Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification

<p>Abstract</p> <p>Background</p> <p>During primary infection of its human host, Herpes Simplex Virus Type-1 (HSV-1) establishes latency in neurons where the viral genome is maintained in a circular form associated with nucleosomes in a chromatin configration. During latency, most viral genes are silenced, although the molecular mechanisms responsible for this are unclear. We hypothesized that neuronal factors repress HSV-1 gene expression during latency. A search of the HSV-1 DNA sequence for potential regulatory elements identified a Repressor Element-1/Neuronal Restrictive Silencer Element (RE-1/NRSE) located between HSV-1 genes ICP22 and ICP4. We predicted that the Repressor Element Silencing Transcription Factor/Neuronal Restrictive Silencer Factor (REST/NRSF) regulates expression of ICP22 and ICP4.</p> <p>Results</p> <p>Transient cotransfection indicated that REST/NRSF inhibited the activity of both promoters. In contrast, cotransfection of a mutant form of REST/NRSF encoding only the DNA-binding domain of the protein resulted in less inhibition. Stably transformed cell lines containing episomal reporter plasmids with a chromatin structure showed that REST/NRSF specifically inhibited the ICP4 promoter, but not the ICP22 promoter. REST/NRSF inhibition of the ICP4 promoter was reversed by histone deacetylase (HDAC) inhibitor Trichostatin A (TSA). Additionally, chromatin immuno-precipitation (ChIP) assays indicated that the corepressor CoREST was recruited to the proximity of ICP4 promoter and that acetylation of histone H4 was reduced in the presence of REST/NRSF.</p> <p>Conclusion</p> <p>Since the ICP4 protein is a key transactivator of HSV-1 lytic cycle genes, these results suggest that REST/NRSF may have an important role in the establishment and/or maintenance of HSV-1 gene silencing during latency by targeting ICP4 expression.</p

Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification-3

<p><b>Copyright information:</b></p><p>Taken from "Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification"</p><p>http://www.virologyj.com/content/4/1/56</p><p>Virology Journal 2007;4():56-56.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1906746.</p><p></p> nM TSA. 1. transfected with control plasmid. 2. transfected with 1 μg of pFLAG-REST. 3. transfected with 1 μg of pCMVp73. The values represent Student's tests in pairwise comparisons to the control without TSA. . Stable 293HEK cells containing pICP4 (293HEK-pICP4) was transfected with control plasmid, pFLAG-REST, or pCMVp73 in the presence (white bar) or absence (black bar) of 100 nM TSA. 1. transfected with control plasmid. 2. transfected with 1 μg of pFLAG-REST. 3. transfected with 1 μg of pCMVp73. The values represent Student's tests in pairwise comparisons to the control without TSA. . Effect of TSA on REST/NRSF and p73 transcriptions in 293HEK-pICP4. M: 100 bp ladder. 1. Transfected with 1 μg control plasmid. 2. Transfected with 1 μg pFLAG-REST. 3. Transfected with 1 μg pCMVp73. 4. Transfected with 1 μg control plasmid with 100 nM TSA. 5. Transfected with 1 μg pFLAG-REST with 100 nM TSA. 6. Transfected with 1 μg pCMVp73 with 100 nM TSA. The cDNA from REST/NRSF and actin were marked by arrows

Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification-1

<p><b>Copyright information:</b></p><p>Taken from "Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification"</p><p>http://www.virologyj.com/content/4/1/56</p><p>Virology Journal 2007;4():56-56.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1906746.</p><p></p> 1. pICP22 and control plasmid. 2. pICP22 and pFLAG-REST (1:1). 3. pICP22 and pFLAG-REST (1:2). 4. pICP22 and pCMVp73 (1:1). 5. pICP22 and pCMVp73 (1:2). The asterisk values represent Student's tests in pairwise comparisons to the Lane 1 pICP22 + Control plasmid. The error bars represent standard deviations. The data were calculated and graphed using Microsoft Excel. B. Cotransfection of pICP4 with different amount of expression plasmids was performed followed by SEAP assay to analyze the regulatory effect of REST/NRSF on ICP4 promoter. 1. pICP4 and control plasmid. 2. pICP4 and pFLAG-REST (1:1). 3. pICP4 and pFLAG-REST (1:2). 4. pICP4 and pCMVp73 (1:1). 5. pICP4 and pCMVp73 (1:2). The values represent Student's tests in pairwise comparisons to the Lane 1 pICP4 + Control plasmid. The error bars represent standard deviations. The data were calculated and graphed using Microsoft Excel. C. Plasmid pSG28 and pH4-2 were cotransfected with pFLAG-REST or pCMVp73 followed by RNA isolation and RT-PCR. 1. pSG28 and control plasmid (1:1). 2. pSG28 and pFLAG-REST (1:1). 3. pSG28 and pCMVp73 (1:1). 4. pH4-2 and control plasmid (1:1). 5. pH4-2 and pFLAG-REST (1:1). 6. pH4-2 and pCMVp73 (1:1). D. The RT-PCR results were quantified by Kodak Gel-Logic 100 system to measure the sum intensity of each band representing the regulatory effect of REST/NRSF on ICP4 transcription

Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification-4

<p><b>Copyright information:</b></p><p>Taken from "Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification"</p><p>http://www.virologyj.com/content/4/1/56</p><p>Virology Journal 2007;4():56-56.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1906746.</p><p></p>AG-REST. Immunoblot was performed using anti-REST antibody. . EMSA using transfected cell extract. Lane 1. Labeled HSV-1 RE-1 ds oligo incubated with extract isolated from cells transfected with control plasmid. Lane 2. Labeled HSV-1 RE-1 ds oligo incubated with extract isolated from cells transfected with pFLAG-REST. Lane 3: Labeled mutant oligo incubated with extract isolated from cells transfected with pFLAG-REST. Lane 4: Labeled HSV-1 RE-1 ds oligo incubated with extract isolated from cells transfected with pCMVp73. Lane 5: Labeled wild-type ds oligo containing 10× unlabeled wild-type oligo incubated with extract isolated from cells transfected with pFLAG-REST. Lane 6: Labeled wild-type ds oligo containing 25× unlabeled wild-type oligo incubated with extract isolated from cells transfected with pFLAG-REST. Noted that endogenous REST/NRSF produced a shifted band (Lane 1). . Analysis of REST/NRSF binding, histone H4 acetylation alteration, and CoREST recruitment by ChIP. 293HEK-pICP4 cell line was transfected with pFLAG-REST and subjected to ChIP assay. Samples prior to the immuno-precipitation are used for input control. The samples were amplified by PCR and subjected to 1.2% agarose electrophoresis staining with ethidium bromide. 1. pICP4 + Control. 2. pICP4 + pFLAG-REST. 3. pICP4 + pCMVp73

Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification-2

<p><b>Copyright information:</b></p><p>Taken from "Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification"</p><p>http://www.virologyj.com/content/4/1/56</p><p>Virology Journal 2007;4():56-56.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1906746.</p><p></p>4, 9, and 10), and pCMVp73 (Lane 5, 6, 11, and 12). Lane 1–6: Ethidium bromide staining. Lane 7–12: plasmid-specific nucleosmal protected ladder detected by vector probe. Lane 13 and 14: Naked plasmid digestion control. . Analysis of nucleosomal formation on episomal plasmids. The nuclei from stable 293HEK-pICP22 were subjected to MNase digestion followed by Southern blot hybridization. Lane 1–3: ethidium bromide staining of the genomic DNA nucleosomal protected ladder. Lane 4–6: episomal plasmid-specific nucleosmal protected ladder detected by vector probe. . Examination of episomal status of plasmids in the stable cells harboring pICP4 by Southern hybridization. Lane 1. Total DNA purified from 1.28 × 10stable cells; Lane 2: 10 pg plasmid; Lane 3: 0.1 ng plasmid DNA; 4: 1 ng plasmid DNA; 5: 10 ng plasmid DNA

Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification-5

<p><b>Copyright information:</b></p><p>Taken from "Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF) can regulate HSV-1 immediate-early transcription via histone modification"</p><p>http://www.virologyj.com/content/4/1/56</p><p>Virology Journal 2007;4():56-56.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1906746.</p><p></p>d and terminal repeats (TRand IR). The ICP22 gene is present in Uand one of the two ICP4 is located at the junction of Uand IRsince the genes that are encoded within the repeat sequences are present twice in the viral genome. The HSV-1 RE-1/NRSE is mapped from 132082 to 132103 according to the HSV-1 complete genome sequence accession number X14112. . Putative HSV-1 RE-1/NRSE. The HSV-1 RE-1/NRSE sequence was identified to overlap ICP22 TATA box (Bold) compared to consensus sequence. The core sequence is underlined. The matching result indicated that the homology is 76%. W: A or T; N: any nucleotide; S: G or C; Y: C or T; R: A or G