Sox6 Directly Silences Epsilon Globin Expression in Definitive Erythropoiesis

Sox6 is a member of the Sox transcription factor family that is defined by the conserved high mobility group (HMG) DNA binding domain, first described in the testis determining gene, Sry. Previous studies have suggested that Sox6 plays a role in the development of the central nervous system, cartilage, and muscle. In the Sox6-deficient mouse, p(100H), ɛy globin is persistently expressed, and increased numbers of nucleated red cells are present in the fetal circulation. Transfection assays in GM979 (erythroleukemic) cells define a 36–base pair region of the ɛy proximal promoter that is critical for Sox6 mediated repression. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrate that Sox6 acts as a repressor by directly binding to the ɛy promoter. The normal expression of Sox6 in wild-type fetal liver and the ectopic expression of ɛy in p(100H) homozygous fetal liver demonstrate that Sox6 functions in definitive erythropoiesis. The present study shows that Sox6 is required for silencing of ɛy globin in definitive erythropoiesis and suggests a role for Sox6 in erythroid cell maturation. Thus, Sox6 regulation of ɛy globin might provide a novel therapeutical target in the treatment of hemoglobinopathies such as sickle cell anemia and thalassemia

ChIP Assay

<p>MEL cells (A) and 15.5-dpc fetal liver cells (B) were treated as detailed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020014#s4" target="_blank">Materials and Methods</a>. 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 <i>p</i> 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 Both were negative (unpublished data).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.</p

The Blood and Liver Phenotype of WT and <i>p^100H</i> Homozygous Mice

<div><p>(A) Red blood cells of WT mice (left panels) and <i>p^100H</i> homozygous mice (right panels) are shown at the indicated ages.</p><p>(B) Liver cells of WT mice (left panels) and <i>p^100H</i> homozygous mice (right panels) are shown at the indicated ages.</p></div

Real-Time PCR of Globin Genes

<p>The levels of expression of ɛy, βh1, zeta, and βmaj/min were measured at 15.5 dpc and 18.5 dpc in homozygous WT and <i>p^100H</i> mutant littermates by real-time PCR (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020014#s4" target="_blank">Materials and Methods</a>). 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.</p

The Effect of Sox6 on the ɛy Promoter

<div><p>(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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020014#s4" target="_blank">Materials and Methods</a>). Sox6 expression is driven by the CMV promoter.</p><p>(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).</p><p>(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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020014#s4" target="_blank">Materials and Methods</a>), were engineered in reporter constructs as in (A) and were transfected along with CMV driven Sox6 to GM979 cells (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020014#s4" target="_blank">Materials and Methods</a> for details). The relative repression by Sox6 on the activity of the different reporter constructs is shown. All experiments were done in triplicate.</p></div

Analysis of the Minimal Region (36 bp) of the Proximal ɛy Promoter Responsive to Sox6

<div><p>(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.</p><p>(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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020014#s4" target="_blank">Materials and Methods</a>). 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.</p><p>(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).</p><p>(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).</p><p>(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<i>.</i> 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).</p><p>(F) Both consensus Sox/Sox6 binding sites are required for Sox6 responsiveness. GM979 cells were transfected with a reporter construct (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020014#pgen-0020014-g002" target="_blank">Figure 2</a>A) 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.</p></div

Expression Pattern of Sox6 by Northern Blot and In Situ Assays

<div><p>(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.</p><p>(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).</p><p>The size bars represent 100 μm.</p></div