Hypermethylated LTR retrotransposon exhibits enhancer activity

<p>LTR retrotransposons are repetitive DNA elements comprising ∼10% of the human genome. They are silenced by hypermethylation of cytosines in CpG dinucleotides and are considered parasitic DNA serving no useful function for the host genome. However, hypermethylated LTRs contain enhancer and promoter sequences and can promote tissue-specific transcription of <i>cis</i>-linked genes. To resolve the apparent paradox of hypermethylated LTRs possessing transcriptional activities, we studied the ERV-9 LTR retrotransposon located at the 5′ border of the transcriptionally active β<i>-globin</i> gene locus in human erythroid progenitor and erythroleukemia K562 cells. We found that the ERV-9 LTR, containing 65 CpGs in 1.7 kb DNA, was hypermethylated (with > 90% methylated CpGs). Hypermethylated LTR possessed transcriptional enhancer activity, since <i>in vivo</i> deletion of the LTR by CRISPR-cas9 suppressed transcription of the globin genes by > 50%. ChIP-qPCR and ChIP-seq studies showed that the hypermethylated LTR enhancer spanning recurrent CCAATCG and GATA motifs associated respectively with key transcription factors (TFs) NF-Y and GATA-1 and -2 at reduced levels, compared with the unmethylated LTR in transfected LTR-reporter gene plasmids. Electrophoretic mobility shift assays with methylated LTR enhancer probe showed that the methylated probe bound both NF-Y and GATA-1 and -2 with lower affinities than the unmethylated enhancer probe. Thus, hypermethylation drastically reduced, but did not totally abolish, the binding affinities of the enhancer motifs to the key TFs to assemble the LTR-pol II transcription complex that activated transcription of <i>cis</i>-linked genes at reduced efficiency.</p

Over-expression of NF-YA, GATA-2, -1 or BCL11A activates or represses γ-globin promoter activity and induces corresponding changes in the <i>in vivo</i> assembly of proximal γ-globin promoter complex.

<p><b>A.</b> Fluorescent K562 cells over-expressing NF-YA long isoform (YAL) with co-expressed GFP sorted by FACS. Lower right quadrant: sorted GFP-fluorescent cells comprising 33% of total cell population used for analyses in panels <b>B–E.</b> FACS sorting of K562 and D14 cells over-expressing the other three transcription factors (not shown). B. Western blots of proteins isolated from K562 cells over-expressing NF-YAL, GATA-2, -1 or BCL-XL and K562 cells in which NF-YA or GATA-1 was knocked down by the respective siRNA, siNF-YA or siGATA-1. +Vector: control K562 cells trasduced or transfected by the empty vector. <b>C & D.</b> Effects of over-expression/knockdown of NF-YAL, GATA-2, -1 or various isoforms of BCL11A on the transcription level of endogenous γ-globin mRNA in K562 and D14 adult erythroid cells determined by real-time RT-PCR. The level of γ-globin mRNA in K562 and D14 cells transduced/transfected with the vector was set at 100. Levels of γ-globin mRNA in test samples were averages of two independently transduced/transfected cells. Effects of over-expressing NF-YA short isoform, NF-YAS, on transcription of γ-globin mRNA in K562 and D14 cells were similar to the effects of over-expressing NF-YAL (not shown). <b>E & F</b>. ChIP assays of the endogenous γ-globin promoter in K562 cells over-expressing NF-YAL, BCL-XL, GATA-2 or -1. The NF-YA and BCL11A antibodies used in the ChIP assays recognized all isoforms of the respective transcription factor (Fig. 1C) and should pull down chromatin associated with all the isoforms of NF-YA and BCL11A. ChIP values were averages of two independent pull-down assays.</p

NF-Y Recruits Both Transcription Activator and Repressor to Modulate Tissue- and Developmental Stage-Specific Expression of Human γ-Globin Gene

<div><p>The human embryonic, fetal and adult β-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.</p> </div

Function of the tandem CCAAT, GATA and GGCCGG motifs in regulating proximal γ-globin promoter activity, determined in GFP reporter plasmids by transfection/transduction assays.

<p><b>A.</b> Plasmid maps of Wt and mutant long 1.3 kb and short, proximal 0.13 kb γ-globin promoter coupled to GFP gene. Mutated bases in the motifs were shown in parentheses. <b>B.</b> Transfection into K562 cells of GFP plasmids containing the long and the short promoters, left and right panels respectively. Numbers on top of the bars: fluorescence levels of GFP expressed from the mutant plasmids compared to those from Wt plasmids set at 100. Values were averages of two independent transcfection experiments. <b>C.</b> Transduction into adult erythroid cells of recombinant lentiviruses containing the Wt and mutant proximal γ-globin promoters coupled to GFP gene. Cells were transduced on day 3 and GFP fluorescence was determined on day 7, 10 and 14 of culture, as shown in the 3 panels respectively. The GFP level in control cells transduced with the wildtype 0.13 kb γ-globin promoter-GFP lentivirus was set at 100. Values were averages of two independent transduction experiments.</p

Protein interaction network in the proximal γ-globin promoter complex. A.

<p>Western blots of proteins co-immunoprecipitated with tagged NF-YA, GATA-2 or -1 or BCL11A-XL expressed from plasmids transfected into K562 cells. <b>B.</b> Pair-wise in vitro interactions between purified transcription factors and co-factors. Pair-wise interactions within the red frame: in vitro interactions between transcription factors. Pair-wise interactions within the blue frame: in vitro interactions between transcription factors and co-factors. ++, +, −: strong, average or no interaction between the proteins. Interactions marked with ∗: the proteins in the pair-wise interaction were isolated from both K562 and HEK293 transfected with the expression plasmids for the target proteins. Otherwise, the proteins were isolated from transfected HEK293 cells.</p

Identification of transcription factors that bind to the proximal γ-globin promoter as determined by EMSA. A, B & E.

<p>Wt proximal γ-globin promoter probe with nuclear extracts from human fetal liver, K562 cells and D14 adult erythroid cells, respectively. Top labels: Competitors and antibodies used in EMSAs. Self, self CCAAT, self GATA, (GATA)7 and self GC were respectively 100x molar excess of unlabeled probe, distal −115 and proximal −88 CCAAT motifs, the −73 GATA motif, seven tandem GATA motifs (29), and GC-rich bases spanning GGCCGG motif. Margin labels: bands generated by the transcription factors as marked. <b>C & F.</b> Mutant CCAAT proximal promoter probe with K562 and D14 nuclear extracts; all other labels were the same as in <b>A</b>. <b>D.</b> Binding affinity of COUP-TFII to its cognate site overlapping the distal CCAAT motif in the proximal γ-globin promoter: Competition EMSAs of proximal γ-globin promoter probe spanning the tandem CCAAT motifs to the GATA site (base positions: −120 to −62 in Fig. 1A) with K562 nuclear extract. Competitors d, p and d+p CCAAT: distal and proximal CCAAT motif and distal+proximal CCAAT motifs in the γ-globin promoter; E1CCAAT: CCAAT motif in the ERV-9 LTR enhancer upstream of the β-globin gene locus (25). For sequences of the competitors, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047175#pone.0047175.s005" target="_blank">Methods S1</a>. <b>G.</b> Binding affinities of GATA-1 and -2 to the proximal γ-globin promoter: EMSA of Wt proximal promoter probe with D14 nuclear extract. Lanes 2 & 6, 3 & 7, 4 & 8: myc-tagged GATA-1, G-1(myc), and myc-tagged GATA-2, G-2(myc), added respectively to D14 nuclear extract alone, and with GATA-1 or -2 antibody. <b>H.</b> Binding of GATA-1 and -2 to the proximal γ-globin promoter required presence of NF-Y determined by EMSA with immuno-depleted K562 nuclear extract (ID-EMSA). Upper panel: Western blots of K562 nuclear extracts immuno-depleted with antibody to NF-YA, GATA-1 and -2, ΔNF-YA, Δ GATA-1 and ΔGATA-2 respectively. Lower panel: EMSA of Wt proximal promoter probe spanning the tandem CCAAT motifs to the −73 GATA site with wildtype, ΔNF-YA, Δ GATA-1 and ΔGATA-2 K562 nuclear extracts respectively.</p

Models of molecular assemblies of the active and repressed proximal γ-globin promoter complexes. A

<p>. Active complex. Blue ribbon: Proximal γ-globin promoter DNA containing transcription activator-binding motifs, CCAAT and GATA (red bars) and the repressor-binding motif GGCCGG (green bar); blue rectangle with angled arrow: γ-globin gene and transcriptional direction of γ-globin mRNA. Red colored proteins: transcription activators; pink colored proteins: co-activators. Dark green and green colored proteins: transcription repressors; BCL-M: BCL11A median-isoforms expressed predominantly in human fetal and K562 erythroid cells; light green colored protein: the HDAC1 co-repressor complex. NF-Y bound at each of the tandem CCAAT motifs bends the DNA by ∼70^o (3) to form the pocket for assembly of the proximal promoter complex. <b>B.</b> Repressed complex. Larger sizes of BCL, COUPTFII and HDAC1 complex and smaller sizes of YA, GATA-2 and -1 and the co-activators represent higher and lower levels of the respective proteins–additionally indicated also by the upward or downward arrows–in the repressed promoter complex as compared to the levels of these proteins in the active complex in <b>A</b>; BCL: all sized-isoforms expressed in D14 erythroid cells (Fig. 1C); dotted angled arrow: lower transcription level of γ-globin mRNA from the repressed promoter.</p

Molecular assemblies of the active and repressed proximal γ-globin promoter complexes in human fetal and adult erythroid cells.

<p><b>A.</b> ChIP assays of the proximal γ-globin promoter complexes in fetal brain, fetal liver and adult D14 erythroid cells, FB, FL and D14, respectively. Values were averages of two independent pull-down assays. <b>B–E.</b> Re-ChIP assays of fetal liver and D14 adult erythroid cells, left and right panels respectively: The antibodies to the respective transcription factors used in first ChIP is shown on the Y-axis. The amount of γ-globin promoter pulled down by antibodies in the 1st ChIP was set at 100 to serve as the reference for comparing the amount of promoter pulled down in the 2nd ChIP by antibodies to proteins shown on the X-axis. Values were averages of two independent re-ChIP assays.</p

Expression profiles of human globin genes and transcription factors that bind to the proximal γ-globin promoter in human fetal and adult erythroid cells.

<p><b>A.</b> Sequence of the proximal γ-globin promoter, which is identical in both GΓ- and Aγ-globin promoters. Shaded and/or underlined bases: DNA motifs that bind transcription factors as marked. Numbers in parentheses: first base positions in the motifs relative to the transcription start site. <b>B.</b> Transcription profiles of globin genes and transcription factors determined by quantitative real-time RT-PCR in total cellular RNAs isolated from human fetal liver, K562, and adult erythroid cells cultured from CD34+ cells for 3–14 days and non-erythroid human fetal brain cells, FL, K, D3–D14 and FB, respectively. In BCL11A panel, the PCR primer pair amplified all 4 different isoforms detected in Fig. 1C. The level of 18S ribosomal RNA set at 10⁶ served as the reference for comparison. RNA levels in K562, fetal liver and D14 cells were averages of two separate RNA preparations; RNA levels in other cells were averages of two RT-PCR reactions. <b>C.</b> Protein expression profiles of transcription factors determined by Western blots. Numbers in left margin: sizes of proteins in Kd.</p

DataSheet_1_Impaired meningeal lymphatic drainage in Listeria monocytogenes infection.pdf

Previous studies have demonstrated an association between lymphatic vessels and diseases caused by bacterial infections. Listeria monocytogenes (LM) bacterial infection can affect multiple organs, including the intestine, brain, liver and spleen, which can be fatal. However, the impacts of LM infection on morphological and functional changes of lymphatic vessels remain unexplored. In this study, we found that LM infection not only induces meningeal and mesenteric lymphangiogenesis in mice, but also impairs meningeal lymphatic vessels (MLVs)-mediated macromolecules drainage. Interestingly, we found that the genes associated with lymphatic vessel development and function, such as Gata2 and Foxc2, were downregulated, suggesting that LM infection may affect cellular polarization and valve development. On the other hand, photodynamic ablation of MLVs exacerbated inflammation and bacterial load in the brain of mice with LM infection. Overall, our findings indicate that LM infection induces lymphangiogenesis and may affect cell polarization, cavity formation, and valve development during lymphangiogenesis, ultimately impairing MLVs drainage.</p