Hippo Pathway Effector Tead1 Induces Cardiac Fibroblast to Cardiomyocyte Reprogramming

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

The stem cell factor SALL4 is an essential transcriptional regulator in mixed lineage leukemia-rearranged leukemogenesis

Abstract Background The 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. Methods Using 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. Results In 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. Conclusions Our 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

MTORC1-independent TFEB activation via Akt inhibition promotes cellular clearance in neurodegenerative storage diseases

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Additional file 2: of The stem cell factor SALL4 is an essential transcriptional regulator in mixed lineage leukemia-rearranged leukemogenesis

Full list of SALL4-bound genes. (PDF 334 kb

CLN8 is an endoplasmic reticulum cargo receptor that regulates lysosome biogenesis

Organelle biogenesis requires proper transport of proteins from their site of synthesis to their target subcellular compartment1-3. Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and traffic through the Golgi complex before being transferred to the endolysosomal system4-6, but how they are transferred from the ER to the Golgi is unknown. Here, we show that ER-to-Golgi transfer of lysosomal enzymes requires CLN8, an ER-associated membrane protein whose loss of function leads to the lysosomal storage disorder, neuronal ceroid lipofuscinosis 8 (a type of Batten disease)7. ER-to-Golgi trafficking of CLN8 requires interaction with the COPII and COPI machineries via specific export and retrieval signals localized in the cytosolic carboxy terminus of CLN8. CLN8 deficiency leads to depletion of soluble enzymes in the lysosome, thus impairing lysosome biogenesis. Binding to lysosomal enzymes requires the second luminal loop of CLN8 and is abolished by some disease-causing mutations within this region. Our data establish an unanticipated example of an ER receptor serving the biogenesis of an organelle and indicate that impaired transport of lysosomal enzymes underlies Batten disease caused by mutations in CLN8

mTORC1-independent TFEB activation via Akt inhibition promotes cellular clearance in neurodegenerative storage diseases.

Neurodegenerative diseases characterized by aberrant accumulation of undigested cellular components represent unmet medical conditions for which the identification of actionable targets is urgently needed. Here we identify a pharmacologically actionable pathway that controls cellular clearance via Akt modulation of transcription factor EB (TFEB), a master regulator of lysosomal pathways. We show that Akt phosphorylates TFEB at Ser467 and represses TFEB nuclear translocation independently of mechanistic target of rapamycin complex 1 (mTORC1), a known TFEB inhibitor. The autophagy enhancer trehalose activates TFEB by diminishing Akt activity. Administration of trehalose to a mouse model of Batten disease, a prototypical neurodegenerative disease presenting with intralysosomal storage, enhances clearance of proteolipid aggregates, reduces neuropathology and prolongs survival of diseased mice. Pharmacological inhibition of Akt promotes cellular clearance in cells from patients with a variety of lysosomal diseases, thus suggesting broad applicability of this approach. These findings open new perspectives for the clinical translation of TFEB-mediated enhancement of cellular clearance in neurodegenerative storage diseases

CLN8 is an endoplasmic reticulum cargo receptor that regulates lysosome biogenesis

Organelle biogenesis requires proper transport of proteins from their site of synthesis to their target subcellular compartment1-3. Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and traffic through the Golgi complex before being transferred to the endolysosomal system4-6, but how they are transferred from the ER to the Golgi is unknown. Here, we show that ER-to-Golgi transfer of lysosomal enzymes requires CLN8, an ER-associated membrane protein whose loss of function leads to the lysosomal storage disorder, neuronal ceroid lipofuscinosis 8 (a type of Batten disease)7. ER-to-Golgi trafficking of CLN8 requires interaction with the COPII and COPI machineries via specific export and retrieval signals localized in the cytosolic carboxy terminus of CLN8. CLN8 deficiency leads to depletion of soluble enzymes in the lysosome, thus impairing lysosome biogenesis. Binding to lysosomal enzymes requires the second luminal loop of CLN8 and is abolished by some disease-causing mutations within this region. Our data establish an unanticipated example of an ER receptor serving the biogenesis of an organelle and indicate that impaired transport of lysosomal enzymes underlies Batten disease caused by mutations in CLN8