Hanneke Vlaming

The upstreams and downstreams of H3K79 methylation by DOT1L

Histone modifications regulate key processes of eukaryotic genomes. Misregulation of the enzymes that place these modifications can lead to disease. An example of this is DOT1L, the enzyme that can mono-, di-, and trimethylate the nucleosome core on lysine 79 of histone H3 (H3K79). DOT1L plays a role in development and its misregulation has been implicated in several cancers, most notably leukemias caused by a rearrangement of the MLL gene. A DOT1L inhibitor is in clinical trials for these leukemias and shows promising results, yet we are only beginning to understand DOT1L’s function and regulation in the cell. Here, we review what happens upstream and downstream of H3K79 methylation. H3K79 methylation levels are highest in transcribed genes, where H2B ubiquitination can promote DOT1L activity. In addition, DOT1L can be targeted to transcribed regions of the genome by several of its interaction partners. Although methylation levels strongly correlate with transcription, the mechanistic link between the two is unclear and probably context-dependent. Methylation of H3K79 may act through recruiting or repelling effector proteins, but we do not yet know which effectors mediate DOT1L’s functions. Understanding DOT1L biology better will help us to understand the effects of DOT1L inhibitors and may allow the development of alternative strategies to target the DOT1L pathway.

Flexibility in crosstalk between H2B ubiquitination and H3 methylation in vivo

Histone H2B ubiquitination is a dynamic modification that promotes methylation of histone H3K79 and H3K4. This crosstalk is important for the DNA damage response and has been implicated in cancer. Here, we show that in engineered yeast strains, ubiquitins tethered to every nucleosome promote H3K79 and H3K4 methylation from a proximal as well as a more distal site, but only if in a correct orientation. This plasticity indicates that the exact location of the attachment site, the native ubiquitin‐lysine linkage and ubiquitination cycles are not critical for trans‐histone crosstalk in vivo. The flexibility in crosstalk also indicates that other ubiquitination events may promote H3 methylation.

Yeast PP4 Interacts with ATR Homolog Ddc2-Mec1 and Regulates Checkpoint Signaling

Mec1-Ddc2 (ATR-ATRIP) controls the DNA damage checkpoint and shows differential cell-cycle regulation in yeast. To find regulators of Mec1-Ddc2, we exploited a mec1 mutant that retains catalytic activity in G2 and recruitment to stalled replication forks, but which is compromised for the intra-S phase checkpoint. Two screens, one for spontaneous survivors and an E-MAP screen for synthetic growth effects, identified loss of PP4 phosphatase, pph3Δ and psy2Δ, as the strongest suppressors of mec1-100 lethality on HU. Restored Rad53 phosphorylation accounts for part, but not all, of the pph3Δ-mediated survival. Phosphoproteomic analysis confirmed that 94% of the mec1-100-compromised targets on HU are PP4 regulated, including a phosphoacceptor site within Mec1 itself, mutation of which confers damage sensitivity. Physical interaction between Pph3 and Mec1, mediated by cofactors Psy2 and Ddc2, is shown biochemically and through FRET in subnuclear repair foci. This establishes a physical and functional Mec1-PP4 unit for regulating the checkpoint response.

HDAC1 and HDAC2 collectively regulate intestinal stem cell homeostasis

Histone deacetylases (HDACs) are post‐translational modifiers that deacetylate proteins. Despite their crucial role in numerous biological processes, the use of broad‐range HDAC inhibitors (HDACi), has shown clinical efficacy. However, undesired side effects highlight the necessity to better understand the biology of different HDACs and target the relevant HDACs. Using a novel mouse model, in which HDAC1 and HDAC2 can be simultaneously deleted in the intestine of adult mice, we show that the simultaneous deletion of HDAC1 and HDAC2 leads to a rapid loss of intestinal homeostasis. Importantly, this deletion cannot be sustained, and 8 days after initial ablation, stem cells that have escaped HDAC1 or HDAC2 deletion swiftly repopulate the intestinal lining. In vitro ablation of HDAC1 and HDAC2 using intestinal organoid cultures resulted in a down‐regulation of multiple intestinal stem cell markers and functional loss of clonogenic capacity. Importantly, treatment of wild‐type organoids with class I‐specific HDACi MS‐275 also induced a similar loss of stemness, providing a possible rationale for the gastrointestinal side effects often observed in HDACi‐treated patients. In conclusion, these data show that HDAC1 and HDAC2 have a redundant function and are essential to maintain intestinal homeostasis.—Zimberlin, C. D., Lancini, C., Sno, R., Rosekrans, S. L., McLean, C. M., Vlaming, H., van den Brink, G. R., Bots, M., Medema, J. P., Dannenberg, J.‐H. HDAC1 and HDAC2 collectively regulate intestinal stem cell homeostasis. FASEB J. 29, 2070‐2080 (2015). www.fasebj.org

Crosstalk between aging and the epigenome

implicated several additional histone modifiers in life span determination [6,7]. In addition to the organism’s aging pro� gram, stochastic events can also induce epi� genomic changes over time. For example, human monozygotic twins have very similar DNA methylation patterns at birth, but they also show differences that can increase as they get older [8]. These changes may be attributed to environmental in� uences. Another possi� bility is that unavoidable errors occur during the maintenance of epigenetic patterns due to the disruptive nature of transactions at the genome. Transcription by RNA polymerases has the potential to reshuf�e or even erase epi� genetic signals because it requires the transient eviction and subsequent reassembly of histones in the wake of the polymerase. Indeed, several studies suggest that transcription destabilizes chromatin and leads to the partial eviction of (modified) histones and replacement by newly synthesized (unmodified) ones [9]. It is worth noting however, that the introduction of many of the histone modifications in active regions of the genome is promoted by the process of tran� scription initiation or elongation. Therefore, these marks can be maintained even when the histones carrying the marks are evicted [10]. DNA replication is another potential source of epigenetic rearrangements. Ahead of the replication fork, old modified histones dissoci� ate from the DNA and behind the replication fork, histones reassemble on the two daughter strands. The chromatin gaps on the duplicated DNA are complemented by a set of newly synthesized unmodified histones. In order to maintain an epigenetic identity, the cells need to re�establish the modification pattern of the mother cell by modifying the newly synthe� sized histones in the daughter cells. The speed at which this occurs can substantially differ Epigenetic states help maintain cell identity but they are also dynamic entities that respond to signals. Indeed, cells undergoing developmental changes are characterized by global rearrange� ments of the epigenetic landscape. Recent stud� ies suggest that aging is one such epigenome� shifting developmental event. What is more, epigenetic regulators seem to in�uence the aging process. Aging can occur in different contexts. Here we discuss the emerging evidence that both organismal and cellular aging, as well as histone protein aging, have intimate connections to the epigenome. Striking links between organismal aging and epigenome alterations have recently been identi� fied in humans and metazoan model organisms [1]. For example, tissues and cells of aging organ� isms show increased levels or redistribution of heterochromatic marks such as tri �methylation of lysine 9 on histone H3 (H3K9me3) [2,3]. The cause and biological significance of these changes are still unclear; however, a driver for epigenome changes could be an aging pro� gram that changes the expression of chroma� tin�modifying or �demodifying enzymes. For example, loss of H3K27me3 in (prematurely) aging human brains is accompanied by the increased expression of the H3K27�specific demethylase UTX [4]. Aging somatic cells in Caenorhabditis elegans show a similar decline in H3K27me3 and increase in UTX�1 expression [4,5]. Importantly, genetic inactivation of C. elegans UTX�1 prevents the age�induced loss of H3K27me3 and extends life span of the worm via the insulin�signaling pathway, a major life span regulator [4,5]. These findings suggest that loss of H3K27me3, a repressive mark associated with regions of facultative heterochromatin, is not only associated with aging but may in fact be causally involved in the aging process. Recent genetic studies in �ies and worms have

Dot1 histone methyltransferases share a distributive mechanism but have highly diverged catalytic properties

The conserved histone methyltransferase Dot1 establishes an H3K79 methylation pattern consisting of mono-, di- and trimethylation states on histone H3 via a distributive mechanism. This mechanism has been shown to be important for the regulation of the different H3K79 methylation states in yeast. Dot1 enzymes in yeast, Trypanosoma brucei (TbDot1A and TbDot1B, which methylate H3K76) and human (hDot1L) generate very divergent methylation patterns. To understand how these species-specific methylation patterns are generated, the methylation output of the Dot1 enzymes was compared by expressing them in yeast at various expression levels. Computational simulations based on these data showed that the Dot1 enzymes have highly distinct catalytic properties, but share a distributive mechanism. The mechanism of methylation and the distinct rate constants have implications for the regulation of H3K79/K76 methylation. A mathematical model of H3K76 methylation during the trypanosome cell cycle suggests that temporally-regulated consecutive action of TbDot1A and TbDot1B is required for the observed regulation of H3K76 methylation states.

Direct screening for chromatin status on DNA barcodes in yeast delineates the regulome of H3K79 methylation by Dot1

Given the frequent misregulation of chromatin in cancer, it is important to understand the cellular mechanisms that regulate chromatin structure. However, systematic screening for epigenetic regulators is challenging and often relies on laborious assays or indirect reporter read-outs. Here we describe a strategy, Epi-ID, to directly assess chromatin status in thousands of mutants. In Epi-ID, chromatin status on DNA barcodes is interrogated by chromatin immunoprecipitation followed by deep sequencing, allowing for quantitative comparison of many mutants in parallel. Screening of a barcoded yeast knock-out collection for regulators of histone H3K79 methylation by Dot1 identified all known regulators as well as novel players and processes. These include histone deposition, homologous recombination, and adenosine kinase, which influences the methionine cycle. Gcn5, the acetyltransferase within the SAGA complex, was found to regulate histone methylation and H2B ubiquitination. The concept of Epi-ID is widely applicable and can be readily applied to other chromatin features. DOI: http://dx.doi.org/10.7554/eLife.18919.001

Dot1 promotes H2B ubiquitination by a methyltransferase-independent mechanism

Abstract The histone methyltransferase Dot1 is conserved from yeast to human and methylates lysine 79 of histone H3 (H3K79) on the core of the nucleosome. H3K79 methylation by Dot1 affects gene expression and the response to DNA damage, and is enhanced by monoubiquitination of the C-terminus of histone H2B (H2Bub1). To gain more insight into the functions of Dot1, we generated genetic interaction maps of increased-dosage alleles of DOT1. We identified a functional relationship between increased Dot1 dosage and loss of the DUB module of the SAGA co-activator complex, which deubiquitinates H2Bub1 and thereby negatively regulates H3K79 methylation. Increased Dot1 dosage was found to promote H2Bub1 in a dose-dependent manner and this was exacerbated by the loss of SAGA-DUB activity, which also caused a negative genetic interaction. The stimulatory effect on H2B ubiquitination was mediated by the N-terminus of Dot1, independent of methyltransferase activity. Our findings show that Dot1 and H2Bub1 are subject to bi-directional crosstalk and that Dot1 possesses chromatin regulatory functions that are independent of its methyltransferase activity.

Modeling Distributive Histone Modification by Dot1 Methyltransferases: From Mechanism to Biological Insights

Methylation of histone proteins plays a crucial role controlling genome activity. To understand how the responsible histone methyltransferases are regulated it is important to know their fundamental biochemical properties within the cell and relate these to cellular methylation dynamics. Repeated experiment-modeling cycles have led to insights into the in vivo dynamics of methylation of lysine 79 on histone H3 (H3K79) by the methyltransferase Disruptor of Telomeric Silencing 1 (Dot1). Genetic perturbation in yeast, quantitative measurements, and computational modeling were combined to show that Dot1 employs an uncommon, distributive methylation mechanism. A steady-state in vivo methylation model using this information has provided validated explanations for methylation defects in mutants. Subsequent single-cell models have provided insights into the dynamics of H3K79 methylation throughout the cell cycle and uncovered a role for histone protein aging. These integrated modeling approaches will aid in understanding how regulatory mechanisms influence Dot1’s role in gene expression, cell cycle progression, and cancer and can be applied to other methylation systems.