Retrovirus silencing in stem cells produces silent or variegated provirus. Additional memory and extinction mechanisms act during differentiation. Here we show that retrovirus is silent or variegated in mouse embryonic stem (ES) cells that are de novo methyltransferase (dnmt3a and dnmt3b) null. Memory is maintained during differentiation, and extinction occurs on variegated retrovirus, indicating that DNA methylation is dispensable for all forms of retrovirus silencing. Silent and variegated provirus are marked by hypoacetylated histone H3 and bound H1. In wild-type ES cells, silent and variegated proviruses are methylated and bound by hypoacetylated H3, MeCP2, and less H1. Silencing, variegation, and extinction are partially reactivated by 5-AzaC in this context. Lentivirus vectors are also silent or variegated, marked by silent chromatin, and exhibit memory and extinction. We conclude that the universal epigenetic mark of retrovirus silencing is silent chromatin established via the dynamic interplay of multiple epigenetic modifications that include but do not require DNA methylation. A molecular mechanism of competitive H1 and MeCP2 binding may account for this epigenetic interplay, and a model for variegation is discussed.

Silencing of retrovirus vectors poses a significant obstacle to genetic manipulation of stem cells and their use in gene therapy. We describe a mammalian silencer blocking assay using insulator elements positioned between retrovirus silencer elements and an LCRbeta-globin reporter transgene. In transgenic mice, we show that retrovirus silencers are blocked by the cHS4 insulator. Silencer blocking is independent of the CTCF binding site and is most effective when flanking the internal reporter transgene. These data distinguish silencer blocking activity by cHS4 from its enhancer blocking activity. Retrovirus vectors can be created at high titer with one but not two internal dimer cHS4 cores. cHS4 in the LTRs has no effect on expression in transduced F9 cells, suggesting that position effect blocking is not sufficient to escape silencing. The Drosophila insulators gypsy and Scs fail to block silencing in transgenic mice, but gypsy stimulates vector expression 2-fold when located in the LTRs of an infectious retrovirus. The silencer blocking assay complements existing insulator assays in mammalian cells, provides new insight into mechanisms of insulation and is a valuable tool to identify additional silencer blocking insulators that cooperate with cHS4 to improve stem cell retrovirus vector design.

BACKGROUND.The authors have previously shown that overexpression of claudin 1 (CLDN1) is associated with advanced disease stage in oral squamous cell carcinomas (OSCCs). Their goal was to examine CLDN1 expression in a large series of primary OSCCs and to further investigate whether CLDN1 overexpression plays a role in invasion in OSCC.METHODS.CLDN1 gene expression levels were determined by quantitative real‐time reverse transcription polymerase chain reaction (QRT‐PCR) in 100 primary OSCCs. CLDN1 protein expression was examined by immunohistochemistry in 70 of 100 OSCCs. E‐Cadherin protein levels were also assessed in 58 OSCCs. The authors performed a transwell Matrigel invasion assay for assessment of the invasive potential of CLDN1 overexpressing oral carcinoma cells. Western blotting and QRT‐PCR were used to assess CLDN1 expression in transfected cells and controls.RESULTS.CLDN1 mRNA was increased (median = 18.5) in 79 of 100 OSCCs, compared with normal oral mucosa (expression = 1.0). CLDN1 overexpression was associated with angiolymphatic (P = .037) and perineural invasion (P = .051). CLDN1 was highly expressed in 48 of 70 (68%) OSCCs. E‐Cadherin was lost or underexpressed in 49 of 58 (84%) OSCCs. The invasion assay showed that cells overexpressing CLDN1 have increased invasive potential, whereas small interfering RNA–mediated depletion of CLDN1 decreased the invasive potential of cells.CONCLUSIONS.CLDN1 overexpression is associated with angiolymphatic and perineural invasion, consistent with aggressive tumor behavior. Overexpression of CLDN1 protein is associated with increased invasiveness of oral carcinoma cells. Cancer 2008. © 2008 American Cancer Society.

The human beta-globin locus is home to five genes that are regulated in a tissue-specific and developmental stage-specific manner. While the exact mode of expression remains somewhat enigmatic, a significant effort has been focused at the locus control region (LCR). The LCR is marked by five DNase I-hypersensitive sites (HS) approximately 15 kb upstream of the epsilon-globin gene. Nuclear matrix-associated regions (MARs) organize chromatin into functional domains and at least one of the HS appears bound to the nuclear matrix. We have employed an in vivo based PCR MAR assay to investigate the role of MAR-mediated regulation of the beta-globin locus. This was facilitated with a novel reaction efficiency based quantitative real-time PCR analysis software tool, Target Analysis Quantification. Using a log-linear regression strategy, discordances were eliminated. This allowed us to reliably estimate the relative amount of initial template associated with the nuclear matrix at 15 unique regions spanning the beta-globin locus in both non-expressing and expressing cell lines. A dynamic association dependent on expression status was revealed both at the LCR/5'HS region and within the second intron of the beta-globin gene. These results provide the first evidence that nuclear matrix association dynamically mediates the looping of the beta-globin locus to achieve transcriptional control.