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Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias., (© 2024. The Author(s).)

The adoptive transfer of regulatory T cells is a promising strategy to prevent graft-versus-host disease after allogeneic bone marrow transplantation. Here, we use a major histocompatibility complex-mismatched mouse model to follow the fate of in vitro expanded donor regulatory T cells upon migration to target organs. Employing comprehensive gene expression and repertoire profiling, we show that they retain their suppressive function and plasticity after transfer. Upon entering non-lymphoid tissues, donor regulatory T cells acquire organ-specific gene expression profiles resembling tissue-resident cells and activate hallmark suppressive and cytotoxic pathways, most evidently in the colon, when co-transplanted with graft-versus-host disease-inducing conventional T cells. Dominant T cell receptor clonotypes overlap between organs and across recipients and their relative abundance correlates with protection efficacy. Thus, this study reveals donor regulatory T cell selection and adaptation mechanisms in target organs and highlights protective features of Treg to guide the development of improved graft-versus-host disease prevention strategies., (© 2024. The Author(s).)

Cohesin is a major structural component of mammalian genomes and is required to maintain loop structures. While acute depletion in short-term culture models suggests a limited importance of cohesin for steady-state transcriptional circuits, long-term studies are hampered by essential functions of cohesin during replication. Here, we study genome architecture in a postmitotic differentiation setting, the differentiation of human blood monocytes (MO). We profile and compare epigenetic, transcriptome and 3D conformation landscapes during MO differentiation (either into dendritic cells or macrophages) across the genome and detect numerous architectural changes, ranging from higher level compartments down to chromatin loops. Changes in loop structures correlate with cohesin-binding, as well as epigenetic and transcriptional changes during differentiation. Functional studies show that the siRNA-mediated depletion of cohesin (and to a lesser extent also CTCF) markedly disturbs loop structures and dysregulates genes and enhancers that are primarily regulated during normal MO differentiation. In addition, gene activation programs in cohesin-depleted MO-derived macrophages are disturbed. Our findings implicate an essential function of cohesin in controlling long-term, differentiation- and activation-associated gene expression programs., (© 2022. The Author(s).)

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.

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.

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.

The transcriptional regulator Rbpj is involved in T-helper (T H ) subset polarization, but its function in T reg cells remains unclear. Here we show that T reg -specific Rbpj deletion leads to splenomegaly and lymphadenopathy despite increased numbers of T reg cells with a polyclonal TCR repertoire. A specific defect of Rbpj-deficient T reg cells in controlling T H 2 polarization and B cell responses is observed, leading to the spontaneous formation of germinal centers and a T H 2-associated immunoglobulin class switch. The observed phenotype is environment-dependent and can be induced by infection with parasitic nematodes. Rbpj-deficient T reg cells adopt open chromatin landscapes and gene expression profiles reminiscent of tissue-derived T H 2-polarized T reg cells, with a prevailing signature of the transcription factor Gata-3. Taken together, our study suggests that T reg cells require Rbpj to specifically restrain T H 2 responses, including their own excessive T H 2-like differentiation potential.