PMC

In unstimulated cells, NELF binds the nascent mRNA and RNAPII. The SERPINB2 enhancer transcribes eRNAs after LPS treatment. These eRNAs regulate the histone modifications in the enhancer and promoter regions. The eRNAs interact with NELF and CDK9 (P-TEFb). P-TEFb phosphorylates NELF and RNAPII serine 2. Once released from NELF-induced pausing, RNAPII is able to enter into productive elongation and SERPINB2 mRNA is induced.

MonoMac6 cells were treated with 1µg/ml LPS for the indicated time. ChIP assays were performed with antibodies to P-TEFb (CDK9) (A) or NELF-A (B). LPS led to increased CDK9 at the promoter and enhancer by 10 minutes and decreased NELF binding at the promoter and enhancer with slower kinetics (n =4, * p<0.05, ** p<0.01). (C) Over-expression of 774 and 775 led to diminished NELF binding at the promoter even without LPS treatment. GAPDH is included as a non-LPS-inducible control and pc-DNA3.1 (PC) is a vector control. D) Knock-down of eRNAs did not alter the resting levels of NELF on the enhancer and promoter but there was a trend to preservation of NELF on DNA after LPS stimulation. GAPDH, in contrast, was unaffected by the knock-downs.

(A) ChIP-seq tracks from new data for LPS and control primary monocytes showed increased H3K4me2 and H3K27ac at the enhancer and promoter after LPS treatment. (B) Enhanceratlas.org tracks (accessed December, 2016) display chromatin loops observed in CD14 monocytes at this locus, providing a three-dimensional framework for potential enhancer-promoter interactions []. Knock-down of 774 and 775 affected acquisition of H3K4me2 (C), H3K27ac (D), H3K4me3 (E), and H3K4me2 (F) at Enhancer 1 (left panels) and to a lesser degree extent at the promoter (middle panels) after LPS. The GAPDH promoter was not affected by LPS or the knock-downs (right panels). These experiments were performed in MonoMac6 cells (n =4, * p<0.05, ** p<0.01).

Primary monocytes from healthy controls were pretreated with the 10 µM iBET (A), 40µM DRB (B) or 20 µM C646 (C) for 30 minutes before adding 1 µg/ml LPS.
Total RNA was isolated and qRT-PCR was used to measure the ncRNA or mRNA level (n=4, error bars represent SD, * indicates p<0.05, ** p<0.01, *** p<0.001). The y-axis represents fold change over untreated cells. Each inhibitor treatment was associated with diminished SERPINB2 mRNA as well as diminished levels of eRNAs. SERPINB10 mRNA levels were enhanced by inhibitor treatment, a paradoxical response. To define effects on transcription factor binding, eRNAs 774 and 775 were over-expressed and c-JUN binding was determined by ChIP- assay (D). 775 over-expression was associated with increased c-JUN at the enhancer. GST binding was unaffected (n=3, error bars represent SD, * indicates p<0.05).

(A) LPS-induced SERPINB2 RNA expression kinetic analysis. The eRNA and mRNA expression levels were measured using qRT-PCR at various time-points and normalized to 18S in MonoMac6 cells. SERPINB10 is minimally LPS-inducible while SERPINB2 responds to LPS. GAPDH is a non-inducible control. The eRNAs appear temporally before the mRNAs. (B) Knock-down of SERPINB2 eRNAs decreased the LPS-induced SERPINB2 mRNA. MonoMac6 cells were transfected with the indicated 20-bp phosphorothioate antisense oligonucleotides (ASO) designed to knock-down 774, 775 (two different ASO) or GFP for 3 hours and stimulated with or without 1 µg/ml LPS for 60 minutes. The eRNAs and SERPINB2 mRNAs were measured using qRT-PCR. The antisense knock-down was specific and effective for each eRNA (left panel). SERPINB2 expression was inhibited while that of SERPINB10 (right panel) was not affected (n=4, * p<0.05, ** p<0.01). (C) Over-expression of SERPINB2 eRNA led to increased SERPINB2 mRNA. The constructs pcDNA3.1+(PC), pcDNA3.1-s774 (PC-774) and pcDNA3.1-775 (PC-775) were the eRNA over-expression vectors and pcDNA3.1-477 (PC-477) was the off-target control. Constructs were transfected into MonoMac6 cells and stimulated with 1 µg/ml LPS for 60 minutes. Total RNA was isolated and qRT-PCR performed. The left panel confirms over-expression by the constructs while the right panel demonstrates that 774 and 775 over-expression drives increased SERPINB2 expression but not SERPINB10 expression. Results are the means with standard deviation (n=3, * p<0.05, ** p<0.01, *** p<0.001).

MonoMac6 cells were treated with 1 µg/ml LPS for 0, 10, 30 and 60 minutes. RNA-IPs were performed using NELF-A or CDK9 antibodies. Bound RNAs were detected by qRT-PCR. Input RNA was used for normalization. eRNAs were observed to significantly bind to CDK9 upon LPS stimulation with rapid kinetics. The effect was specific as neither the 896 nor 571 RNA species had significant induction of binding with LPS. The short mRNA, immediately downstream of the TSS (B2+80), exhibited a slight increase that did not reach significance. NELF, in contrast, bound eRNAs at baseline and increased binding after LPS, peaking at 30 minutes. The eRNAs remained associated with chromatin, as demonstrated by the H4ac RNA-IP. B2+80 is the first 80 nucleotides of the SERPINB2 mRNA and 896 is an off-target control RNA from a regulatory region that is not LPS inducible. 571 is from an upstream region of SERPINB2 that does nto appear to represent an enhancer. Results are the means + standard deviation (n=3, * p<0.05).

(A) Histone marks and DNase hypersensitive sites (DS) in various human cells from ENCODE [] data indicated that these ncRNAs are located within active enhancer regions, termed Enhancer 1 and Enhancer 2. The eRNAs described in the text are listed above the tracks. (B) The sequencing depth of H3K4me3 at the SERPINB2 gene is displayed from a previously published dataset [, ]. The depth is the normalized average of 6 control or 6 SLE samples. The plotted data is in log2 scale. The magenta color indicates the overlap, with pink and blue representing the mean for SLE and control, respectively. H3K4me3 is higher in SLE patients at SERPINB2 both upstream and downstream of the transcription start site (0). (C) Quantitative RT-PCR was used to measure the difference of SERPINB2 mRNA level in 15 SLE patient monocyte samples and 13 healthy control monocyte samples. The RNA samples came from a previously published cohort []. SERPINB2 mRNA levels are significantly higher (**** p<0.0001). Results were normalized to 18S expression.

(A) The schematic diagram shows the luciferase reporter vector driven by a TK promoter. The enhancer DNA from 775 was subcloned downstream of luciferase in the antisense orientation relative to the SERPINB2 gene.
(B) The firefly luciferase vectors were co-transfected with a constitutive Renilla control pRL-TK into HEK293 cell or K562 cells. Construct 172 is a negative control, which is an LPS-induced non-coding RNA but not an eRNA and not located in the SERPIN locus. PGL3-TK-A7 is a positive ncRNA enhancer control from near the UBE2VI gene (48). Inducible firefly luciferase was normalized to the Renilla signal. Results are the means + standard deviation (n=3, * p<0.05, ** p<0.01). The 775 construct induced expression in this transient transfection assay. (C) The SERPINB2 eRNAs were mainly expressed in monocytes. The primary monocytes (M), T and B cells were purified from healthy people by Dynal bead purification and RNA was extracted and analyzed with qRT-PCR. Results were normalized to 18S expression (n= 3, error bars in panels denote SD). (D) The SERPINB2 eRNAs were localized primarily to the chromatin fraction. RNA was extracted from MonoMac6 cytoplasm, nucleoplasm, and chromatin fractions. PCR was performed after reverse transcription (n= 3, error bars in panels denote SD). Actin and HOTAIR are shown as positive controls for cytoplasmic and chromatin fractions, respectively. (E, F, G) Primary monocytes were purified by adherence and stimulated. RNA was harvested after 90 minutes of LPS treatment or LPS with the indicated inhibitors. SERPINB2 eRNAs (E), SERPINB2 mRNA (F), and SERPINB10 mRNA (G) were quantitated. Error bars are standard deviations (n=3). The y-axis represents fold change over mock-treated cells (control). RNAs from the SERPINB2 Enhancer 1 region and the SERPINB2 mRNA were comparably regulated while SERPINB10 behaved differently.