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

Mantle cell lymphoma and its management: where are we now?

Abstract Mantle cell lymphoma is a relatively new recognized hematological malignant disease, comprising of 2.5–6% non-Hodgkin’s lymphomas. The complexity of its clinical presentations (nodular pattern, diffuse pattern, and blastoid variant), variety in disease progression, and treatment response, make this disease a research focus to both experimental oncology and clinical oncology. Overexpression of cyclin D1 and chromosome t(11,14) translocation are the known molecular biomarkers of this disease. Mantle cell international prognostic index (MIPI), ki-67 proliferation index, and TP53 mutation are emerging as the prognostic biomarkers. Epigenetic profile variance and SOX11 gene expression profile correlate with treatment response. Over the years, the treatment strategy has been gradually evolving from combination chemotherapy to combination of targeted therapy, epigenetic modulation therapy, and immunotherapy. In a surprisingly short period of time, FDA specifically approved 4 drugs for treating mantle cell lymphoma: lenalidomide, an immunomodulatory agent; Bortezomib, a proteasome inhibitor; and Ibrutinib and acalabrutinib, both Bruton kinase inhibitors. Epigenetic agents (e.g. Cladribine and Vorinostat) and mTOR inhibitors (e.g. Temsirolimus and Everolimus) have been showing promising results in several clinical trials. However, treating aggressive variants of this disease that appear to be refractory/relapse to multiple lines of treatment, even after allogeneic stem cell transplant, is still a serious challenge. Developing a personalized, precise therapeutic strategy combining targeted therapy, immunotherapy, epigenetic modulating therapy, and cellular therapy is the direction of finding a curative therapy for this subgroup of patients

Replication Initiation Patterns in the β-Globin Loci of Totipotent and Differentiated Murine Cells: Evidence for Multiple Initiation Regions

The replication initiation pattern of the murine β-globin locus was analyzed in totipotent embryonic stem cells and in differentiated cell lines. Initiation events in the murine β-globin locus were detected in a region extending from the embryonic Ey gene to the adult βminor gene, unlike the restricted initiation observed in the human locus. Totipotent and differentiated cells exhibited similar initiation patterns. Deletion of the region between the adult globin genes did not prevent initiation in the remainder of the locus, suggesting that the potential to initiate DNA replication was not contained exclusively within the primary sequence of the deleted region. In addition, a deletion encompassing the six identified 5′ hypersensitive sites in the mouse locus control region had no effect on initiation from within the locus. As this deletion also did not affect the chromatin structure of the locus, we propose that the sequences determining both chromatin structure and replication initiation lie outside the hypersensitive sites removed by the deletion

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