Involvement of chromatin and histone deacetylation in SV40 T antigen transcription regulation

Simian Virus 40 (SV40) large T antigen (T Ag) is a multifunctional viral oncoprotein that regulates viral and cellular transcriptional activity. However, the mechanisms by which such regulation occurs remain unclear. Here we show that T antigen represses CBP-mediated transcriptional activity. This repression is concomitant with histone H3 deacetylation and is TSA sensitive. Moreover, our results demonstrate that T antigen interacts with HDAC1 in vitro in an Rb-independent manner. In addition, the overexpression of HDAC1 cooperates with T antigen to antagonize CBP transactivation function and correlates with chromatin deacetylation of the TK promoter. Finally, decreasing HDAC1 levels with small interfering RNA (siRNA) partially abolishes T antigen-induced repression. These findings highlight the importance of the histone acetylation/deacetylation balance in the cellular transformation mediated by oncoviral proteins

Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders.

Genetic variants in chromatin regulators are frequently found in neurodevelopmental disorders, but their effect in disease etiology is rarely determined. Here, we uncover and functionally define pathogenic variants in the chromatin modifier EZH1 as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals. EZH1 encodes one of the two alternative histone H3 lysine 27 methyltransferases of the PRC2 complex. Unlike the other PRC2 subunits, which are involved in cancers and developmental syndromes, the implication of EZH1 in human development and disease is largely unknown. Using cellular and biochemical studies, we demonstrate that recessive variants impair EZH1 expression causing loss of function effects, while dominant variants are missense mutations that affect evolutionarily conserved aminoacids, likely impacting EZH1 structure or function. Accordingly, we found increased methyltransferase activity leading to gain of function of two EZH1 missense variants. Furthermore, we show that EZH1 is necessary and sufficient for differentiation of neural progenitor cells in the developing chick embryo neural tube. Finally, using human pluripotent stem cell-derived neural cultures and forebrain organoids, we demonstrate that EZH1 variants perturb cortical neuron differentiation. Overall, our work reveals a critical role of EZH1 in neurogenesis regulation and provides molecular diagnosis for previously undefined neurodevelopmental disorders

Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders

Genetic variants in chromatin regulators are frequently found in neurodevelopmental disorders, but their effect in disease etiology is rarely determined. Here, we uncover and functionally define pathogenic variants in the chromatin modifier EZH1 as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals. EZH1 encodes one of the two alternative histone H3 lysine 27 methyltransferases of the PRC2 complex. Unlike the other PRC2 subunits, which are involved in cancers and developmental syndromes, the implication of EZH1 in human development and disease is largely unknown. Using cellular and biochemical studies, we demonstrate that recessive variants impair EZH1 expression causing loss of function effects, while dominant variants are missense mutations that affect evolutionarily conserved aminoacids, likely impacting EZH1 structure or function. Accordingly, we found increased methyltransferase activity leading to gain of function of two EZH1 missense variants. Furthermore, we show that EZH1 is necessary and sufficient for differentiation of neural progenitor cells in the developing chick embryo neural tube. Finally, using human pluripotent stem cell-derived neural cultures and forebrain organoids, we demonstrate that EZH1 variants perturb cortical neuron differentiation. Overall, our work reveals a critical role of EZH1 in neurogenesis regulation and provides molecular diagnosis for previously undefined neurodevelopmental disorders

The histone acetyltransferases CBP/p300 are degraded in NIH 3T3 cells by activation of Ras signalling pathway

The CBP [CREB (cAMP-response-element-binding protein)-binding protein]/p300 acetyltransferases function as transcriptional co-activators and play critical roles in cell differentiation and proliferation. Accumulating evidence shows that alterations of the CBP/p300 protein levels are linked to human tumours. In the present study, we show that the levels of the CBP/p300 co-activators are decreased dramatically by continuous PDGF (platelet-derived growth factor) and Ras signalling pathway activation in NIH 3T3 fibroblasts. This effect occurs by reducing the expression levels of the CBP/p300 genes. In addition, CBP and p300 are degraded by the 26 S proteasome pathway leading to an overall decrease in the levels of the CBP/p300 proteins. Furthermore, we provide evidence that Mdm2 (murine double minute 2), in the presence of active H-Ras or N-Ras, induces CBP/p300 degradation in NIH 3T3 cells. These findings support a novel mechanism for modulating other signalling transduction pathways that require these common co-activators

The SV40 T antigen modulates CBP histone acetyltransferase activity

Histone acetyltransferases (HATs) play a key role in transcription control, cell proliferation and differentiation by modulating chromatin structure; however, little is known about their own regulation. Here we show that expression of the viral oncoprotein SV40 T antigen increases histone acetylation and global cellular HAT activities. In addition, it enhances CREB-binding protein HAT activity and modulates its transcriptional activity. Finally, we show that inhibition of cellular histone deacetylases by trichostatin A increases the SV40 infectivity rate. These findings highlight the importance of histone acetylation in the regulation of the cell cycle by oncoviral proteins

Regulation of E2F1 activity by acetylation

During the G(1) phase of the cell cycle, an E2F–RB complex represses transcription, via the recruitment of histone deacetylase activity. Phosphorylation of RB at the G(1)/S boundary generates a pool of ‘free’ E2F, which then stimulates transcription of S-phase genes. Given that E2F1 activity is stimulated by p300/CBP acetylase and repressed by an RB-associated deacetylase, we asked if E2F1 was subject to modification by acetylation. We show that the p300/CBP-associated factor P/CAF, and to a lesser extent p300/CBP itself, can acetylate E2F1 in vitro and that intracellular E2F1 is acetylated. The acetylation sites lie adjacent to the E2F1 DNA-binding domain and involve lysine residues highly conserved in E2F1, 2 and 3. Acetylation by P/CAF has three functional consequences on E2F1 activity: increased DNA-binding ability, activation potential and protein half-life. These results suggest that acetylation stimulates the functions of the non-RB bound ‘free’ form of E2F1. Consistent with this, we find that the RB-associated histone deacetylase can deacetylate E2F1. These results identify acetylation as a novel regulatory modification that stimulates E2F1's activation functions

HDAC1 acts in tandem with T antigen to antagonize the transcriptional activity of CBP

<p><b>Copyright information:</b></p><p>Taken from "Involvement of chromatin and histone deacetylation in SV40 T antigen transcription regulation"</p><p></p><p>Nucleic Acids Research 2007;35(6):1958-1968.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1874590.</p><p>© 2007 The Author(s)</p> HeLa cells were transfected with 2 μg of the Gal4-TK-luciferase reporter, 2 μg of Gal-CBP (FL), 2 μg of pSG5-T antigen, 1 μg of pcDNA3-HDAC1 and 1 μg of renilla reporter vector. Whole cell extracts were used in the luciferase-renilla assay. The activity derived from the Gal4-TK-luciferase reporter was normalized to 1.0 and the other activities are expressed relative to this. The data represent an average of at least five independent transfections. A schematic representation of the constructs used is shown at the bottom of the figure. HeLa cells were transfected with 2 μg of Gal4-TK-luciferase reporter (lane 1), 2 μg of Gal-CBP (FL) (lane 2), 2 μg of pSG5-T antigen (lane 3) and 1 μg of pcDNA3-HDAC1 (lane 4) and comparative ChIP analysis were performed in parallel with the same number of cells using antibodies that specifically recognize acetylated histone H3 (K 9 and 14), poly-acetylated histone H4 and HDAC1. The immunoprecipitates were analyzed by quantitative PCR as in A. Quantification of the acetyl-H3 and acetyl-H4 ChIPs bands (as described in Materials and Methods) is shown at the bottom part of the figure. HeLa cells were transfected with 2 μg of the Gal4-TK-luciferase reporter, 1 μg of renilla reporter, 2 μg of Gal-CBP (FL), 2 μg of pSG5-T antigen and 1 μg of pcDNA3-HDAC1 together with 8 μg of HDAC1(1, 2 and 3) or control siRNA vectors. Total cell extracts were prepared 48 h after transfection and used in the luciferase-renilla assay. The activity derived from the Gal4-TK-luciferase reporter in the presence of the control siRNA vectors was normalized to 1.0 and the other activities are expressed relative to this. The diagrams show the relative protein levels obtained from three independent experiments. HeLa cells were transfected with 8 μg of control siRNA or different HDAC1 siRNA (1, 2 or 3) alone or in combination. Total cells extracts were prepared 80 h after transfection and the levels of endogenous HDAC1 protein were detected by western blot analysis. The decrease of HDAC1 levels reached 90%; however, when the HDAC1 levels were analyzed 48 h after transfection the observed decrease was modest (20–30%) (data not shown). The experiment in (C) was performed 48 h after transfection due to the low activation of TK promoter by Gal-CBP after that time. HeLa cells were transfected as in (A) and the levels of T antigen protein in the presence of control siRNA or HDAC1 siRNA were analyzed by Western blot