Control epigenético de la hematopoyesis

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ICO - Germans Trias i Pujol

Josep Carreras Leukaemia Research Institute
Can Ruti Campus
Ctra de Can Ruti, Camí de les Escoles s/n
08916 Badalona, Barcelona
Spain

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Summary

Cell fate decisions, such as haematopoietic differentiation, are typically initiated by transcription factors that regulate gene expression in concert with epigenetic modifications, which include histone and DNA modifications. DNA methylation related genes including DNMT3A or TET2 are among the most frequently mutated genes in blood malignancies. Traditionally, studies aimed at understanding the effect of aberrant DNA methylation in cancer patients have focused on gene promoters and gene bodies. However, we and others have recently described that the most DNA methylation-dynamic regions are located distally from the genes (at enhancer elements), coinciding with their preferential binding by DNMT3A and TET2. Therefore, how aberrant DNA methylation dynamics impact on the chromatin structure at distal regulatory regions during blood cancer onset and progression remains to be fully elucidated. 

Research

Research in our group is aimed to uncover the epigenetic mechanisms governing haematopoietic cell fate decisions, with a special interest in those leading to the onset and development of blood malignancies (including leukaemia and myelodysplastic syndromes among others)

Lines of research:

1. Study of the interplay between DNA (hydroxy)methylation and chromatin dynamics at distal gene regulatory regions during hematopoietic cell fate decisions.

2. Uncovering the role of TET2 in the epigenetic control of the chromatin at distal gene regulatory regions during leukemic onset and progression.

3. Deciphering the molecular mechanisms underlying 5hmC-mediated chromatin compaction during cell fate decisions.

4. Study of the role of mRNA methylation-mediated post-transcriptional control during myeloid cell differentiation.

We are currently looking for highly motivated Postdocs to join our group. Please contact José Luis Sardina (jsardina@carrerasresearch.orgfor further information

Awards

2019   Contract: Miguel Servet (Tipo I)

People

Selected publications

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 Res 28 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.
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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 Res 29 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.
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Di Stefano B, Sardina JL, van Oevelen C, Collombet S, Kallin EM, Vicent GP, Lu J, Thieffry D, Beato M, Graf T

C/EBPα poises B cells for rapid reprogramming into induced pluripotent stem cells.

Nature 13 Feb 2014, 506 (7487) 235-9. Epub 15 Dic 2013
CCAAT/enhancer binding protein-α (C/EBPα) induces transdifferentiation of B cells into macrophages at high efficiencies and enhances reprogramming into induced pluripotent stem (iPS) cells when co-expressed with the transcription factors Oct4 (Pou5f1), Sox2, Klf4 and Myc (hereafter called OSKM). However, how C/EBPα accomplishes these effects is unclear. Here we find that in mouse primary B cells transient C/EBPα expression followed by OSKM activation induces a 100-fold increase in iPS cell reprogramming efficiency, involving 95% of the population. During this conversion, pluripotency and epithelial-mesenchymal transition genes become markedly upregulated, and 60% of the cells express Oct4 within 2 days. C/EBPα acts as a 'path-breaker' as it transiently makes the chromatin of pluripotency genes more accessible to DNase I. C/EBPα also induces the expression of the dioxygenase Tet2 and promotes its translocation to the nucleus where it binds to regulatory regions of pluripotency genes that become demethylated after OSKM induction. In line with these findings, overexpression of Tet2 enhances OSKM-induced B-cell reprogramming. Because the enzyme is also required for efficient C/EBPα-induced immune cell conversion, our data indicate that Tet2 provides a mechanistic link between iPS cell reprogramming and B-cell transdifferentiation. The rapid iPS reprogramming approach described here should help to fully elucidate the process and has potential clinical applications.
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Bueno C, Sardina JL, Di Stefano B, Romero-Moya D, Muñoz-López A, Ariza L, Chillón MC, Balanzategui A, Castaño J, Herreros A, Fraga MF, Fernández A, Granada I, Quintana-Bustamante O, Segovia JC, Nishimura K, Ohtaka M, Nakanishi M, Graf T, Menendez P.

Reprogramming human B cells into induced pluripotent stem cells and its enhancement by C/EBPα.

Leukemia. 2016 Mar;30(3):674-82 26 Oct 2015, .
B cells have been shown to be refractory to reprogramming and B-cell-derived induced pluripotent stem cells (iPSC) have only been generated from murine B cells engineered to carry doxycycline-inducible Oct4, Sox2, Klf4 and Myc (OSKM) cassette in every tissue and from EBV/SV40LT-immortalized lymphoblastoid cell lines. Here, we show for the first time that freshly isolated non-cultured human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20+ B cells can be reprogrammed to iPSCs carrying complete VDJH immunoglobulin (Ig) gene monoclonal rearrangements using non-integrative tetracistronic, but not monocistronic, OSKM-expressing Sendai Virus. Co-expression of C/EBPα with OSKM facilitates iPSC generation from both CB- and PB-derived B cells. We also demonstrate that myeloid cells are much easier to reprogram than B and T lymphocytes. Differentiation potential back into the cell type of their origin of B-cell-, T-cell-, myeloid- and fibroblast-iPSCs is not skewed, suggesting that their differentiation does not seem influenced by 'epigenetic memory'. Our data reflect the actual cell-autonomous reprogramming capacity of human primary B cells because biased reprogramming was avoided by using freshly isolated primary cells, not exposed to cytokine cocktails favoring proliferation, differentiation or survival. The ability to reprogram CB/PB-derived primary human B cells offers an unprecedented opportunity for studying developmental B lymphopoiesis and modeling B-cell malignancies.
Aleksey Lazarenkov, José Luis Sardina

Dissecting TET2 Regulatory Networks in Blood Differentiation and Cancer

Cancers 2022, 14(3), 830; https://doi.org/10.3390/cancers14030830 6 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.
Tian TV, Sardina JL

Uncovering Sequence-Specific Transcription Factors Interacting with TET2.

Methods Mol Biol 20 May 2021, 2272 239-250.
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.
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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.

Blood. 2020 Jan 9;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 Biol Jul 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.
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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 Cell 1 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.
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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 A 6 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.
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