Profiling of open chromatin in developing pig (Sus scrofa) muscle to identify regulatory regions

There is very little information about how the genome is regulated in domestic pigs (Sus scrofa). This lack of knowledge hinders efforts to define and predict the effects of genetic variants in pig breeding programs. To address this knowledge gap, we need to identify regulatory sequences in the pig genome starting with regions of open chromatin. We used the "Improved Protocol for the Assay for Transposase-Accessible Chromatin (Omni-ATAC-Seq)" to identify putative regulatory regions in flash-frozen semitendinosus muscle from 24 male piglets. We collected samples from the smallest-, average-, and largest-sized male piglets from each litter through five developmental time points. Of the 4661 ATAC-Seq peaks identified that represent regions of open chromatin, >50% were within 1 kb of known transcription start sites. Differential read count analysis revealed 377 ATAC-Seq defined genomic regions where chromatin accessibility differed significantly across developmental time points. We found regions of open chromatin associated with downregulation of genes involved in muscle development that were present in small-sized fetal piglets but absent in large-sized fetal piglets at day 90 of gestation. The dataset that we have generated provides a resource for studies of genome regulation in pigs and contributes valuable functional annotation information to filter genetic variants for use in genomic selection in pig breeding programs

Pro-Inflammatory Cytokine Induction of 11β-hydroxysteroid Dehydrogenase Type 1 in A549 Cells Requires Phosphorylation of C/EBPβ at Thr235

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts inert glucocorticoids into active forms, thereby increasing intracellular glucocorticoid levels, important to restrain acute inflammation. 11β-HSD1 is induced by pro-inflammatory cytokines in a variety of cells. Here, we show 11β-HSD1 expression in human A549 epithelial cells is increased by pro-inflammatory cytokines (IL-1α/TNFα) via the P2 promoter of the HSD11B1 gene. Inhibition of p38 MAPK attenuated the pro-inflammatory cytokine induction of mRNA encoding 11β-HSD1 as well as that encoding C/EBPβ. IL-1α/TNFα-induced phosphorylation of C/EBPβ at Thr235 was also attenuated by p38 MAPK inhibition suggesting involvement of a p38 MAPK-C/EBPβ pathway. siRNA-mediated knock-down of C/EBPβ and NF-κB/RelA implicated both transcription factors in the IL-1α/TNFα induction of HSD11B1 mRNA. Transient transfections of HSD11B1 promoter-reporter constructs identified the proximal region of the P2 promoter of HSD11B1 as essential for this induction. IL-1α increased binding of C/EBPβ to the HSD11B1 P2 promoter, but this was not observed for NF-κB/RelA, suggesting indirect regulation by NF-κB/RelA. Ectopic expression of mutant chicken C/EBPβ constructs unable to undergo phosphorylation at the threonine equivalent to Thr235 attenuated the IL-1α-induction of HSD11B1, whereas mimicking constitutive phosphorylation of Thr235 (by mutation to aspartate) increased basal expression of HSD11B1 mRNA without affecting IL-1α-induced levels. These data clearly demonstrate a role for both C/EBPβ and NF-κB/RelA in the pro-inflammatory cytokine induction of HSD11B1 in human epithelial cells and show that p38 MAPK-induced phosphorylation of C/EBPβ at Thr235 is critical in this

The proximal region is required for pro-inflammatory cytokine induction of the P2 promoter of <i>HSD11B1</i>.

<p>(<b>A</b>) Transiently transfected A549 cells showing IL-1α-induction of luciferase reporter constructs encoding the human <i>HSD11B1</i> P2 promoter; H11β1(-2700/+78) and H11β1(-440/+78), and the rat <i>Hsd11b1</i> P2 promoter; R11β1(-1799/+49) and R11β1(-196/+49). (<b>B</b>) Internal deletions of the rat <i>Hsd11b1</i> P2 promoter (-1799/+49) that removed -1290 to -125 R∆11β1(-1290/-125) or -311 to -125 R∆11β1(-311/-125) abolished IL-1α induction of promoter activity. (<b>C</b>) Mutation of FP3 and/or FP4 within the proximal promoter (-196/+49) of the rat <i>Hsd11b1</i> promoter abolished induction by IL-1α. Black bars, IL-1α-treated cells; white bars, untreated. Promoter activity is expressed as luciferase/β-galactosidase (internal control) activity. Values are fold induction relative to untreated cells transfected with the same plasmid (means ± SEM; n≥6). Absolute luciferase/β-galactosidase values (without normalization) are shown in the bars of the graph. *, significantly different from untreated cells transfected with the same plasmid. **, p<0.001; ***, p<0.0001.</p

Chromatin immunoprecipitation assays show binding of C/EBPβ, but not of NF-κB/RelA, to the <i>HSD11B1</i> P2 promoter following IL-1α treatment.

<p>(<b>A</b>) Sequence of the region of the P2 promoter of the <i>HSD11B1</i> gene that is essential for induction by IL-1α, which includes the previously described C/EBP binding sites, FP3 and FP4 [10] (boxed). The transcription start site (+1) is indicated by a bent arrow. Putative NF-κB binding sites (predicted using AliBaba 2.1 software; Biobase, Biological databases) are underlined. Primers used in ChIP assays are indicated by arrows under the sequence. (<b>B</b>) qPCR quantification of ChIP assays showed C/EBPβ but not NF-κB binding to the P2 promoter of <i>HSD11B1</i> in cells treated with IL-1α (black bars). White bars, untreated cells. Values, in arbitrary units (AU), are mean ± SEM; n=5. *, p<0.05. (<b>C</b>) qPCR measurements of levels of mRNA encoding 11β-HSD1 in untreated (white bars) or following IL-1α treatment (black bars) of A549 cells transiently transfected with plasmids (50ng) encoding wild-type (WT) chicken C/EBPβ or mutants C/EBPβ(T220A) or C/EBPβ(T220D), abolishing or mimicking, respectively, constitutive phosphorylation of T220 (equivalent to T235 in human C/EBPβ). Data, in arbitrary units (AU), are expressed relative to levels in IL-1α-treated cells transfected with WT C/EBPβ, arbitrarily set to 100 and are mean ± SEM; n=6. **, p<0.001 compared to untreated cells transfected with WT C/EBPβ; #, p<0.05 compared to cells transfected with C/EBPβ(T220A) and treated with IL-1α.</p

Inhibition of p38 MAPK attenuates IL-1α induction of mRNAs encoding 11β-HSD1 and C/EBPβ and prevents IL-1α induced C/EBPβ phosphorylation.

<p>(<b>A</b>, <b>B</b>) qPCR measurement of levels of mRNA encoding 11β-HSD1 in A549 cells without inhibitor (-) or pre-incubated for 1h with inhibitors of p38 MAPK (p38, SB203580; 10µM), JNK II (SP600125; 20µM) or MEK I (PD0325901; 0.5µM) prior to 24h treatment with IL-1α (<b>A</b>) or TNFα (<b>B</b>) (black bars). White bars; no cytokine treatment. All data are expressed relative to levels in untreated cells (no inhibitor, no cytokine), arbitrarily set to 1, and are mean ± SEM; n≥3. *, significantly different from IL-1α-stimulated control. *, p<0.05; **p<0.001. (<b>C</b>) qPCR measurements of levels of mRNA encoding C/EBPβ in cells treated for 24h with IL-1α with (p38 inhibitor), or without (-) prior addition of p38 MAPK inhibitor. Data are expressed relative to levels in untreated control cells (no inhibitor, no cytokine), arbitrarily set to 1, and are mean ± SEM; n≥6. *, significantly different from IL-1α-stimulated control. *, p<0.05. #, significantly different from unstimulated cells. ##, p<0.001. (<b>D</b>) Western blot showing levels of phosphorylated C/EBPβ-LAP (P-C/EBPβ-LAP); upper image) in untreated A549 cells (-p38 MAPK, -IL-1α; white bar), or following 15min treatment with IL-1α alone (-p38 MAPK, +IL-1α; black bar) or with prior addition of p38 MAPK inhibitor for 1h (+p38 MAPK, +IL-1α; grey bar; SB203580, 50µM). The blot (20µg protein/lane) was stripped and reprobed with C/EBPβ antibody (bottom membrane) to detect total C/EBPβ-LAP. Protein bands were quantified using the Odyssey Infrared Imaging System and the ratio of P-C/EBPβ-LAP/total C/EBPβ-LAP calculated. Data are expressed relative to levels in untreated cells (no inhibitor, no cytokine), arbitrarily set to 1, and are mean ± SEM; n=3. *, p<0.05; **, p<0.001. (<b>E</b>) Western blot showing increased levels of C/EBPβ-LAP and C/EBPβ-LIP isoforms after IL-1α treatment for up to 6h, compared to control untreated cells (C). (<b>F</b>) qPCR measurement of levels of mRNA encoding 11β-HSD1 in A549 cells incubated for 1h with CHX (+) or without CHX (-) followed by IL-1α/TNFα treatment for 4h or control (Con). Data are expressed relative to levels in untreated cells (no CHX, no cytokine), arbitrarily set to 1, and are mean ± SEM; n=3. *, p<0.05 significant effect of CHX. #, significantly different to untreated cells (no CHX, no cytokine). #, p<0.05; # #, p<0.001.</p

Pro-inflammatory cytokines increase levels of <i>HSD11B1</i> mRNA from the P2 promoter in A549 cells.

<p>(<b>A</b>) qPCR measurement of <i>HSD11B1</i> mRNA following 24h treatment of A549 cells with IL-1α (10ng/ml) or TNFα (20ng/ml) (black bars). Controls (white bars) were untreated. All values are expressed relative to levels in untreated cells, arbitrarily set to 1 and are mean ± SEM; n≥6. *, significantly different from control. ***, p<0.0001. (<b>B</b>) Schematic representation of the <i>HSD11B1</i> gene showing exons 1A, 1B, 2 and 3 (open boxes) and the corresponding start of transcription for the P1 and P2 promoters (bent arrows). The forward primers used to amplify transcripts from P1 (pP1), P2 (pP2) or a region common to transcripts originating from both P1 and P2 promoters (pC) are indicated. The reverse primer was used for all PCR reactions. (<b>C</b>) Gel showing PCR products from transcripts initiating at P1 (<i>1</i>, 267bp), P2 (<i>2</i>, 285bp) and a region common to transcripts originating from either promoter (<i>C</i>; 152bp) produced using cDNA from untreated (Con) A549 cells or following 24h treatment with IL-1α or TNFα. CCRF-CEM human leukaemia cells, which express <i>HSD11B1</i> predominantly from the P1 promoter, served as a positive control for P1-initiated transcripts. All PCR products were run together and in the same gel.</p

C/EBPβ and NF-κB/RelA are located in the nucleus in IL-1α/TNFα treated A549 cells.

<p>C/EBPβ is located in the nucleus in untreated and cytokine-treated A549 cells whereas NF-κB/RelA rapidly translocates to the nucleus in response to cytokine treatment. Micrographs show A549 cells 15min or 24h after addition of IL-1α (upper panels) or TNFα (lower panels), compared to untreated (control) cells. Red fluorescence shows immunodetection of C/EBPβ (A-C and J-L) or NF-κB/RelA (D-I and M-O). DAPI (blue) was used to stain nuclei. All micrographs were obtained with an objective magnification of x40.</p

Proinflammatory Cytokine Induction of 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) in Human Adipocytes Is Mediated by MEK, C/EBPβ, and NF-κB/RelA

Context:Levels of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which regenerates active glucocorticoids, are selectively elevated in adipose tissue in human obesity and metabolic syndrome, both conditions associated with chronic low-grade inflammation. 11β-HSD1 expression is induced by proinflammatory cytokines in a variety of cell types, including in human adipocytes differentiated in vitro.Objective:Our objective was to determine the mechanisms by which proinflammatory cytokines induce 11β-HSD1 in human adipocytes.Results:The proinflammatory cytokines IL-1α (10 ng/mL) and TNFα (20 ng/mL) increased 11β-HSD1 mRNA levels in human primary adipocyte fractions and Simpson-Golabi-Behmel syndrome (SGBS) adipocytes (P &lt; .001). Inhibition of the MAPK/ERK kinase (MEK) attenuated CCAAT/enhancer binding protein (C/EBP) β phosphorylation at Thr235 and IL-1α/TNFα induction of 11β-HSD1 (P ≤ .007). The small interfering RNA-mediated knockdown of C/EBPβ and nuclear factor (NF)-κB/RelA or inhibition of NF-κB/RelA also attenuated cytokine induction of 11β-HSD1 (P ≤ .001). Moreover, induction of 11β-HSD1 by IL-1α in SGBS cells was associated with nuclear localization of C/EBPβ and NF-κB/RelA. Chromatin immunoprecipitation experiments showed C/EBPβ and NF-κB/RelA located to the 11β-HSD1 promoter in human adipose tissue. Treatment of adipocyte fractions or SGBS adipocytes with metformin or acetylsalicylic acid, which target C/EBPβ and NF-κB/RelA signaling, attenuated the IL-1α induction of 11β-HSD1 (P ≤ .002).Conclusions:Increased proinflammatory signaling in inflamed adipose tissue may mediate elevated 11β-HSD1 expression at this site via MEK, C/EBPβ, and NF-κB/RelA. These molecules/signaling pathways are, therefore, potential targets for drugs, including metformin and acetylsalicylic acid, to prevent/decreased up-regulation of 11β-HSD1 in human obese/metabolic syndrome adipose tissue