A highly conserved SOX6 double binding site mediates SOX6 gene downregulation in erythroid cells

The Sox6 transcription factor plays critical roles in various cell types, including erythroid cells. Sox6-deficient mice are anemic due to impaired red cell maturation and show inappropriate globin gene expression in definitive erythrocytes. To identify new Sox6 target genes in erythroid cells, we used the known repressive double Sox6 consensus within the εy-globin promoter to perform a bioinformatic genome-wide search for similar, evolutionarily conserved motifs located within genes whose expression changes during erythropoiesis. We found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in vitro and in vivo, and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells and in primary erythroblasts. The binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied, in erythroid cells, by strong downregulation of the endogenous Sox6 transcript and by decreased in vivo chromatin accessibility of this region to the PstI restriction enzyme. These observations suggest that the negative Sox6 autoregulation, mediated by the double Sox6 binding site within its own promoter, may be relevant to control the Sox6 transcriptional downregulation that we observe in human erythroid cultures and in mouse bone marrow cells in late erythroid maturation

Green fluorescent protein transgene driven by Kit regulatory sequences is expressed in hematopoietic stem cells

The expression of Kit in multiple types of stem cells suggests that common transcriptional programs might regulate this gene in different stem cells. In this work, the authors used mouse lines expressing transgenic green fluorescent protein under the control of Kit promoter/first intron regulatory elements. This study provides the basis for the elucidation of DNA sequences regulating a stem cell gene in multiple types of stem cells

Identification of new murine dystrophic-fibres associated-miRNAs in muscle biopsies of DMD subjects.

<p>(A) The age and type of muscle of healthy subjects (12) and DMD patients (18) were listed. Moreover the type of muscle from which the biopsies were isolated were also reported. (B–C) MiR-15b, miR-128a, miR-206, miR-17 and miR-27a were quantified by absolute Q-PCR in muscle biopsies of control and dystrophic subjects listed in A. The absolute values (pg) of tested miRNAs were represented in the histogram for healthy (white bars) and dystrophic single fibres (black bars). (B) Quantitative analysis showed that only miR-128a, miR-206 and miR-17 were up-regulated in human dystrophic single fibres independently of the muscle type. (Two-tail parametric t-test; *p value <0, 05; **p value <0, 01; ***p value <0,001).</p

MyomiR-206 modulates Hmgb3 expression during myogenesis.

<p>(A) Hek cells were co-transfected with mimic-miR-206 at a concentration of 25 nM for 48 h and with pGLO-control or pGLOHmgb3 or pGLOHmgb3Mut. MiR-206 down-regulated the expression of luciferase of 32% (p = 0, 0151) in Hek cells co-transfected with pGLOHmgb3. Instead no down-regulation happened when the binding site of miR-206 was mutated. (B) 3T3 cells were transfected with mimic miR-206 at a concentration of 25 nM for 48 h, evidencing a strong down-regulation of endogenous Hmgb3 mRNAs. (C) Quantification of Hmgb3 mRNA by qRT-PCR in single fibres isolated from CTX-injured TA at day 2, 5, 7 and 10 from the injection evidenced a strong down-regulation of Hmgb3 during muscle regeneration. (D–E) Hmgb3 mRNA (D) and miR-206 (E) were respectively quantified by relative qRT-PCR and by absolute Q-PCR in proliferating C₁C₁₂ myoblasts cell line (MB) versus differentiated C₁C₁₂ myotubes (MT). The quantification analysis highlighted an opposite expression trend for Hmgb3 and miR-206, further validating inhibition of Hmgb3 by miR-206 (AU = arbitrary units). (Two-tail parametric t-test; *p value <0, 05; **p value <0,01; ***p value <0,001).</p

Acute muscle damage activates miRNA machinery in isolated myofibers.

<p>Dystrophic-fibres associated miRNAs were quantified by Q-PCR in single fibres isolated from TA of c57bl mice (n = 10) after CTX-injection. Mice were sacrified at the day 2, 5, 7, 10 after CTX-injury. In the table are represented the fold change values of tested miRNAs in CTX-injected muscle compare to controlateral not injected TA. MiR-206, miR-31, miR-21, miR-335-5p, miR-27a, miR-142-5p and miR-223 were significantly up-regulated afterwards muscle damage respect damaged muscle. Otherwise the other 7 miRNAs were not triggered upon CTX-injection.</p><p>(Two-tail parametric t-test; *p value <0, 05; **p value <0, 01; ***p value <0,001).</p

Hmgb3 expression-pattern in single fibres.

<p>(A) Immunoblotting experiments confirmed the presence of Hmgb3 protein in single muscle fibers isolated from the TA and VA of c57bl (n = 3) and mdx (n = 3) mice. (B) Densitometric analysis on WB bands evidenced a decreased expression of Hmgb3 in the TA and VA (p = 0.0133) of mdx mice. (C) Quantification of Hmgb3 mRNA by qRT-PCR in single fibres isolated from the TA and VA of mdx mice (n = 10) and of c57bl (n = 10) mice confirmed a down-regulation of Hmgb3 in dystrophic muscle. (Two-tail parametric t-test; *p value <0, 05; **p value <0, 01; ***p value <0,001).</p

MiRNA profile of single fibres isolated from the mdx mouse.

<p>(A) Clustered heat map showing the expression ratios of miRNAs in TA, DIA and VA of age- and sex-matched c57bl (n = 3) and mdx (n = 3) mice. Expression data were normalized on Universal Reference. A total of 14 miRNAs were found over-expressed in dystrophic samples with distinction among myofibres isolated from different muscle type. (B) Fold change values of the 14 up-regulated miRNAs were reported in the table (ns = no significant).</p

Dystrophin absence is not responsible for the over-expression of several muscle-enriched miRNAs in the mdx mouse.

<p>(A) MiRNAs that were observed over-expressed in the adult mdx mouse (3½ months-old) by the array analysis were quantified by Q-PCR in single fibres isolated from the hind limb of newborn mdx (n = 10) and c5bl (n = 10) mice. The absolute quantity (pg) of each miRNA was represented in the histogram as white columns for single fibres of c57bl mice and black bars for dystrophic muscle fibres. Single fibres of newborn mdx mice were characterized by control levels of muscle-enriched miRNAs, demonstrating no correlation with the genetic defect of DMD. (B) MiRNAs that were observed over-expressed in the adult mdx mouse (3½ months-old) by the array analysis were quantified by Q-PCR in single fibres isolated from the TA, DIA and VA of 6 months-old mdx mice and normalized on control samples. In the table were represented the fold change values of tested miRNAs in myofibres of 3½ and 6 months-old mdx mice in comparison to c57bl myofibers. Tested miRNAs showed a heterogeneous behaves depending on the muscle type and/or disease progression. (Two-wail parametric t-test; p value <0, 05 **p value <0, 01 ***p value <0,001).</p