HBO1, a MYSTerious KAT and its links to cancer.

The histone acetyltransferase HBO1, also known as KAT7, is a major chromatin modifying enzyme responsible for H3 and H4 acetylation. It is found within two distinct tetrameric complexes, the JADE subunit-containing complex and BRPF subunit-containing complex. The HBO1-JADE complex acetylates lysine 5, 8 and 12 of histone H4, and the HBO1-BRPF complex acetylates lysine 14 of histone H3. HBO1 regulates gene transcription, DNA replication, DNA damage repair, and centromere function. It is involved in diverse signaling pathways and plays crucial roles in development and stem cell biology. Recent work has established a strong relationship of HBO1 with the histone methyltransferase MLL/KMT2A in acute myeloid leukemia. Here, we discuss functional and pathological links of HBO1 to cancer, highlighting the underlying mechanisms that may pave the way to the development of novel anti-cancer therapies.

ASXLs binding to the PHD2/3 fingers of MLL4 provides a mechanism for the recruitment of BAP1 to active enhancers.

The human methyltransferase and transcriptional coactivator MLL4 and its paralog MLL3 are frequently mutated in cancer. MLL4 and MLL3 monomethylate histone H3K4 and contain a set of uncharacterized PHD fingers. Here, we report a novel function of the PHD2 and PHD3 (PHD2/3) fingers of MLL4 and MLL3 that bind to ASXL2, a component of the Polycomb repressive H2AK119 deubiquitinase (PR-DUB) complex. The structure of MLL4 PHD2/3 in complex with the MLL-binding helix (MBH) of ASXL2 and mutational analyses reveal the molecular mechanism which is conserved in homologous ASXL1 and ASXL3. The native interaction of the Trithorax MLL3/4 complexes with the PR-DUB complex in vivo depends solely on MBH of ASXL1/2, coupling the two histone modifying activities. ChIP-seq analysis in embryonic stem cells demonstrates that MBH of ASXL1/2 is required for the deubiquitinase BAP1 recruitment to MLL4-bound active enhancers. Our findings suggest an ASXL1/2-dependent functional link between the MLL3/4 and PR-DUB complexes.

The Functional Relationship Between RNA Splicing and the Chromatin Landscape.

Chromatin is a highly regulated and dynamic structure that has been shown to play an essential role in transcriptional and co-transcriptional regulation. In the context of RNA splicing, early evidence suggested a loose connection between the chromatin landscape and splicing. More recently, it has been shown that splicing occurs in a co-transcriptional manner, meaning that the splicing process occurs in the context of chromatin. Experimental and computational evidence have also shown that chromatin dynamics can influence the splicing process and vice versa. However, much of this evidence provides mainly correlative relationships between chromatin and splicing with just a few concrete examples providing defined molecular mechanisms by which these two processes are functionally related. Nevertheless, it is clear that chromatin and RNA splicing are tightly interconnected to one another. In this review, we highlight the current state of knowledge of the relationship between chromatin and splicing.

Guiding the HBO1 complex function through the JADE subunit.

JADE is a core subunit of the HBO1 acetyltransferase complex that regulates developmental and epigenetic programs and promotes gene transcription. Here we describe the mechanism by which JADE facilitates recruitment of the HBO1 complex to chromatin and mediates its enzymatic activity. Structural, genomic and complex assembly in vivo studies show that the PZP (PHD1-zinc-knuckle-PHD2) domain of JADE engages the nucleosome through binding to histone H3 and DNA and is necessary for the association with chromatin targets. Recognition of unmethylated H3K4 by PZP directs enzymatic activity of the complex toward histone H4 acetylation, whereas H3K4 hypermethylation alters histone substrate selectivity. We demonstrate that PZP contributes to leukemogenesis, augmenting transforming activity of the NUP98-JADE2 fusion. Our findings highlight biological consequences and the impact of the intact JADE subunit on genomic recruitment, enzymatic function and pathological activity of the HBO1 complex.

The immediate-early protein 1 of human herpesvirus 6B interacts with NBS1 and inhibits ATM signaling.

Viral infection often trigger an ATM serine/threonine kinase (ATM)-dependent DNA damage response in host cells that suppresses viral replication. Viruses evolved different strategies to counteract this antiviral surveillance system. Here, we report that human herpesvirus 6B (HHV-6B) infection causes genomic instability by suppressing ATM signaling in host cells. Expression of immediate-early protein 1 (IE1) phenocopies this phenotype and blocks homology-directed double-strand break repair. Mechanistically, IE1 interacts with NBS1, and inhibits ATM signaling through two distinct domains. HHV-6B seems to efficiently inhibit ATM signaling as further depletion of either NBS1 or ATM do not significantly boost viral replication in infected cells. Interestingly, viral integration of HHV-6B into the host's telomeres is not strictly dependent on NBS1, challenging current models where integration occurs through homology-directed repair. Given that spontaneous IE1 expression has been detected in cells of subjects with inherited chromosomally-integrated form of HHV-6B (iciHHV-6B), a condition associated with several health conditions, our results raise the possibility of a link between genomic instability and the development of iciHHV-6-associated diseases.

JAZF1: A metabolic actor subunit of the NuA4/TIP60 chromatin modifying complex.

The multisubunit NuA4/TIP60 complex is a lysine acetyltransferase, chromatin modifying factor and gene co-activator involved in diverse biological processes. The past decade has seen a growing appreciation for its role as a metabolic effector and modulator. However, molecular insights are scarce and often contradictory, underscoring the need for further mechanistic investigation. A particularly exciting route emerged with the recent identification of a novel subunit, JAZF1, which has been extensively linked to metabolic homeostasis. This review summarizes the major findings implicating NuA4/TIP60 in metabolism, especially in light of JAZF1 as part of the complex.

MORF and MOZ acetyltransferases target unmethylated CpG islands through the winged helix domain.

Human acetyltransferases MOZ and MORF are implicated in chromosomal translocations associated with aggressive leukemias. Oncogenic translocations involve the far amino terminus of MOZ/MORF, the function of which remains unclear. Here, we identified and characterized two structured winged helix (WH) domains, WH1 and WH2, in MORF and MOZ. WHs bind DNA in a cooperative manner, with WH1 specifically recognizing unmethylated CpG sequences. Structural and genomic analyses show that the DNA binding function of WHs targets MORF/MOZ to gene promoters, stimulating transcription and H3K23 acetylation, and WH1 recruits oncogenic fusions to HOXA genes that trigger leukemogenesis. Cryo-EM, NMR, mass spectrometry and mutagenesis studies provide mechanistic insight into the DNA-binding mechanism, which includes the association of WH1 with the CpG-containing linker DNA and binding of WH2 to the dyad of the nucleosome. The discovery of WHs in MORF and MOZ and their DNA binding functions could open an avenue in developing therapeutics to treat diseases associated with aberrant MOZ/MORF acetyltransferase activities.

A balancing act: interactions within NuA4/TIP60 regulate picNuA4 function in Saccharomyces cerevisiae and humans.

The NuA4 lysine acetyltransferase complex acetylates histone and nonhistone proteins and functions in transcription regulation, cell cycle progression, and DNA repair. NuA4 harbors an interesting duality in that its catalytic module can function independently and distinctly as picNuA4. At the molecular level, picNuA4 anchors to its bigger brother via physical interactions between the C-terminus of Epl1 and the HSA domain of Eaf1, the NuA4 central scaffolding subunit. This is reflected at the regulatory level, as picNuA4 can be liberated genetically from NuA4 by disrupting the Epl1-Eaf1 interaction. As such, removal of either Eaf1 or the Epl1 C-terminus offers a unique opportunity to elucidate the contributions of Eaf1 and Epl1 to NuA4 biology and in turn their roles in balancing picNuA4 and NuA4 activities. Using high-throughput genetic and gene expression profiling, and targeted functional assays to compare eaf1Δ and epl1-CΔ mutants, we found that EAF1 and EPL1 had both overlapping and distinct roles. Strikingly, loss of EAF1 or its HSA domain led to a significant decrease in the amount of picNuA4, while loss of the Epl1 C-terminus increased picNuA4 levels, suggesting starkly opposing effects on picNuA4 regulation. The eaf1Δ epl1-CΔ double mutants resembled the epl1-CΔ single mutants, indicating that Eaf1's role in picNuA4 regulation depended on the Epl1 C-terminus. Key aspects of this regulation were evolutionarily conserved, as truncating an Epl1 homolog in human cells increased the levels of other picNuA4 subunits. Our findings suggested a model in which distinct aspects of the Epl1-Eaf1 interaction regulated picNuA4 amount and activity.

Binding of the SARS-CoV-2 envelope E protein to human BRD4 is essential for infection.

Emerging new variants of SARS-CoV-2 and inevitable acquired drug resistance call for the continued search of new pharmacological targets to fight the potentially fatal infection. Here, we describe the mechanisms by which the E protein of SARS-CoV-2 hijacks the human transcriptional regulator BRD4. We found that SARS-CoV-2 E is acetylated in vivo and co-immunoprecipitates with BRD4 in human cells. Bromodomains (BDs) of BRD4 bind to the C-terminus of the E protein, acetylated by human acetyltransferase p300, whereas the ET domain of BRD4 recognizes the unmodified motif of the E protein. Inhibitors of BRD4 BDs, JQ1 or OTX015, decrease SARS-CoV-2 infectivity in lung bronchial epithelial cells, indicating that the acetyllysine binding function of BDs is necessary for the virus fitness and that BRD4 represents a potential anti-COVID-19 target. Our findings provide insight into molecular mechanisms that contribute to SARS-CoV-2 pathogenesis and shed light on a new strategy to block SARS-CoV-2 infection.

Recurrent chromosomal translocations in sarcomas create a megacomplex that mislocalizes NuA4/TIP60 to Polycomb target loci.

Chromosomal translocations frequently promote carcinogenesis by producing gain-of-function fusion proteins. Recent studies have identified highly recurrent chromosomal translocations in patients with endometrial stromal sarcomas (ESSs) and ossifying fibromyxoid tumors (OFMTs), leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a coactivator that acetylates chromatin and loads the H2A.Z histone variant, PHF1 is part of the Polycomb repressive complex 2 (PRC2) linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27. In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation. The chimeric protein assembles a megacomplex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks in the genome, in particular over an entire topologically associating domain including part of the HOXD cluster. This is linked to aberrant gene expression-most notably increased expression of PRC2 target genes. Furthermore, we show that JAZF1-implicated with a PRC2 component in the most frequent translocation in ESSs, JAZF1-SUZ12-is a potent transcription activator that physically associates with NuA4/TIP60, its fusion creating outcomes similar to those of EPC1-PHF1 Importantly, the specific increased expression of PRC2 targets/HOX genes was also confirmed with ESS patient samples. Altogether, these results indicate that most chromosomal translocations linked to these sarcomas use the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes, leading to mislocalization of histone marks and aberrant Polycomb target gene expression.

Oncogenic ZMYND11-MBTD1 fusion protein anchors the NuA4/TIP60 histone acetyltransferase complex to the coding region of active genes.

A recurrent chromosomal translocation found in acute myeloid leukemia leads to an in-frame fusion of the transcription repressor ZMYND11 to MBTD1, a subunit of the NuA4/TIP60 histone acetyltransferase complex. To understand the abnormal molecular events that ZMYND11-MBTD1 expression can create, we perform a biochemical and functional characterization comparison to each individual fusion partner. ZMYND11-MBTD1 is stably incorporated into the endogenous NuA4/TIP60 complex, leading to its mislocalization on the body of genes normally bound by ZMYND11. This can be correlated to increased chromatin acetylation and altered gene transcription, most notably on the MYC oncogene, and alternative splicing. Importantly, ZMYND11-MBTD1 expression favors Myc-driven pluripotency during embryonic stem cell differentiation and self-renewal of hematopoietic stem/progenitor cells. Altogether, these results indicate that the ZMYND11-MBTD1 fusion functions primarily by mistargeting the NuA4/TIP60 complex to the body of genes, altering normal transcription of specific genes, likely driving oncogenesis in part through the Myc regulatory network.

MRG Proteins Are Shared by Multiple Protein Complexes With Distinct Functions.

MRG15/MORF4L1 is a highly conserved protein in eukaryotes that contains a chromodomain (CHD) recognizing methylation of lysine 36 on histone H3 (H3K36me3) in chromatin. Intriguingly, it has been reported in the literature to interact with several different factors involved in chromatin modifications, gene regulation, alternative mRNA splicing, and DNA repair by homologous recombination. To get a complete and reliable picture of associations in physiological conditions, we used genome editing and tandem affinity purification to analyze the stable native interactome of human MRG15, its paralog MRGX/MORF4L2 that lacks the CHD, and MRGBP (MRG-binding protein) in isogenic K562 cells. We found stable interchangeable association of MRG15 and MRGX with the NuA4/TIP60 histone acetyltransferase/chromatin remodeler, Sin3B histone deacetylase/demethylase, ASH1L histone methyltransferase, and PALB2-BRCA2 DNA repair protein complexes. These associations were further confirmed and analyzed by CRISPR tagging of endogenous proteins and comparison of expressed isoforms. Importantly, based on structural information, point mutations could be introduced that specifically disrupt MRG15 association with some complexes but not others. Most interestingly, we also identified a new abundant native complex formed by MRG15/X-MRGBP-BRD8-EP400NL (EP400 N-terminal like) that is functionally similar to the yeast TINTIN (Trimer Independent of NuA4 for Transcription Interactions with Nucleosomes) complex. Our results show that EP400NL, being homologous to the N-terminal region of NuA4/TIP60 subunit EP400, creates TINTIN by competing for BRD8 association. Functional genomics indicate that human TINTIN plays a role in transcription of specific genes. This is most likely linked to the H4ac-binding bromodomain of BRD8 along the H3K36me3-binding CHD of MRG15 on the coding region of transcribed genes. Taken together, our data provide a complete detailed picture of human MRG proteins-associated protein complexes, which are essential to understand and correlate their diverse biological functions in chromatin-based nuclear processes.

New insights into the DNA repair pathway choice with NuA4/TIP60.

In eukaryotic cells, DNA double-strand breaks (DSBs) can be repaired through two main pathways, non-homologous end-joining (NHEJ) or homologous recombination (HR). The selection of the repair pathway choice is governed by an antagonistic relationship between repair factors specific to each pathway, in a cell cycle-dependent manner. The molecular mechanisms of this decision implicate post-translational modifications of chromatin surrounding the break. Here, we discuss the recent advances regarding the function of the NuA4/TIP60 histone acetyltransferase/chromatin remodeling complex during DSBs repair. In particular, we emphasise the contribution of NuA4/TIP60 in repair pathway choice, in collaboration with the SAGA acetyltransferase complex, and how they regulate chromatin dynamics, modify non-histone substrates to allow DNA end resection and recombination.

Rap1 regulates TIP60 function during fate transition between two-cell-like and pluripotent states.

In mammals, the conserved telomere binding protein Rap1 serves a diverse set of nontelomeric functions, including activation of the NF-kB signaling pathway, maintenance of metabolic function in vivo, and transcriptional regulation. Here, we uncover the mechanism by which Rap1 modulates gene expression. Using a separation-of-function allele, we show that Rap1 transcriptional regulation is largely independent of TRF2-mediated binding to telomeres and does not involve direct binding to genomic loci. Instead, Rap1 interacts with the TIP60/p400 complex and modulates its histone acetyltransferase activity. Notably, we show that deletion of Rap1 in mouse embryonic stem cells increases the fraction of two-cell-like cells. Specifically, Rap1 enhances the repressive activity of Tip60/p400 across a subset of two-cell-stage genes, including Zscan4 and the endogenous retrovirus MERVL. Preferential up-regulation of genes proximal to MERVL elements in Rap1-deficient settings implicates these endogenous retroviral elements in the derepression of proximal genes. Altogether, our study reveals an unprecedented link between Rap1 and the TIP60/p400 complex in the regulation of pluripotency.

NuA4 and H2A.Z control environmental responses and autotrophic growth in Arabidopsis.

Nucleosomal acetyltransferase of H4 (NuA4) is an essential transcriptional coactivator in eukaryotes, but remains poorly characterized in plants. Here, we describe Arabidopsis homologs of the NuA4 scaffold proteins Enhancer of Polycomb-Like 1 (AtEPL1) and Esa1-Associated Factor 1 (AtEAF1). Loss of AtEAF1 results in inhibition of growth and chloroplast development. These effects are stronger in the Atepl1 mutant and are further enhanced by loss of Golden2-Like (GLK) transcription factors, suggesting that NuA4 activates nuclear plastid genes alongside GLK. We demonstrate that AtEPL1 is necessary for nucleosomal acetylation of histones H4 and H2A.Z by NuA4 in vitro. These chromatin marks are diminished genome-wide in Atepl1, while another active chromatin mark, H3K9 acetylation (H3K9ac), is locally enhanced. Expression of many chloroplast-related genes depends on NuA4, as they are downregulated with loss of H4ac and H2A.Zac. Finally, we demonstrate that NuA4 promotes H2A.Z deposition and by doing so prevents spurious activation of stress response genes.

The MOZ-BRPF1 acetyltransferase complex in epigenetic crosstalk linked to gene regulation, development, and human diseases.

Acetylation of lysine residues on histone tails is an important post-translational modification (PTM) that regulates chromatin dynamics to allow gene transcription as well as DNA replication and repair. Histone acetyltransferases (HATs) are often found in large multi-subunit complexes and can also modify specific lysine residues in non-histone substrates. Interestingly, the presence of various histone PTM recognizing domains (reader domains) in these complexes ensures their specific localization, enabling the epigenetic crosstalk and context-specific activity. In this review, we will cover the biochemical and functional properties of the MOZ-BRPF1 acetyltransferase complex, underlining its role in normal biological processes as well as in disease progression. We will discuss how epigenetic reader domains within the MOZ-BRPF1 complex affect its chromatin localization and the histone acetyltransferase specificity of the complex. We will also summarize how MOZ-BRPF1 is linked to development via controlling cell stemness and how mutations or changes in expression levels of MOZ/BRPF1 can lead to developmental disorders or cancer. As a last touch, we will review the latest drug candidates for these two proteins and discuss the therapeutic possibilities.

Characterization of multiple interactions between the envelope E protein of SARS-CoV-2 and human BRD4.

The SARS-CoV-2 envelope (E) protein hijacks human BRD4 (bromodomain and extra-terminal domain protein 4). Here, we describe a protocol to characterize the interaction of the acetylated E protein with BRD4 in vivo. We detail steps to use NMR spectroscopy to map the binding interface and include steps to monitor the effect of BRD4 inhibitors in SARS-CoV-2-infected human lung bronchial epithelial cells. This approach could be applied to study interactions involving other viral and human proteins. For complete details on the use and execution of this protocol, please refer to Vann et al. (2022).1.

Approaches to Study Native Chromatin-Modifying Complex Activities and Functions.

The modification of histones-the structural components of chromatin-is a central topic in research efforts to understand the mechanisms regulating genome expression and stability. These modifications frequently occur through associations with multisubunit complexes, which contain active enzymes and additional components that orient their specificity and read the histone modifications that comprise epigenetic signatures. To understand the functions of these modifications it is critical to study the enzymes and substrates involved in their native contexts. Here, we describe experimental approaches to purify native chromatin modifiers complexes from mammalian cells and to produce recombinant nucleosomes that are used as substrates to determine the activity of the complex. In addition, we present a novel approach, similar to the yeast anchor-away system, to study the functions of essential chromatin modifiers by quickly inducing their depletion from the nucleus. The step-by-step protocols included will help standardize these approaches in the research community, enabling convincing conclusions about the specificities and functions of these crucial regulators of the eukaryotic genome.

Antagonistic relationship of NuA4 with the non-homologous end-joining machinery at DNA damage sites.

The NuA4 histone acetyltransferase complex, apart from its known role in gene regulation, has also been directly implicated in the repair of DNA double-strand breaks (DSBs), favoring homologous recombination (HR) in S/G2 during the cell cycle. Here, we investigate the antagonistic relationship of NuA4 with non-homologous end joining (NHEJ) factors. We show that budding yeast Rad9, the 53BP1 ortholog, can inhibit NuA4 acetyltransferase activity when bound to chromatin in vitro. While we previously reported that NuA4 is recruited at DSBs during the S/G2 phase, we can also detect its recruitment in G1 when genes for Rad9 and NHEJ factors Yku80 and Nej1 are mutated. This is accompanied with the binding of single-strand DNA binding protein RPA and Rad52, indicating DNA end resection in G1 as well as recruitment of the HR machinery. This NuA4 recruitment to DSBs in G1 depends on Mre11-Rad50-Xrs2 (MRX) and Lcd1/Ddc2 and is linked to the hyper-resection phenotype of NHEJ mutants. It also implicates NuA4 in the resection-based single-strand annealing (SSA) repair pathway along Rad52. Interestingly, we identified two novel non-histone acetylation targets of NuA4, Nej1 and Yku80. Acetyl-mimicking mutant of Nej1 inhibits repair of DNA breaks by NHEJ, decreases its interaction with other core NHEJ factors such as Yku80 and Lif1 and favors end resection. Altogether, these results establish a strong reciprocal antagonistic regulatory function of NuA4 and NHEJ factors in repair pathway choice and suggests a role of NuA4 in alternative repair mechanisms in situations where some DNA-end resection can occur in G1.

DNA Damage-Induced Phosphorylation of Histone H2A at Serine 15 Is Linked to DNA End Resection.

The repair of DNA double-strand breaks (DSBs) occurs in chromatin, and several histone posttranslational modifications have been implicated in the process. Modifications of the histone H2A N-terminal tail have also been linked to DNA damage response, through acetylation or ubiquitination of lysine residues that regulate repair pathway choice. Here, we characterize a new DNA damage-induced phosphorylation on chromatin, at serine 15 of H2A in yeast. We show that this SQ motif functions independently of the classical S129 C-terminal site (γ-H2A) and that mutant-mimicking constitutive phosphorylation increases cell sensitivity to DNA damage. H2AS129ph is induced by Tel1ATM and Mec1ATR, and the loss of Lcd1ATRIP or Mec1 signaling decreases γ-H2A spreading distal to the DSB. In contrast, H2AS15ph is completely dependent on Lcd1ATRIP, indicating that this modification only happens when end resection is engaged. This is supported by an increase in replication protein A (RPA) and a decrease in DNA signal near the DSB in H2A-S15E phosphomimic mutants, indicating higher resection. In mammals, this serine is replaced by a lysine (H2AK15) which undergoes an acetyl-monoubiquityl switch to regulate binding of 53BP1 and resection. This regulation seems functionally conserved with budding yeast H2AS15 and 53BP1-homolog Rad9, using different posttranslational modifications between organisms but achieving the same function.