RNA transcripts, miRNA-sized fragments and proteins produced from D4Z4 units: new candidates for the pathophysiology of facioscapulohumeral dystrophy.

Deletion of a subset of the D4Z4 macrosatellite repeats in the subtelomeric region of chromosome 4q causes facioscapulohumeral muscular dystrophy (FSHD) when occurring on a specific haplotype of 4qter (4qA161). Several genes have been examined as candidates for causing FSHD, including the DUX4 homeobox gene in the D4Z4 repeat, but none have been definitively shown to cause the disease, nor has the full extent of transcripts from the D4Z4 region been carefully characterized. Using strand-specific RT-PCR, we have identified several sense and antisense transcripts originating from the 4q D4Z4 units in wild-type and FSHD muscle cells. Consistent with prior reports, we find that the DUX4 transcript from the last (most telomeric) D4Z4 unit is polyadenylated and has two introns in its 3-prime untranslated region. In addition, we show that this transcript generates (i) small si/miRNA-sized fragments, (ii) uncapped, polyadenylated 3-prime fragments that encode the conserved C-terminal portion of DUX4 and (iii) capped and polyadenylated mRNAs that contain the double-homeobox domain of DUX4 but splice-out the C-terminal portion. Transfection studies demonstrate that translation initiation at an internal methionine can produce the C-terminal polypeptide and developmental studies show that this peptide inhibits myogenesis at a step between MyoD transcription and the activation of MyoD target genes. Together, we have identified new sense and anti-sense RNA transcripts, novel mRNAs and mi/siRNA-sized RNA fragments generated from the D4Z4 units that are new candidates for the pathophysiology of FSHD.

MyoD inhibits Fstl1 and Utrn expression by inducing transcription of miR-206.

Terminal differentiation of distinct cell types requires the transcriptional activation of differentiation-specific genes and the suppression of genes associated with the precursor cell. For example, the expression of utrophin (Utrn) is suppressed during skeletal muscle differentiation, and it is replaced at the sarcolemma by the related dystrophin protein. The MyoD transcription factor directly activates the expression of a large number of skeletal muscle genes, but also suppresses the expression of many genes. To characterize a mechanism of MyoD-mediated suppression of gene expression, we investigated two genes that are suppressed in fibroblasts converted to skeletal muscle by MyoD, follistatin-like 1 (Fstl1) and Utrn. MyoD directly activates the expression of a muscle-specific microRNA (miRNA), miR-206, which targets sequences in the Fstl1 and Utrn RNA, and these sequences are sufficient to suppress gene expression in the presence of miR-206. These findings demonstrate that MyoD, in addition to activating muscle-specific genes, induces miRNAs that repress gene expression during skeletal muscle differentiation.

Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis.

Mesenchymal stem cells (MSCs) transplanted at sites of nerve injury are thought to promote functional recovery by producing trophic factors that induce survival and regeneration of host neurons. To evaluate this phenomenon further, we quantified in human MSCs neurotrophin expression levels and their effects on neuronal cell survival and neuritogenesis. Screening a human MSC cDNA library revealed expressed transcripts encoding BDNF and beta-NGF but not NT-3 and NT-4. Immunostaining demonstrated that BDNF and beta-NGF proteins were restricted to specific MSC subpopulations, which was confirmed by ELISA analysis of 56 separate subclones. Using a co-culture assay, we also demonstrated that BDNF expression levels correlated with the ability of MSC populations or subclones to induce survival and neurite outgrowth in the SH-SY5Y neuroblastoma cell line. However, these MSC-induced effects were only partially inhibited by a neutralizing anti-BDNF antibody. MSCs were also shown to promote neurite outgrowth within dorsal root ganglion explants despite secreting 25-fold lower level of beta-NGF required exogenously to produce a similar effect. Interrogation of the human MSC transcriptome identified expressed mRNAs encoding various neurite-inducing factors, axon guidance and neural cell adhesion molecules. Moreover, a subset of these transcripts was shown to correlate with BDNF expression in MSC subclones. Collectively, these studies reveal the existence of MSC subpopulations that co-express neurotrophins and other potent neuro-regulatory molecules, which contribute to MSC-induced effects on neuronal cell survival and nerve regeneration. These subpopulations may represent more potent vectors for treating a variety of neurological disorders.