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2_test

{"project":"2_test","denotations":[{"id":"16462943-11702786-85760988","span":{"begin":328,"end":330},"obj":"11702786"},{"id":"16462943-10760285-85760989","span":{"begin":1243,"end":1245},"obj":"10760285"},{"id":"16462943-1701019-85760990","span":{"begin":1697,"end":1699},"obj":"1701019"},{"id":"16462943-1848707-85760991","span":{"begin":2832,"end":2834},"obj":"1848707"},{"id":"16462943-2748594-85760992","span":{"begin":2939,"end":2941},"obj":"2748594"},{"id":"16462943-12677004-85760993","span":{"begin":3308,"end":3310},"obj":"12677004"},{"id":"16462943-11504872-85760994","span":{"begin":4993,"end":4994},"obj":"11504872"},{"id":"16462943-11504872-85760995","span":{"begin":5298,"end":5299},"obj":"11504872"},{"id":"16462943-11504872-85760996","span":{"begin":5801,"end":5802},"obj":"11504872"},{"id":"16462943-283312-85760997","span":{"begin":9863,"end":9865},"obj":"283312"},{"id":"T32068","span":{"begin":328,"end":330},"obj":"11702786"},{"id":"T50025","span":{"begin":1243,"end":1245},"obj":"10760285"},{"id":"T75422","span":{"begin":1697,"end":1699},"obj":"1701019"},{"id":"T87948","span":{"begin":2832,"end":2834},"obj":"1848707"},{"id":"T97917","span":{"begin":2939,"end":2941},"obj":"2748594"},{"id":"T50696","span":{"begin":3308,"end":3310},"obj":"12677004"},{"id":"T36890","span":{"begin":4993,"end":4994},"obj":"11504872"},{"id":"T88558","span":{"begin":5298,"end":5299},"obj":"11504872"},{"id":"T4443","span":{"begin":5801,"end":5802},"obj":"11504872"},{"id":"T6698","span":{"begin":9863,"end":9865},"obj":"283312"}],"text":"Results\n\nPersistent Expression of the Embryonic Globin, ɛy, in Sox6-Deficient Mice\nThe ɛy globin gene was initially identified as an upregulated transcript in the p 100H mouse using subtractive hybridization to identify Sox6 downstream targets. This initial observation was confirmed in an independent knock-out allele of Sox6 [13] using real-time polymerase chain reaction (PCR) (unpublished data). Real-time PCR was used to quantitate the expression levels of other globin genes in p100H mutant and WT mouse livers at two developmental stages, 15.5 dpc and 18.5 dpc. As shown in Figure 1, the ɛy gene is expressed at high levels at both time points in mutant mice, in contrast to the decline in expression observed in WT mice. No difference was seen between WT and heterozygous mice (unpublished data). Interestingly, the expression levels of the other two embryonic globin genes (ζ and βh1) are also higher in p100H homozygous mice, compared with WT mice, but to a much lesser extent than seen for ɛy globin at 18.5 dpc (Figure 1). Moreover, the expression level of adult β globin is also somewhat higher in p100H homozygous mice than in WT mice at 18.5 dpc (Figure 1). Perinatal lethality of mutant mice (presumably from the heart defect [14]) precludes us from evaluating postnatal globin expression. The graphs in Figure 1 illustrate real time PCR results that were performed in triplicate (standard deviation of the data is shown by error bars). Because all of the assays were performed at the same time with the same internal control, the levels shown are relative levels and are thus comparable across all samples and are in agreement with previously published results for WT fetal mice [29]. We note that the level of ɛy expression in the livers of 15.5 dpc and 18.5 dpc homozygous mutant mice is statistically equivalent to the level of βmaj/min expression in the livers of 15.5 dpc and 18.5 dpc homozygous WT mice.\nFigure 1 Real-Time PCR of Globin Genes\nThe levels of expression of ɛy, βh1, zeta, and βmaj/min were measured at 15.5 dpc and 18.5 dpc in homozygous WT and p100H mutant littermates by real-time PCR (see Materials and Methods). Relative expression levels in the livers of each genotype are graphed for each globin gene (performed in triplicate and normalized with GAPDH). Standard deviation is indicated by bars.\n\nTransfection Studies Using GM979 Cells Indicate That Sox6 Directly Represses the ɛy Gene Promoter at the Transcriptional Level\nReal-time PCR assays (Figure 1) measure steady-state levels of ɛy mRNA, not transcriptional activity of the ɛy promoter. To investigate whether Sox6 directly acts on the ɛy gene promoter at the transcriptional level, we used an in vitro transient transfection assay and GM979 cells, a murine erythroleukemic cell line that expresses both ɛy and adult beta globins [30]. We generated an ɛy promoter reporter construct (E-Luc) by fusing a micro-LCR (μLCR) element (2.5 kb) [31] to the ɛy proximal promoter (2.2 kb), followed by the luciferase reporter gene, as shown in Figure 2A (detailed in Materials and Methods). Overexpression of Sox6 in GM979 cells by transient transfection leads to a dosage-dependent repression of E-Luc reporter activity (Figure 2B). In contrast, overexpression of a truncated Sox6 protein that lacks its HMG domain [32] (similar to the p 100H mouse allele) fails to repress E-Luc activity (Figure 2B). These data indicate that Sox6 acts to repress the ɛy promoter at the transcriptional level.\nFigure 2 The Effect of Sox6 on the ɛy Promoter\n(A) Constructs of the ɛy promoter reporter (E-luc) and Sox6 overexpression vector. The E-luc reporter construct consists of a 2.5-kb μLCR element, a 2.2-kb ɛy proximal promoter, and the luciferase reporter in the pGL-3 basic plasmid (see Materials and Methods). Sox6 expression is driven by the CMV promoter.\n(B) Sox6 represses ɛy promoter activity in a dosage-dependent manner. In GM979 cells, the E-Luc ɛy promoter reporter construct was co-transfected (1) without overexpression of Sox6; (2–4) with increasing amounts of CMV-Sox6 overexpression vector; (5) with a truncated version of Sox6 that lacks its HMG domain; (6) with a mutant version of Sox6 (L386H) that has previously been shown to abolish interaction with CtBP2; or (7) with an empty reporter plasmid (without ɛy promoter and μLCR element).\n(C) Promoter deletion analyses to delimit the critical sequence. The 2.2-kb proximal promoter or deletions of it, as indicated on the left (numbering relative to +1 = the transcription start site of ɛy globin, see Materials and Methods), were engineered in reporter constructs as in (A) and were transfected along with CMV driven Sox6 to GM979 cells (see Materials and Methods for details). The relative repression by Sox6 on the activity of the different reporter constructs is shown. All experiments were done in triplicate. Sox6 has been shown to act as a repressor and to interact with a widely expressed co-repressor, CtBP2, on the fgf-3 promoter [5]. CtBP2 is expressed in GM979 cells (unpublished data). To investigate whether the interaction with CtBP2 is required for Sox6 repression of the ɛy promoter, we introduced a point mutation (L386H) in the Sox6 protein that has been previously reported to be sufficient to abolish Sox6-CtBP2 interaction [5]. This amino acid change is not in the HMG DNA binding domain. However, this mutant version of Sox6 retains the ability to repress the ɛy promoter in the transfection assay (Figure 2B), indicating that Sox6 represses the ɛy promoter in a CtBP2-independent manner. Deletion analysis of the ɛy promoter, as shown in Figure 2C, defined a region (−63 to −37) within the ɛy proximal promoter that is critical for Sox6 repression. Analysis of this short region reveals two Sox/Sox6 consensus binding sites [5] (Figure 3A).\nFigure 3 Analysis of the Minimal Region (36 bp) of the Proximal ɛy Promoter Responsive to Sox6\n(A) The sequence of the 36-bp fragment and its mutant versions used in EMSA. The WT 36-bp DNA sequence (−63 to −28) of the ɛy globin proximal promoter contains two Sox/Sox6 consensus binding sites, shown in bold underline. Versions with truncation of this sequence (M1) or mutation of one of the two consensus binding sites (M2 and M3) are also shown.\n(B) EMSA with c-Myc-tagged Sox6. EMSA was performed using the 36-bp radio-labeled WT probe (as shown in (A)) and c-Myc tagged Sox6 translated in vitro using reticulocyte lysate (see Materials and Methods). Lane 1: radio-labeled free probe (run out of the gel); Lane 2: no competition, no antibody; Lane 3: competition with 200-fold excess cold probe, no antibody; Lane 4: no competition, c-Myc antibody (producing a supershift); Lane 5: no competition, Sox6 antibody (producing a supershift); Lane 6: no competion, no antibody using in vitro translated vector containing c-Myc, but not Sox6.\n(C) EMSA with HA-tagged Sox6. EMSA was performed similarly as in (B) using HA-tagged Sox6 translated in vitro. Lane 1: radio-labeled free probe (run out of the gel); Lane 2: no competition, no antibody; Lane 3: competition with 200-fold excess cold probe, no antibody; Lane 4: no competition, HA antibody (producing a supershift).\n(D) EMSA using MEL cell nuclear extracts and the 36-bp WT probe. Lane 1: radio-labeled free probe (run out of the gel); Lane 2: no competition, no antibody; Lane 3: competition with 200-fold excess cold probe, no antibody; Lane 4: no competition, Sox6 antibody (producing a supershift).\n(E) EMSA with c-Myc-tagged Sox6, WT and mutant versions of the 36-bp fragment in competition. EMSA was performed using the radio-labeled 36-bp WT probe and the c-Myc tagged Sox6 translated in vitro. Lane 1: radio-labeled free probe; Lane 2: no competition, no antibody. Competition was performed using 200-fold excess cold probes corresponding to WT (Lane 3), M1 (Lane 4), M2 (Lane 5), and M3 (Lane 6).\n(F) Both consensus Sox/Sox6 binding sites are required for Sox6 responsiveness. GM979 cells were transfected with a reporter construct (Figure 2A) containing −63 to +45 of the ɛy proximal promoter together with the CMV-Sox6 overexpression vector. Mutations of the consensus binding sites were also tested (M3, M2, M2 plus M3, see (A)). The fold repression of Sox6 with the WT or mutant constructs is shown. The baseline activities of the mutagenized reporter constructs are comparable to the WT construct.\n\nEMSA and ChIP Assays Show that Sox6 Directly Binds to the ɛy Promoter\nSox6 might repress the ɛy promoter, either through direct physical contact with the promoter or by regulating intermediates affecting the ɛy promoter. To investigate whether Sox6 is directly associated with the ɛy promoter, we first performed electrophoretic mobility shift assays (EMSA) using a c-Myc-tagged Sox6 in a reticulocyte lysate-based transcription/translation in vitro system. The probes used are listed in Figure 3A. The 36–base pair (bp) WT probe corresponds to the critical region of the ɛy promoter defined in our promoter deletion analyses. This probe contains two consensus Sox/Sox6 binding sites. Also included in our EMSA are three mutated probes that are, either truncated (M1), or mutated (M2 and M3) in Sox/Sox6 binding sites (Figure 3A). Sox6 is able to physically associate with the 36-bp region (Figure 3B) within the ɛy promoter defined by the deletion analysis experiments (Figure 2C). The 36-bp probe was shifted by the tagged Sox6 protein. Moreover, both c-Myc and Sox6 antibodies supershift the band, indicating that the binding is Sox6-specific. To rule out the possibility that the c-Myc tag itself binds to the probe, an HA-tagged Sox6 was used in another EMSA that confirmed these results (Figure 3C).\nNext, nuclear extracts from MEL cells were used in EMSA employing the same 36-bp probe. MEL cells, a murine erythroleukemic cell line, express adult β globins, but not ɛy [33]. Sox6 directly binds to this DNA sequence in MEL cells (Figure 3D). The intact consensus Sox/Sox6 binding sites of the DNA probe are required for the binding, as shown in the competition assay (Figure 3E). Ablation of putative Sox/Sox6 binding sites (M1 and M3) abolish their ability to compete in EMSA (Figure 3E). The M2 mutant probe may compete partially with WT binding.\nTo investigate the functional significance of the intact Sox/Sox6 binding sites, the ɛy promoter reporter constructs with mutagenized Sox/Sox6 binding sites were co-transfected with the Sox6 overexpression vector into GM979 cells. Consistent with the EMSA results, the mutant ɛy promoter reporter constructs (with either one or both Sox/Sox6 binding sites mutagenized) do not result in significant promoter repression in transfection studies (Figure 3F). Thus, both sites are required for maximal repression of ɛy by Sox6, but not to the same degree.\nWe also tested whether Sox6 binds to the ɛy promoter in vivo using chromatin immunoprecipitation (ChIP) (Figure 4). The Sox6-containing complex was immunoprecipitated from MEL cells or from liver cells of 15.5 dpc WT mice using Sox6 antibody. Figure 4 shows that the ɛy proximal promoter is readily immunoprecipitated with Sox6 antibody in both MEL cells and liver cells. Normal IgG was used as a negative control (Figure 4A). The above data (Figures 3 and 4) clearly indicate that Sox6 acts as a repressor of the ɛy gene by directly binding to the ɛy promoter, probably as a dimer.\nFigure 4 ChIP Assay\nMEL cells (A) and 15.5-dpc fetal liver cells (B) were treated as detailed in Materials and Methods. 10% of the sample was saved as total input (Inp); remaining samples were divided: plus Sox6 antibody (Ab+), minus Sox6 antibody (Ab−), as well as no DNA (DNA−) and normal rabbit IgG (IgG) that served as negative controls. Other controls for these experiments included PCR within the promoter of the α-globin gene and intron 24 of the p gene. Both were negative (unpublished data). PCR was carried out using primer pairs flanking the Sox/Sox6 binding sites (see Material and Methods) of the ɛy proximal promoter. For all reactions, we used 2 μl of immuno-precipitated DNA and 2 μl of 1/100 total input. Semiquantitative PCR was done within the exponential range. Multiple independent experiments were done.\n\nThe Persistent Expression of ɛy Globin in Sox6-Deficient Mice Is Due to a Defect in the ɛy-Gene–Silencing Mechanism in Definitive Erythroid Cells\nNormally, the ɛy globin gene is exclusively expressed in primitive erythrocytes and silenced in definitive erythrocytes. To determine whether the persistent expression of ɛy globin is due to residual primitive erythrocytes or is due to ectopic expression of ɛy globin in definitive erythrocytes, we examined the spatial pattern of ɛy globin transcripts in mouse embryos by in situ hybridization (Figure 5). As expected, ɛy globin is not expressed in the WT 14.5-dpc liver, the site of definitive erythropoiesis in the fetus. In contrast, abundant ectopic ɛy mRNA expression is seen in the liver of 14.5-dpc mutants (Figure 5 A–D). However, the expression of βmaj/min globin is equally abundant in both WT and p100H mutant mice (Figure 5 E and F). These data demonstrate that the persistent high levels of ɛy are due to ectopic expression in the definitive erythroid cells that mature in the fetal liver, suggesting that there is an intrinsic defect of the ɛy silencing mechanism in Sox6-null mice.\nFigure 5 In Situ Hybridization of ɛy and βmaj/min transcripts in WT and p100H Mutant Mice at 14.5 dpc\nExpression of ɛy globin (A–D) and β globin (E and F) in sagittal sections of 14.5-dpc fetuses is shown in pseudocolor (red).\n(A) WT fetus.\n(B) p100H Homozygous mutant fetus.\n(C and D) Inset boxes are shown. Prominent expression of ɛy globin is only detected in the liver of mutant mice.\n(E and F) In contrast, both WT and mutant mouse livers express adult βmaj/min, respectively. No signals were detected above background using sense probes (unpublished data).\nThe size bars represent 1 mm for (A) and (B); 100 μm for (C–F).\n\nThe Expression Pattern of Sox6 Suggests a Role in Definitive Erythropoiesis\nThe observation that Sox6-deficient mice ectopically express ɛy globin in liver, where definitive erythroid cells mature, suggests that Sox6 is an important regulator in definitive erythropoiesis. To determine the temporal and spatial expression pattern of Sox6, Northern blot and in situ hybridization assays were employed. As shown in Figure 6A, Sox6 is detectable by Northern blot beginning at 10.5 dpc, coincident with the temporal onset of definitive erythropoiesis in the liver. Furthermore, in situ hybridization shows that Sox6 is highly transcribed in 12.5-dpc liver, but not in yolk sac blood islands at 7.5 dpc (Figure 6B). Therefore, Sox6 expression is temporally and spatially coincident with definitive, but not primitive, erythropoiesis. These data, taken together with the observation that ɛy globin is highly expressed in the liver cells of 14.5-dpc p100H mutant mice (Figure 5), demonstrate that Sox6 functions in definitive erythropoiesis.\nFigure 6 Expression Pattern of Sox6 by Northern Blot and In Situ Assays\n(A) Sox6 expression during embryonic development shown by Northern blot. Each lane contains 20 μg of total RNA from embryos whose ages are listed above each lane as dpc. The filter was hybridized with a 32P-labeled 575-bp mouse Sox6 cDNA fragment (nucleotides 1353–1927). Numbers on the left are sizes of standard marker fragments in kb.\n(B) Sox6 expression shown by in situ hybridization. Panel i: Sagittal section through an E12.5 mouse embryo using antisense Sox6. mRNA distribution is represented by pseudocolored red signal superimposed on the counterstained specimen. Sox6 transcripts are detected primarily in the fetal liver, developing nervous system, chondrocytes and craniofacial area. Panel ii: The sense control probe shows no signal above background. Panel iii: E7.5 embryo hybridized to antisense probe for Sox6. No signal is detected above background specifically in blood islands (or with the sense probe, unpublished data).\nThe size bars represent 100 μm.\n\nMutant p100H Mice Have Higher Numbers of Nucleated Red Cells\nAmong the other Sox6 effects in erythropoiesis, we have noticed that there are more nucleated red blood cells circulating in p100H mutant mice than in WT mice at 14.5 dpc and 18.5 dpc (Figure 7A). However, at postnatal day 10.5, we do not see circulating nucleated red cells in either WT or mutant mice, suggesting that this may be a transient effect. In addition, the mutant liver shows a significant increase in hematopoietic precursor cells including nucleated erythrocytes at 18.5 dpc (Figure 7B). This alteration is noted as early as 14.5 dpc. These observations suggest that besides silencing the ɛy globin gene, Sox6 may affect red cell maturation.\nFigure 7 The Blood and Liver Phenotype of WT and p100H Homozygous Mice\n(A) Red blood cells of WT mice (left panels) and p100H homozygous mice (right panels) are shown at the indicated ages.\n(B) Liver cells of WT mice (left panels) and p100H homozygous mice (right panels) are shown at the indicated ages."}

pmc-enju-pas

bionlp-st-ge-2016-coref

{"project":"bionlp-st-ge-2016-coref","denotations":[{"id":"T7238","span":{"begin":10575,"end":10597},"obj":"Antecedent"},{"id":"T7237","span":{"begin":10703,"end":10713},"obj":"Anaphor"},{"id":"T7236","span":{"begin":9262,"end":9274},"obj":"Antecedent"},{"id":"T7235","span":{"begin":9891,"end":9908},"obj":"Anaphor"}],"relations":[{"id":"R4979","pred":"boundBy","subj":"T7235","obj":"T7236"},{"id":"R4980","pred":"boundBy","subj":"T7237","obj":"T7238"}],"namespaces":[{"prefix":"_base","uri":"http://bionlp.dbcls.jp/ontology/ge.owl#"}],"text":"Results\n\nPersistent Expression of the Embryonic Globin, ɛy, in Sox6-Deficient Mice\nThe ɛy globin gene was initially identified as an upregulated transcript in the p 100H mouse using subtractive hybridization to identify Sox6 downstream targets. This initial observation was confirmed in an independent knock-out allele of Sox6 [13] using real-time polymerase chain reaction (PCR) (unpublished data). Real-time PCR was used to quantitate the expression levels of other globin genes in p100H mutant and WT mouse livers at two developmental stages, 15.5 dpc and 18.5 dpc. As shown in Figure 1, the ɛy gene is expressed at high levels at both time points in mutant mice, in contrast to the decline in expression observed in WT mice. No difference was seen between WT and heterozygous mice (unpublished data). Interestingly, the expression levels of the other two embryonic globin genes (ζ and βh1) are also higher in p100H homozygous mice, compared with WT mice, but to a much lesser extent than seen for ɛy globin at 18.5 dpc (Figure 1). Moreover, the expression level of adult β globin is also somewhat higher in p100H homozygous mice than in WT mice at 18.5 dpc (Figure 1). Perinatal lethality of mutant mice (presumably from the heart defect [14]) precludes us from evaluating postnatal globin expression. The graphs in Figure 1 illustrate real time PCR results that were performed in triplicate (standard deviation of the data is shown by error bars). Because all of the assays were performed at the same time with the same internal control, the levels shown are relative levels and are thus comparable across all samples and are in agreement with previously published results for WT fetal mice [29]. We note that the level of ɛy expression in the livers of 15.5 dpc and 18.5 dpc homozygous mutant mice is statistically equivalent to the level of βmaj/min expression in the livers of 15.5 dpc and 18.5 dpc homozygous WT mice.\nFigure 1 Real-Time PCR of Globin Genes\nThe levels of expression of ɛy, βh1, zeta, and βmaj/min were measured at 15.5 dpc and 18.5 dpc in homozygous WT and p100H mutant littermates by real-time PCR (see Materials and Methods). Relative expression levels in the livers of each genotype are graphed for each globin gene (performed in triplicate and normalized with GAPDH). Standard deviation is indicated by bars.\n\nTransfection Studies Using GM979 Cells Indicate That Sox6 Directly Represses the ɛy Gene Promoter at the Transcriptional Level\nReal-time PCR assays (Figure 1) measure steady-state levels of ɛy mRNA, not transcriptional activity of the ɛy promoter. To investigate whether Sox6 directly acts on the ɛy gene promoter at the transcriptional level, we used an in vitro transient transfection assay and GM979 cells, a murine erythroleukemic cell line that expresses both ɛy and adult beta globins [30]. We generated an ɛy promoter reporter construct (E-Luc) by fusing a micro-LCR (μLCR) element (2.5 kb) [31] to the ɛy proximal promoter (2.2 kb), followed by the luciferase reporter gene, as shown in Figure 2A (detailed in Materials and Methods). Overexpression of Sox6 in GM979 cells by transient transfection leads to a dosage-dependent repression of E-Luc reporter activity (Figure 2B). In contrast, overexpression of a truncated Sox6 protein that lacks its HMG domain [32] (similar to the p 100H mouse allele) fails to repress E-Luc activity (Figure 2B). These data indicate that Sox6 acts to repress the ɛy promoter at the transcriptional level.\nFigure 2 The Effect of Sox6 on the ɛy Promoter\n(A) Constructs of the ɛy promoter reporter (E-luc) and Sox6 overexpression vector. The E-luc reporter construct consists of a 2.5-kb μLCR element, a 2.2-kb ɛy proximal promoter, and the luciferase reporter in the pGL-3 basic plasmid (see Materials and Methods). Sox6 expression is driven by the CMV promoter.\n(B) Sox6 represses ɛy promoter activity in a dosage-dependent manner. In GM979 cells, the E-Luc ɛy promoter reporter construct was co-transfected (1) without overexpression of Sox6; (2–4) with increasing amounts of CMV-Sox6 overexpression vector; (5) with a truncated version of Sox6 that lacks its HMG domain; (6) with a mutant version of Sox6 (L386H) that has previously been shown to abolish interaction with CtBP2; or (7) with an empty reporter plasmid (without ɛy promoter and μLCR element).\n(C) Promoter deletion analyses to delimit the critical sequence. The 2.2-kb proximal promoter or deletions of it, as indicated on the left (numbering relative to +1 = the transcription start site of ɛy globin, see Materials and Methods), were engineered in reporter constructs as in (A) and were transfected along with CMV driven Sox6 to GM979 cells (see Materials and Methods for details). The relative repression by Sox6 on the activity of the different reporter constructs is shown. All experiments were done in triplicate. Sox6 has been shown to act as a repressor and to interact with a widely expressed co-repressor, CtBP2, on the fgf-3 promoter [5]. CtBP2 is expressed in GM979 cells (unpublished data). To investigate whether the interaction with CtBP2 is required for Sox6 repression of the ɛy promoter, we introduced a point mutation (L386H) in the Sox6 protein that has been previously reported to be sufficient to abolish Sox6-CtBP2 interaction [5]. This amino acid change is not in the HMG DNA binding domain. However, this mutant version of Sox6 retains the ability to repress the ɛy promoter in the transfection assay (Figure 2B), indicating that Sox6 represses the ɛy promoter in a CtBP2-independent manner. Deletion analysis of the ɛy promoter, as shown in Figure 2C, defined a region (−63 to −37) within the ɛy proximal promoter that is critical for Sox6 repression. Analysis of this short region reveals two Sox/Sox6 consensus binding sites [5] (Figure 3A).\nFigure 3 Analysis of the Minimal Region (36 bp) of the Proximal ɛy Promoter Responsive to Sox6\n(A) The sequence of the 36-bp fragment and its mutant versions used in EMSA. The WT 36-bp DNA sequence (−63 to −28) of the ɛy globin proximal promoter contains two Sox/Sox6 consensus binding sites, shown in bold underline. Versions with truncation of this sequence (M1) or mutation of one of the two consensus binding sites (M2 and M3) are also shown.\n(B) EMSA with c-Myc-tagged Sox6. EMSA was performed using the 36-bp radio-labeled WT probe (as shown in (A)) and c-Myc tagged Sox6 translated in vitro using reticulocyte lysate (see Materials and Methods). Lane 1: radio-labeled free probe (run out of the gel); Lane 2: no competition, no antibody; Lane 3: competition with 200-fold excess cold probe, no antibody; Lane 4: no competition, c-Myc antibody (producing a supershift); Lane 5: no competition, Sox6 antibody (producing a supershift); Lane 6: no competion, no antibody using in vitro translated vector containing c-Myc, but not Sox6.\n(C) EMSA with HA-tagged Sox6. EMSA was performed similarly as in (B) using HA-tagged Sox6 translated in vitro. Lane 1: radio-labeled free probe (run out of the gel); Lane 2: no competition, no antibody; Lane 3: competition with 200-fold excess cold probe, no antibody; Lane 4: no competition, HA antibody (producing a supershift).\n(D) EMSA using MEL cell nuclear extracts and the 36-bp WT probe. Lane 1: radio-labeled free probe (run out of the gel); Lane 2: no competition, no antibody; Lane 3: competition with 200-fold excess cold probe, no antibody; Lane 4: no competition, Sox6 antibody (producing a supershift).\n(E) EMSA with c-Myc-tagged Sox6, WT and mutant versions of the 36-bp fragment in competition. EMSA was performed using the radio-labeled 36-bp WT probe and the c-Myc tagged Sox6 translated in vitro. Lane 1: radio-labeled free probe; Lane 2: no competition, no antibody. Competition was performed using 200-fold excess cold probes corresponding to WT (Lane 3), M1 (Lane 4), M2 (Lane 5), and M3 (Lane 6).\n(F) Both consensus Sox/Sox6 binding sites are required for Sox6 responsiveness. GM979 cells were transfected with a reporter construct (Figure 2A) containing −63 to +45 of the ɛy proximal promoter together with the CMV-Sox6 overexpression vector. Mutations of the consensus binding sites were also tested (M3, M2, M2 plus M3, see (A)). The fold repression of Sox6 with the WT or mutant constructs is shown. The baseline activities of the mutagenized reporter constructs are comparable to the WT construct.\n\nEMSA and ChIP Assays Show that Sox6 Directly Binds to the ɛy Promoter\nSox6 might repress the ɛy promoter, either through direct physical contact with the promoter or by regulating intermediates affecting the ɛy promoter. To investigate whether Sox6 is directly associated with the ɛy promoter, we first performed electrophoretic mobility shift assays (EMSA) using a c-Myc-tagged Sox6 in a reticulocyte lysate-based transcription/translation in vitro system. The probes used are listed in Figure 3A. The 36–base pair (bp) WT probe corresponds to the critical region of the ɛy promoter defined in our promoter deletion analyses. This probe contains two consensus Sox/Sox6 binding sites. Also included in our EMSA are three mutated probes that are, either truncated (M1), or mutated (M2 and M3) in Sox/Sox6 binding sites (Figure 3A). Sox6 is able to physically associate with the 36-bp region (Figure 3B) within the ɛy promoter defined by the deletion analysis experiments (Figure 2C). The 36-bp probe was shifted by the tagged Sox6 protein. Moreover, both c-Myc and Sox6 antibodies supershift the band, indicating that the binding is Sox6-specific. To rule out the possibility that the c-Myc tag itself binds to the probe, an HA-tagged Sox6 was used in another EMSA that confirmed these results (Figure 3C).\nNext, nuclear extracts from MEL cells were used in EMSA employing the same 36-bp probe. MEL cells, a murine erythroleukemic cell line, express adult β globins, but not ɛy [33]. Sox6 directly binds to this DNA sequence in MEL cells (Figure 3D). The intact consensus Sox/Sox6 binding sites of the DNA probe are required for the binding, as shown in the competition assay (Figure 3E). Ablation of putative Sox/Sox6 binding sites (M1 and M3) abolish their ability to compete in EMSA (Figure 3E). The M2 mutant probe may compete partially with WT binding.\nTo investigate the functional significance of the intact Sox/Sox6 binding sites, the ɛy promoter reporter constructs with mutagenized Sox/Sox6 binding sites were co-transfected with the Sox6 overexpression vector into GM979 cells. Consistent with the EMSA results, the mutant ɛy promoter reporter constructs (with either one or both Sox/Sox6 binding sites mutagenized) do not result in significant promoter repression in transfection studies (Figure 3F). Thus, both sites are required for maximal repression of ɛy by Sox6, but not to the same degree.\nWe also tested whether Sox6 binds to the ɛy promoter in vivo using chromatin immunoprecipitation (ChIP) (Figure 4). The Sox6-containing complex was immunoprecipitated from MEL cells or from liver cells of 15.5 dpc WT mice using Sox6 antibody. Figure 4 shows that the ɛy proximal promoter is readily immunoprecipitated with Sox6 antibody in both MEL cells and liver cells. Normal IgG was used as a negative control (Figure 4A). The above data (Figures 3 and 4) clearly indicate that Sox6 acts as a repressor of the ɛy gene by directly binding to the ɛy promoter, probably as a dimer.\nFigure 4 ChIP Assay\nMEL cells (A) and 15.5-dpc fetal liver cells (B) were treated as detailed in Materials and Methods. 10% of the sample was saved as total input (Inp); remaining samples were divided: plus Sox6 antibody (Ab+), minus Sox6 antibody (Ab−), as well as no DNA (DNA−) and normal rabbit IgG (IgG) that served as negative controls. Other controls for these experiments included PCR within the promoter of the α-globin gene and intron 24 of the p gene. Both were negative (unpublished data). PCR was carried out using primer pairs flanking the Sox/Sox6 binding sites (see Material and Methods) of the ɛy proximal promoter. For all reactions, we used 2 μl of immuno-precipitated DNA and 2 μl of 1/100 total input. Semiquantitative PCR was done within the exponential range. Multiple independent experiments were done.\n\nThe Persistent Expression of ɛy Globin in Sox6-Deficient Mice Is Due to a Defect in the ɛy-Gene–Silencing Mechanism in Definitive Erythroid Cells\nNormally, the ɛy globin gene is exclusively expressed in primitive erythrocytes and silenced in definitive erythrocytes. To determine whether the persistent expression of ɛy globin is due to residual primitive erythrocytes or is due to ectopic expression of ɛy globin in definitive erythrocytes, we examined the spatial pattern of ɛy globin transcripts in mouse embryos by in situ hybridization (Figure 5). As expected, ɛy globin is not expressed in the WT 14.5-dpc liver, the site of definitive erythropoiesis in the fetus. In contrast, abundant ectopic ɛy mRNA expression is seen in the liver of 14.5-dpc mutants (Figure 5 A–D). However, the expression of βmaj/min globin is equally abundant in both WT and p100H mutant mice (Figure 5 E and F). These data demonstrate that the persistent high levels of ɛy are due to ectopic expression in the definitive erythroid cells that mature in the fetal liver, suggesting that there is an intrinsic defect of the ɛy silencing mechanism in Sox6-null mice.\nFigure 5 In Situ Hybridization of ɛy and βmaj/min transcripts in WT and p100H Mutant Mice at 14.5 dpc\nExpression of ɛy globin (A–D) and β globin (E and F) in sagittal sections of 14.5-dpc fetuses is shown in pseudocolor (red).\n(A) WT fetus.\n(B) p100H Homozygous mutant fetus.\n(C and D) Inset boxes are shown. Prominent expression of ɛy globin is only detected in the liver of mutant mice.\n(E and F) In contrast, both WT and mutant mouse livers express adult βmaj/min, respectively. No signals were detected above background using sense probes (unpublished data).\nThe size bars represent 1 mm for (A) and (B); 100 μm for (C–F).\n\nThe Expression Pattern of Sox6 Suggests a Role in Definitive Erythropoiesis\nThe observation that Sox6-deficient mice ectopically express ɛy globin in liver, where definitive erythroid cells mature, suggests that Sox6 is an important regulator in definitive erythropoiesis. To determine the temporal and spatial expression pattern of Sox6, Northern blot and in situ hybridization assays were employed. As shown in Figure 6A, Sox6 is detectable by Northern blot beginning at 10.5 dpc, coincident with the temporal onset of definitive erythropoiesis in the liver. Furthermore, in situ hybridization shows that Sox6 is highly transcribed in 12.5-dpc liver, but not in yolk sac blood islands at 7.5 dpc (Figure 6B). Therefore, Sox6 expression is temporally and spatially coincident with definitive, but not primitive, erythropoiesis. These data, taken together with the observation that ɛy globin is highly expressed in the liver cells of 14.5-dpc p100H mutant mice (Figure 5), demonstrate that Sox6 functions in definitive erythropoiesis.\nFigure 6 Expression Pattern of Sox6 by Northern Blot and In Situ Assays\n(A) Sox6 expression during embryonic development shown by Northern blot. Each lane contains 20 μg of total RNA from embryos whose ages are listed above each lane as dpc. The filter was hybridized with a 32P-labeled 575-bp mouse Sox6 cDNA fragment (nucleotides 1353–1927). Numbers on the left are sizes of standard marker fragments in kb.\n(B) Sox6 expression shown by in situ hybridization. Panel i: Sagittal section through an E12.5 mouse embryo using antisense Sox6. mRNA distribution is represented by pseudocolored red signal superimposed on the counterstained specimen. Sox6 transcripts are detected primarily in the fetal liver, developing nervous system, chondrocytes and craniofacial area. Panel ii: The sense control probe shows no signal above background. Panel iii: E7.5 embryo hybridized to antisense probe for Sox6. No signal is detected above background specifically in blood islands (or with the sense probe, unpublished data).\nThe size bars represent 100 μm.\n\nMutant p100H Mice Have Higher Numbers of Nucleated Red Cells\nAmong the other Sox6 effects in erythropoiesis, we have noticed that there are more nucleated red blood cells circulating in p100H mutant mice than in WT mice at 14.5 dpc and 18.5 dpc (Figure 7A). However, at postnatal day 10.5, we do not see circulating nucleated red cells in either WT or mutant mice, suggesting that this may be a transient effect. In addition, the mutant liver shows a significant increase in hematopoietic precursor cells including nucleated erythrocytes at 18.5 dpc (Figure 7B). This alteration is noted as early as 14.5 dpc. These observations suggest that besides silencing the ɛy globin gene, Sox6 may affect red cell maturation.\nFigure 7 The Blood and Liver Phenotype of WT and p100H Homozygous Mice\n(A) Red blood cells of WT mice (left panels) and p100H homozygous mice (right panels) are shown at the indicated ages.\n(B) Liver cells of WT mice (left panels) and p100H homozygous mice (right panels) are shown at the indicated ages."}

bionlp-st-ge-2016-test-proteins

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UBERON-AE

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BioNLP16_DUT

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DLUT931

bionlp-st-ge-2016-test-ihmc

bionlp-st-ge-2016-spacy-parsed

bionlp-st-ge-2016-test-tees

testone

test3

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