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

Sox6 regulation of cardiac myocyte development

A mouse mutation (p(100H)/p(100H)) has been identified that is associated with cardioskeletal myopathy, heart block, delayed growth and early postnatal death. The gene that is disrupted in this mutation encodes the transcription factor Sox6. P19CL6 cells were used as an in vitro cardiomyocyte differentiation system and revealed that Sox6 is expressed exclusively when the cells are committed to differentiate to beating cardiac myocytes. We used the yeast two-hybrid system to identify the Prtb (Proline-rich transcript of the brain) protein as a Sox6 interactor, and subsequently confirmed the interaction by co-immunoprecipitation. Prtb expression in P19CL6 cells increased with differentiation to beating cardiomyocytes. Using the P19CL6 cells stably transfected with noggin, an antagonist of BMP (Bone Morphogenic Protein), we found that BMP expression is required for Sox6 expression in cardiomyocyte differentiation. Surprisingly, the expression of the α(1c)-subunit gene of the L-type Ca(2+) channel decreased in P19CL6 cells as they differentiated to beating cardiac cells. Ectopic expression of Sox6 or Prtb alone in P19CL6 cells caused down-regulation of L-type Ca(2+) α(1c) expression, but when Sox6 and Prtb were co-transfected to the cells, L-type Ca(2+) α(1c) remained at basal levels. A similar relationship of Sox6 and L-type Ca(2+) α(1c) expression was seen in vivo (comparing wild-type and p(100H)/p(100H) mutant mice). Thus, Sox6 is within the BMP pathway in cardiac differentiation, interacts with Prtb and may play a critical role in the regulation of a cardiac L-type Ca(2+) channel

Mutations in the Human Orthologue of the Mouse underwhite Gene (uw) Underlie a New Form of Oculocutaneous Albinism, OCA4

Oculocutaneous albinism (OCA) affects ∼1/20,000 people worldwide. All forms of OCA exhibit generalized hypopigmentation. Reduced pigmentation during eye development results in misrouting of the optic nerves, nystagmus, alternating strabismus, and reduced visual acuity. Loss of pigmentation in the skin leads to an increased risk for skin cancer. Two common forms and one infrequent form of OCA have been described. OCA1 (MIM 203100) is associated with mutations of the TYR gene encoding tyrosinase (the rate-limiting enzyme in the production of melanin pigment) and accounts for ∼40% of OCA worldwide. OCA2 (MIM 203200), the most common form of OCA, is associated with mutations of the P gene and accounts for ∼50% of OCA worldwide. OCA3 (MIM 203290), a rare form of OCA and also known as “rufous/red albinism,” is associated with mutations in TYRP1 (encoding tyrosinase-related protein 1). Analysis of the TYR and P genes in patients with OCA suggests that other genes may be associated with OCA. We have identified the mouse underwhite gene (uw) and its human orthologue, which underlies a new form of human OCA, termed “OCA4.” The encoded protein, MATP (for “membrane-associated transporter protein”) is predicted to span the membrane 12 times and likely functions as a transporter

A 122.5-Kilobase Deletion of the P Gene Underlies the High Prevalence of Oculocutaneous Albinism Type 2 in the Navajo Population

Oculocutaneous albinism (OCA) is a genetically heterogeneous disorder. There are four known types of OCA: OCA1–OCA4. The clinical manifestations of all types of OCA include skin and hair hypopigmentation and visual impairment. Although there are a few documented observations of high frequency of albinism among Native Americans, including the Hopi, Zuni, Kuna, Jemez, Laguna, San Juan, and Navajo, no causative molecular defect has been previously reported. In the present study, we show that albinism in one Native American population, the Navajo, is caused by a LINE-mediated 122.5-kilobase deletion of the P gene, thus demonstrating that albinism in this population is OCA2. This deletion appears to be Navajo specific, because this allele was not detected in 34 other individuals with albinism who listed other Native American origins, nor has it been reported in any other ethnic group. The molecular characterization of this deletion allele allowed us to design a three-primer polymerase chain reaction system to estimate the carrier frequency in the Navajo population by screening 134 unrelated normally pigmented Navajos. The carrier frequency was found to be ∼4.5%. The estimated prevalence of OCA2 in Navajos is between ∼1 per 1,500 and 1 per 2,000. We further estimate that this mutation originated 400–1,000 years ago from a single founder

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

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