Morante-Palacios O, Godoy-Tena G, Calafell-Segura J, Ciudad L, Martínez-Cáceres EM, Sardina JL, Ballestar E
Vitamin C enhances NF-κB-driven epigenomic reprogramming and boosts the immunogenic properties of dendritic cells.
Nucleic Acids Res28 Oct 2022, . Epub 28 Oct 2022
Dendritic cells (DCs), the most potent antigen-presenting cells, are necessary for effective activation of naïve T cells. DCs' immunological properties are modulated in response to various stimuli. Active DNA demethylation is crucial for DC differentiation and function. Vitamin C, a known cofactor of ten-eleven translocation (TET) enzymes, drives active demethylation. Vitamin C has recently emerged as a promising adjuvant for several types of cancer; however, its effects on human immune cells are poorly understood. In this study, we investigate the epigenomic and transcriptomic reprogramming orchestrated by vitamin C in monocyte-derived DC differentiation and maturation. Vitamin C triggers extensive demethylation at NF-κB/p65 binding sites, together with concordant upregulation of antigen-presentation and immune response-related genes during DC maturation. p65 interacts with TET2 and mediates the aforementioned vitamin C-mediated changes, as demonstrated by pharmacological inhibition. Moreover, vitamin C increases TNFβ production in DCs through NF-κB, in concordance with the upregulation of its coding gene and the demethylation of adjacent CpGs. Finally, vitamin C enhances DC's ability to stimulate the proliferation of autologous antigen-specific T cells. We propose that vitamin C could potentially improve monocyte-derived DC-based cell therapies.
Azagra A, Meler A, de Barrios O, Tomás-Daza L, Collazo O, Monterde B, Obiols M, Rovirosa L, Vila-Casadesús M, Cabrera-Pasadas M, Gusi-Vives M, Graf T, Varela I, Sardina JL, Javierre BM, Parra M
The HDAC7-TET2 epigenetic axis is essential during early B lymphocyte development.
Nucleic Acids Res29 Jul 2022, . Epub 29 Jul 2022
Correct B cell identity at each stage of cellular differentiation during B lymphocyte development is critically dependent on a tightly controlled epigenomic landscape. We previously identified HDAC7 as an essential regulator of early B cell development and its absence leads to a drastic block at the pro-B to pre-B cell transition. More recently, we demonstrated that HDAC7 loss in pro-B-ALL in infants associates with a worse prognosis. Here we delineate the molecular mechanisms by which HDAC7 modulates early B cell development. We find that HDAC7 deficiency drives global chromatin de-condensation, histone marks deposition and deregulates other epigenetic regulators and mobile elements. Specifically, the absence of HDAC7 induces TET2 expression, which promotes DNA 5-hydroxymethylation and chromatin de-condensation. HDAC7 deficiency also results in the aberrant expression of microRNAs and LINE-1 transposable elements. These findings shed light on the mechanisms by which HDAC7 loss or misregulation may lead to B cell-based hematological malignancies.
Dissecting TET2 Regulatory Networks in Blood Differentiation and Cancer
Cancers 2022, 14(3), 830; https://doi.org/10.3390/cancers140308306 Feb 2022, .
Cytosine methylation (5mC) of CpG is the major epigenetic modification of mammalian DNA, playing essential roles during development and cancer. Although DNA methylation is generally associated with transcriptional repression, its role in gene regulation during cell fate decisions remains poorly understood. DNA demethylation can be either passive or active when initiated by TET dioxygenases. During active demethylation, transcription factors (TFs) recruit TET enzymes (TET1, 2, and 3) to specific gene regulatory regions to first catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and subsequently to higher oxidized cytosine derivatives. Only TET2 is frequently mutated in the hematopoietic system from the three TET family members. These mutations initially lead to the hematopoietic stem cells (HSCs) compartment expansion, eventually evolving to give rise to a wide range of blood malignancies. This review focuses on recent advances in characterizing the main TET2-mediated molecular mechanisms that activate aberrant transcriptional programs in blood cancer onset and development. In addition, we discuss some of the key outstanding questions in the field.
Justin Brumbaugh, Bruno Di Stefano, José Luis Sardina
Editorial: Chromatin Regulation in Cell Fate Decisions
Front. Cell Dev. Biol. | doi: 10.3389/fcell.2021.73402012 Aug 2021, .
Many epigenetic marks change dramatically during embryogenesis, suggesting that they have a fundamental role in developmental decisions. For example, the early embryo experiences an initial, widespread loss of DNA methylation that is reacquired around the time of implantation. In this edition, Maxim Greenberg reviews the mechanisms responsible for these dynamics, with a focus on key regions that escape DNA methylation during its reestablishment. It remains unresolved whether this early round of DNA demethylation is passive (i.e., DNA methylation is diluted over multiple rounds of cell division) or active (i.e., resulting from enzymatic action). In any case, the Ten-eleven translocation (TET) class of DNA demethylases, and specifically TET2, is crucial for exiting pluripotency and initiating differentiation (Dai et al., 2016). Garcia-Outeiral and colleagues provide an updated perspective on TET2's role in these processes as well as reprogramming, and discuss the possibility that TET2 is also responsible for oxidizing methylation on RNA. Together, these publications highlight the importance of DNA/RNA methylation in the early embryo.[...]
Dynamics of alternative splicing during somatic cell reprogramming reveals functions for RNA-binding proteins CPSF3, hnRNP UL1, and TIA1.
Genome Biol3 Jun 2021, 22(1)171. Epub 3 Jun 2021
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.
Uncovering Sequence-Specific Transcription Factors Interacting with TET2.
Methods Mol Biol20 May 2021, 2272239-250.
Ten-eleven Translocation (TET) enzymes are methylcytosine dioxygenases that are involved in multiple cellular processes, including cellular differentiation and forced cell fate conversions. However, deciphering the molecular mechanisms underlying epigenetic control exerted by these proteins has been hampered by technical limitations, which prevent the identification of essential partners that work in concert with these enzymes to modulate gene expression. In this chapter, we provide a comprehensive description of cutting-edge methods designed to assess physical interactions between sequence-specific transcription factors and the TET2 enzyme.
Xiu Y, Dong Q, Fu L, Bossler A, Tang X, Boyce B, Borcherding N, Leidinger M, Sardina JL, Xue HH, Li Q, Feldman A, Aifantis I, Boccalatte F, Wang L, Jin M, Khoury J, Wang W, Hu S, Yuan Y, Wang E, Yuan J, Janz S, Colgan J, Habelhah H, Waldschmidt T, Müschen M, Bagg A, Darbro B, Zhao C
Coactivation of NF-κB and Notch signaling is sufficient to induce B-cell transformation and enables B-myeloid conversion.
Blood9 Jan 2020, 135(2)108-120.
NF-κB and Notch signaling can be simultaneously activated in a variety of B-cell lymphomas. Patients with B-cell lymphoma occasionally develop clonally related myeloid tumors with poor prognosis. Whether concurrent activation of both pathways is sufficient to induce B-cell transformation and whether the signaling initiates B-myeloid conversion in a pathological context are largely unknown. Here, we provide genetic evidence that concurrent activation of NF-κB and Notch signaling in committed B cells is sufficient to induce B-cell lymphomatous transformation and primes common progenitor cells to convert to myeloid lineage through dedifferentiation, not transdifferentiation. Intriguingly, the converted myeloid cells can further transform, albeit at low frequency, into myeloid leukemia. Mechanistically, coactivation of NF-κB and Notch signaling endows committed B cells with the ability to self renew. Downregulation of BACH2, a lymphoma and myeloid gene suppressor, but not upregulation of CEBPα and/or downregulation of B-cell transcription factors, is an early event in both B-cell transformation and myeloid conversion. Interestingly, a DNA hypomethylating drug not only effectively eliminated the converted myeloid leukemia cells, but also restored the expression of green fluorescent protein, which had been lost in converted myeloid leukemia cells. Collectively, our results suggest that targeting NF-κB and Notch signaling will not only improve lymphoma treatment, but also prevent the lymphoma-to-myeloid tumor conversion. Importantly, DNA hypomethylating drugs might efficiently treat these converted myeloid neoplasms.
Tian TV, Di Stefano B, Stik G, Vila-Casadesús M, Sardina JL, Vidal E, Dasti A, Segura-Morales C, De Andrés-Aguayo L, Gómez A, Goldmann J, Jaenisch R, Graf T
Whsc1 links pluripotency exit with mesendoderm specification.
Nat Cell BiolJul 2019, 21(7)824-834. Epub 24 Jun 2019
How pluripotent stem cells differentiate into the main germ layers is a key question of developmental biology. Here, we show that the chromatin-related factor Whsc1 (also known as Nsd2 and MMSET) has a dual role in pluripotency exit and germ layer specification of embryonic stem cells. On induction of differentiation, a proportion of Whsc1-depleted embryonic stem cells remain entrapped in a pluripotent state and fail to form mesendoderm, although they are still capable of generating neuroectoderm. These functions of Whsc1 are independent of its methyltransferase activity. Whsc1 binds to enhancers of the mesendodermal regulators Gata4, T (Brachyury), Gata6 and Foxa2, together with Brd4, and activates the expression of these genes. Depleting each of these regulators also delays pluripotency exit, suggesting that they mediate the effects observed with Whsc1. Our data indicate that Whsc1 links silencing of the pluripotency regulatory network with activation of mesendoderm lineages.
Sardina JL, Collombet S, Tian TV, Gómez A, Di Stefano B, Berenguer C, Brumbaugh J, Stadhouders R, Segura-Morales C, Gut M, Gut IG, Heath S, Aranda S, Di Croce L, Hochedlinger K, Thieffry D, Graf T
Transcription Factors Drive Tet2-Mediated Enhancer Demethylation to Reprogram Cell Fate.
Cell Stem Cell1 Nov 2018, 23(5)727-741.e9. Epub 13 Sep 2018
Here, we report DNA methylation and hydroxymethylation dynamics at nucleotide resolution using C/EBPα-enhanced reprogramming of B cells into induced pluripotent cells (iPSCs). We observed successive waves of hydroxymethylation at enhancers, concomitant with a decrease in DNA methylation, suggesting active demethylation. Consistent with this finding, ablation of the DNA demethylase Tet2 almost completely abolishes reprogramming. C/EBPα, Klf4, and Tfcp2l1 each interact with Tet2 and recruit the enzyme to specific DNA sites. During reprogramming, some of these sites maintain high levels of 5hmC, and enhancers and promoters of key pluripotency factors become demethylated as early as 1 day after Yamanaka factor induction. Surprisingly, methylation changes precede chromatin opening in distinct chromatin regions, including Klf4 bound sites, revealing a pioneer factor activity associated with alternation in DNA methylation. Rapid changes in hydroxymethylation similar to those in B cells were also observed during compound-accelerated reprogramming of fibroblasts into iPSCs, highlighting the generality of our observations.
Collombet S, van Oevelen C, Sardina Ortega JL, Abou-Jaoudé W, Di Stefano B, Thomas-Chollier M, Graf T, Thieffry D
Logical modeling of lymphoid and myeloid cell specification and transdifferentiation.
Proc Natl Acad Sci U S A6 Jun 2017, 114(23)5792-5799.
Blood cells are derived from a common set of hematopoietic stem cells, which differentiate into more specific progenitors of the myeloid and lymphoid lineages, ultimately leading to differentiated cells. This developmental process is controlled by a complex regulatory network involving cytokines and their receptors, transcription factors, and chromatin remodelers. Using public data and data from our own molecular genetic experiments (quantitative PCR, Western blot, EMSA) or genome-wide assays (RNA-sequencing, ChIP-sequencing), we have assembled a comprehensive regulatory network encompassing the main transcription factors and signaling components involved in myeloid and lymphoid development. Focusing on B-cell and macrophage development, we defined a qualitative dynamical model recapitulating cytokine-induced differentiation of common progenitors, the effect of various reported gene knockdowns, and the reprogramming of pre-B cells into macrophages induced by the ectopic expression of specific transcription factors. The resulting network model can be used as a template for the integration of new hematopoietic differentiation and transdifferentiation data to foster our understanding of lymphoid/myeloid cell-fate decisions.