3D printing biocompatible materials with Multi Jet Fusion for bioreactor applications.

In the evolving three-dimensional (3D) printing technology, the involvement of different materials in any new 3D printing process necessitates a thorough evaluation of the product's biocompatibility for biomedical application. Here, we examined the ability of Multi Jet Fusion (MJF)-printed PA-12 to support cell proliferation and osteogenesis. Our results show that leachate from MJF-printed PA-12 does not inhibit the growth of L929 fibroblast and MC3T3e1 osteoblast. The substrate supports the attachment and proliferation of both cell types, though not at a level comparable to conventional polystyrene culture plate. Neither plasma treatment, poly-D-lysine, nor collagen coatings narrowed the gap substantially, suggesting the possible influence of other limiting factors. The substrate can also support MC3T3e1 osteogenesis. However, MJF-printed PA-12 exhibits varying ability in supporting the proliferation of different cell types, especially in subsequent passages. While L929's proliferation is comparable from passage-to-passage, MC3T3e1's growth ability is noticeably compromised. Interestingly, our results show that L929 subcultured back to polystyrene plate retains the ability to grow as robustly as those on the conventional plate, suggesting that MJF-printed PA-12 does not permanently impair cell proliferation. In addition, we have shown the successful culture of bacterial Escherichia coli on MJF-printed PA-12. Together, our study demonstrated the potential of MJF-printed PA-12 for biological applications.

Probing p300/CBP associated factor (PCAF)-dependent pathways with a small molecule inhibitor.

PCAF (KAT2B) belongs to the GNAT family of lysine acetyltransferases (KAT) and specifically acetylates the histone H3K9 residue and several nonhistone proteins. PCAF is also a transcriptional coactivator. Due to the lack of a PCAF KAT-specific small molecule inhibitor, the exclusive role of the acetyltransferase activity of PCAF is not well understood. Here, we report that a natural compound of the hydroxybenzoquinone class, embelin, specifically inhibits H3Lys9 acetylation in mice and inhibits recombinant PCAF-mediated acetylation with near complete specificity in vitro. Furthermore, using embelin, we have identified the gene networks that are regulated by PCAF during muscle differentiation, further highlighting the broader regulatory functions of PCAF in muscle differentiation in addition to the regulation via MyoD acetylation.

G9a mediates Sharp-1-dependent inhibition of skeletal muscle differentiation.

Sharp-1, a basic helix-loop-helix transcription factor, is a potent repressor of skeletal muscle differentiation and is dysregulated in muscle pathologies. However, the mechanisms by which it inhibits myogenesis are not fully understood. Here we show that G9a, a lysine methyltransferase, is involved in Sharp-1-mediated inhibition of muscle differentiation. We demonstrate that G9a directly interacts with Sharp-1 and enhances its ability to transcriptionally repress the myogenin promoter. Concomitant with a differentiation block, G9a-dependent histone H3 lysine 9 dimethylation (H3K9me2) and MyoD methylation are apparent upon Sharp-1 overexpression in muscle cells. RNA interference-mediated reduction of G9a or pharmacological inhibition of its activity erases these repressive marks and rescues the differentiation defect imposed by Sharp-1. Our findings provide new insights into Sharp-1-dependent regulation of myogenesis and identify epigenetic mechanisms that could be targeted in myopathies characterized by elevated Sharp-1 levels.

SUMO modification of Stra13 is required for repression of cyclin D1 expression and cellular growth arrest.

Stra13, a basic helix-loop-helix (bHLH) transcription factor is involved in myriad biological functions including cellular growth arrest, differentiation and senescence. However, the mechanisms by which its transcriptional activity and function are regulated remain unclear. In this study, we provide evidence that post-translational modification of Stra13 by Small Ubiquitin-like Modifier (SUMO) dramatically potentiates its ability to transcriptionally repress cyclin D1 and mediate G(1) cell cycle arrest in fibroblast cells. Mutation of SUMO acceptor lysines 159 and 279 located in the C-terminal repression domain has no impact on nuclear localization; however, it abrogates association with the co-repressor histone deacetylase 1 (HDAC1), attenuates repression of cyclin D1, and prevents Stra13-mediated growth suppression. HDAC1, which promotes cellular proliferation and cell cycle progression, antagonizes Stra13 sumoylation-dependent growth arrest. Our results uncover an unidentified regulatory axis between Stra13 and HDAC1 in progression through the G(1)/S phase of the cell cycle, and provide new mechanistic insights into regulation of Stra13-mediated transcriptional repression by sumoylation.

Role of misfolded N-CoR mediated transcriptional deregulation of Flt3 in acute monocytic leukemia (AML)-M5 subtype.

The nuclear receptor co-repressor (N-CoR) is a key component of the generic multi-protein complex involved in transcriptional control. Flt3, a key regulator of hematopoietic cell growth, is frequently deregulated in AML (acute myeloid leukemia). Here, we report that loss of N-CoR-mediated transcriptional control of Flt3 due to misfolding, contributes to malignant growth in AML of the M5 subtype (AML-M5). An analysis of hematopoietic genes in AML cells led to the identification of Flt3 as a transcriptional target of N-CoR. Flt3 level was inversely related to N-CoR status in various leukemia cells. N-CoR was associated with the Flt3 promoter in-vivo, and a reporter driven by the Flt3 promoter was effectively repressed by N-CoR. Blocking N-CoR loss with Genistein; an inhibitor of N-CoR misfolding, significantly down-regulated Flt3 levels regardless of the Flt3 receptor mutational status and promoted the differentiation of AML-M5 cells. While stimulation of the Flt3 receptor with the Flt3 ligand triggered N-CoR loss, Flt3 antibody mediated blockade of Flt3 ligand-receptor binding led to N-CoR stabilization. Genetic ablation of N-CoR potentiated Flt3 ligand induced proliferation of BA/F3 cells. These findings suggest that N-CoR-induced repression of Flt3 might be crucial for limiting the contribution of the Flt3 signaling pathway on the growth potential of leukemic cells and its deregulation due to N-CoR loss in AML-M5, could contribute to malignant growth by conferring a proliferative advantage to the leukemic blasts. Therapeutic restoration of N-CoR function could thus be a useful approach in restricting the contribution of the Flt3 signaling pathway in AML-M5 pathogenesis.

Lysine methyltransferase G9a methylates the transcription factor MyoD and regulates skeletal muscle differentiation.

Skeletal muscle cells have served as a paradigm for understanding mechanisms leading to cellular differentiation. The proliferation and differentiation of muscle precursor cells require the concerted activity of myogenic regulatory factors including MyoD. In addition, chromatin modifiers mediate dynamic modifications of histone tails that are vital to reprogramming cells toward terminal differentiation. Here, we provide evidence for a unique dimension to epigenetic regulation of skeletal myogenesis. We demonstrate that the lysine methyltransferase G9a is dynamically expressed in myoblasts and impedes differentiation in a methyltransferase activity-dependent manner. In addition to mediating histone H3 lysine-9 di-methylation (H3K9me2) on MyoD target promoters, endogenous G9a interacts with MyoD in precursor cells and directly methylates it at lysine 104 (K104) to constrain its transcriptional activity. Mutation of K104 renders MyoD refractory to inhibition by G9a and enhances its myogenic activity. Interestingly, MyoD methylation is critical for G9a-mediated inhibition of myogenesis. These findings provide evidence of an unanticipated role for methyltransferases in cellular differentiation states by direct posttranslational modification of a transcription factor.