Identification and Validation of a Putative Polycomb Responsive Element in the Human Genome

Epigenetic cellular memory mechanisms that involve polycomb and trithorax group of proteins are well conserved across metazoans. The cis-acting elements interacting with these proteins, however, are poorly understood in mammals. In a directed search we identified a potential polycomb responsive element with 25 repeats of YY1 binding motifthatwe designate PRE-PIK3C2B as it occurs in the first intron of human PIK3C2B gene. It down regulates reporter gene expression in HEK cells and the repression is dependent on polycomb group of proteins (PcG). We demonstrate that PRE-PIK3C2B interacts directly with YY1 in vitro and recruits PRC2 complex in vivo. The localization of PcG proteins including YY1 to PRE-PIK3C2B in HEK cells is decreased on knock-down of either YY1 or SUZ12. Endogenous PRE-PIK3C2B shows bivalent marking having H3K27me3 and H3K4me3 for repressed and active state respectively. In transgenic Drosophila, PRE-PIK3C2B down regulates mini-white expression, exhibits variegation and pairing sensitive silencing (PSS), which has not been previously demonstrated for mammalian PRE. Taken together, our results strongly suggest that PRE-PIK3C2B functions as a site of interaction for polycomb proteins.     

Redox state of tumor suppressor p53 regulates its sequence-specific DNA binding in DNA-damaged cells by cysteine 277

Using a bio-oligo pull-down DNA-binding assay we investigated the binding capacity of endogenous, DNA damage-induced p53 in human diploid fibroblasts to several p53-responsive elements (REs) present in p53-regulated genes. During the course of p53 accumulation, we observed a decrease in p53 binding to the GADD45 but not to the p21^WAF1/CIP1 RE. Using mutated GADD45 sequences we show that this change is dependent on the presence of cytosines at position 3 in RE pentamers and on the p53 redox state. Site-directed mutagenesis experiments demonstrated that Cys277 (a residue directly contacting base 3 in a RE pentamer) is critical for differential regulation of GADD45 in DNA-damaged cells. These data represent a novel mechanism for differential affinity of p53 to distinct REs.     

YY1 regulates the neural crest-associated slug gene in Xenopus laevis

slug gene expression is associated with the specification and migration of neural crest cells in the African clawed frog Xenopus laevis. We provide evidence that the protein Ying-Yang 1 (YY1) regulates the slug gene expression both indirectly and directly, via a YY1 cis-element in the slug promoter, during Xenopus development. The ability of the YY1 to bind this YY1 cis-element was confirmed by electromobility shift assays and reporter assays. YY1 was detected in the nuclei of ectodermal cells contemporaneously with the process of neural crest specification. The injection of anti-YY1 morpholino, which targeted both YY1alpha and YY1beta gene products, depleted YY1 expression below 20% and was lethal at gastrulation. Sublethal depletion of YY1 reduced the length of the anterior-posterior axis and severely inhibited the expression of the neural marker Nrp1 and of the slug gene. Overexpression of YY1 or mutation of the YY1 cis-element reduced the restricted spatial expression of the slug reporter gene in the neural ectoderm border and provoked its expression in the nonneural ectoderm. Chromatin immunoprecipitation indicated that endogenous YY1 interacts directly with the YY1 cis-element of the endogenous slug gene and with the slug gene reporter sequence injected into embryos. The results suggest that YY1 is essential for Xenopus development; is necessary for neural ectoderm differentiation, a prerequisite for neural crest specification; and restricts which cells can form neural crest mesenchyme through directly blocking slug gene activity.     

Transcriptional and post-transcriptional regulation of retrotransposons IAP and MuERV-L affect pluripotency of mice ES cells

Background  

In the mouse, culture of embryonic stem (ES) cells may decrease their pluripotency and give rise to foetal abnormalities in recipient embryos. These abnormalities are frequently associated with both, chromosome abnormalities or epigenetic alteration of imprinting genes; however, little is known about the epigenetic stability of endogenous retrotransposable elements (REs). In our laboratory, we came across a R1 ES cell line, which at passage 27, lost the ability of germline transmission and started inducing the kinky tail phenotype in all chimeric animals produced with it.     

YY1 activates Msx2 gene independent of bone morphogenetic protein signaling

Msx2 is a homeobox gene expressed in multiple embryonic tissues which functions as a key mediator of numerous developmental processes. YY1 is a bi-functional zinc finger protein that serves as a repressor or activator to a variety of promoters. The role of YY1 during embryogenesis remains unknown. In this study, we report that Msx2 is regulated by YY1 through protein–DNA interactions. During embryogenesis, the expression pattern of YY1 was observed to overlap in part with that of Msx2. Most notably, during first branchial arch and limb development, both YY1 and Msx2 were highly expressed, and their patterns were complementary. To test the hypothesis that YY1 regulates Msx2 gene expression, P19 embryonal cells were used in a number of expression and binding assays. We discovered that, in these cells, YY1 activated endogenous Msx2 gene expression as well as Msx2 promoter–luciferase fusion gene activity. These biological activities were dependent on both the DNA binding and activation domains of YY1. In addition, YY1 bound specifically to three YY1 binding sites on the proximal promoter of Msx2 that accounted for this transactivation. Mutations introduced to these sites reduced the level of YY1 transactivation. As bone morphogenetic protein type 4 (BMP4) regulates Msx2 expression in embryonic tissues and in P19 cells, we further tested whether YY1 is the mediator of this BMP4 activity. BMP4 did not induce the expression of YY1 in early mouse mandibular explants, nor in P19 cells, suggesting that YY1 is not a required mediator of the BMP4 pathway in these tissues at this developmental stage. Taken together, these findings suggest that YY1 functions as an activator for the Msx2 gene, and that this regulation, which is independent of the BMP4 pathway, may be required during early mouse craniofacial and limb morphogenesis.     

A novel, efficient method to derive bovine and mouse embryonic stem cells with in vivo differentiation potential by treatment with 5-azacytidine

Pluripotent embryonic stem cells (ESCs) were first isolated nearly three decades ago from mice, yet efficient ESC isolation has been limited to rodents and primates to date. We report a novel and robust technique for isolating ESCs from mammalian pre-implantation embryos by altering the epigenotype of embryonic explants and using pressed zona pellucida-free blastocysts. We first examined this technique for murine ESC derivation. Compared with controls, murine ESCs were efficiently derived when explants were exposed to 1μM 5-azacytidine, an epigenetic modifier that causes DNA demethylation (56.1% vs 31.6%; P < 0.01). Mouse ESCs stained positively for alkaline phosphatase, expressed markers of pluripotency including Oct4, Rex1 and SSEA1 and formed teratomas when injected into Severe Combined Immuno-Deficient (SCID) mice. The approach was subsequently used for bovine ESC derivation. In bovine a higher concentration of 5-azacytidine (5 μM) was required to elicit a response. This technique resulted in up to 18 times more efficient isolation of pluripotent cells than traditional methods (71.4% vs 4.0%; P < 0.001). These putative bovine ESCs expressed OCT4, REX1 mRNA and SSEA-1 and SSEA-4 proteins; and were able to form embryoid bodies in vitro and teratomas when injected in Severe Combined Immuno Deficient (SCID) mice. This is the first report on derivation of ESCs with both in vitro and in vivo differentiation potential in a livestock species.     

Rex1p deficiency leads to accumulation of precursor initiator tRNAMet and polyadenylation of substrate RNAs in Saccharomyces cerevisiae

A synthetic genetic array was used to identify lethal and slow-growth phenotypes produced when a mutation in TRM6, which encodes a tRNA modification enzyme subunit, was combined with the deletion of any non-essential gene in Saccharomyces cerevisiae. We found that deletion of the REX1 gene resulted in a slow-growth phenotype in the trm6-504 strain. Previously, REX1 was shown to be involved in processing the 3′ ends of 5S rRNA and the dimeric tRNA^Arg-tRNA^Asp. In this study, we have discovered a requirement for Rex1p in processing the 3′ end of tRNA_i^Met precursors and show that precursor tRNA_i^Met accumulates in a trm6-504 rex1Δ strain. Loss of Rex1p results in polyadenylation of its substrates, including tRNA_i^Met, suggesting that defects in 3′ end processing can activate the nuclear surveillance pathway. Finally, purified Rex1p displays Mg²⁺-dependent ribonuclease activity in vitro, and the enzyme is inactivated by mutation of two highly conserved amino acids.     

Conformations of p53 response elements in solution deduced using site-directed spin labeling and Monte Carlo sampling

The tumor suppressor protein p53 regulates numerous signaling pathways by specifically recognizing diverse p53 response elements (REs). Understanding the mechanisms of p53-DNA interaction requires structural information on p53 REs. However, such information is limited as a 3D structure of any RE in the unbound form is not available yet. Here, site-directed spin labeling was used to probe the solution structures of REs involved in p53 regulation of the p21 and Bax genes. Multiple nanometer distances in the p21-RE and BAX-RE, measured using a nucleotide-independent nitroxide probe and double-electron-electron-resonance spectroscopy, were used to derive molecular models of unbound REs from pools of all-atom structures generated by Monte-Carlo simulations, thus enabling analyses to reveal sequence-dependent DNA shape features of unbound REs in solution. The data revealed distinct RE conformational changes on binding to the p53 core domain, and support the hypothesis that sequence-dependent properties encoded in REs are exploited by p53 to achieve the energetically most favorable mode of deformation, consequently enhancing binding specificity. This work reveals mechanisms of p53-DNA recognition, and establishes a new experimental/computational approach for studying DNA shape in solution that has far-reaching implications for studying protein–DNA interactions.