Panobinostat Effectively Increases Histone Acetylation and Alters Chromatin Accessibility Landscape in Canine Embryonic Fibroblasts but Does Not Enhance Cellular Reprogramming

Robust and reproducible protocols to efficiently reprogram adult canine cells to induced pluripotent stem cells are still elusive. Somatic cell reprogramming requires global chromatin remodeling that is finely orchestrated spatially and temporally. Histone acetylation and deacetylation are key regulators of chromatin condensation, mediated by histone acetyltransferases and histone deacetylases (HDACs), respectively. HDAC inhibitors have been used to increase histone acetylation, chromatin accessibility, and somatic cell reprogramming in human and mice cells. We hypothesized that inhibition of HDACs in canine fibroblasts would increase their reprogramming efficiency by altering the epigenomic landscape and enabling greater chromatin accessibility. We report that a combined treatment of panobinostat (LBH589) and vitamin C effectively inhibits HDAC function and increases histone acetylation in canine embryonic fibroblasts in vitro, with no significant cytotoxic effects. We further determined the effect of this treatment on global chromatin accessibility via Assay for Transposase-Accessible Chromatin using sequencing. Finally, the treatment did not induce any significant increase in cellular reprogramming efficiency. Although our data demonstrate that the unique epigenetic landscape of canine cells does not make them amenable to cellular reprogramming through the proposed treatment, it provides a rationale for a targeted, canine-specific, reprogramming approach by enhancing the expression of transcription factors such as CEBP.

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Ascorbic acid in epigenetic reprogramming

Current Stem Cell Research & Therapy ◽

10.2174/1574888x16666210714152730 ◽

2021 ◽

Vol 16 ◽

Author(s):

Xinhui Liu ◽

Aamir Khan ◽

Huan Li ◽

Shensen Wang ◽

Xuechai Chen ◽

...

Keyword(s):

Ascorbic Acid ◽

Vitamin C ◽

Somatic Cell ◽

Epigenetic Modification ◽

Critical Role ◽

Cellular Reprogramming ◽

Epigenetic Reprogramming ◽

Cell Reprogramming ◽

Histone Demethylases ◽

Somatic Cell Reprogramming

: Emerging evidence suggests that ascorbic acid (vitamin C) enhances the reprogramming process by multiple mechanisms. This is primarily due to its cofactor role in Fe(II) and 2-oxoglutarate-dependent dioxygenases, including the DNA demethylases Ten Eleven Translocase (TET) and histone demethylases. Epigenetic variations have been shown to play a critical role in somatic cell reprogramming. DNA methylation and histone methylation are extensively recognized as barriers to somatic cell reprogramming. N6-methyladenosine (m6A), known as RNA methylation, is an epigenetic modification of mRNAs and has also been shown to play a role in regulating cellular reprogramming. Multiple cofactors are reported to promote the activity of demethylases, including vitamin C. This review focuses on examining the evidence and mechanism of vitamin C in DNA and histone demethylation and highlights its potential involvement in regulating m6A demethylation. It also shows the significant contribution of vitamin C in epigenetic regulation and the affiliation of demethylases with vitamin C-facilitated epigenetic reprogramming.

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Dynamics of alternative splicing during somatic cell reprogramming reveals functions for RNA-binding proteins CPSF3, hnRNP UL1, and TIA1

Genome Biology ◽

10.1186/s13059-021-02372-5 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Claudia Vivori ◽

Panagiotis Papasaikas ◽

Ralph Stadhouders ◽

Bruno Di Stefano ◽

Anna Ribó Rubio ◽

...

Keyword(s):

Alternative Splicing ◽

Somatic Cell ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Cell Reprogramming ◽

Splicing Regulation ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Somatic Cell Reprogramming

Abstract Background Somatic cell reprogramming is the process that allows differentiated cells to revert to a pluripotent state. In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we study the dynamics of alternative splicing changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells and compare them to those occurring during reprogramming of mouse embryonic fibroblasts. Results We observe a significant overlap between alternative splicing changes detected in the two reprogramming systems, which are generally uncoupled from changes in transcriptional levels. Correlation between gene expression of potential regulators and specific clusters of alternative splicing changes enables the identification and subsequent validation of CPSF3 and hnRNP UL1 as facilitators, and TIA1 as repressor of mouse embryonic fibroblasts reprogramming. We further find that these RNA-binding proteins control partially overlapping programs of splicing regulation, involving genes relevant for developmental and morphogenetic processes. Conclusions Our results reveal common programs of splicing regulation during reprogramming of different cell types and identify three novel regulators of this process and their targets.

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AF10 (MLLT10) prevents somatic cell reprogramming through regulation of H3K79 methylation

10.1101/2020.12.22.423908 ◽

2020 ◽

Author(s):

Deniz Uğurlu-Çimen ◽

Deniz Odluyurt ◽

Kenan Sevinç ◽

Nazlı Ezgi Özkan-Küçük ◽

Burcu Özçimen ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cells ◽

Cell Reprogramming ◽

Interacting Proteins ◽

Cell Identity ◽

Somatic Cell Reprogramming ◽

Proteomic Approach ◽

Reprogramming Efficiency ◽

Induced Pluripotent ◽

H3k79 Methylation

SummaryThe histone H3 lysine 79 (H3K79) methyltransferase DOT1L is a key chromatin-based barrier to somatic cell reprogramming. However, the mechanisms by which DOT1L safeguards cell identity and somatic-specific transcriptional programs remain unknown. Here, we employed a proteomic approach using proximity-based labeling to identify DOT1L-interacting proteins and investigated their effects on reprogramming. Among DOT1L interactors, suppression of AF10 (MLLT10) via RNA interference or CRISPR/Cas9, significantly increases reprogramming efficiency. In somatic cells and induced pluripotent stem cells (iPSCs) H3K79 di-methylation is dependent on AF10 expression. In AF10 knockout cells, re-expression wildtype AF10, but not a mutant defective in DOT1L binding, rescues overall H3K79 methylation and reduces reprogramming efficiency. Transcriptomic analyses during reprogramming show that AF10 suppression results in downregulation of fibroblast-specific genes and accelerates the activation of pluripotency-associated genes. Our findings establish AF10 as a novel barrier to reprogramming by regulating H3K79 methylation and thereby sheds light on the mechanism by which cell identity is maintained in somatic cells.

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Enhanced human somatic cell reprogramming efficiency by fusion of the MYC transactivation domain and OCT4

Stem Cell Research ◽

10.1016/j.scr.2017.10.014 ◽

2017 ◽

Vol 25 ◽

pp. 88-97 ◽

Cited By ~ 8

Author(s):

Ling Wang ◽

Delun Huang ◽

Chang Huang ◽

Yexuan Yin ◽

Kaneha Vali ◽

...

Keyword(s):

Somatic Cell ◽

Cell Reprogramming ◽

Transactivation Domain ◽

Human Somatic Cell ◽

Somatic Cell Reprogramming ◽

Reprogramming Efficiency

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Chromatin accessibility in canine stromal cells and its implications for canine somatic cell reprogramming

Stem Cells Translational Medicine ◽

10.1002/sctm.20-0278 ◽

2020 ◽

Author(s):

Maria Questa ◽

Maryam Moshref ◽

Robert J. Jimenez ◽

Veronica Lopez‐Cervantes ◽

Charles K. Crawford ◽

...

Keyword(s):

Somatic Cell ◽

Stromal Cells ◽

Chromatin Accessibility ◽

Cell Reprogramming ◽

Somatic Cell Reprogramming

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ALKBH5 regulates somatic cell reprogramming in a phase specific manner

10.1101/2021.05.10.443389 ◽

2021 ◽

Author(s):

Sherif Mahrous Khodeer ◽

Arne Klungland ◽

John Arne Dahl

Keyword(s):

Somatic Cell ◽

Late Phase ◽

Somatic Cells ◽

Cell Reprogramming ◽

Somatic Cell Reprogramming ◽

Promising Tool ◽

Mesenchymal To Epithelial Transition ◽

Reprogramming Efficiency ◽

Specific Manner ◽

Induced Pluripotent

Establishment of the pluripotency regulatory network in somatic cells by introducing four transcriptional factors, (Octamer binding transcription factor 4 (OCT4), SRY (sex determining region Y)-box 2 (SOX2), Kruppel - like factor 4 (KLF4), and cellular-Myelocytomatosis (c-MYC) provides a promising tool for cell-based therapies in regenerative medicine. Still, the mechanisms at play when generating induced pluripotent stem cells from somatic cells is only partly understood. Here we show that the RNA specific N6-methyladenosine (m6A) demethylase ALKBH5 regulates somatic cell reprogramming in a stage specific manner. Knockdown or knockout of Alkbh5 in the early reprogramming phase impairs the reprogramming efficiency by reducing the proliferation rate through arresting the cells at G2/M phase and decreasing the mesenchymal-to-epithelial transition (MET) rate. However, there is no significant change in reprogramming efficiency when Alkbh5 is depleted at the late phase of reprogramming. On the other hand, ALKBH5 overexpression at the earlyreprogramming phase has no significant impact on reprogramming efficiency, while overexpression at the late phase enhances the reprogramming by stabilizing Nanog transcripts resulting in upregulated Nanog expression. Our study provides mechanistic insight into the crucial dynamic role of ALKBH5 in regulating somatic cell reprogramming at the posttranscriptional level.

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AF10 (MLLT10) prevents somatic cell reprogramming through regulation of DOT1L-mediated H3K79 methylation

Epigenetics & Chromatin ◽

10.1186/s13072-021-00406-7 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Deniz Uğurlu-Çimen ◽

Deniz Odluyurt ◽

Kenan Sevinç ◽

Nazlı Ezgi Özkan-Küçük ◽

Burcu Özçimen ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cells ◽

Cell Reprogramming ◽

Cell Identity ◽

Knock Out ◽

Somatic Cell Reprogramming ◽

Proteomic Approach ◽

Reprogramming Efficiency ◽

Induced Pluripotent ◽

H3k79 Methylation

Abstract Background The histone H3 lysine 79 (H3K79) methyltransferase DOT1L is a key chromatin-based barrier to somatic cell reprogramming. However, the mechanisms by which DOT1L safeguards cell identity and somatic-specific transcriptional programs remain unknown. Results We employed a proteomic approach using proximity-based labeling to identify DOT1L-interacting proteins and investigated their effects on reprogramming. Among DOT1L interactors, suppression of AF10 (MLLT10) via RNA interference or CRISPR/Cas9, significantly increases reprogramming efficiency. In somatic cells and induced pluripotent stem cells (iPSCs) higher order H3K79 methylation is dependent on AF10 expression. In AF10 knock-out cells, re-expression wild-type AF10, but not a DOT1L binding-impaired mutant, rescues overall H3K79 methylation and reduces reprogramming efficiency. Transcriptomic analyses during reprogramming show that AF10 suppression results in downregulation of fibroblast-specific genes and accelerates the activation of pluripotency-associated genes. Conclusions Our findings establish AF10 as a novel barrier to reprogramming by regulating H3K79 methylation and thereby sheds light on the mechanism by which cell identity is maintained in somatic cells.

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