Publications by authors named "Michael Rehli"

91 Publications

Aberrant DNA methylation patterns in microsatellite stable human colorectal cancers define a new marker panel for the CpG island methylator phenotype.

Int J Cancer 2021 Sep 30. Epub 2021 Sep 30.

Regensburg Center for Interventional Immunology (RCI), University Regensburg and University Medical Center Regensburg, Regensburg, Germany.

A distinct group of colorectal carcinomas (CRCs) referred to as the "CpG island methylator phenotype" (CIMP) shows an extremely high incidence of de novo DNA methylation and may share common pathological, clinical or molecular features. However, there is limited consensus about which CpG islands (CGIs) define a CIMP, particularly in microsatellite stable (MSS) carcinomas. To study this phenotype in a systematic manner, we analyzed genome-wide CGI DNA methylation profiles of 19 MSS CRC using methyl-CpG immunoprecipitation (MCIp) and hybridization on 244K CGI oligonucleotide microarrays, determined KRAS and BRAF mutation status and compared disease-related DNA methylation changes to chromosomal instability as detected by microarray-based comparative genomic hybridization. Results were validated using mass spectrometry analysis of bisulfite-converted DNA at a subset of 76 individual CGIs in 120 CRC and 43 matched normal tissue samples. Both genome-wide profiling and CpG methylation fine mapping segregated a group of CRC showing pronounced and frequent de novo DNA methylation of a distinct group of CGIs that only partially overlapped with previously established classifiers. The CIMP group defined in our study revealed significant association with colon localization, either KRAS or BRAF mutation, and mostly minor chromosomal losses but no association with known histopathological features. Our data provide a basis for defining novel marker panels that may enable a more reliable classification of CIMP in all CRCs, independently of the MS status.
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http://dx.doi.org/10.1002/ijc.33831DOI Listing
September 2021

Methyl donor micronutrients, CD40-ligand methylation and disease activity in systemic lupus erythematosus: A cross-sectional association study.

Lupus 2021 Jul 20:9612033211034559. Epub 2021 Jul 20.

Medical Faculty, Dept. & Hiller Research Unit for Rheumatology, Heinrich-Heine-University, Düsseldorf, Germany.

Objective: Hypomethylation of CD40-ligand (CD40L) in T-cells is associated with increased disease activity in systemic lupus erythematosus (SLE). We therefore investigated possible associations of dietary methyl donors and products with CD40L methylation status in SLE.

Methods: Food frequency questionnaires were employed to calculate methyl donor micronutrients in 61 female SLE patients (age 45.7 ± 12.0 years, disease duration 16.2 ± 8.4 years) and compared to methylation levels of previously identified key DNA methylation sites (CpG17 and CpG22) within CD40L promotor of T-cells using quantitative DNA methylation analysis on the EpiTYPER mass spectrometry platform. Disease activity was assessed by SLE Disease Activity Index (SLEDAI). Linear regression modelling was used. P values were adjusted according to Benjamini & Hochberg.

Results: Amongst the micronutrients assessed (g per day), methionine and cysteine were associated with methylation of CpG17 (β = 5.0 (95%CI: 0.6-9.4), p = 0.04; and β = 2.4 (0.6-4.1), p = 0.02, respectively). Methionine, choline, and cysteine were additionally associated with the mean methylation of the entire (β = 9.5 (1.0-18.0), p = 0.04; β = 1.6 (0.4-3.0), p = 0.04; and β = 4.3 (0.9-7.7), p = 0.02, respectively). Associations of the SLEDAI with hypomethylation were confirmed for CpG17 (β=-32.6 (-60.6 to -4.6), p = 0.04) and CpG22 (β=-38.3 (-61.2 to -15.4), p = 0.004), but not the mean methylation of . Dietary products with the highest impact on methylation included meat, ice cream, white bread, and cooked potatoes.

Conclusions: Dietary methyl donors may influence DNA methylation levels and thereby disease activity in SLE.
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http://dx.doi.org/10.1177/09612033211034559DOI Listing
July 2021

Allele-specific expression of GATA2 due to epigenetic dysregulation in CEBPA double-mutant AML.

Blood 2021 07;138(2):160-177

Department of Hematology, Erasmus Medical Center (MC) Cancer Institute, Rotterdam, The Netherlands.

Transcriptional deregulation is a central event in the development of acute myeloid leukemia (AML). To identify potential disturbances in gene regulation, we conducted an unbiased screen of allele-specific expression (ASE) in 209 AML cases. The gene encoding GATA binding protein 2 (GATA2) displayed ASE more often than any other myeloid- or cancer-related gene. GATA2 ASE was strongly associated with CEBPA double mutations (DMs), with 95% of cases presenting GATA2 ASE. In CEBPA DM AML with GATA2 mutations, the mutated allele was preferentially expressed. We found that GATA2 ASE was a somatic event lost in complete remission, supporting the notion that it plays a role in CEBPA DM AML. Acquisition of GATA2 ASE involved silencing of 1 allele via promoter methylation and concurrent overactivation of the other allele, thereby preserving expression levels. Notably, promoter methylation was also lost in remission along with GATA2 ASE. In summary, we propose that GATA2 ASE is acquired by epigenetic mechanisms and is a prerequisite for the development of AML with CEBPA DMs. This finding constitutes a novel example of an epigenetic hit cooperating with a genetic hit in the pathogenesis of AML.
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http://dx.doi.org/10.1182/blood.2020009244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8489178PMC
July 2021

Single-cell chromatin accessibility landscape identifies tissue repair program in human regulatory T cells.

Immunity 2021 04 30;54(4):702-720.e17. Epub 2021 Mar 30.

Regensburg Center for Interventional Immunology (RCI); Chair for Immunology, University Regensburg, 93053 Regensburg, Germany. Electronic address:

Murine regulatory T (Treg) cells in tissues promote tissue homeostasis and regeneration. We sought to identify features that characterize human Treg cells with these functions in healthy tissues. Single-cell chromatin accessibility profiles of murine and human tissue Treg cells defined a conserved, microbiota-independent tissue-repair Treg signature with a prevailing footprint of the transcription factor BATF. This signature, combined with gene expression profiling and TCR fate mapping, identified a population of tissue-like Treg cells in human peripheral blood that expressed BATF, chemokine receptor CCR8 and HLA-DR. Human BATFCCR8 Treg cells from normal skin and adipose tissue shared features with nonlymphoid T follicular helper-like (Tfh-like) cells, and induction of a Tfh-like differentiation program in naive human Treg cells partially recapitulated tissue Treg regenerative characteristics, including wound healing potential. Human BATFCCR8 Treg cells from healthy tissue share features with tumor-resident Treg cells, highlighting the importance of understanding the context-specific functions of these cells.
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http://dx.doi.org/10.1016/j.immuni.2021.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050210PMC
April 2021

The epigenetic pioneer EGR2 initiates DNA demethylation in differentiating monocytes at both stable and transient binding sites.

Nat Commun 2021 03 10;12(1):1556. Epub 2021 Mar 10.

Department of Internal Medicine III, University Hospital Regensburg, 93053, Regensburg, Germany.

The differentiation of human blood monocytes (MO), the post-mitotic precursors of macrophages (MAC) and dendritic cells (moDC), is accompanied by the active turnover of DNA methylation, but the extent, consequences and mechanisms of DNA methylation changes remain unclear. Here, we profile and compare epigenetic landscapes during IL-4/GM-CSF-driven MO differentiation across the genome and detect several thousand regions that are actively demethylated during culture, both with or without accompanying changes in chromatin accessibility or transcription factor (TF) binding. We further identify TF that are globally associated with DNA demethylation processes. While interferon regulatory factor 4 (IRF4) is found to control hallmark dendritic cell functions with less impact on DNA methylation, early growth response 2 (EGR2) proves essential for MO differentiation as well as DNA methylation turnover at its binding sites. We also show that ERG2 interacts with the 5mC hydroxylase TET2, and its consensus binding sequences show a characteristic DNA methylation footprint at demethylated sites with or without detectable protein binding. Our findings reveal an essential role for EGR2 as epigenetic pioneer in human MO and suggest that active DNA demethylation can be initiated by the TET2-recruiting TF both at stable and transient binding sites.
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http://dx.doi.org/10.1038/s41467-021-21661-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946903PMC
March 2021

The IKZF1-IRF4/IRF5 Axis Controls Polarization of Myeloma-Associated Macrophages.

Cancer Immunol Res 2021 03 9;9(3):265-278. Epub 2021 Feb 9.

Department of Internal Medicine 5, Hematology and Oncology, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Erlangen, Germany.

The bone marrow niche has a pivotal role in progression, survival, and drug resistance of multiple myeloma cells. Therefore, it is important to develop means for targeting the multiple myeloma bone marrow microenvironment. Myeloma-associated macrophages (MAM) in the bone marrow niche are M2 like. They provide nurturing signals to multiple myeloma cells and promote immune escape. Reprogramming M2-like macrophages toward a tumoricidal M1 phenotype represents an intriguing therapeutic strategy. This is especially interesting in view of the successful use of mAbs against multiple myeloma cells, as these therapies hold the potential to trigger macrophage-mediated phagocytosis and cytotoxicity. In this study, we observed that MAMs derived from patients treated with the immunomodulatory drug (IMiD) lenalidomide skewed phenotypically and functionally toward an M1 phenotype. Lenalidomide is known to exert its beneficial effects by modulating the CRBN-CRL4 E3 ligase to ubiquitinate and degrade the transcription factor IKAROS family zinc finger 1 (IKZF1). In M2-like MAMs, we observed enhanced IKZF1 levels that vanished through treatment with lenalidomide, yielding MAMs with a bioenergetic profile, T-cell stimulatory properties, and loss of tumor-promoting capabilities that resemble M1 cells. We also provide evidence that IMiDs interfere epigenetically, via degradation of IKZF1, with IFN regulatory factors 4 and 5, which in turn alters the balance of M1/M2 polarization. We validated our observations using the Crbn mouse model that recapitulates the IMiD-triggered IKZF1 degradation. These data show a role for IKZF1 in macrophage polarization and can provide explanations for the clinical benefits observed when combining IMiDs with therapeutic antibodies..
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0555DOI Listing
March 2021

Associations of site-specific CD4-T-cell hypomethylation within CD40-ligand promotor and enhancer regions with disease activity of women with systemic lupus erythematosus.

Lupus 2021 01 20;30(1):45-51. Epub 2020 Oct 20.

Medical Faculty, Department & Hiller Research Unit for Rheumatology, Heinrich-Heine-University, Düsseldorf, Germany.

Objective: To comprehensively assess associations of site-specific CD4-T-cell hypomethylation of the CD40-Ligand gene () with disease activity of women with systemic lupus erythematosus (SLE).

Methods: CpG-sites within the DNA of the promotor and two enhancer regions (n = 22) of were identified and numbered consecutively. The rate of methylated DNA in isolated CD4-T-cells of women with SLE were quantified for each methylation site by MALDI-TOF. Disease activity was assessed by SLE Disease Activity Index (SLEDAI). Associations of site-specific methylation rates with the SLEDAI scores were assessed by linear regression modelling. P values were adjusted according to Bonferroni-Holm as indicated.

Results: 60 female SLE patients participated in the study (age 45.7 ± 11.1 years, disease duration 17.0 ± 8.3 years). Significant associations to the SLEDAI were noted for CpG22 hypomethylation of the promotor (β = -40.1, p = 0.017, adjusted p = 0.027), trends were noted for CpG17 hypomethylation of the promotor (β = -30.5, p = 0.032, adjusted p = 0.6), and for CpG11 hypermethylation of the second enhancer (β = 15.0, p = 0.046, adjusted p = 0.8).

Conclusion: Site-specific hypomethylation of the promotor in CD4-T-cells show associations with disease activity in female SLE patients.
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http://dx.doi.org/10.1177/0961203320965690DOI Listing
January 2021

Mechanisms governing the pioneering and redistribution capabilities of the non-classical pioneer PU.1.

Nat Commun 2020 01 21;11(1):402. Epub 2020 Jan 21.

Department of Internal Medicine III, University Hospital Regensburg, 93053, Regensburg, Germany.

Establishing gene regulatory networks during differentiation or reprogramming requires master or pioneer transcription factors (TFs) such as PU.1, a prototype master TF of hematopoietic lineage differentiation. To systematically determine molecular features that control its activity, here we analyze DNA-binding in vitro and genome-wide in vivo across different cell types with native or ectopic PU.1 expression. Although PU.1, in contrast to classical pioneer factors, is unable to access nucleosomal target sites in vitro, ectopic induction of PU.1 leads to the extensive remodeling of chromatin and redistribution of partner TFs. De novo chromatin access, stable binding, and redistribution of partner TFs both require PU.1's N-terminal acidic activation domain and its ability to recruit SWI/SNF remodeling complexes, suggesting that the latter may collect and distribute co-associated TFs in conjunction with the non-classical pioneer TF PU.1.
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http://dx.doi.org/10.1038/s41467-019-13960-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972792PMC
January 2020

Precursors for Nonlymphoid-Tissue Treg Cells Reside in Secondary Lymphoid Organs and Are Programmed by the Transcription Factor BATF.

Immunity 2020 02 7;52(2):295-312.e11. Epub 2020 Jan 7.

Regensburg Center for Interventional Immunology (RCI), Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany; Chair for Immunology, Regensburg University, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany; Immune Tolerance Group, Tumor Immunology Program, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Electronic address:

Specialized regulatory T (Treg) cells accumulate and perform homeostatic and regenerative functions in nonlymphoid tissues. Whether common precursors for nonlymphoid-tissue Treg cells exist and how they differentiate remain elusive. Using transcription factor nuclear factor, interleukin 3 regulated (Nfil3) reporter mice and single-cell RNA-sequencing (scRNA-seq), we identified two precursor stages of interleukin 33 (IL-33) receptor ST2-expressing nonlymphoid tissue Treg cells, which resided in the spleen and lymph nodes. Global chromatin profiling of nonlymphoid tissue Treg cells and the two precursor stages revealed a stepwise acquisition of chromatin accessibility and reprogramming toward the nonlymphoid-tissue Treg cell phenotype. Mechanistically, we identified and validated the transcription factor Batf as the driver of the molecular tissue program in the precursors. Understanding this tissue development program will help to harness regenerative properties of tissue Treg cells for therapy.
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http://dx.doi.org/10.1016/j.immuni.2019.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026712PMC
February 2020

C-Jun drives melanoma progression in PTEN wild type melanoma cells.

Cell Death Dis 2019 08 5;10(8):584. Epub 2019 Aug 5.

Institute of Biochemistry (Emil-Fischer Center), Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany.

Due to the critical impact of active AP-1 transcription factors in melanoma, it is important to define their target genes and to identify and ultimately inhibit oncogenic signals. Here we mapped the genome-wide occupancy of the AP-1 family member c-Jun in different melanoma cells and correlated AP-1 binding with transcriptome data to detect genes in melanoma regulated by c-Jun. Our analysis shows that c-Jun supports the malignant phenotype by deregulating genes in cancer-relevant signaling pathways, such as mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways. Moreover, we demonstrate that the importance of c-Jun depends on melanoma stage and mutation status of the tumor suppressor PTEN. Our study reveals that activation of c-Jun overrules the tumor suppressive effect of PTEN in early melanoma development. These findings help to understand the relevance of c-Jun within cancer pathways in different melanoma cell types, especially in relation to MAPK and PI3K pathways, which are commonly deregulated in melanomas. Consequently, targeting c-Jun in PTEN melanoma cells may represent a promising therapeutic strategy to inhibit survival of melanoma cells to prevent the development of a metastatic phenotype.
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http://dx.doi.org/10.1038/s41419-019-1821-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6680049PMC
August 2019

Rbpj expression in regulatory T cells is critical for restraining T2 responses.

Nat Commun 2019 04 8;10(1):1621. Epub 2019 Apr 8.

Chair for Immunology, University Regensburg and University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93053, Regensburg, Germany.

The transcriptional regulator Rbpj is involved in T-helper (T) subset polarization, but its function in T cells remains unclear. Here we show that T-specific Rbpj deletion leads to splenomegaly and lymphadenopathy despite increased numbers of T cells with a polyclonal TCR repertoire. A specific defect of Rbpj-deficient T cells in controlling T2 polarization and B cell responses is observed, leading to the spontaneous formation of germinal centers and a T2-associated immunoglobulin class switch. The observed phenotype is environment-dependent and can be induced by infection with parasitic nematodes. Rbpj-deficient T cells adopt open chromatin landscapes and gene expression profiles reminiscent of tissue-derived T2-polarized T cells, with a prevailing signature of the transcription factor Gata-3. Taken together, our study suggests that T cells require Rbpj to specifically restrain T2 responses, including their own excessive T2-like differentiation potential.
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http://dx.doi.org/10.1038/s41467-019-09276-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453958PMC
April 2019

Temporal autoregulation during human PU.1 locus SubTAD formation.

Blood 2018 12 12;132(25):2643-2655. Epub 2018 Oct 12.

Institute of Molecular Tumor Biology and.

Epigenetic control of gene expression occurs within discrete spatial chromosomal units called topologically associating domains (TADs), but the exact spatial requirements of most genes are unknown; this is of particular interest for genes involved in cancer. We therefore applied high-resolution chromosomal conformation capture sequencing to map the three-dimensional (3D) organization of the human locus encoding the key myeloid transcription factor PU.1 in healthy monocytes and acute myeloid leukemia (AML) cells. We identified a dynamic ∼75-kb unit (SubTAD) as the genomic region in which spatial interactions between PU.1 gene regulatory elements occur during myeloid differentiation and are interrupted in AML. Within this SubTAD, proper initiation of the spatial chromosomal interactions requires PU.1 autoregulation and recruitment of the chromatin-adaptor protein LDB1 (LIM domain-binding protein 1). However, once these spatial interactions have occurred, LDB1 stabilizes them independently of PU.1 autoregulation. Thus, our data support that PU.1 autoregulates its expression in a "hit-and-run" manner by initiating stable chromosomal loops that result in a transcriptionally active chromatin architecture.
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http://dx.doi.org/10.1182/blood-2018-02-834721DOI Listing
December 2018

Profiling of aberrant DNA methylation in acute myeloid leukemia reveals subclasses of CG-rich regions with epigenetic or genetic association.

Leukemia 2019 01 20;33(1):26-36. Epub 2018 Jun 20.

Department of Internal Medicine III, University Hospital Regensburg, 93042, Regensburg, Germany.

Malignant transformation is frequently associated with disease-specific epigenetic alterations, but the underlying mechanisms and pathophysiological consequences remain poorly understood. Here, we used global comparative DNA methylation profiling at CG-rich regions of 27 acute myeloid leukemia (AML) samples to select a subset of aberrantly methylated CG-rich regions (~400 regions, ~15,000 CpGs) for quantitative DNA methylation profiling in a large cohort of AML patients (n = 196) using MALDI-TOF analysis of bisulfite-treated DNA. Meta-analysis separated a subgroup of CG-rich regions showing highly correlated DNA methylation changes that were marked by histone H3 lysine 27 trimethylation in normal hematopoietic progenitor cells. While the group of non-polycomb group (PcG) target regions displayed methylation patterns that correlated well with molecular and cytogenetic markers, PcG target regions displayed a much weaker association with genetic features. However, the degree of methylation gain across the latter panel showed significant correlation with active DNMT3A levels and with overall survival. Our study suggests that both epigenetic as well as genetic aberrations underlay AML-related changes in DNA methylation at CG-rich regions and that the former may provide a marker to improve classification and prognostication of adult AML patients.
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http://dx.doi.org/10.1038/s41375-018-0165-2DOI Listing
January 2019

Shared activity patterns arising at genetic susceptibility loci reveal underlying genomic and cellular architecture of human disease.

PLoS Comput Biol 2018 03 1;14(3):e1005934. Epub 2018 Mar 1.

Broad Institute of Harvard and MIT, Cambridge, United States of America.

Genetic variants underlying complex traits, including disease susceptibility, are enriched within the transcriptional regulatory elements, promoters and enhancers. There is emerging evidence that regulatory elements associated with particular traits or diseases share similar patterns of transcriptional activity. Accordingly, shared transcriptional activity (coexpression) may help prioritise loci associated with a given trait, and help to identify underlying biological processes. Using cap analysis of gene expression (CAGE) profiles of promoter- and enhancer-derived RNAs across 1824 human samples, we have analysed coexpression of RNAs originating from trait-associated regulatory regions using a novel quantitative method (network density analysis; NDA). For most traits studied, phenotype-associated variants in regulatory regions were linked to tightly-coexpressed networks that are likely to share important functional characteristics. Coexpression provides a new signal, independent of phenotype association, to enable fine mapping of causative variants. The NDA coexpression approach identifies new genetic variants associated with specific traits, including an association between the regulation of the OCT1 cation transporter and genetic variants underlying circulating cholesterol levels. NDA strongly implicates particular cell types and tissues in disease pathogenesis. For example, distinct groupings of disease-associated regulatory regions implicate two distinct biological processes in the pathogenesis of ulcerative colitis; a further two separate processes are implicated in Crohn's disease. Thus, our functional analysis of genetic predisposition to disease defines new distinct disease endotypes. We predict that patients with a preponderance of susceptibility variants in each group are likely to respond differently to pharmacological therapy. Together, these findings enable a deeper biological understanding of the causal basis of complex traits.
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http://dx.doi.org/10.1371/journal.pcbi.1005934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849332PMC
March 2018

In-vitro blockade of the CD4 receptor co-signal in antigen-specific T-cell stimulation cultures induces the outgrowth of potent CD4 independent T-cell effectors.

J Immunol Methods 2018 03 14;454:80-85. Epub 2017 Nov 14.

Department of Internal Medicine III, Hematology and Oncology, University Hospital Regensburg, Germany; Regensburg Center for Interventional Immunology, University of Regensburg, Regensburg, Germany. Electronic address:

T-cell receptor (TCR) redirected T cells are promising tools for adoptive cancer immunotherapy. Since not only CD8 but also CD4 T cells are key players for efficient antitumor responses, the targeted redirection of both subsets with the same antigen-specific TCR comes more and more into focus. Although rapidly evolving technologies enable the reliable genetic re-programming of T cells, the limited availability of TCRs that induce T-cell activation in both T-cell subsets without CD4/CD8 co-receptor contribution hampers the broad application of this approach. We developed a novel stimulation approach, which drives the activation and proliferation of CD4 T-cell populations capable of inducing effector functions in a CD4-independent manner. Naive-enriched CD4 T cells were stimulated against dendritic cells (DC) expressing allogeneic HLA-DP antigens upon RNA transfection and CD4/HLA interactions were blocked by the addition of CD4 binding antibody. Evolving CD4 T-cell populations were specifically activated independent of the CD4 co-signal and induced strong TCR-mediated IFN-γ secretion as well as cytolysis upon recognition of leukemia cells expressing HLA-DP antigen. Our novel stimulation approach may facilitate the generation of CD4 T cells as source for co-receptor independent TCRs for future immunotherapies.
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http://dx.doi.org/10.1016/j.jim.2017.11.006DOI Listing
March 2018

Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation.

Nucleic Acids Res 2017 Sep;45(16):e147

Helmholtz Centre for Infection Research, RG Model Systems for Infection and Immunity (MSYS), Braunschweig, Germany.

Faithful expression of transgenes in cell cultures and mice is often challenged by locus dependent epigenetic silencing. We investigated silencing of Tet-controlled expression cassettes within the mouse ROSA26 locus. We observed pronounced DNA methylation of the Tet promoter concomitant with loss of expression in mES cells as well as in differentiated cells and transgenic animals. Strikingly, the ROSA26 promoter remains active and methylation free indicating that this silencing mechanism specifically affects the transgene, but does not spread to the host's chromosomal neighborhood. To reactivate Tet cassettes a synthetic fusion protein was constructed and expressed in silenced cells. This protein includes the enzymatic domains of ten eleven translocation methylcytosine dioxygenase 1 (TET-1) as well as the Tet repressor DNA binding domain. Expression of the synthetic fusion protein and Doxycycline treatment allowed targeted demethylation of the Tet promoter in the ROSA26 locus and in another genomic site, rescuing transgene expression in cells and transgenic mice. Thus, inducible, reversible and site-specific epigenetic modulation is a promising strategy for reactivation of silenced transgene expression, independent of the integration site.
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http://dx.doi.org/10.1093/nar/gkx601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766184PMC
September 2017

FANTOM5 CAGE profiles of human and mouse samples.

Sci Data 2017 08 29;4:170112. Epub 2017 Aug 29.

Scottish Centre for Regenerative Medicine, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, UK.

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.
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http://dx.doi.org/10.1038/sdata.2017.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574368PMC
August 2017

An integrated expression atlas of miRNAs and their promoters in human and mouse.

Nat Biotechnol 2017 Sep 21;35(9):872-878. Epub 2017 Aug 21.

Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Japan.

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions.
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http://dx.doi.org/10.1038/nbt.3947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767576PMC
September 2017

Analysis of the human monocyte-derived macrophage transcriptome and response to lipopolysaccharide provides new insights into genetic aetiology of inflammatory bowel disease.

PLoS Genet 2017 03 6;13(3):e1006641. Epub 2017 Mar 6.

The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, United Kingdom.

The FANTOM5 consortium utilised cap analysis of gene expression (CAGE) to provide an unprecedented insight into transcriptional regulation in human cells and tissues. In the current study, we have used CAGE-based transcriptional profiling on an extended dense time course of the response of human monocyte-derived macrophages grown in macrophage colony-stimulating factor (CSF1) to bacterial lipopolysaccharide (LPS). We propose that this system provides a model for the differentiation and adaptation of monocytes entering the intestinal lamina propria. The response to LPS is shown to be a cascade of successive waves of transient gene expression extending over at least 48 hours, with hundreds of positive and negative regulatory loops. Promoter analysis using motif activity response analysis (MARA) identified some of the transcription factors likely to be responsible for the temporal profile of transcriptional activation. Each LPS-inducible locus was associated with multiple inducible enhancers, and in each case, transient eRNA transcription at multiple sites detected by CAGE preceded the appearance of promoter-associated transcripts. LPS-inducible long non-coding RNAs were commonly associated with clusters of inducible enhancers. We used these data to re-examine the hundreds of loci associated with susceptibility to inflammatory bowel disease (IBD) in genome-wide association studies. Loci associated with IBD were strongly and specifically (relative to rheumatoid arthritis and unrelated traits) enriched for promoters that were regulated in monocyte differentiation or activation. Amongst previously-identified IBD susceptibility loci, the vast majority contained at least one promoter that was regulated in CSF1-dependent monocyte-macrophage transitions and/or in response to LPS. On this basis, we concluded that IBD loci are strongly-enriched for monocyte-specific genes, and identified at least 134 additional candidate genes associated with IBD susceptibility from reanalysis of published GWA studies. We propose that dysregulation of monocyte adaptation to the environment of the gastrointestinal mucosa is the key process leading to inflammatory bowel disease.
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http://dx.doi.org/10.1371/journal.pgen.1006641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358891PMC
March 2017

Molecular dissection of the valproic acid effects on glioma cells.

Oncotarget 2016 Sep;7(39):62989-63002

Department of Neuropathology, Regensburg University Hospital, Regensburg, Germany.

Many glioblastoma patients suffer from seizures why they are treated with antiepileptic agents. Valproic acid (VPA) is a histone deacetylase inhibitor that apart from its anticonvulsive effects in some retrospective studies has been suggested to lead to a superior outcome of glioblastoma patients. However, the exact molecular effects of VPA treatment on glioblastoma cells have not yet been deciphered. We treated glioblastoma cells with VPA, recorded the functional effects of this treatment and performed a global and unbiased next generation sequencing study on the chromatin (ChIP) and RNA level. 1) VPA treatment clearly sensitized glioma cells to temozolomide: A protruding VPA-induced molecular feature in this context was the transcriptional upregulation/reexpression of numerous solute carrier (SLC) transporters that was also reflected by euchromatinization on the histone level and a reexpression of SLC transporters in human biopsy samples after VPA treatment. DNA repair genes were adversely reduced. 2) VPA treatment, however, also reduced cell proliferation in temozolomide-naive cells: On the molecular level in this context we observed a transcriptional upregulation/reexpression and euchromatinization of several glioblastoma relevant tumor suppressor genes and a reduction of stemness markers, while transcriptional subtype classification (mesenchymal/proneural) remained unaltered. Taken together, these findings argue for both temozolomide-dependent and -independent effects of VPA. VPA might increase the uptake of temozolomide and simultaneously lead to a less malignant glioblastoma phenotype. From a mere molecular perspective these findings might indicate a surplus value of VPA in glioblastoma therapy and could therefore contribute an additional ratio for clinical decision making.
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http://dx.doi.org/10.18632/oncotarget.11379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5325342PMC
September 2016

Transcriptional Regulation and Macrophage Differentiation.

Microbiol Spectr 2016 06;4(3)

University Hospital Regensburg, Department of Internal Medicine III, D-93047 Regensburg, Germany.

Monocytes and macrophages are professional phagocytes that occupy specific niches in every tissue of the body. Their survival, proliferation, and differentiation are controlled by signals from the macrophage colony-stimulating factor receptor (CSF-1R) and its two ligands, CSF-1 and interleukin-34. In this review, we address the developmental and transcriptional relationships between hematopoietic progenitor cells, blood monocytes, and tissue macrophages as well as the distinctions from dendritic cells. A huge repertoire of receptors allows monocytes, tissue-resident macrophages, or pathology-associated macrophages to adapt to specific microenvironments. These processes create a broad spectrum of macrophages with different functions and individual effector capacities. The production of large transcriptomic data sets in mouse, human, and other species provides new insights into the mechanisms that underlie macrophage functional plasticity.
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http://dx.doi.org/10.1128/microbiolspec.MCHD-0024-2015DOI Listing
June 2016

An autonomous CEBPA enhancer specific for myeloid-lineage priming and neutrophilic differentiation.

Blood 2016 06 10;127(24):2991-3003. Epub 2016 Mar 10.

Department of Hematology.

Neutrophilic differentiation is dependent on CCAAT enhancer-binding protein α (C/EBPα), a transcription factor expressed in multiple organs including the bone marrow. Using functional genomic technologies in combination with clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 genome editing and in vivo mouse modeling, we show that CEBPA is located in a 170-kb topological-associated domain that contains 14 potential enhancers. Of these, 1 enhancer located +42 kb from CEBPA is active and engages with the CEBPA promoter in myeloid cells only. Germ line deletion of the homologous enhancer in mice in vivo reduces Cebpa levels exclusively in hematopoietic stem cells (HSCs) and myeloid-primed progenitor cells leading to severe defects in the granulocytic lineage, without affecting any other Cebpa-expressing organ studied. The enhancer-deleted progenitor cells lose their myeloid transcription program and are blocked in differentiation. Deletion of the enhancer also causes loss of HSC maintenance. We conclude that a single +42-kb enhancer is essential for CEBPA expression in myeloid cells only.
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http://dx.doi.org/10.1182/blood-2016-01-695759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043424PMC
June 2016

Identification and characterization of the specific murine NK cell subset supporting graft--leukemia- and reducing graft--host-effects.

Oncoimmunology 2015 Jan 3;4(1):e981483. Epub 2015 Feb 3.

Department of Internal Medicine 5; Hematology and Oncology; Friedrich-Alexander Universität Erlangen-Nürnberg; University Hospital Erlangen ; Erlangen, Germany ; Childrens Hospital; Department of Pediatric Hematology and Oncology; Johann Wolfgang Goethe University ; Frankfurt, Germany ; LOEWE Center for Cell and Gene Therapy; Johann Wolfgang Goethe University ; Frankfurt, Germany.

Clinical studies investigating the impact of natural killer (NK) cells in allogeneic hematopoietic stem cell transplantation settings have yielded promising results. However, NK cells are a functionally and phenotypically heterogeneous population. Therefore, we addressed the functional relevance of specific NK cell subsets distinguished by expression of CD117, CD27 and CD11b surface markers in graft-versus-leukemia (GVL)-reaction and graft-versus-host-disease (GVHD). Our results clearly demonstrate that the subset of c-KitCD27CD11b NK cells expressed multiple cytotoxic pathway genes and provided optimal graft-versus-leukemia-effects, while significantly reducing T cell proliferation induced by allogeneic dendritic cells. Furthermore, these NK cells migrated to inflamed intestinal tissues where graft-versus-host-colitis was efficiently mitigated. For the first time, we identified the c-KitCD27CD11b NK cell population as the specific effector NK cell subset capable of significantly diminishing GVHD in fully mismatched bone marrow transplantation settings. In conclusion, the subset of c-KitCD27CD11b NK cells not only supports GVL, but also plays a unique role in the protection against GVHD by migrating to the peripheral GVHD target organs where they exert efficient immunoregulatory activities. These new insights demonstrate the importance of selecting the optimal NK cell subset for cellular immunotherapy following allogeneic hematopoietic stem cell transplantation.
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http://dx.doi.org/10.4161/2162402X.2014.981483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4368119PMC
January 2015

Gateways to the FANTOM5 promoter level mammalian expression atlas.

Genome Biol 2015 Jan 5;16:22. Epub 2015 Jan 5.

The FANTOM5 project investigates transcription initiation activities in more than 1,000 human and mouse primary cells, cell lines and tissues using CAGE. Based on manual curation of sample information and development of an ontology for sample classification, we assemble the resulting data into a centralized data resource (http://fantom.gsc.riken.jp/5/). This resource contains web-based tools and data-access points for the research community to search and extract data related to samples, genes, promoter activities, transcription factors and enhancers across the FANTOM5 atlas.
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http://dx.doi.org/10.1186/s13059-014-0560-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310165PMC
January 2015

Technical Advance: Transcription factor, promoter, and enhancer utilization in human myeloid cells.

J Leukoc Biol 2015 May 25;97(5):985-995. Epub 2015 Feb 25.

*The Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Scotland, United Kingdom; RIKEN Preventive Medicine and Diagnosis Innovation Program, Tsurumi-ku, Yokohama, Japan; RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Tsurumi-ku, Yokohama, Japan; Department of Biosciences and Nutrition, Karolinska Institute, Huddinge, Sweden; Department of Dermatology and Allergy, Charité Universitätsmedizin Berlin, Germany; Department of Internal Medicine III, University Hospital, University of Regensburg, Germany; Department of Experimental Immunology, Academic Medical Center, Amsterdam, the Netherlands; and **RIKEN Omics Science Center, Tsurumi-ku, Yokohama, Japan

The generation of myeloid cells from their progenitors is regulated at the level of transcription by combinatorial control of key transcription factors influencing cell-fate choice. To unravel the global dynamics of this process at the transcript level, we generated transcription profiles for 91 human cell types of myeloid origin by use of CAGE profiling. The CAGE sequencing of these samples has allowed us to investigate diverse aspects of transcription control during myelopoiesis, such as identification of novel transcription factors, miRNAs, and noncoding RNAs specific to the myeloid lineage. We further reconstructed a transcription regulatory network by clustering coexpressed transcripts and associating them with enriched cis-regulatory motifs. With the use of the bidirectional expression as a proxy for enhancers, we predicted over 2000 novel enhancers, including an enhancer 38 kb downstream of and an intronic enhancer in the gene locus. Finally, we highlighted relevance of these data to dissect transcription dynamics during progressive maturation of granulocyte precursors. A multifaceted analysis of the myeloid transcriptome is made available (www.myeloidome.roslin.ed.ac.uk). This high-quality dataset provides a powerful resource to study transcriptional regulation during myelopoiesis and to infer the likely functions of unannotated genes in human innate immunity.
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http://dx.doi.org/10.1189/jlb.6TA1014-477RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4398258PMC
May 2015

Lactic acid delays the inflammatory response of human monocytes.

Biochem Biophys Res Commun 2015 Feb 9;457(3):412-8. Epub 2015 Jan 9.

Department of Internal Medicine III, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg, Germany; RCI Regensburg Center for Interventional Immunology, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg, Germany. Electronic address:

Lactic acid (LA) accumulates under inflammatory conditions, e.g. in wounds or tumors, and influences local immune cell functions. We previously noted inhibitory effects of LA on glycolysis and TNF secretion of human LPS-stimulated monocytes. Here, we globally analyze the influence of LA on gene expression during monocyte activation. To separate LA-specific from lactate- or pH-effects, monocytes were treated for one or four hours with LPS in the presence of physiological concentrations of LA, sodium lactate (NaL) or acidic pH. Analyses of global gene expression profiles revealed striking effects of LA during the early stimulation phase. Up-regulation of most LPS-induced genes was significantly delayed in the presence of LA, while this inhibitory effect was attenuated in acidified samples and not detected after incubation with NaL. LA targets included genes encoding for important monocyte effector proteins like cytokines (e.g. TNF and IL-23) or chemokines (e.g. CCL2 and CCL7). LA effects were validated for several targets by quantitative RT-PCR and/or ELISA. Further analysis of LPS-signaling pathways revealed that LA delayed the phosphorylation of protein kinase B (AKT) as well as the degradation of IκBα. Consistently, the LPS-induced nuclear accumulation of NFκB was also diminished in response to LA. These results indicate that the broad effect of LA on gene expression and function of human monocytes is at least partially caused by its interference with immediate signal transduction events after activation. This mechanism might contribute to monocyte suppression in the tumor environment.
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http://dx.doi.org/10.1016/j.bbrc.2015.01.005DOI Listing
February 2015

Genome-wide identification of hypoxia-inducible factor-1 and -2 binding sites in hypoxic human macrophages alternatively activated by IL-10.

Biochim Biophys Acta 2015 Jan 1;1849(1):10-22. Epub 2014 Nov 1.

Institute of Biochemistry I/ZAFES, Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany.

Macrophages (MΦ) often accumulate in hypoxic areas, where they significantly influence disease progression. Anti-inflammatory cytokines, such as IL-10, generate alternatively activated macrophages that support tumor growth. To understand how alternative activation affects the transcriptional profile of hypoxic macrophages, we globally mapped binding sites of hypoxia-inducible factor (HIF)-1α and HIF-2α in primary human monocyte-derived macrophages prestimulated with IL-10. 713 HIF-1 and 795 HIF-2 binding sites were identified under hypoxia. Pretreatment with IL-10 altered the binding pattern, with 120 new HIF-1 and 188 new HIF-2 binding sites emerging. HIF-1 binding was most prominent in promoters, while HIF-2 binding was more abundant in enhancer regions. Comparison of ChIP-seq data obtained in other cells revealed a highly cell type specific binding of HIF. In MΦ HIF binding occurred preferentially in already active enhancers or promoters. To assess the roles of HIF on gene expression, primary human macrophages were treated with siRNA against HIF-1α or HIF-2α, followed by genome-wide gene expression analysis. Comparing mRNA expression to the HIF binding profile revealed a significant enrichment of hypoxia-inducible genes previously identified by ChIP-seq. Analysis of gene expression under hypoxia alone and hypoxia/IL-10 showed the enhanced induction of a set of genes including PLOD2 and SLC2A3, while another group including KDM3A and ADM remained unaffected or was reduced by IL-10. Taken together IL-10 influences the DNA binding pattern of HIF and the level of gene induction.
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http://dx.doi.org/10.1016/j.bbagrm.2014.10.006DOI Listing
January 2015

Early aberrant DNA methylation events in a mouse model of acute myeloid leukemia.

Genome Med 2014 30;6(4):34. Epub 2014 Apr 30.

Department of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany.

Background: Aberrant DNA methylation is frequently found in human malignancies including acute myeloid leukemia (AML). While most studies focus on later disease stages, the onset of aberrant DNA methylation events and their dynamics during leukemic progression are largely unknown.

Methods: We screened genome-wide for aberrant CpG island methylation in three disease stages of a murine AML model that is driven by hypomorphic expression of the hematopoietic transcription factor PU.1. DNA methylation levels of selected genes were correlated with methylation levels of CD34+ cells and lineage negative, CD127-, c-Kit+, Sca-1+ cells; common myeloid progenitors; granulocyte-macrophage progenitors; and megakaryocyte-erythroid progenitors.

Results: We identified 1,184 hypermethylated array probes covering 762 associated genes in the preleukemic stage. During disease progression, the number of hypermethylated genes increased to 5,465 in the late leukemic disease stage. Using publicly available data, we found a significant enrichment of PU.1 binding sites in the preleukemic hypermethylated genes, suggesting that shortage of PU.1 makes PU.1 binding sites in the DNA accessible for aberrant methylation. Many known AML associated genes such as RUNX1 and HIC1 were found among the preleukemic hypermethylated genes. Nine novel hypermethylated genes, FZD5, FZD8, PRDM16, ROBO3, CXCL14, BCOR, ITPKA, HES6 and TAL1, the latter four being potential PU.1 targets, were confirmed to be hypermethylated in human normal karyotype AML patients, underscoring the relevance of the mouse model for human AML.

Conclusions: Our study identified early aberrantly methylated genes as potential contributors to onset and progression of AML.
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http://dx.doi.org/10.1186/gm551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4062060PMC
June 2014
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