An insulator element located at the cyclin B1 interacting protein 1 gene locus is highly conserved among mammalian species. Yoshida, W; Tomikawa, J; Inaki, M; Kimura, H; Onodera, M; Hata, K; Nakabayashi, K PloS one
10
e0131204
2015
Show Abstract
Insulators are cis-elements that control the direction of enhancer and silencer activities (enhancer-blocking) and protect genes from silencing by heterochromatinization (barrier activity). Understanding insulators is critical to elucidate gene regulatory mechanisms at chromosomal domain levels. Here, we focused on a genomic region upstream of the mouse Ccnb1ip1 (cyclin B1 interacting protein 1) gene that was methylated in E9.5 embryos of the C57BL/6 strain, but unmethylated in those of the 129X1/SvJ and JF1/Ms strains. We hypothesized the existence of an insulator-type element that prevents the spread of DNA methylation within the 1.8 kbp segment, and actually identified a 242-bp and a 185-bp fragments that were located adjacent to each other and showed insulator and enhancer activities, respectively, in reporter assays. We designated these genomic regions as the Ccnb1ip1 insulator and the Ccnb1ip1 enhancer. The Ccnb1ip1 insulator showed enhancer-blocking activity in the luciferase assays and barrier activity in the colony formation assays. Further examination of the Ccnb1ip1 locus in other mammalian species revealed that the insulator and enhancer are highly conserved among a wide variety of species, and are located immediately upstream of the transcriptional start site of Ccnb1ip1. These newly identified cis-elements may be involved in transcriptional regulation of Ccnb1ip1, which is important in meiotic crossing-over and G2/M transition of the mitotic cell cycle. | | | 26110280
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Ezh2 mediated H3K27me3 activity facilitates somatic transition during human pluripotent reprogramming. Rao, RA; Dhele, N; Cheemadan, S; Ketkar, A; Jayandharan, GR; Palakodeti, D; Rampalli, S Scientific reports
5
8229
2015
Show Abstract
Factor induced reprogramming of fibroblasts is an orchestrated but inefficient process. At the epigenetic level, it results in drastic chromatin changes to erase the existing somatic "memory" and to establish the pluripotent state. Accordingly, alterations of chromatin regulators including Ezh2 influence iPSC generation. While the role of individual transcription factors in resetting the chromatin landscape during iPSC generation is increasingly evident, their engagement with chromatin modulators remains to be elucidated. In the current study, we demonstrate that histone methyl transferase activity of Ezh2 is required for mesenchymal to epithelial transition (MET) during human iPSC generation. We show that the H3K27me3 activity favors induction of pluripotency by transcriptionally targeting the TGF-β signaling pathway. We also demonstrate that the Ezh2 negatively regulates the expression of pro-EMT miRNA's such as miR-23a locus during MET. Unique association of Ezh2 with c-Myc was required to silence the aforementioned circuitry. Collectively, our findings provide a mechanistic understanding by which Ezh2 restricts the somatic programme during early phase of cellular reprogramming and establish the importance of Ezh2 dependent H3K27me3 activity in transcriptional and miRNA modulation during human iPSC generation. | | | 25648270
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Histone monoubiquitination by Clock-Bmal1 complex marks Per1 and Per2 genes for circadian feedback. Tamayo, AG; Duong, HA; Robles, MS; Mann, M; Weitz, CJ Nature structural & molecular biology
22
759-66
2015
Show Abstract
Circadian rhythms in mammals are driven by a feedback loop in which the transcription factor Clock-Bmal1 activates expression of Per and Cry proteins, which together form a large nuclear complex (Per complex) that represses Clock-Bmal1 activity. We found that mouse Clock-Bmal1 recruits the Ddb1-Cullin-4 ubiquitin ligase to Per (Per1 and Per2), Cry (Cry1 and Cry2) and other circadian target genes. Histone H2B monoubiquitination at Per genes was rhythmic and depended on Bmal1, Ddb1 and Cullin-4a. Depletion of Ddb1-Cullin-4a or an independent decrease in H2B monoubiquitination caused defective circadian feedback and decreased the association of the Per complex with DNA-bound Clock-Bmal1. Clock-Bmal1 thus covalently marks Per genes for subsequent recruitment of the Per complex. Our results reveal a chromatin-mediated signal from the positive to the negative limb of the clock that provides a licensing mechanism for circadian feedback. | | | 26323038
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Characterization of the mammalian miRNA turnover landscape. Guo, Y; Liu, J; Elfenbein, SJ; Ma, Y; Zhong, M; Qiu, C; Ding, Y; Lu, J Nucleic acids research
43
2326-41
2015
Show Abstract
Steady state cellular microRNA (miRNA) levels represent the balance between miRNA biogenesis and turnover. The kinetics and sequence determinants of mammalian miRNA turnover during and after miRNA maturation are not fully understood. Through a large-scale study on mammalian miRNA turnover, we report the co-existence of multiple cellular miRNA pools with distinct turnover kinetics and biogenesis properties and reveal previously unrecognized sequence features for fast turnover miRNAs. We measured miRNA turnover rates in eight mammalian cell types with a combination of expression profiling and deep sequencing. While most miRNAs are stable, a subset of miRNAs, mostly miRNA*s, turnovers quickly, many of which display a two-step turnover kinetics. Moreover, different sequence isoforms of the same miRNA can possess vastly different turnover rates. Fast turnover miRNA isoforms are enriched for 5' nucleotide bias against Argonaute-(AGO)-loading, but also additional 3' and central sequence features. Modeling based on two fast turnover miRNA*s miR-222-5p and miR-125b-1-3p, we unexpectedly found that while both miRNA*s are associated with AGO, they strongly differ in HSP90 association and sensitivity to HSP90 inhibition. Our data characterize the landscape of genome-wide miRNA turnover in cultured mammalian cells and reveal differential HSP90 requirements for different miRNA*s. Our findings also implicate rules for designing stable small RNAs, such as siRNAs. | | | 25653157
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Splicing function of mitotic regulators links R-loop-mediated DNA damage to tumor cell killing. Wan, Y; Zheng, X; Chen, H; Guo, Y; Jiang, H; He, X; Zhu, X; Zheng, Y The Journal of cell biology
209
235-46
2015
Show Abstract
Although studies suggest that perturbing mitotic progression leads to DNA damage and p53 activation, which in turn lead to either cell apoptosis or senescence, it remains unclear how mitotic defects trigger p53 activation. We show that BuGZ and Bub3, which are two mitotic regulators localized in the interphase nucleus, interact with the splicing machinery and are required for pre-mRNA splicing. Similar to inhibition of RNA splicing by pladienolide B, depletion of either BuGZ or Bub3 led to increased formation of RNA-DNA hybrids (R-loops), which led to DNA damage and p53 activation in both human tumor cells and primary cells. Thus, R-loop-mediated DNA damage and p53 activation offer a mechanistic explanation for apoptosis of cancer cells and senescence of primary cells upon disruption of the dual-function mitotic regulators. This demonstrates the importance of understanding the full range of functions of mitotic regulators to develop antitumor drugs. | | | 25918225
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Profiling post-transcriptionally networked mRNA subsets using RIP-Chip and RIP-Seq. Jayaseelan, S; Doyle, F; Tenenbaum, SA Methods
67
13-9
2014
Show Abstract
Post-transcriptional regulation of messenger RNA contributes to numerous aspects of gene expression. The key component to this level of regulation is the interaction of RNA-binding proteins (RBPs) and their associated target mRNA. Splicing, stability, localization, translational efficiency, and alternate codon use are just some of the post-transcriptional processes regulated by RBPs. Central to our understanding of these processes is the need to characterize the network of RBP-mRNA associations and create a map of this functional post-transcriptional regulatory system. Here we provide a detailed methodology for mRNA isolation using RBP immunoprecipitation (RIP) as a primary partitioning approach followed by microarray (Chip) or next generation sequencing (NGS) analysis. We do this by using specific antibodies to target RBPs for the capture of associated RNA cargo. RIP-Chip/Seq has proven to be is a versatile, genomic technique that has been widely used to study endogenous RBP-RNA associations. | | | 24257445
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p53-dependent gene repression through p21 is mediated by recruitment of E2F4 repression complexes. Benson, EK; Mungamuri, SK; Attie, O; Kracikova, M; Sachidanandam, R; Manfredi, JJ; Aaronson, SA Oncogene
33
3959-69
2014
Show Abstract
The p53 tumor suppressor protein is a major sensor of cellular stresses, and upon stabilization, activates or represses many genes that control cell fate decisions. While the mechanism of p53-mediated transactivation is well established, several mechanisms have been proposed for p53-mediated repression. Here, we demonstrate that the cyclin-dependent kinase inhibitor p21 is both necessary and sufficient for the downregulation of known p53-repression targets, including survivin, CDC25C, and CDC25B in response to p53 induction. These same targets are similarly repressed in response to p16 overexpression, implicating the involvement of the shared downstream retinoblastoma (RB)-E2F pathway. We further show that in response to either p53 or p21 induction, E2F4 complexes are specifically recruited onto the promoters of these p53-repression targets. Moreover, abrogation of E2F4 recruitment via the inactivation of RB pocket proteins, but not by RB loss of function alone, prevents the repression of these genes. Finally, our results indicate that E2F4 promoter occupancy is globally associated with p53-repression targets, but not with p53 activation targets, implicating E2F4 complexes as effectors of p21-dependent p53-mediated repression. | Western Blotting | | 24096481
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BMI1 reprogrammes histone acetylation and enhances c-fos pathway via directly binding to Zmym3 in malignant myeloid progression. Shen, H; Chen, Z; Ding, X; Qi, X; Cen, J; Wang, Y; Yao, L; Chen, Y Journal of cellular and molecular medicine
18
1004-17
2014
Show Abstract
The polycomb group BMI1 is proved to be crucial in malignant myeloid progression. However, the underlying mechanism of the action of BMI1 in myeloid malignant progression was not well characterized. In this study, we found that the patients of both myelodysplastic syndromes and chronic myeloid leukaemia with BMI1 overexpression had a higher risk in malignant myeloid progression. In vitro gene transfection studies showed that BMI1 inhibited cell myeloid and erythroid differentiation induced by 12-O-tetradecanoyl phorbol-13-acetate (TPA) and histone deacetylase inhibitor sodium butyrate respectively. BMI1 also resisted apoptosis induced by arsenic trioxide. Moreover, the transcript levels of Runx1 and Pten were down-regulated in Bmi1-transfected cells in company with histone deacetylation modification. By using chromatin immunoprecipitation (ChIP) collaborated with secondary generation sequencing and verified by ChIP-PCR, we found that BMI1 directly bound to the promoter region of Zmym3, which encodes a component of histone deacetylase-containing complexes. In addition, as one of the downstream target genes of this complex, c-fos was activated with increasing histone acetylation when ZMYM3 was suppressed in the Bmi1-transfected cells. These results suggested that BMI1 may reprogramme the histone acetylation profile in multiple genes through either indirect or direct binding effects which probably contributes to the malignant progression of myeloid progenitor cells. | | | 24571310
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A long non-coding RNA promotes full activation of adult gene expression in the chicken α-globin domain. Arriaga-Canon, C; Fonseca-Guzmán, Y; Valdes-Quezada, C; Arzate-Mejía, R; Guerrero, G; Recillas-Targa, F Epigenetics
9
173-81
2014
Show Abstract
Long non-coding RNAs (lncRNAs) were recently shown to regulate chromatin remodelling activities. Their function in regulating gene expression switching during specific developmental stages is poorly understood. Here we describe a nuclear, non-coding transcript responsive for the stage-specific activation of the chicken adult α(D) globin gene. This non-coding transcript, named α-globin transcript long non-coding RNA (lncRNA-αGT) is transcriptionally upregulated in late stages of chicken development, when active chromatin marks the adult α(D) gene promoter. Accordingly, the lncRNA-αGT promoter drives erythroid-specific transcription. Furthermore, loss of function experiments showed that lncRNA-αGT is required for full activation of the α(D) adult gene and maintenance of transcriptionally active chromatin. These findings uncovered lncRNA-αGT as an important part of the switching from embryonic to adult α-globin gene expression, and suggest a function of lncRNA-αGT in contributing to the maintenance of adult α-globin gene expression by promoting an active chromatin structure. | | | 24196393
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Oxidative stress and inflammation modulate Rev-erbα signaling in the neonatal lung and affect circadian rhythmicity. Yang, G; Wright, CJ; Hinson, MD; Fernando, AP; Sengupta, S; Biswas, C; La, P; Dennery, PA Antioxid Redox Signal
21
17-32
2014
Show Abstract
The response to oxidative stress and inflammation varies with diurnal rhythms. Nevertheless, it is not known whether circadian genes are regulated by these stimuli. We evaluated whether Rev-erbα, a key circadian gene, was regulated by oxidative stress and/or inflammation in vitro and in a mouse model.A unique sequence consisting of overlapping AP-1 and nuclear factor kappa B (NFκB) consensus sequences was identified on the mouse Rev-erbα promoter. This sequence mediates Rev-erbα promoter activity and transcription in response to oxidative stress and inflammation. This region serves as an NrF2 platform both to receive oxidative stress signals and to activate Rev-erbα, as well as an NFκB-binding site to repress Rev-erbα with inflammatory stimuli. The amplitude of the rhythmicity of Rev-erbα was altered by pre-exposure to hyperoxia or disruption of NFκB in a cell culture model of circadian simulation. Oxidative stress overcame the inhibitory effect of NFκB binding on Rev-erbα transcription. This was confirmed in neonatal mice exposed to hyperoxia, where hyperoxia-induced lung Rev-erbα transcription was further increased with NFκB disruption. Interestingly, this effect was not observed in similarly exposed adult mice.These data provide novel mechanistic insights into how key circadian genes are regulated by oxidative stress and inflammation in the neonatal lung.Rev-erbα transcription and circadian oscillation are susceptible to oxidative stress and inflammation in the neonate. Due to Rev-erbα's role in cellular metabolism, this could contribute to lung cellular function and injury from inflammation and oxidative stress. | | | 24252172
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