Reprogramming Transcription Factors Research Articles

Expression of Atoh1, Gfi1, and Pou4f3 in the mature cochlea reprograms nonsensory cells into hair cells

Mechanosensory hair cells of the mature mammalian organ of Corti do not regenerate; consequently, loss of hair cells leads to permanent hearing loss. Although nonmammalian vertebrates can regenerate hair cells from neighboring supporting cells, many humans with severe hearing loss lack both hair cells and supporting cells, with the organ of Corti being replaced by a flat epithelium of nonsensory cells. To determine whether the mature cochlea can produce hair cells in vivo, we reprogrammed nonsensory cells adjacent to the organ of Corti with three hair cell transcription factors: Gfi1, Atoh1, and Pou4f3. We generated numerous hair cell-like cells in nonsensory regions of the cochlea and new hair cells continued to be added over a period of 9 wk. Significantly, cells adjacent to reprogrammed hair cells expressed markers of supporting cells, suggesting that transcription factor reprogramming of nonsensory cochlear cells in adult animals can generate mosaics of sensory cells like those seen in the organ of Corti. Generating such sensory mosaics by reprogramming may represent a potential strategy for hearing restoration in humans.

Transcription Factors in Brain Regeneration: A Potential Novel Therapeutic Target.

Transcription factors play a crucial role in providing identity to each cell population. To maintain cell identity, it is essential to balance the expression of activator and inhibitor transcription factors. Cell plasticity and reprogramming offer great potential for future therapeutic applications, as they can regenerate damaged tissue. Specific niche factors can modify gene expression and differentiate or transdifferentiate the target cell to the required fate. Ongoing research is being carried out on the possibilities of transcription factors in regenerating neurons, with neural stem cells (NSCs) being considered the preferred cells for generating new neurons due to their epigenomic and transcriptome memory. NEUROD1/ASCL1, BRN2, MYTL1, and other transcription factors can induce direct reprogramming of somatic cells, such as fibroblasts, into neurons. However, the molecular biology of transcription factors in reprogramming and differentiation still needs to be fully understood.

Master Transcription Factor Reprogramming Unleashes Selective Translation Promoting Castration Resistance and Immune Evasion in Lethal Prostate Cancer.

Our study shows that specialized eIF3B-dependent translation of specific mRNAs released upon downregulation of the master transcription factor MITF confers castration resistance and immune evasion in lethal prostate cancer. Pharmacologic targeting of this mechanism delays castration resistance and increases immune-checkpoint efficacy. This article is featured in Selected Articles from This Issue, p. 2489.

A universal deep-learning model for zinc finger design enables transcription factor reprogramming

Cys2His2 zinc finger (ZF) domains engineered to bind specific target sequences in the genome provide an effective strategy for programmable regulation of gene expression, with many potential therapeutic applications. However, the structurally intricate engagement of ZF domains with DNA has made their design challenging. Here we describe the screening of 49 billion protein–DNA interactions and the development of a deep-learning model, ZFDesign, that solves ZF design for any genomic target. ZFDesign is a modern machine learning method that models global and target-specific differences induced by a range of library environments and specifically takes into account compatibility of neighboring fingers using a novel hierarchical transformer architecture. We demonstrate the versatility of designed ZFs as nucleases as well as activators and repressors by seamless reprogramming of human transcription factors. These factors could be used to upregulate an allele of haploinsufficiency, downregulate a gain-of-function mutation or test the consequence of regulation of a single gene as opposed to the many genes that a transcription factor would normally influence.

Non-genetic adaptive resistance to KRASG12C inhibition: EMT is not the only culprit.

Adaptions to therapeutic pressures exerted on cancer cells enable malignant progression of the tumor, culminating in escape from programmed cell death and development of resistant diseases. A common form of cancer adaptation is non-genetic alterations that exploit mechanisms already present in cancer cells and do not require genetic modifications that can also lead to resistance mechanisms. Epithelial-to-mesenchymal transition (EMT) is one of the most prevalent mechanisms of adaptive drug resistance and resulting cancer treatment failure, driven by epigenetic reprogramming and EMT-specific transcription factors. A recent breakthrough in cancer treatment is the development of KRASG12C inhibitors, which herald a new era of therapy by knocking out a unique substitution of an oncogenic driver. However, these highly selective agents targeting KRASG12C, such as FDA-approved sotorasib (AMG510) and adagrasib (MRTX849), inevitably encounter multiple mechanisms of drug resistance. In addition to EMT, cancer cells can hijack or rewire the sophisticated signaling networks that physiologically control cell proliferation, growth, and differentiation to promote malignant cancer cell phenotypes, suggesting that inhibition of multiple interconnected signaling pathways may be required to block tumor progression on KRASG12C inhibitor therapy. Furthermore, the tumor microenvironment (TME) of cancer cells, such as tumor-infiltrating lymphocytes (TILs), contribute significantly to immune escape and tumor progression, suggesting a therapeutic approach that targets not only cancer cells but also the TME. Deciphering and targeting cancer adaptions promises mechanistic insights into tumor pathobiology and improved clinical management of KRASG12C-mutant cancer. This review presents recent advances in non-genetic adaptations leading to resistance to KRASG12C inhibitors, with a focus on oncogenic pathway rewiring, TME, and EMT.

Direct Cell Conversion of Somatic Cells into Dopamine Neurons: Achievements and Perspectives

In the last decade, direct reprogramming has emerged as a novel strategy to obtain mature and functional dopamine neurons from somatic cells. This approach could overcome issues linked to the use of human pluripotent stem cells such as ethical concerns and safety problems that can arise from the overgrowth of undifferentiated cells after transplantation. Several conversion methodologies have been developed to obtain induced DA neurons (iDANs) or induced DA neuron progenitors (iDPs). iDANs have also proved to successfully integrate in mice striatum, alleviating Parkinson's disease (PD) motor symptoms. In the next decade, human iDANs and/or iDPs could be translated to clinic to achieve a patient-tailored therapy, but current critical issues hinder this goal, such as the low conversion rate of adult human fibroblasts and the risks associated with lentiviral delivery of conversion factors. In this study, we summarize the strategies and recent improvements developed for the generation of mouse and human iDANs/iDPs. Furthermore, we discuss the more recent application of in vivo direct conversion, which may enable clinical therapies for PD by means of brain in situ delivery of dopaminergic reprogramming transcription factors.

Exploring glioblastoma stem cell heterogeneity: Immune microenvironment modulation and therapeutic opportunities.

Despite its growing use in cancer treatment, immunotherapy has been virtually ineffective in clinical trials for gliomas. The inherently cold tumor immune microenvironment (TIME) in gliomas, characterized by a high ratio of pro-tumor to anti-tumor immune cell infiltrates, acts as a seemingly insurmountable barrier to immunotherapy. Glioma stem cells (GSCs) within these tumors are key contributors to this cold TIME, often functioning indirectly through activation and recruitment of pro-tumor immune cell types. Furthermore, drivers of GSC plasticity and heterogeneity (e.g., reprogramming transcription factors, epigenetic modifications) are associated with induction of immunosuppressive cell states. Recent studies have identified GSC-intrinsic mechanisms, including functional mimicry of immune suppressive cell types, as key determinants of anti-tumor immune escape. In this review, we cover recent advancements in our understanding of GSC-intrinsic mechanisms that modulate GSC-TIME interactions and discuss cutting-edge techniques and bioinformatics platforms available to study immune modulation at high cellular resolution with exploration of both malignant (i.e., GSC) and non-malignant (i.e., immune) cell fractions. Finally, we provide insight into the therapeutic opportunities for targeting immunomodulatory GSC-intrinsic mechanisms to potentiate immunotherapy response in gliomas.

Genetic Mechanism Study of Auditory Phoenix Spheres and Transcription Factors Prediction for Direct Reprogramming by Bioinformatics.

Background: Hearing loss is the most common irreversible sensory disorder. By delivering regenerative cells into the cochlea, cell-based therapy provides a novel strategy for hearing restoration. Recently, newly-identified phoenix cells have drawn attention due to their nearly unlimited self-renewal and neural differentiation capabilities. They are a promising cell source for cell therapy and a potential substitute for induced pluripotent stem cells (iPSCs) in many in vitro applications. However, the underlying genomic mechanism of their self-renewal capabilities is largely unknown. The aim of this study was to identify hub genes and potential molecular mechanisms between differentiated and undifferentiated phoenix cells and predict transcription factors (TFs) for direct reprogramming. Material and Methods: The datasets were downloaded from the ArrayExpress database. Samples of differentiated and undifferentiated phoenix cells with three biological replicates were utilised for bioinformatic analysis. Differentially expressed genes (DEGs) were screened and the Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were investigated. The gene set enrichment analysis (GSEA) was conducted to verify the enrichment of four self-defined gene set collections, followed by protein-protein interaction (PPI) network construction and subcluster analysis. The prediction of TFs for direct reprogramming was performed based on the TRANSFAC database. Results: Ten hub genes were identified to be the key candidates for self-renewal. Ten TFs were predicted as the direct reprogramming factors. This study provides a theoretical foundation for understanding phoenix cells and clues for direct reprogramming, which would stimulate further experiments and clinical applications in hearing research and treatment.

Establishment of pluripotent stem cell line induced by PATL2 heterozygous mutation in patients with oocyte maturation defect-4

Oocyte maturation defect-4 (OOMD4) is an autosomal recessive disease characterized by oocyte maturation arrest. On chromosome 15q21, the PATL2 gene is mutated, resulting in OOMD4 in either a homozygous or compound heterozygous form. Herein, the peripheral blood mononuclear cells (PBMCs) were obtained from a female patient who was heterozygous for OOMD4 due to a PALT2 gene mutation. Then we obtained the induced pluripotent stem cell (iPSC) by using episomal vectors with transcription factors for reprogramming. The teratoma assay revealed that the iPSC line exhibited pluripotency with the ability to differentiate into three germ layers, namely ectoderm, mesoderm, and endoderm, with positive expression of their markers, such as TUJ, SMA, and AFP, respectively. Furthermore, a normal karyotype (46, XX) was observed. In this view, iPSCs can be a valuable tool for conducting extensive research on the OOMD4, establishing models, and identifying potential therapeutic targets.

Mechanical signals induces reprogramming of mature adipocytes through the YAP/TAZ-binding motif

Tissue engineering technology will be the main approach to tissue regeneration in the future and are promising for the treatment of large-area burns and refractory wounds. Dedifferentiated fat cells (DFAT) derived from mature adipocytes (MAs) as a seed cell have great potential in cell therapy and tissue engineering for the treatment of a variety of clinical diseases because of their wider availability, stronger proliferation ability, multidirectional redifferentiation potential, higher cell purity, lower heterogeneity, and greater biosafety profile. However, the triggering mechanism for MAs reprogramming in vitro is unclear. In this study, MAs were successfully induced to dedifferentiate into DFAT in a short time in vitro using an “improved ceiling culture method". Flow cytometry, adipogenic, and osteogenic differentiation experiments verified that DFAT cells present the biological characteristics of stem cells. In addition, changing the stiffness of the extracellular matrix can inhibit the dedifferentiation of MAs to DFAT, and increase the expression of Yes-associated protein/transcriptional co-activator with the PDZ-binding motif (YAP/TAZ), nuclear translocation, and the expression of reprogramming transcription factors. In conclusion, extracellular matrix stiffness can induce MAs to dedifferentiate into DFAT in vitro, and can directly transmit mechanical force signals to the nucleus via YAP/TAZ binding to trigger the expression of stem cell-related reprogramming factors.

The co-modulation of RAV transcription factors in ROS burst and extensive transcriptional reprogramming underlies disease resistance in cassava.

MeRAVs positively regulate ROS burst and the expression of downstream disease resistance-related genes, which underlie improved disease resistance to Xam. Cassava (Manihot esculenta Crantz) is an important food crop and energy crop, but its yield is seriously affected by cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam). Related to ABI3/VP1 (RAV) transcription factor family belongs to the APETALA2/Ethylene-Responsive Factor (AP2/ERF) family, which plays an important role in plant growth, development and response to biotic and abiotic stresses. In this study, we found that MeRAVs positively co-regulates the resistance to Xam and stimulates the innate immune response by regulating reactive oxygen species (ROS) burst in cassava. Dual-luciferase assay showed that seven MeRAVs exhibited transcriptional activate activity by binding CAACA motif and CACCTG motif. A large number of differentially expressed genes (DEGs) were identified through RNA-seq analysis of MeRAVs-silenced lines, and the DEGs co-regulated by seven MeRAVs accounted for more than 45% of the total DEGs. In addition, seven MeRAVs positively regulate expression of disease resistance-related genes through directly binding to their promoters. In summary, MeRAVs co-regulate ROS burst and the expression of downstream disease resistance-related genes, which underlie improved disease resistance to Xam.

Improving prediction of core transcription factors for cell reprogramming and transdifferentiation

Identification of transcription factors (TFs) that could induce and direct cell conversion remains a challenge. Though several hundreds of TFs are usually transcribed in each cell type, the identity of a cell is controlled and can be achieved through the ectopic overexpression of only a small subset of so-called core TFs. Currently, the experimental identification of the core TFs for a broad spectrum of cell types remains challenging. Computational solutions to this problem would provide a better understanding of the mechanisms controlling cell identity during natural embryonic or malignant development, as well as give a foundation for cell-based therapy. Herein, we propose a computational approach based on over-enrichment of transcription factors binding sites (TFBS) in differentially accessible chromatin regions that could identify the potential core TFs for a variety of primary human cells involved in hematopoiesis. Our approach enables the integration of both transcriptomic (single-cell RNA sequencing, scRNA-seq) and epigenenomic (single-cell assay for transposable-accessible chromatin, scATAC-seq) data at the single-cell resolution to search for core TFs, and can be scalable to predict subsets of core TFs and their role in a given conversion between cells.

Retraction Note to: Transient Activation of Reprogramming Transcription Factors Using Protein Transduction Facilitates Conversion of Human Fibroblasts Toward Cardiomyocyte-Like Cells.

Retraction Note to: Transient Activation of Reprogramming Transcription Factors Using Protein Transduction Facilitates Conversion of Human Fibroblasts Toward Cardiomyocyte-Like Cells.

Direct Reprogramming of Mouse Fibroblasts into Melanocytes.

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

Overexpression of OsERF83, a Vascular Tissue-Specific Transcription Factor Gene, Confers Drought Tolerance in Rice.

Abiotic stresses severely affect plant growth and productivity. To cope with abiotic stresses, plants have evolved tolerance mechanisms that are tightly regulated by reprogramming transcription factors (TFs). APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors are known to play an important role in various abiotic stresses. However, our understanding of the molecular mechanisms remains incomplete. In this study, we identified the role of OsERF83, a member of the AP2/ERF transcription factor family, in response to drought stress. OsERF83 is a transcription factor localized to the nucleus and induced in response to various abiotic stresses, such as drought and abscisic acid (ABA). Overexpression of OsERF83 in transgenic plants (OsERF83OX) significantly increased drought tolerance, with higher photochemical efficiency in rice. OsERF83OX was also associated with growth retardation, with reduced grain yields under normal growth conditions. OsERF83 is predominantly expressed in the vascular tissue of all organs. Transcriptome analysis revealed that OsERF83 regulates drought response genes, which are related to the transporter (OsNPF8.10, OsNPF8.17, OsLH1), lignin biosynthesis (OsLAC17, OsLAC10, CAD8D), terpenoid synthesis (OsTPS33, OsTPS14, OsTPS3), cytochrome P450 family (Oscyp71Z4, CYP76M10), and abiotic stress-related genes (OsSAP, OsLEA14, PCC13-62). OsERF83 also up-regulates biotic stress-associated genes, including PATHOGENESIS-RELATED PROTEIN (PR), WALL-ASSOCIATED KINASE (WAK), CELLULOSE SYNTHASE-LIKE PROTEIN E1 (CslE1), and LYSM RECEPTOR-LIKE KINASE (RLK) genes. Our results provide new insight into the multiple roles of OsERF83 in the cross-talk between abiotic and biotic stress signaling pathways.

Forkhead box family transcription factors as versatile regulators for cellular reprogramming to pluripotency

Forkhead box (Fox) transcription factors play important roles in mammalian development and disease. However, their function in mouse somatic cell reprogramming remains unclear. Here, we report that FoxD subfamily and FoxG1 accelerate induced pluripotent stem cells (iPSCs) generation from mouse fibroblasts as early as day4 while FoxA and FoxO subfamily impede this process obviously. More importantly, FoxD3, FoxD4 and FoxG1 can replace Oct4 respectively and generate iPSCs with germline transmission together with Sox2 and Klf4. On the contrary, FoxO6 almost totally blocks reprogramming through inhibiting cell proliferation, suppressing the expression of pluripotent genes and hindering the process of mesenchymal to epithelial transition (MET). Thus, our study uncovers unexpected roles of Fox transcription factors in reprogramming and offers new insights into cell fate transition.

Histone H2B ubiquitination mediated chromatin relaxation is essential for the induction of somatic cell reprogramming.

ObjectivesCell reprogramming has significant impacts on their potential application in regenerative medicine. Chromatin remodelling plays a very important role in cell reprogramming, but its underlying mechanism remains poorly understood.Materials and methodsRNA‐seq, quantitative RT‐PCR and western blot analysis were applied to study the role of RNF20 and H2B ubiquitination during mouse somatic cell reprogramming. Chromatin structure and the recruitment of transcription factors were analysed by ChIP‐seq, micrococcal nuclease sensitivity assays and immunofluorescence staining.ResultsWe show that RNF20 is highly expressed at the early stage of reprogramming along with the accumulation of H2B ubiquitination at the same stage, and Rnf20 knockout results in the failure of reprogramming at the initial stage but not the maturation and stabilization stages. RNA‐seq showed that Rnf20 knockout mainly affects the early stage of cell reprogramming by impairing the transcription of MET‐related genes and early pluripotency genes. Importantly, Rnf20 knockout results in a more compacted chromosomes structure in reprogrammable cells, suppressing the recruitment of reprogramming transcription factors to their proper locations on the chromosomes, and finally resulting in the failure of pluripotent gene network establishment.ConclusionsHistone H2B ubiquitination mediated chromatin relaxation is essential for the induction of somatic cell reprogramming.

Transcriptional dynamics of transposable elements when converting fibroblast cells of Macaca mulatta to neuroepithelial stem cells

BackgroundTransposable elements (TE) account for more than 50% of human genome. It has been reported that some types of TEs are dynamically regulated in the reprogramming of human cell lines. However, it is largely unknown whether some TEs in Macaca mulatta are also regulated during the reprogramming of cell lines of monkey.ResultsHere, we systematically examined the transcriptional activities of TEs during the conversion of Macaca mulatta fibroblast cells to neuroepithelial stem cells (NESCs). Hundreds of TEs were dynamically regulated during the reprogramming of Macaca mulatta fibroblast cells. Furthermore, 48 Long Terminal Repeats (LTRs), as well as some integrase elements, of Macaca endogenous retrovirus 3 (MacERV3) were transiently activated during the early stages of the conversion process, some of which were further confirmed with PCR experiments. These LTRs were potentially bound by critical transcription factors for reprogramming, such as KLF4 and ETV5.ConclusionThese results suggest that the transcription of TEs are delicately regulated during the reprogramming of Macaca mulatta fibroblast cells. Although the family of ERVs activated during the reprogramming of fibroblast cells in Macaca mulatta is different from those in the reprogramming of human fibroblast cells, our results suggest that the activation of some ERVs is a conserved mechanism in primates for converting fibroblast cells to stem cells.

A multi-herb-combined remedy to overcome hyper-inflammatory response by reprogramming transcription factor profile and shaping monocyte subsets

Traditional Chinese multi-herb-combined prescriptions usually show better performance than a single agent since a group of effective compounds interfere multiple disease-relevant targets simultaneously. Huang-Lian-Jie-Du decoction is a remedy made of four herbs that are widely used to treat oral ulcers, gingivitis, and periodontitis. However, the active ingredients and underlying mechanisms are not clear. To address these questions, we prepared a water extract solution of Huang-Lian-Jie-Du decoction (HLJDD), called it as WEH (Water Extract Solution of HLJDD), and used it to treat LPS-induced systemic inflammation in mice. We observed that WEH attenuated inflammatory responses including reducing production of cytokines, chemokines and interferons (IFNs), further attenuating emergency myelopoiesis, and preventing mice septic lethality. Upon LPS stimulation, mice pretreated with WEH increased circulating Ly6C- patrolling and splenic Ly6C+ inflammatory monocytes. The acute myelopoiesis related transcriptional factor profile was rearranged by WEH. Mechanistically we confirmed that WEH interrupted LPS/TLR4/CD14 signaling-mediated downstream signaling pathways through its nine principal ingredients, which blocked LPS stimulated divergent signaling cascades, such as activation of NF-κB, p38 MAPK, and ERK1/2. We conclude that the old remedy blunts LPS-induced “danger” signal recognition and transduction process at multiple sites. To translate our findings into clinical applications, we refined the crude extract into a pure multicomponent drug by directly mixing these nine chemical entities, which completely reproduced the effect of protecting mice from lethal septic shock. Finally, we reduced a large number of compounds within a multi-herb water extract to seven-chemical combination that exhibited superior therapeutic efficacy compared with WEH.

Transcription Factor Reprogramming in the Inner Ear: Turning on Cell Fate Switches to Regenerate Sensory Hair Cells.

Non-mammalian vertebrates can restore their auditory and vestibular hair cells naturally by triggering the regeneration of adjacent supporting cells. The transcription factor ATOH1 is a key regulator of hair cell development and regeneration in the inner ear. Following the death of hair cells, supporting cells upregulate ATOH1 and give rise to new hair cells. However, in the mature mammalian cochlea, such natural regeneration of hair cells is largely absent. Transcription factor reprogramming has been used in many tissues to convert one cell type into another, with the long-term hope of achieving tissue regeneration. Reprogramming transcription factors work by altering the transcriptomic and epigenetic landscapes in a target cell, resulting in a fate change to the desired cell type. Several studies have shown that ATOH1 is capable of reprogramming cochlear non-sensory tissue into cells resembling hair cells in young animals. However, the reprogramming ability of ATOH1 is lost with age, implying that the potency of individual hair cell-specific transcription factors may be reduced or lost over time by mechanisms that are still not clear. To circumvent this, combinations of key hair cell transcription factors have been used to promote hair cell regeneration in older animals. In this review, we summarize recent findings that have identified and studied these reprogramming factor combinations for hair cell regeneration. Finally, we discuss the important questions that emerge from these findings, particularly the feasibility of therapeutic strategies using reprogramming factors to restore human hearing in the future.