BackgroundThe stem cell factor spalt-like transcription factor 4 (SALL4) plays important roles in normal hematopoiesis and also in leukemogenesis. We previously reported that SALL4 exerts its effect by recruiting important epigenetic factors such as DNA methyltransferases DNMT1 and lysine-specific demethylase 1 (LSD1/KDM1A). Both of these proteins are critically involved in mixed lineage leukemia (MLL)-rearranged (MLL-r) leukemia, which has a very poor clinical prognosis. Recently, SALL4 has been further linked to the functions of MLL and its target gene homeobox A9 (HOXA9). However, it remains unclear whether SALL4 is indeed a key player in MLL-r leukemia pathogenesis.MethodsUsing a mouse bone marrow retroviral transduction/ transplantation approach combined with tamoxifen-inducible, CreERT2-mediated Sall4 gene deletion, we studied SALL4 functions in leukemic transformation that was induced by MLL-AF9—one of the most common MLL-r oncoproteins found in patients. In addition, the underlying transcriptional and epigenetic mechanisms were explored using chromatin immunoprecipitation (ChIP) sequencing (ChIP-Seq), mRNA microarray, qRT-PCR, histone modification, co-immunoprecipitation (co-IP), cell cycle, and apoptosis assays. The effects of SALL4 loss on normal hematopoiesis in mice were also investigated.ResultsIn vitro and in vivo studies revealed that SALL4 expression is critically required for MLL-AF9-induced leukemic transformation and disease progression in mice. Loss of SALL4 in MLL-AF9-transformed cells induced apoptosis and cell cycle arrest at G1. ChIP-Seq assay identified that Sall4 binds to key MLL-AF9 target genes and important MLL-r or non-MLL-r leukemia-related genes. ChIP-PCR assays indicated that SALL4 affects the levels of the histone modification markers H3K79me2/3 and H3K4me3 at MLL-AF9 target gene promoters by physically interacting with DOT1-like histone H3K79 methyltransferase (DOT1l) and LSD1/KDM1A, and thereby regulates transcript expression. Surprisingly, normal Sall4 f/f/CreERT2 mice treated with tamoxifen or vav-Cre-mediated (hematopoietic-specific) Sall4 −/− mice were healthy and displayed no significant hematopoietic defects.ConclusionsOur findings indicate that SALL4 critically contributes to MLL-AF9-induced leukemia, unraveling the underlying transcriptional and epigenetic mechanisms in this disease and suggesting that selectively targeting the SALL4 pathway may be a promising approach for managing human MLL-r leukemia.Electronic supplementary materialThe online version of this article (10.1186/s13045-017-0531-y) contains supplementary material, which is available to authorized users.

Background Given known inefficiencies in reprogramming of fibroblasts into mature induced cardiomyocytes ( iCM s), we sought to identify small molecules that would overcome these barriers to cardiac cell transdifferentiation. Methods and Results We screened alternative combinations of compounds known to impact cell reprogramming using morphologic and functional cell differentiation assays in vitro. After screening 6 putative reprogramming factors, we found that a combination of the histone deacetylase inhibitor sodium butyrate, the WNT inhibitor ICG ‐001, and the cardiac growth regulator retinoic acid (RA) maximally enhanced iCM generation from primary rat cardiac fibroblasts when combined with administration of the cardiodifferentiating transcription factors Gata4, Mef2C, and Tbx5 ( GMT ) compared with GMT administration alone (23±1.5% versus 3.3±0.2%; P <0.0001). Expression of the cardiac markers cardiac troponin T, Myh6, and Nkx2.5 was upregulated as early as 10 days after GMT –sodium butyrate, ICG‐001, and RA treatment. Human iCM generation was likewise enhanced when administration of the human cardiac reprogramming factors GMT , Hand2, and Myocardin plus miR‐590 was combined with sodium butyrate, ICG‐001, and RA compared with GMT , Hand2, and Myocardin plus miR‐590 treatment alone (25±1.3% versus 5.7±0.4%; P <0.0001). Rat and human iCM s also more frequently demonstrated spontaneous beating in coculture with neonatal cardiomyocytes with the addition of sodium butyrate, ICG‐001, and RA to transcription factor cocktails compared with transcription factor treatment alone. Conclusions The combined administration of histone deacetylase and WNT inhibitors with RA enhances rat and human iCM generation induced by transcription factor administration alone. These findings suggest opportunities for improved translational approaches for cardiac regeneration.

Downregulation of p63 facilitates direct cardiac cellular reprogramming and may help overcome the resistance of human cells to reprogramming.

Objective The administration of a variety of reprogramming factor cocktails has now been shown to reprogram cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs). Reductions in ventricular fibrosis observed after reprogramming factor administration, however, seem to far exceed the extent of iCM generation in vivo. We therefore investigated whether reprogramming factor administration might primarily play a role in activating anti-fibrotic molecular pathways. Methods Adult rat cardiac fibroblasts (RCFs) were infected with lentivirus encoding the transcription factors Gata4, Mef2c or Tbx5, all three vectors (GMT) or a GFP control vector. Gene and protein expression assays were performed to identify relevant anti-fibrotic targets of these factors. The anti-fibrotic effects of these factors were then investigated in a rat coronary ligation model. Results GMT administration to RCFs in vitro significantly downregulated expression of Snail, and the pro-fibrotic factors, CTGF, Collagen1a1, and Fibronectin. Of these factors, Gata4 was shown to be the one responsible for the downregulation of the pro-fibrotic factors, and Snail (mRNA expression fold change relative to GFP for Snail, Gata4: 0.5 ± 0.3, Mef2c: 1.3 ± 1.0, Tbx5: 0.9 ± 0.5, GMT: 0.6 ± 0.2, p<0.05). ChIP qPCR identified Gata4 binding sites in the Snail promoter. In a rat coronary ligation model, only Gata4 administration alone also improved post– infarct ventricular function and also reduced the extent of post-infarct fibrosis. Conclusions Gata4 administration reduces post–infarct ventricular fibrosis and improves ventricular function in a rat coronary ligation model, potentially as a result of Gata4 mediated downregulation of the pro-fibrotic mediator Snail.

Background The conversion of fibroblasts into induced cardiomyocytes may regenerate myocardial tissue from cardiac scar through in situ cell transdifferentiation. The efficiency transdifferentiation is low, especially for human cells. We explored the leveraging of Hippo pathway intermediates to enhance induced cardiomyocyte generation. Methods and Results We screened Hippo effectors Yap (yes‐associated protein), Taz (transcriptional activator binding domain), and Tead1 (TEA domain transcription factor 1; Td) for their reprogramming efficacy with cardio‐differentiating factors Gata4, Mef2C, and Tbx5 (GMT). Td induced nearly 3‐fold increased expression of cardiomyocyte marker cTnT (cardiac troponin T) by mouse embryonic and adult rat fibroblasts versus GMT administration alone ( P <0.0001), while Yap and Taz failed to enhance cTnT expression. Serial substitution demonstrated that Td replacement of TBX5 induced the greatest cTnT expression enhancement and sarcomere organization in rat fibroblasts treated with all GMT substitutions (GMTd versus GMT: 17±1.2% versus 5.4±0.3%, P <0.0001). Cell contractility (beating) was seen in 6% of GMTd‐treated cells by 4 weeks after treatment, whereas no beating GMT‐treated cells were observed. Human cardiac fibroblasts likewise demonstrated increased cTnT expression with GMTd versus GMT treatment (7.5±0.3% versus 3.0±0.3%, P <0.01). Mechanistically, GMTd administration increased expression of the trimethylated lysine 4 of histone 3 (H3K4me3) mark at the promoter regions of cardio‐differentiation genes and mitochondrial biogenesis regulator genes in rat and human fibroblast, compared with GMT. Conclusions These data suggest that the Hippo pathway intermediate Tead1 is an important regulator of cardiac reprogramming that increases the efficiency of maturate induced cardiomyocytes generation and may be a vital component of human cardiodifferentiation strategies.