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DOT1L-controlled cell-fate determination and transcription elongation are independent of H3K79 methylation.

ABSTRACT: Actively transcribed genes in mammals are decorated by H3K79 methylation, which is correlated with transcription levels and is catalyzed by the histone methyltransferase DOT1L. DOT1L is required for mammalian development, and the inhibition of its catalytic activity has been extensively studied for cancer therapy; however, the mechanisms underlying DOT1L's functions in normal development and cancer pathogenesis remain elusive. To dissect the relationship between H3K79 methylation, cellular differentiation, and transcription regulation, we systematically examined the role of DOT1L and its catalytic activity in embryonic stem cells (ESCs). DOT1L is dispensable for ESC self-renewal but is required for establishing the proper expression signature of neural progenitor cells, while catalytic inactivation of DOT1L has a lesser effect. Furthermore, DOT1L loss, rather than its catalytic inactivation, causes defects in glial cell specification. Although DOT1L loss by itself has no major defect in transcription elongation, transcription elongation defects seen with the super elongation complex inhibitor KL-2 are exacerbated in DOT1L knockout cells, but not in catalytically dead DOT1L cells, revealing a role of DOT1L in promoting productive transcription elongation that is independent of H3K79 methylation. Taken together, our study reveals a catalytic-independent role of DOT1L in modulating cell-fate determination and in transcriptional elongation control.

SUBMITTER: Cao K

PROVIDER: S-EPMC7959542 | biostudies-literature |

REPOSITORIES: biostudies-literature

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DOT1L-controlled cell-fate determination and transcription elongation are independent of H3K79 methylation

Project description:Actively transcribed genes in mammals are decorated by H3K79 methylation, which is correlated with transcription levels and is catalyzed by the histone methyltransferase DOT1L. DOT1L is required for mammalian development, and the inhibition of its catalytic activity has been extensively studied for cancer therapy; however, the mechanisms underlying DOT1L’s functions in normal development and cancer pathogenesis remain elusive. To dissect the relationship between H3K79 methylation, cellular differentiation, and transcription regulation, we systematically examined the role of DOT1L and its catalytic activity in embryonic stem cells (ESCs). DOT1L is dispensable for ESC self-renewal but is required for establishing the proper expression signature of neural progenitor cells, while catalytic inactivation of DOT1L has a lesser effect. Furthermore, DOT1L loss, rather than its catalytic inactivation, causes defects in glial cell specification. Although DOT1L loss by itself has no major defect in transcription elongation, transcription elongation defects seen with the super elongation complex inhibitor KL-2 are exacerbated in DOT1L knockout cells, but not in catalytically dead DOT1L cells, revealing a role of DOT1L in promoting productive transcription elongation that is independent of H3K79 methylation. Taken together, our study reveals a catalytic-independent role of DOT1L in modulating cell-fate determination and in transcriptional elongation control.

2020-10-21 | GSE134083 | GEO

DOT1L-controlled cell-fate determination and transcription elongation are independent of H3K79 methylation

Project description:DOT1L-controlled cell-fate determination and transcription elongation are independent of H3K79 methylation

| PRJNA553753 | ENA

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

Project description:BackgroundThe 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.ResultsWe 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.ConclusionsOur 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.

| S-EPMC8254283 | biostudies-literature

MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L.

Project description:The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations of the Mixed Lineage Leukemia (MLL) gene. We identified the MLL-fusion targets in an MLL-AF9 leukemia model, and conducted epigenetic profiling for H3K79me2, H3K4me3, H3K27me3, and H3K36me3 in hematopoietic progenitor and leukemia stem cells (LSCs). We found abnormal profiles only for H3K79me2 on MLL-AF9 fusion target loci in LSCs. Inactivation of Dot1l led to downregulation of direct MLL-AF9 targets and an MLL translocation-associated gene expression signature, whereas global gene expression remained largely unaffected. Suppression of MLL translocation-associated gene expression corresponded with dependence of MLL-AF9 leukemia on Dot1l in vivo. These data point to DOT1L as a potential therapeutic target in MLL-rearranged leukemia.

| S-EPMC3329803 | biostudies-literature

AF9 YEATS domain links histone acetylation to DOT1L-mediated H3K79 methylation.

Project description:The recognition of modified histones by "reader" proteins constitutes a key mechanism regulating gene expression in the chromatin context. Compared with the great variety of readers for histone methylation, few protein modules that recognize histone acetylation are known. Here, we show that the AF9 YEATS domain binds strongly to histone H3K9 acetylation and, to a lesser extent, H3K27 and H3K18 acetylation. Crystal structural studies revealed that AF9 YEATS adopts an eight-stranded immunoglobin fold and utilizes a serine-lined aromatic "sandwiching" cage for acetyllysine readout, representing a novel recognition mechanism that is distinct from that of known acetyllysine readers. ChIP-seq experiments revealed a strong colocalization of AF9 and H3K9 acetylation genome-wide, which is important for the chromatin recruitment of the H3K79 methyltransferase DOT1L. Together, our studies identified the evolutionarily conserved YEATS domain as a novel acetyllysine-binding module and established a direct link between histone acetylation and DOT1L-mediated H3K79 methylation in transcription control.

| S-EPMC4344132 | biostudies-literature

DOT1L inhibition reveals a distinct subset of enhancers dependent on H3K79 methylation.

Project description:Enhancer elements are a key regulatory feature of many important genes. Several general features including the presence of specific histone modifications are used to demarcate potentially active enhancers. Here we reveal that putative enhancers marked with H3 lysine 79 (H3K79) di or trimethylation (me2/3) (which we name H3K79me2/3 enhancer elements or KEEs) can be found in multiple cell types. Mixed lineage leukemia gene (MLL) rearrangements (MLL-r) such as MLL-AF4 are a major cause of incurable acute lymphoblastic leukemias (ALL). Using the DOT1L inhibitor EPZ-5676 in MLL-AF4 leukemia cells, we show that H3K79me2/3 is required for maintaining chromatin accessibility, histone acetylation and transcription factor binding specifically at KEEs but not non-KEE enhancers. We go on to show that H3K79me2/3 is essential for maintaining enhancer-promoter interactions at a subset of KEEs. Together, these data implicate H3K79me2/3 as having a functional role at a subset of active enhancers in MLL-AF4 leukemia cells.

| S-EPMC6594956 | biostudies-literature

Role of Dot1L and H3K79 methylation in regulating somatic hypermutation of immunoglobulin genes.

Project description:Somatic hypermutation (SHM) and class-switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation-induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome, and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt's lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than on the constant (C) region, which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sμ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of polymerase II (Pol II) and its S2 phosphorylated form at the IgH locus. Knockout of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of the P-TEFb complex) on the V region, and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreases SHM and the abundance of Pol II S2P at the IgH locus. Since all these factors play a role in transcription elongation, our studies reinforce the idea that the chromatin context and dynamics of transcription are critical for SHM.

| S-EPMC8307847 | biostudies-literature

DOT1L interaction partner AF10 controls patterning of H3K79 methylation and RNA polymerase II to maintain cell identity.

Project description:Histone 3 lysine 79 methylation (H3K79me) is enriched on gene bodies proportional to gene expression levels and serves as a strong barrier for the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs). DOT1L is the sole histone methyltransferase that deposits all three orders-mono (me1), di (me2), and tri (me3) methylation-at H3K79. Here, we leverage genetic and chemical approaches to parse the specific functions of orders of H3K79me in maintaining cell identity. DOT1L interacts with AF10 (Mllt10), which recognizes unmodified H3K27 and boosts H3K79me2/3 methylation. AF10 deletion evicts H3K79me2/3 and reorganizes H3K79me1 to the transcription start site to facilitate iPSC formation in the absence of steady-state transcriptional changes. Instead, AF10 loss redistributes RNA polymerase II to a uniquely pluripotent pattern at highly expressed, rapidly transcribed housekeeping genes. Taken together, we reveal a specific mechanism for H3K79me2/3 located at the gene body in reinforcing cell identity.

| S-EPMC10724070 | biostudies-literature

Dynamics of DOT1L localization and H3K79 methylation during meiotic prophase I in mouse spermatocytes.

Project description:During meiotic prophase I, interactions between maternal and paternal chromosomes, under checkpoint surveillance, establish connections between homologs that promote their accurate distribution to meiotic progeny. In human, faulty meiosis causes aneuploidy resulting in miscarriages and genetic diseases. Meiotic processes occur in the context of chromatin; therefore, histone post-translational modifications are expected to play important roles. Here, we report the cytological distribution of the evolutionarily conserved DOT1L methyltransferase and the different H3K79 methylation states resulting from its activity (mono-, di- and tri-methylation; H3K79me1, me2 and me3, respectively) during meiotic prophase I in mouse spermatocytes. In the wild type, whereas low amounts of H3K79me1 are rather uniformly present throughout prophase I, levels of DOT1L, H3K79me2 and H3K79me3 exhibit a notable increase from pachynema onwards, but with differential subnuclear distribution patterns. The heterochromatic centromeric regions and the sex body are enriched for H3K79me3. In contrast, H3K79me2 is present all over the chromatin, but is largely excluded from the sex body despite the accumulation of DOT1L. In meiosis-defective mouse mutants, the increase of DOT1L and H3K79me is blocked at the same stage where meiosis is arrested. H3K79me patterns, combined with the cytological analysis of the H3.3, γH2AX, macroH2A and H2A.Z histone variants, are consistent with a differential role for these epigenetic marks in male mouse meiotic prophase I. We propose that H3K79me2 is related to transcriptional reactivation on autosomes during pachynema, whereas H3K79me3 may contribute to the maintenance of repressive chromatin at centromeric regions and the sex body.

| S-EPMC3969405 | biostudies-literature

Transcription elongation regulator 1 is a co-integrator of the cell fate determination factor Dachshund homolog 1.

Project description:DACH1 (Dachshund homolog 1) is a key component of the retinal determination gene network and regulates gene expression either indirectly as a co-integrator or through direct DNA binding. The current studies were conducted to understand, at a higher level of resolution, the mechanisms governing DACH1-mediated transcriptional repression via DNA sequence-specific binding. DACH1 repressed gene transcription driven by the DACH1-responsive element (DRE). Recent genome-wide ChIP-Seq analysis demonstrated DACH1 binding sites co-localized with Forkhead protein (FOX) binding sites. Herein, DACH1 repressed, whereas FOX proteins enhanced, both DRE and FOXA-responsive element-driven gene expression. Reduced DACH1 expression using a shRNA approach enhanced FOX protein activity. As DACH1 antagonized FOX target gene expression and attenuated FOX signaling, we sought to identify limiting co-integrator proteins governing DACH1 signaling. Proteomic analysis identified transcription elongation regulator 1 (TCERG1) as the transcriptional co-regulator of DACH1 activity. The FF2 domain of TCERG1 was required for DACH1 binding, and the deletion of FF2 abolished DACH1 trans-repression function. The carboxyl terminus of DACH1 was necessary and sufficient for TCERG1 binding. Thus, DACH1 represses gene transcription through direct DNA binding to the promoter region of target genes by recruiting the transcriptional co-regulator, TCERG1.

| S-EPMC3001014 | biostudies-literature

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