Adenovirus E1A Activation Domain Regulates H3 Acetylation Affecting Varied Steps in Transcription at Different Viral Promoters.

How histone acetylation promotes transcription is not clearly understood. Here, we confirm an interaction between p300 and the adenovirus 2 large E1A activation domain (AD) and map the interacting regions in E1A by observing colocalization at an integrated lacO array of fusions of LacI-mCherry to E1A fragments with YFP-p300. Viruses with mutations in E1A subdomains were constructed and analyzed for kinetics of early viral RNA expression and association of acetylated H3K9, K18, K27, TBP, and RNA polymerase II (Pol II) across the viral genome. The results indicate that this E1A interaction with p300 is required for H3K18 and H3K27 acetylation at the E2early, E3, and E4 promoters and is required for TBP and Pol II association with the E2early promoter. In contrast, H3K18/27 acetylation was not required for TBP and Pol II association with the E3 and E4 promoters but was required for E4 transcription at a step subsequent to Pol II preinitiation complex assembly.IMPORTANCE Despite a wealth of data associating promoter and enhancer region histone N-terminal tail lysine acetylation with transcriptional activity, there are relatively few examples of studies that establish causation between these histone posttranslational modifications and transcription. While hypoacetylation of histone H3 lysines 18 and 27 is associated with repression, the step(s) in the overall process of transcription that is blocked at a hypoacetylated promoter is not clearly established in most instances. Studies presented here confirm that the adenovirus 2 large E1A protein activation domain interacts with p300, as reported previously (P. Pelka, J. N. G. Ablack, J. Torchia, A. S. Turnell, R. J. A. Grand, J. S. Mymryk, Nucleic Acids Res 37:1095-1106, 2009, https://doi.org/10.1093/nar/gkn1057), and that the resulting acetylation of H3K18/27 affects varied steps in transcription at different viral promoters

Transcriptomic and ChIP-sequence interrogation of EGFR signaling in HER2+ breast cancer cells reveals a dynamic chromatin landscape and S100 genes as targets.

BACKGROUND:The Human Epidermal Growth Factor Receptor (EGFR/HER1) can be activated by several ligands including Transforming Growth Factor alpha (TGF-α) and Epidermal Growth Factor (EGF). Following ligand binding, EGFR heterodimerizes with other HER family members, such as HER2 (human epidermal growth factor receptor-2). Previously, we showed that the EGFR is upregulated in trastuzumab resistant HER2 positive (HER2+) breast cancer cells. This study is aimed to determine the downstream effects on transcription following EGFR upregulation in HER2+ breast cancer cells. METHODS:RNA-sequence and ChIP-sequence for H3K18ac and H3K27ac (Histone H3 lysine K18 and K27 acetylation) were conducted following an Epidermal Growth Factor (EGF) treatment time course in HER2+ breast cancer cells, SKBR3. The levels of several proteins of interest were confirmed by western blot analysis. The cellular localization of proteins of interest was examined using biochemically fractionated lysates followed by western blot analysis. RESULTS:Over the course of 24 h, EGFR stimulation resulted in the modulation of over 4000 transcripts. Moreover, our data demonstrates that EGFR/HER2 signaling regulates the epigenome, with global H3K18ac and H3K27ac oscillating as a function of time following EGF treatment. RNA-sequence data demonstrates the activation of immediate early genes (IEGs) and delayed early genes (DEGs) within 1 h of EGF treatment. More importantly, we have identified members of the S100 (S100 Calcium Binding Protein) gene family as likely direct targets of EGFR signaling as H3K18ac, H3K27ac and pol2 (RNA polymerase II) increase near the transcription start sites of some of these genes. CONCLUSIONS:Our data suggests that S100 proteins, which act as Ca2+ sensors, could play a role in EGF induced tumor cell growth and metastasis, contribute to trastuzumab resistance and cell migration and that they are likely drug targets in HER2+ breast cancer

Conserved and divergent gene regulatory programs of the mammalian neocortex

Divergence of cis-regulatory elements drives species-specific trait

The Adenovirus E1A C Terminus Suppresses a Delayed Antiviral Response and Modulates RAS Signaling

The N-terminal half of adenovirus e1a assembles multimeric complexes with host proteins that repress innate immune responses and force host cells into S-phase. In contrast, the functions of e1a's C-terminal interactions with FOXK, DCAF7, and CtBP are unknown. We found that these interactions modulate RAS signaling, and that a single e1a molecule must bind all three of these host proteins to suppress activation of a subset of IFN-stimulated genes (ISGs). These ISGs were otherwise induced in primary respiratory epithelial cells at 12 hr p.i. This delayed activation of ISGs required IRF3 and coincided with an ∼10-fold increase in IRF3 from protein stabilization. The induced IRF3 bound to chromatin and localized to the promoters of activated ISGs. While IRF3, STAT1/2, and IRF9 all greatly increased in concentration, there were no corresponding mRNA increases, suggesting that e1a regulates the stabilities of these key activators of innate immune responses, as shown directly for IRF3

Adenovirus E1A Activation Domain Regulates H3 Acetylation Affecting Varied Steps in Transcription at Different Viral Promoters.

How histone acetylation promotes transcription is not clearly understood. Here, we confirm an interaction between p300 and the adenovirus 2 large E1A activation domain (AD) and map the interacting regions in E1A by observing colocalization at an integrated lacO array of fusions of LacI-mCherry to E1A fragments with YFP-p300. Viruses with mutations in E1A subdomains were constructed and analyzed for kinetics of early viral RNA expression and association of acetylated H3K9, K18, K27, TBP, and RNA polymerase II (Pol II) across the viral genome. The results indicate that this E1A interaction with p300 is required for H3K18 and H3K27 acetylation at the E2early, E3, and E4 promoters and is required for TBP and Pol II association with the E2early promoter. In contrast, H3K18/27 acetylation was not required for TBP and Pol II association with the E3 and E4 promoters but was required for E4 transcription at a step subsequent to Pol II preinitiation complex assembly.IMPORTANCE Despite a wealth of data associating promoter and enhancer region histone N-terminal tail lysine acetylation with transcriptional activity, there are relatively few examples of studies that establish causation between these histone posttranslational modifications and transcription. While hypoacetylation of histone H3 lysines 18 and 27 is associated with repression, the step(s) in the overall process of transcription that is blocked at a hypoacetylated promoter is not clearly established in most instances. Studies presented here confirm that the adenovirus 2 large E1A protein activation domain interacts with p300, as reported previously (P. Pelka, J. N. G. Ablack, J. Torchia, A. S. Turnell, R. J. A. Grand, J. S. Mymryk, Nucleic Acids Res 37:1095-1106, 2009, https://doi.org/10.1093/nar/gkn1057), and that the resulting acetylation of H3K18/27 affects varied steps in transcription at different viral promoters

Transcriptomic and ChIP-sequence interrogation of EGFR signaling in HER2+ breast cancer cells reveals a dynamic chromatin landscape and S100 genes as targets.

BACKGROUND:The Human Epidermal Growth Factor Receptor (EGFR/HER1) can be activated by several ligands including Transforming Growth Factor alpha (TGF-α) and Epidermal Growth Factor (EGF). Following ligand binding, EGFR heterodimerizes with other HER family members, such as HER2 (human epidermal growth factor receptor-2). Previously, we showed that the EGFR is upregulated in trastuzumab resistant HER2 positive (HER2+) breast cancer cells. This study is aimed to determine the downstream effects on transcription following EGFR upregulation in HER2+ breast cancer cells. METHODS:RNA-sequence and ChIP-sequence for H3K18ac and H3K27ac (Histone H3 lysine K18 and K27 acetylation) were conducted following an Epidermal Growth Factor (EGF) treatment time course in HER2+ breast cancer cells, SKBR3. The levels of several proteins of interest were confirmed by western blot analysis. The cellular localization of proteins of interest was examined using biochemically fractionated lysates followed by western blot analysis. RESULTS:Over the course of 24 h, EGFR stimulation resulted in the modulation of over 4000 transcripts. Moreover, our data demonstrates that EGFR/HER2 signaling regulates the epigenome, with global H3K18ac and H3K27ac oscillating as a function of time following EGF treatment. RNA-sequence data demonstrates the activation of immediate early genes (IEGs) and delayed early genes (DEGs) within 1 h of EGF treatment. More importantly, we have identified members of the S100 (S100 Calcium Binding Protein) gene family as likely direct targets of EGFR signaling as H3K18ac, H3K27ac and pol2 (RNA polymerase II) increase near the transcription start sites of some of these genes. CONCLUSIONS:Our data suggests that S100 proteins, which act as Ca2+ sensors, could play a role in EGF induced tumor cell growth and metastasis, contribute to trastuzumab resistance and cell migration and that they are likely drug targets in HER2+ breast cancer

Adenovirus Small E1A Employs the Lysine Acetylases p300/CBP and Tumor Suppressor Rb to Repress Select Host Genes and Promote Productive Virus Infection

Oncogenic transformation by adenovirus small e1a depends on simultaneous interactions with the host lysine acetylases p300/CBP and the tumor suppressor RB. How these interactions influence cellular gene expression remains unclear. We find that e1a displaces RBs from E2F transcription factors and promotes p300 acetylation of RB1 K873/K874 to lock it into a repressing conformation that interacts with repressive chromatin-modifying enzymes. These repressing p300-e1a-RB1 complexes specifically interact with host genes that have unusually high p300 association within the gene body. The TGF-β, TNF-, and interleukin-signaling pathway components are enriched among such p300-targeted genes. The p300-e1a-RB1 complex condenses chromatin in a manner dependent on HDAC activity, p300 lysine acetylase activity, the p300 bromodomain, and RB K873/K874 and e1a K239 acetylation to repress host genes that would otherwise inhibit productive virus infection. Thus, adenovirus employs e1a to repress host genes that interfere with viral replication

Adenovirus Small E1A Employs the Lysine Acetylases p300/CBP and Tumor Suppressor Rb to Repress Select Host Genes and Promote Productive Virus Infection

Oncogenic transformation by adenovirus small e1a depends on simultaneous interactions with the host lysine acetylases p300/CBP and the tumor suppressor RB. How these interactions influence cellular gene expression remains unclear. We find that e1a displaces RBs from E2F transcription factors and promotes p300 acetylation of RB1 K873/K874 to lock it into a repressing conformation that interacts with repressive chromatin-modifying enzymes. These repressing p300-e1a-RB1 complexes specifically interact with host genes that have unusually high p300 association within the gene body. The TGF-β, TNF-, and interleukin-signaling pathway components are enriched among such p300-targeted genes. The p300-e1a-RB1 complex condenses chromatin in a manner dependent on HDAC activity, p300 lysine acetylase activity, the p300 bromodomain, and RB K873/K874 and e1a K239 acetylation to repress host genes that would otherwise inhibit productive virus infection. Thus, adenovirus employs e1a to repress host genes that interfere with viral replication