progenitor cells by facilitating histone H3 K4 methylation

Recent studies have unequivocally identified multipotent stem/progenitor cells in mammary glands, offering a tractable model system to unravel genetic and epigenetic regulation of epithelial stem/progenitor cell development and homeostasis. In this study, we show that Pygo2, a member of an evolutionarily conserved family of plant homeo domain–containing proteins, is expressed in embryonic and postnatal mammary progenitor cells. Pygo2 deficiency, which is achieved by complete or epithelia-specific gene ablation in mice, results in defective mammary morphogenesis and regeneration accompanied by severely compromised expansive self-renewal of epithelial progenitor cells. Pygo2 converges with Wnt/β-catenin signaling on progenitor cell regulation and cell cycle gene expression, and loss of epithelial Pygo2 completely rescues β-catenin–induced mammary outgrowth. We further describe a novel molecular function of Pygo2 that is required for mammary progenitor cell expansion, which is to facilitate K4 trimethylation of histone H3, both globally and at Wnt/β-catenin target loci, via direct binding to K4-methyl histone H3 and recruiting histone H3 K4 methyltransferase complexes

Molecular analysis of the human class I alcohol dehydrogenase gene family and nucleotide sequence of the gene encoding the beta subunit.

Human alcohol dehydrogenase (ADH) exists as a heterogeneous group of isozymes capable of oxidizing a wide variety of aliphatic and aromatic alcohols. The five distinct human ADH subunits, each encoded by a separate gene, are differentially expressed during development and are subject to tissue-specific regulation. To analyze the organization and regulation of human ADH genes we first isolated a cDNA clone (pADH12) encoding the 3' portion of the beta ADH gene. In the current study pADH12 was used to screen a human genomic library, and several overlapping and nonoverlapping clones were selected. Hybridization and partial nucleotide sequence analyses of the clones indicated that three full-length human ADH genes encoding the alpha, beta, and gamma subunits were isolated. Human genomic DNA hybridization results indicate that the alpha, beta, and gamma ADH genes form a closely related gene family and suggest that the other known human ADH genes (i.e. those encoding the pi and chi subunits) share a more distant evolutionary relationship. Nucleotide sequence analysis of the beta ADH gene reveals that the coding region is interrupted by eight introns and spans approximately 15 kilobases. A presumptive transcription initiation site for the beta ADH gene was located by S1 nuclease mapping at a position 70 base pairs upstream of the start codon. The 5' flanking region possesses a TATA box promoter element as well as two tandem DNA sequences which display homology to previously examined glucocorticoid-responsive elements

Positive and negative regulatory elements control expression of the yeast retrotransposon Ty3.

We report the results of an analysis of Ty3 transcription and identification of Ty3 regions that mediate pheromone and mating-type regulation to coordinate its expression with the yeast life cycle. A set of strains was constructed which was isogenic except for the number of Ty3 elements, which varied from zero to three. Analysis of Ty3 expression in these strains showed that each of the three elements was transcribed and that each element was regulated. Dissection of the long terminal repeat regulatory region by Northern blot analysis of deletion mutants and reporter gene analysis showed that the upstream junction of Ty3 with flanking chromosomal sequences contained a negative control region. A 19-bp fragment (positions 56-74) containing one consensus copy and one 7 of 8-bp match to the pheromone response element (PRE) consensus was sufficient to mediate pheromone induction in either haploid cell type. Deletion of this region, however, did not abolish expression, indicating that other sequences also activate transcription. A 24-bp block immediately downstream of the PRE region contained a sequence similar to the a1-alpha 2 consensus that conferred mating-type control. A single base pair mutation in the region separating the PRE and a1-alpha 2 sequences blocked pheromone induction, but not mating-type control. Thus, the long terminal repeat of Ty3 is a compact, highly regulated, mobile promoter which is responsive to cell type and mating