The human acetyltransferase complex HBO1 is implicated in cancer and developmental diseases. Here, we report the chromatin association mechanisms for scaffolding subunits of the complex, the JADE and BRPF paralogs. Structural and biochemical studies reveal a distinct paralog-specific engagement of the PZP domains of JADE1/2/3 and BRPF1 with the nucleosome. We show that only JADE1's PZP domain possesses a strong intrinsic DNA binding activity and binds to an almost entire histone H3 tail, forming a tight complex with the nucleosome. In contrast, the BRPF1 PZP domain's association with the nucleosome is a thousand-fold weaker due to its inability to engage with the large portion of H3 tail and a weak binding to DNA. These results suggest that the JADE1 PZP domain can protect the H3 tail from being modified, but the BRPF1 PZP domain cannot, which could contribute to functional differences observed in the HBO1 complexes. We demonstrate non-redundant functions of the paralogs throughout embryonic development and embryonic stem cell pluripotency, during cardiac cell differentiation, and in differentiated liver cells. Our findings provide insights into the diverse mechanisms of action of the HBO1 complex and help to understand how the scaffolding subunits mediate its different cellular functions.

Ribosome biogenesis is a crucial process requiring enormous transcriptional output. In budding yeast, the expression of 138 ribosomal protein (RP) genes and over 200 ribosome biogenesis (RiBi) genes is regulated by an intricate network of factors, including the nutrient-sensitive transcription activator Sfp1 and the NuA4 coactivator/acetyltransferase complex. Nutrient starvation or inhibition of target of rapamycin complex 1 by rapamycin leads to repression of RP and RiBi genes, in part through blocking Sfp1 nuclear localization and NuA4-dependent chromatin acetylation. Here, we demonstrate that Sfp1 physically interacts with NuA4 in a target of rapamycin complex 1-dependent manner. Our results indicate that Sfp1, along with NuA4, regulate the transcription of RiBi and RP genes via distinct mechanisms depending on promoter architectures. Sfp1 promotes histone acetylation at the promoters without affecting NuA4 recruitment. In contrast, NuA4 does impact Sfp1 binding but specifically at two classes of RP genes. Importantly, NuA4 acetylates Sfp1 at lysines 655 and 657, regulating its function. Cells expressing Sfp1 with acetyl-mimicking mutations exhibit increased expression of RiBi genes while RP genes remain stable. However, the same mutants lead to the loss of Sfp1 binding/activity at RiBi genes when cells are under non-optimal growth conditions. Mimicking constitutive acetylation of Sfp1 also limits the transcriptional burst of RP genes upon addition of glucose. Altogether, these results draw an intricate functional relationship between Sfp1 and NuA4 to control ribosome biogenesis, fine-tuning transcription output in different growth conditions.

The epigenetic cofactor ENL (eleven-nineteen-leukemia) and the acetyltransferase MOZ (monocytic leukemia zinc finger) have vital roles in transcriptional regulation and are implicated in aggressive forms of leukemia. Here, we describe the mechanistic basis for the intertwined association of ENL and MOZ. Genomic analysis shows that ENL and MOZ co-occupy active promoters and that MOZ recruits ENL to its gene targets. Structural studies reveal a multivalent assembly of ENL at the intrinsically disordered region (IDR) of MOZ. While the extraterminal (ET) domain of ENL recognizes the canonical ET-binding motif in IDR, the YEATS domains of ENL and homologous AF9 bind to a set of acetylation sites in the MOZ IDR that are generated by the acetyltransferase CBP (CREB-binding protein). Our findings suggest a multifaceted acetylation-dependent and independent coupling of ENL, MOZ and CBP/p300, which may contribute to leukemogenic activities of the ENL-MOZ assembly and chromosomal translocations of ENL, MOZ and CBP/p300.

Histone post-translational modifications (PTMs) are crucial to eukaryotic genome regulation, with a range of reported functions and mechanisms of action. Though often studied individually, it has long been recognized that the modifications function by combinatorial synergy or antagonism. Interplay may involve PTMs on the same histone, within the same nucleosome (containing a histone octamer), or between nucleosomes in higher-order chromatin. Given this, the field must distinguish ever greater complexity, and the context in which it is studied, with brevity and precision. The proteoform was introduced to define individual forms of a protein by sequence and PTMs, followed by the nucleoform to describe the particular gathering of histones within an individual nucleosome. There is now a need to define specific forms of these entities in prose while providing space for experimental nuance. To this end, we introduce a nomenclature that can express discrete PTMs, proteoforms, nucleoforms, or situations where defined PTMs exist in an uncertain context. Though specifically designed for the chromatin field, adaptions of the framework could be used to describe-and thus dissect-how proteoforms are configured in functionally distinct complexes across biology.

The histone H3K36-specific methyltransferase ASH1L plays a critical role in development and is frequently dysregulated in human diseases, particularly cancer. Here, we report on the biological functions of the C-terminal region of ASH1L encompassing a bromodomain (ASH1LBD), a plant homeodomain (ASH1LPHD) finger, and a bromo-adjacent homology (ASH1LBAH) domain, structurally characterize these domains, describe their mechanisms of action, and explore functional crosstalk between them. We find that ASH1LPHD recognizes H3K4me2/3, whereas the neighboring ASH1LBD and ASH1LBAH have DNA binding activities. The DNA binding function of ASH1LBAH is a driving force for the association of ASH1L with the linker DNA in the nucleosome, and the large interface with ASH1LPHD stabilizes the ASH1LBAH fold, merging two domains into a single module. We show that ASH1L is involved in embryonic stem cell differentiation and co-localizes with H3K4me3 but not with H3K36me2 at transcription start sites of target genes and genome wide, and that the interaction of ASH1LPHD with H3K4me3 is inhibitory to the H3K36me2-specific catalytic activity of ASH1L. Our findings shed light on the mechanistic details by which the C-terminal domains of ASH1L associate with chromatin and regulate the enzymatic function of ASH1L.

For functional studies of proteins in cells, effective tools that trigger rapid and efficient inactivation of the proteins are indispensable. The anchor-away (AA) system is designed to deplete nuclear-localized proteins through cellular relocation. The target protein is sequestered by an anchor receptor protein from the nucleus to the cytoplasm. The depletion is achieved through the formation of a stable complex that is solely driven by the addition of rapamycin, between the FRB domain of human mTOR and the FKBP12-binding domain of the FRP protein. The target protein and the receptor protein are fused through genetic recombination with the FRB and FKBP12 domains, respectively, to mediate the interaction. The export of the nuclear protein of interest is executed by the receptor protein, i.e., ribosomal protein RPL13A, which is transiently imported into the nucleus and then exported to the cytoplasm within the pre-ribosome. The proteins of interest can be depleted within minutes although the depleting conditions need to be optimized. AA is a valuable tool to help dissect the biological functions of nuclear proteins, especially ones that are essential for cell viability. In this chapter, we describe the steps to construct the necessary strains and several biochemical and functional experiments to confirm the functional abrogation of target proteins by AA.

Human additional sex combs like (ASXL) proteins are involved in the maintenance of both transcriptional activation and repression through their ability to bind multiple chromatin regulators, including two tumor suppressors: deubiquitinase BAP1 and methyltransferase MLL4 (KMT2D). The ASXL genes are often altered in colorectal cancer (CRC), and ASXL1 is one of the four hub genes related to the pathogenesis of CRC. Here, we show that MLL4 and BAP1 interdependently target specific genomic regions and positively or negatively regulate expression of a subset of genes in the human colon carcinoma HCT116 cells. MLL4 and BAP1 colocalize on a subset of enhancers and promoters in an interdependent manner. Genomic distribution of BAP1 in CRC cells differs from that in ESCs, with substantially more BAP1 binding sites identified on enhancers and promoters in HCT116 cells. MLL4 occupancy on MLL4+ BAP1+ genomic regions depends on functional ASXLs that interact with both MLL4 and BAP1, and CRC-relevant mutations attenuate the formation of the MLL4-ASXL complex. Mutational analysis and binding assays identified CRC hot spot mutations in ASXLs. Our findings suggest that alterations in the genomic distribution of the MLL4-ASXL-BAP1 axis and CRC hot spot mutations in ASXLs perturb normal transcriptional programs and may trigger pathogenic events in colon cancer.

The human NuA4/TIP60 complex is a multi-subunit, dual enzymatic epigenetic factor and gene regulator. It bears histone acetyltransferase (HAT) activity towards the canonical histones H2A and H4 and the histone variant H2A.Z, a function that has been linked to gene activation. It also acts as a chromatin remodeling enzyme through ATP-dependent exchange of nucleosomal H2A-H2B dimers with H2A.Z-H2B, leading to incorporation of H2A.Z into chromatin at gene regulatory elements. NuA4/TIP60 is unique in merging two enzymatic activities targeting H2A.Z. Both NuA4/TIP60-dependent H2A.Z acetylation and remodeling have been linked to several physiological functions and pathologies, but studies have only focused on either one or the other enzymatic activity, and insights on functional coordination between them are lacking. Here, we leverage our EP400 rapid depletion system to explore and untangle the intricate links between H2A.Z acetylation by Tip60 (the HAT subunit) and loading on chromatin by EP400 (the remodeling subunit) through functional genomic and biochemical analyses. Our data support a mechanism in which H2A.Z is first pre-acetylated to allow for H2A.Zac-H2B dimer association with the complex before incorporation into chromatin, particularly at gene promoters to positively regulate transcription. As both H2A.Z-targeted enzymatic functions of NuA4/TIP60 have been linked to disease, albeit separately, our findings hold important implications for therapeutic intervention, where combinatorial targeting is a promising avenue. ### Competing Interest Statement The authors have declared no competing interest. Canadian Institutes of Health Research, https://ror.org/01gavpb45, PJT-183708

Histone post-translational modifications and variants play crucial roles in the adaptability of chromatin structure, facilitating rapid responses necessary for biological processes such as transcription, replication, and DNA damage signaling. Notably, DNA double-strand break (DSB) signaling heavily relies on these histone modifications, with signal amplification and the recruitment of specific DNA repair factors being dictated by them. Among the histones, H2A and its variants are central to this response, with phosphorylation of the variant H2A.X being the initial and most characteristic histone mark deposit upon DNA damage detection. Additional post-translational modifications of H2A and its variants contribute to the selective recruitment of DNA repair factors and influence the choice of DNA repair pathways. This review provides a summary of current knowledge regarding the roles of histone H2A post-translational modifications and variants in DSB signaling and repair, with a particular emphasis on modifications and variants that impact the choice of repair pathways. Additionally, the involvement of histone chaperones, chromatin modifiers, and remodelers in these processes is discussed.

Genomic material exists in the eukaryotic cell in the form of chromatin. The basic building unit of chromatin is the nucleosome. Each nucleosome is composed of two copies of each of four histone proteins (H2A, H2B, H3 and H4) wrapped with DNA. There is plethora of factors that regulate chromatin-associated processes, including replacing canonical histones in the nucleosome with histone variants, and ATP-dependent remodeling of nucleosomes. Thus, characterizing these regulatory processes and their malfunctioning in diseases, like cancer, is critical for understanding fundamentals of chromatin regulation and in the identification of potential therapeutic strategies.

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.

Ribosome biogenesis is a crucial process requiring enormous transcriptional output. In budding yeast, the expression of 138 ribosomal protein (RP) genes and over 200 ribosome biogenesis (RiBi) genes is regulated by and intricate network of factors, including the nutrient-sensitive transcription activator Sfp1 and the NuA4 coactivator/acetyltransferase complex. Nutrient starvation or inhibition of TORC1 by rapamycin leads to repression of RP and RiBi genes, in part through blocking Sfp1 nuclear localization and NuA4-dependent chromatin acetylation. Here, we demonstrate that Sfp1 physically interacts with NuA4 in a TORC1-independent manner. Our results indicate that Sfp1, along with NuA4, regulate transcription of RiBi and RP genes via distinct mechanisms depending on promoter architectures. Sfp1 promotes NuA4-dependent histone acetylation at the promoter of RiBi and RP genes without affecting NuA4 recruitment. In contrast, NuA4 does impact Sfp1 binding but specifically at two classes of RP genes. Importantly, we found that NuA4 acetylates Sfp1 at lysines 655 and lysine 657, regulating its function. Cells expressing Sfp1 with acetyl-mimicking mutations exhibit increased expression of RiBi genes while RP genes remain stable. However, the same mutants lead to the loss of Sfp1 binding/activity at RiBi genes when cells are under non-optimal growth conditions. Mimicking constitutive acetylation of Sfp1 also limits the transcriptional burst of RP genes upon addition of glucose. Altogether, these results draw an intricate functional relationship between Sfp1 and NuA4 to control ribosome biogenesis, fine-tuning transcription output in different growth conditions. ### Competing Interest Statement The authors have declared no competing interest.

The human nucleosome acetyltransferase of histone H4 (NuA4)/Tat-interactive protein, 60 kilodalton (TIP60) coactivator complex, a fusion of the yeast switch/sucrose nonfermentable related 1 (SWR1) and NuA4 complexes, both incorporates the histone variant H2A.Z into nucleosomes and acetylates histones H4, H2A, and H2A.Z to regulate gene expression and maintain genome stability. Our cryo–electron microscopy studies show that, within the NuA4/TIP60 complex, the E1A binding protein P400 (EP400) subunit serves as a scaffold holding the different functional modules in specific positions, creating a distinct arrangement of the actin-related protein (ARP) module. EP400 interacts with the transformation/transcription domain-associated protein (TRRAP) subunit by using a footprint that overlaps with that of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, preventing the formation of a hybrid complex. Loss of the TRRAP subunit leads to mislocalization of NuA4/TIP60, resulting in the redistribution of H2A.Z and its acetylation across the genome, emphasizing the dual functionality of NuA4/TIP60 as a single macromolecular assembly.

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.

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.

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.

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. Human methyltransferase MLL4 mediates embryonic development and is dysregulated in diseases. Zhang et al. found that binding of PHD fingers of MLL4 to ASXL1/2 is required for recruitment of the deubiquitinase BAP1 to MLL4-bound active enhancers in vitro.

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.

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.

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.