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. 2014 Nov 21;15(12):1558–1571. doi: 10.4161/15384101.2014.967094

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© 2016 The Author(s). Published with license by Taylor & Francis Group, LLC

This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.

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Figure 4. — Effect of Alu induction (upon CGGBP1-depletion) on RNA Pol II activity. (A) Measurement of RNA Pol II occupancy on TSS or DS regions of HIST1A, HKII, ACSII and GLUD1 showed that serum stimulation had a consistent enhancing effect on RNA Pol II occupancy at the DS regions. Unlike at the TSS (left panel), at the DS regions (right panel), CGGBP1 depletion reduced the RNA Pol II occupancy. At the DS regions, the RNA Pol II occupancy was not affected by Alu antisense treatment without CGGBP1 depletion, whereas in CGGBP1 depleted samples, a concomitant RNA interference against Alu rescued the RNA Pol II occupancy. Serum treatment also enhanced the RNA Pol II occupancy at the DS regions and at 2 (HIST1A and ACSII) of the 4 housekeeping genes tested, this effect of serum was vanished by depleting CGGBP1 but could be rescued by combining CGGBP1 depletion with Alu antisense. Input-normalized ddCt values are plotted on Y-axis (n = 4 for each bar; for serum 12h data-points all comparisons between Alu scrambled and Alu antisense satisfy T-test with P < .05). (B) Measurement of correlation between mRNA productions per unit amounts of genomic DNA (different amounts derived from different densities of cells harvested). The slopes of correlation curves show lower mRNA production per unit amount of DNA in CGGBP1-depleted cells (Fishers exact test P < .0001). These curves are for cells cultured in 10% serum. (C) Histograms of mRNA/DNA ratios from control or CGGBP1-depleted cells serum-deprived or stimulated with serum for 48h. Interpolations to derive expected mRNA amounts in CGGBP1-depleted cells based on the mRNA/DNA ratios of control cells show no significant reduction under serum deprivation (left panel; Wilcoxon's test p = >0.999, labeled NS), whereas in presence of serum, the actual value of the mRNA production is significantly lower than the expected value of mRNA production (right panel; Wilcoxon's test, p = 0.0313, labeled *).