Trans-tail regulation-mediated suppression of cryptic transcription

Crosstalk between post-translational modifications of histone proteins influences the regulation of chromatin structure and gene expression. Among such crosstalk pathways, the best-characterized example is H2B monoubiquitination-mediated H3K4 and H3K79 methylation, which is referred to as trans-tail regulation. Although many studies have investigated the fragmentary effects of this pathway on silencing and transcription, its ultimate contribution to transcriptional control has remained unclear. Recent advances in molecular techniques and genomics have, however, revealed that the trans-tail crosstalk is linked to a more diverse cascade of histone modifications and has various functions in cotranscriptional processes. Furthermore, H2B monoubiquitination sequentially facilitates H3K4 dimethylation and histone sumoylation, thereby providing a binding platform for recruiting Set3 complex proteins, including two histone deacetylases, to restrict cryptic transcription from gene bodies. The removal of both ubiquitin and SUMO, small ubiquitin-like modifier, modifications from histones also facilitates a change in the phosphorylation pattern of the RNA polymerase II C-terminal domain that is required for subsequent transcriptional elongation. Therefore, this review describes recent findings regarding trans-tail regulation-driven processes to elaborate on their contribution to maintaining transcriptional fidelity.

Neuronal Dot1l is a broad mitochondrial gene-repressor associated with human brain aging via H3K79 hypermethylation

Methylation of histone 3 at lysine 79 (H3K79) and its catalyst, disrupter of telomeric silencing (Dot1l), have been coupled to multiple forms of stress like bioenergetic and ER challenges. However, studies on H3K79 methylation and Dot1l in the aging brain and neurons are very limited. This together with increasing evidence of a dynamic neuroepigenome made us wonder if H3K79 methylation and Dot1l could play unknown roles in brain aging and associated disorders. In aged humans, we found strong and consistent hypermethylation of H3K79 in neurons that accumulate lipofuscine, while neuronal Dot1l transcript abundance reacts to bioenergenic and oxidative challenges. Indeed, in dopaminergic neurons we found rapid global H3K79me turnover (<12h). While shortly after reduction of H3K79 methylation, synaptic transcripts decreased while mitochondrial genes, particularly respiratory chain transcripts increased. Strikingly, 6 months after reduction of Dot1l levels, almost solely a variety of mitochondrial genes linked to aging and Parkinsons disease remained increased. These profiles are in much detail inverse to those described in hallmark PD and aging studies and associate Dot1l and H3K79me with neuronal stress in the aging brain while putting Dot1l forward as dynamic master regulator of mitochondrial transcription in dopamine neurons.

Synthesis and Biological Activity of a Cytostatic Inhibitor of MLLr Leukemia Targeting the DOT1L Protein

Histone methyltransferase DOT1L catalyzes mono-, di- and trimethylation of histone 3 at lysine residue 79 (H3K79) and hypermethylation of H3K79 has been linked to the development of acute leukemias characterized by the MLL (mixed-lineage leukemia) rearrangements (MLLr cells). The inhibition of H3K79 methylation inhibits MLLr cells proliferation, and an inhibitor specific for DOT1L, pinometostat, was in clinical trials (Phase Ib/II). However, the compound showed poor pharmacological properties. Thus, there is a need to find new potent inhibitors of DOT1L for the treatment of rearranged leukemias. Here we present the design, synthesis, and biological evaluation of a small molecule that inhibits in the nM level the enzymatic activity of hDOT1L, H3K79 methylation in MLLr cells with comparable potency to pinometostat, associated with improved metabolic stability and a characteristic cytostatic effect.

Wnt Pathway Regulation by DOT1L Mediated Methylation of H3K79 in Non-Small Cell Lung Cancer

The Wnt pathway is a developmental pathway that is abnormally upregulated in cancer. Our current lab results suggest that decreased H3K79 methylation levels are associated with decreased DNMT1 activity and binding to the WIF1 5’ region, upregulation of WIF1 protein levels, and suppression of Wnt signaling in lung cancer cell lines. We therefore hypothesize that the silencing of the WIF1 gene seen in many lung cancers involves upregulation of H3K79 methylation. We aim to knock down DOT1L and AF10, H3K79me3 mediators, to diminish Wnt expression in non-small cell lung cancers (NSCLCs). NSCLC lines including the A549 and H460 lines will be transfected with shRNAs for DOT1L and AF10; Western blotting will be used to confirm knockdown of DOT1L, AF10, and WIF1 expression. Wnt signaling will be measured by TOPFlash reporter assay, and by western blotting of nuclear β-catenin from total and fractionated cell lysates. We will then measure lung cancer cell growth, apoptosis and senescence.

Non-canonical H3K79me2-dependent pathways promote the survival of MLL-rearranged leukemia

MLL-rearranged leukemia depends on H3K79 methylation. Depletion of this transcriptionally-activating mark by DOT1L deletion or high concentrations of the inhibitor pinometostat downregulates HOXA9 and MEIS1, and consequently reduces leukemia survival. Yet some MLL-rearranged leukemias are inexplicably susceptible to low-dose pinometostat, far below concentrations that downregulate this canonical proliferation pathway. In this context, we define alternative proliferation pathways that more directly derive from H3K79me2 loss. By ICeChIP-seq, H3K79me2 is markedly depleted at pinometostat-downregulated and MLL-fusion targets, with paradoxical increases of H3K4me3 and loss of H3K27me3. Although downregulation of polycomb components accounts for some of the proliferation defect, transcriptional downregulation of FLT3 is the major pathway. Loss-of-FLT3-function recapitulates the cytotoxicity and gene expression consequences of low-dose pinometostat, whereas overexpression of constitutively active STAT5A, a target of FLT3-ITD-signalling, largely rescues these defects. This pathway also depends on MLL1, indicating combinations of DOT1L, MLL1 and FLT3 inhibitors should be explored for treating FLT3-mutant leukemia.

Putting the DOT on IL1A

IL-1α is an upstream component of the senescence-associated secretory phenotype. In this issue, Leon et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202008101) show that DOT1L-mediated H3K79 methylation at the IL1A gene plays a key role in its induction during oncogene-induced senescence.

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

Background The 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. Results We 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. Conclusions Our 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.

Hypoxia induces transcription of DOT1L in articular cartilage to protect against osteoarthritis

Osteoarthritis is the most prevalent joint disease worldwide and a leading source of pain and disability. To date, this disease lacks curative treatment as underlying molecular mechanisms remain largely unknown. The histone methyltransferase DOT1L protects against osteoarthritis, and DOT1L-mediated H3K79 methylation is reduced in human and mouse osteoarthritic joints. Thus, restoring DOT1L function seems to be critical to preserve joint health. However, DOT1L-regulating molecules and networks remain elusive, in the joint and beyond. Here, we identify transcription factors and networks that regulate DOT1L gene expression using a novel bioinformatics pipeline. Thereby, we unravel an undiscovered link between the hypoxia pathway and DOT1L. We provide unprecedented evidence that hypoxia enhances DOT1L expression and H3K79 methylation via hypoxia-inducible factor-1 alpha (HIF-1α). Importantly, we demonstrate that DOT1L contributes to the protective effects of hypoxia in articular cartilage and osteoarthritis. Intra-articular treatment with a selective hypoxia mimetic in mice after surgical induction of osteoarthritis restores DOT1L function and stalls disease progression. Collectively, our data unravel a novel molecular mechanism that protects against osteoarthritis with hypoxia inducing DOT1L transcription in cartilage. Local treatment with a selective hypoxia mimetic in the joint restores DOT1L function and could be an attractive therapeutic strategy for osteoarthritis.

DOT1L Regulates Thermogenic Adipocyte Differentiation and Function via Modulating H3K79 Methylation

Brown and beige adipocytes are characterized as thermogenic adipocytes and have great potential for treating obesity and associated metabolic diseases. Here, we identify a conserved mammalian lysine 79 of histone H3 (H3K79) methyltransferase, disruptor of telomeric silencing -1 like (DOT1L), as a new epigenetic regulator that controls thermogenic adipocyte differentiation and function. We show that deletion of DOT1L in thermogenic adipocytes potently protects mice from diet-induced obesity, improves glucose homeostasis, alleviates hepatic steatosis, and facilitates adaptive thermogenesis<i> in vivo</i>. Loss of DOT1L in primary preadipocytes significantly promotes brown and beige adipogenesis and thermogenesis<i> in vitro</i>. Mechanistically, DOT1L epigenetically regulates the BAT-selective gene program through modulating H3K79 methylation, in particular H3K79me2 modification. Thus, our study demonstrates that DOT1L exerts an important role in energy homeostasis by regulating thermogenic adipocyte differentiation and function.