Human p300 is a transcriptional co-activator and a major acetyltransferase that acetylates histones and other proteins facilitating gene transcription. The activity of p300 relies on the fine-tuned interactome that involves a dozen p300 domains and hundreds of binding partners and links p300 to a wide range of vital signaling events. Here, we report a novel function of the ZZ-type zinc finger (ZZ) of p300 as a reader of histone H3. We show that the ZZ domain and acetyllysine-recognizing bromodomain of p300 play critical roles in modulating p300 enzymatic activity and its association with chromatin. The acetyllysine binding function of bromodomain is essential for acetylation of histones H3 and H4, whereas interaction of the ZZ domain with H3 promotes selective acetylation of the histone H3K27 and H3K18 sites.

Yaf9 is an integral part of the NuA4 acetyltransferase and the SWR1 chromatin remodeling complexes. Here, we show that Yaf9 associates with acetylated histone H3 with high preference for H3K27ac. The crystal structure of the Yaf9 YEATS domain bound to the H3K27ac peptide reveals that the sequence C-terminal to K27ac stabilizes the complex. The side chain of K27ac inserts between two aromatic residues, mutation of which abrogates the interaction in vitro and leads in vivo to phenotypes similar to YAF9 deletion, including loss of SWR1-dependent incorporation of variant histone H2A.Z. Our findings reveal the molecular basis for the recognition of H3K27ac by a YEATS reader and underscore the importance of this interaction in mediating Yaf9 function within the NuA4 and SWR1 complexes.

The assembly and function of the yeast general transcription factor TFIID complex requires specific contacts between its Taf14 and Taf2 subunits, however, the mechanism underlying these contacts remains unclear. Here, we determined the molecular and structural basis by which the YEATS and ET domains of Taf14 bind to the C-terminal tail of Taf2 and identified a unique DNA-binding activity of the linker region connecting the two domains. We show that in the absence of ligands the linker region of Taf14 is occluded by the surrounding domains, and therefore the DNA binding function of Taf14 is autoinhibited. Binding of Taf2 promotes a conformational rearrangement in Taf14, resulting in a release of the linker for the engagement with DNA and the nucleosome. Genetic in vivo data indicate that the association of Taf14 with both Taf2 and DNA is essential for transcriptional regulation. Our findings provide a basis for deciphering the role of individual TFIID subunits in mediating gene transcription.

Direct reprogramming of fibroblasts into cardiomyocytes (CMs) represents a promising strategy to regenerate CMs lost after ischemic heart injury. Overexpression of GATA4, HAND2, MEF2C, TBX5, miR-1, and miR-133 (GHMT2m) along with transforming growth factor beta (TGF-β) inhibition efficiently promote reprogramming. However, the mechanisms by which TGF-β blockade promotes cardiac reprogramming remain unknown. Here, we identify interactions between the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3, the SWI/SNF remodeling complex subunit BRG1, and cardiac transcription factors. Furthermore, canonical TGF-β signaling regulates the interaction between GATA4 and JMJD3. TGF-β activation impairs the ability of GATA4 to bind target genes and prevents demethylation of H3K27 at cardiac gene promoters during cardiac reprogramming. Finally, a mutation in GATA4 (V267M) that is associated with congenital heart disease exhibits reduced binding to JMJD3 and impairs cardiomyogenesis. Thus, we have identified an epigenetic mechanism wherein canonical TGF-β pathway activation impairs cardiac gene programming, in part by interfering with GATA4-JMJD3 interactions.

E2F1 and retinoblastoma (RB) tumor-suppressor protein not only regulate the periodic expression of genes important for cell proliferation, but also localize to DNA double-strand breaks (DSBs) to promote repair. E2F1 is acetylated in response to DNA damage but the role this plays in DNA repair is unknown. Here we demonstrate that E2F1 acetylation creates a binding motif for the bromodomains of the p300/KAT3B and CBP/KAT3A acetyltransferases and that this interaction is required for the recruitment of p300 and CBP to DSBs and the induction of histone acetylation at sites of damage. A knock-in mutation that blocks E2F1 acetylation abolishes the recruitment of p300 and CBP to DSBs and also the accumulation of other chromatin modifying activities and repair factors, including Tip60, BRG1 and NBS1, and renders mice hypersensitive to ionizing radiation (IR). These findings reveal an important role for E2F1 acetylation in orchestrating the remodeling of chromatin structure at DSBs to facilitate repair.

Macrophages promote an early host response to infection by releasing pro-inflammatory cytokines such as interleukin (IL) 1β (IL-1β), tumour necrosis factor (TNF), and IL-6. One of the mechanisms through which cells sense pathogenic microorganisms is through Toll-like receptors (TLRs). IL-1 receptor-associated kinase (IRAK) 1, IRAK2, IRAK3, and IRAK4 are integral to TLR and IL-1 receptor signalling pathways. Recent studies suggest a role for aberrant TLR8 and NLRP3 inflammasome activation during both COVID-19 and HIV-1 infection. Here, we show that pacritinib inhibits the TLR8-dependent pro-inflammatory cytokine response elicited by GU-rich single-stranded RNA derived from SARS-CoV-2 and HIV-1. Using genetic and pharmacologic inhibition, we demonstrate that pacritinib inhibits IRAK1 phosphorylation and ubiquitination which then inhibits the recruitment of the TAK1 complex to IRAK1, thus inhibiting the activation of downstream signalling and the production of pro-inflammatory cytokines.

The core subunit of the MORF acetyltransferase complex BRPF1 contains a unique combination of zinc fingers, including a plant homeodomain (PHD) finger followed by a zinc knuckle and another PHD finger, which together form a PZP domain (BRPF1PZP). BRPF1PZP has been shown to bind to the nucleosome and make contacts with both histone H3 tail and DNA. Here, we describe biophysical and structural methods for characterization of the interactions between BRPF1PZP, H3 tail, DNA, and the intact nucleosome. For complete details on the use and execution of this protocol, please refer to Klein et al. (2020).

Histone acetyltransferases (HATs) assemble into multisubunit complexes in order to target distinct lysine residues on nucleosomal histones. Here, we characterize native HAT complexes assembled by the BRPF family of scaffold proteins. Their plant homeodomain (PHD)-Zn knuckle-PHD domain is essential for binding chromatin and is restricted to unmethylated H3K4, a specificity that is reversed by the associated ING subunit. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as catalytic acetyltransferase subunit. Interestingly, while the previously reported HBO1 complexes containing JADE scaffold proteins target histone H4, the HBO1-BRPF1 complex acetylates only H3 in chromatin. We mapped a small region to the N terminus of scaffold proteins responsible for histone tail selection on chromatin. Thus, alternate choice of subunits associated with HBO1 can switch its specificity between H4 and H3 tails. These results uncover a crucial new role for associated proteins within HAT complexes, previously thought to be intrinsic to the catalytic subunit.

The YEATS domain has been identified as a reader of histone acylation and more recently emerged as a promising anti-cancer therapeutic target. Here, we detail the structural mechanisms for π-π-π stacking involving the YEATS domains of yeast Taf14 and human AF9 and acylated histone H3 peptides and explore DNA-binding activities of these domains. Taf14-YEATS selects for crotonyllysine, forming π stacking with both the crotonyl amide and the alkene moiety, whereas AF9-YEATS exhibits comparable affinities to saturated and unsaturated acyllysines, engaging them through π stacking with the acyl amide. Importantly, AF9-YEATS is capable of binding to DNA, whereas Taf14-YEATS is not. Using a structure-guided approach, we engineered a mutant of Taf14-YEATS that engages crotonyllysine through the aromatic-aliphatic-aromatic π stacking and shows high selectivity for the crotonyl H3K9 modification. Our findings shed light on the molecular principles underlying recognition of acyllysine marks and reveal a previously unidentified DNA-binding activity of AF9-YEATS.

The protein partner of Sans-fille (PPS) and its human homolog DIDO mediate diverse chromatin activities, including the regulation of stemness genes in embryonic stem cells and splicing in Drosophila. Here, we show that the PHD fingers of PPS and DIDO recognize the histone mark H3K4me3 in a pH-dependent manner: the binding is enhanced at high pH values but is decreased at low pH. Structural analysis reveals that the pH dependency is due to the presence of a histidine residue in the K4me3-binding aromatic cage of PPS. The pH-dependent mechanism is conserved in DIDO but is lost in yeast Bye1. Acidification of cells leads to the accelerated efflux of endogenous DIDO, indicating the pH-dependent sensing of H3K4me3 in vivo. This novel mode for the recognition of H3K4me3 establishes the PHD fingers of PPS and DIDO as unique epigenetic readers and high pH sensors and suggests a role for the histidine switch during mitosis.

Macrophages are a reservoir for latent human immunodeficiency type 1 (HIV) infection and a barrier to HIV eradication. In contrast to CD4+ T cells, macrophages are resistant to the cytopathic effects of acute HIV infection. Emerging data suggest a role for TREM1 (triggering receptor expressed on myeloid cells 1) in this resistance to HIV-mediated cytopathogenesis. Here, we show that upon HIV infection, macrophages increase the expression of BCL2, BCLXL, TREM1, mitofusin 1 (MFN1), and MFN2 and the translocation of BCL2L11 (BIM) to the mitochondria and decrease the expression of BCL2-associated agonist of cell death (BAD) and BAX while maintaining a 95% survival rate over 28 days. The HIV proteins Tat and gp120 and the GU-rich single-stranded RNA (ssRNA) (RNA40) from the HIV long terminal repeat region (and a natural Toll-like receptor 8 [TLR8] agonist) induced similar effects. TREM1 silencing in HIV-infected macrophages led to decreased expression of BCL2, BCLXL, MFN1, and MFN2 and increased expression of BAD and BAX. This correlated with a significant increase in apoptosis mediated by a disruption of the mitochondrial membrane potential (Δψm), leading to the release of cytochrome c and caspase 9 cleavage. Exposure of TREM1-silenced macrophages to Tat, gp120, or RNA40 similarly resulted in the disruption of Δψm, cytochrome c release, caspase 9 cleavage, and apoptosis. Thus, our findings identify a mechanism whereby HIV promotes macrophage survival through TREM1-dependent upregulation of BCL2 family proteins and mitofusins that inhibits BCL2L11-mediated disruption of Δψm and subsequent apoptosis. These findings indicate that TREM1 can be a useful target for elimination of the HIV reservoir in macrophages.IMPORTANCE The major challenge to human immunodeficiency virus (HIV) treatment is the development of strategies that lead to viral eradication. A roadblock to accomplishing this goal is the lack of an approach that would safely eliminate HIV from all resting/latent reservoirs, including macrophages. Macrophages are a key part of the innate immune system and are responsible for recognizing invading microbes and sending appropriate signals to other immune cells. Here, we found that HIV induces the upregulation of the protein TREM1 (triggering receptor expressed on myeloid cells 1), which signals an increase in the expression of antiapoptotic proteins, thus promoting survival of HIV-infected macrophages.

Macrophages promote an early host response to infection by releasing pro-inflammatory cytokines such as interleukin-1β (IL-1β), TNF, and IL-6. The bioactivity of IL-1β is classically dependent on NLRP3 inflammasome activation, which culminates in caspase-1 activation and pyroptosis. Recent studies suggest a role for NLRP3 inflammasome activation in lung inflammation and fibrosis in both COVID-19 and SARS, and there is evidence of NLRP3 involvement in HIV-1 disease. Here, we show that GU-rich single-stranded RNA (GU-rich RNA) derived from SARS-CoV-2, SARS-CoV-1, and HIV-1 trigger a TLR8-dependent pro-inflammatory cytokine response from human macrophages in the absence of pyroptosis, with GU-rich RNA from the SARS-CoV-2 spike protein triggering the greatest inflammatory response. Using genetic and pharmacological inhibition, we show that the induction of mature IL-1β is through a non-classical pathway dependent on caspase-1, caspase-8, the NLRP3 inflammasome, potassium efflux, and autophagy while being independent of TRIF (TICAM1), vitamin D3, and pyroptosis.

HIV Nef acts as an anti-autophagic maturation factor through interaction with beclin-1 (BECN1). We report that exposure of macrophages to infectious or non-infectious purified HIV induces toll-like receptor 8 (TLR8) and BECN1 dependent dephosphorylation and nuclear translocation of TFEB and that this correlates with an increase in autophagy markers. RNA interference for ATG13, TFEB, TLR8, or BECN1 inhibits this HIV-induced autophagy. However, once HIV establishes a productive infection, TFEB phosphorylation and cytoplasmic sequestration are increased resulting in decreased autophagy markers. Moreover, by 7 d post-infection, autophagy levels are similar to mock infected controls. Conversely, although Nef deleted HIV similarly induces TFEB dephosphorylation and nuclear localization, and increases autophagy, these levels remain elevated during continued productive infection. Thus, the interaction between HIV and TLR8 serves as a signal for autophagy induction that is dependent upon the dephosphorylation and nuclear translocation of TFEB. During permissive infection, Nef binds BECN1 resulting in mammalian target of rapamycin (MTOR) activation, TFEB phosphorylation and cytosolic sequestration, and the inhibition of autophagy. To our knowledge, this is the first report of a virus modulating TFEB localization and helps to explain how HIV modulates autophagy to promote its own replication and cell survival.

BRPF1 (bromodomain PHD finger 1) is a core subunit of the MOZ histone acetyltransferase (HAT) complex, critical for normal developmental programs and implicated in acute leukemias. BRPF1 contains a unique assembly of zinc fingers, termed a PZP domain, the physiological role of which remains unclear. Here, we elucidate the structure-function relationship of this novel epigenetic reader and detail the biological and mechanistic consequences of its interaction with nucleosomes. PZP has a globular architecture and forms a 2:1 stoichiometry complex with the nucleosome, bivalently interacting with histone H3 and DNA. This binding impacts the nucleosome dynamics, shifting the DNA unwrapping/rewrapping equilibrium toward the unwrapped state and increasing DNA accessibility. We demonstrate that the DNA-binding function of the BRPF1 PZP domain is required for the MOZ-BRPF1-ING5-hEaf6 HAT complex to be recruited to chromatin and to acetylate nucleosomal histones. Our findings reveal a novel link between chromatin dynamics and MOZ-mediated acetylation.

Histone methyltransferase MLL4 is centrally involved in transcriptional regulation and is often mutated in human diseases, including cancer and developmental disorders. MLL4 contains a catalytic SET domain that mono-methylates histone H3K4 and seven PHD fingers of unclear function. Here, we identify the PHD6 finger of MLL4 (MLL4-PHD6) as a selective reader of the epigenetic modification H4K16ac. The solution NMR structure of MLL4-PHD6 in complex with a H4K16ac peptide along with binding and mutational analyses reveal unique mechanistic features underlying recognition of H4K16ac. Genomic studies show that one third of MLL4 chromatin binding sites overlap with H4K16ac-enriched regions in vivo and that MLL4 occupancy in a set of genomic targets depends on the acetyltransferase activity of MOF, a H4K16ac-specific acetyltransferase. The recognition of H4K16ac is conserved in the PHD7 finger of paralogous MLL3. Together, our findings reveal a previously uncharacterized acetyllysine reader and suggest that selective targeting of H4K16ac by MLL4 provides a direct functional link between MLL4, MOF and H4K16 acetylation.

Despite significant advances in the treatment of human immunodeficiency virus type-1 (HIV) infection, antiretroviral therapy only suppresses viral replication but is unable to eliminate infection. Thus, discontinuation of antiretrovirals results in viral reactivation and disease progression. A major reservoir of HIV latent infection resides in resting central memory CD4+ T cells (TCM) that escape clearance by current therapeutic regimens and will require novel strategies for elimination. Here, we evaluated the therapeutic potential of autophagy-inducing peptides, Tat-Beclin 1 and Tat-vFLIP-α2, which can induce a novel Na+/K+-ATPase dependent form of cell death (autosis), to kill latently HIV-infected TCM while preventing virologic rebound. In this study, we encapsulated autophagy inducing peptides into biodegradable lipid-coated hybrid PLGA (poly lactic-co-glycolic acid) nanoparticles for controlled intracellular delivery. A single dose of nanopeptides was found to eliminate latent HIV infection in an in vitro primary model of HIV latency and ex vivo using resting CD4+ T cells obtained from peripheral blood mononuclear cells of HIV-infected patients on antiretroviral with fully suppressed virus for greater than 12 months. Notably, increased LC3B lipidation, SQSTM1/p62 degradation and Na+/K+-ATPase activity characteristic of autosis, were detected in nanopeptide treated latently HIV-infected cells compared to untreated uninfected or infected cells. Nanopeptide-induced cell death could be reversed by knockdown of autophagy proteins, ATG5 and ATG7, and inhibition or knockdown of Na+/K+-ATPase. Importantly, viral rebound was not detected following the induction of the Na+/K+-ATPase dependent form of cell death induced by the Tat-Beclin 1 and Tat-vFLIP-α2 nanopeptides. These findings provide a novel strategy to eradicate HIV latently infected resting memory CD4+ T cells, the major reservoir of HIV latency, through the induction of Na+/K+-ATPase dependent autophagy, while preventing reactivation of virus and new infection of uninfected bystander cells.

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.

Because microglia are a reservoir for HIV and are resistant to the cytopathic effects of HIV infection, they are a roadblock for any HIV cure strategy. We have previously identified that triggering receptor expressed on myeloid cells 1 (TREM1) plays a key role in human macrophage resistance to HIV-mediated cytopathogenesis. In this article, we show that HIV-infected human microglia express increased levels of TREM1 and are resistant to HIV-induced apoptosis. Moreover, upon genetic inhibition of TREM1, HIV-infected microglia undergo cell death in the absence of increased viral or proinflammatory cytokine expression or the targeting of uninfected cells. We also show that the expression of TREM1 is mediated by HIV Tat through a TLR4, TICAM1, PG-endoperoxide synthase 2, PGE synthase, and PGE2-dependent manner. These findings highlight the potential of TREM1 as a therapeutic target to eradicate HIV-infected microglia without inducing a proinflammatory response.

Chromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

PHF20 is a core component of the lysine acetyltransferase complex MOF (male absent on the first)-NSL (non-specific lethal) that generates the major epigenetic mark H4K16ac and is necessary for transcriptional regulation and DNA repair. The role of PHF20 in the complex remains elusive. Here, we report on functional coupling between methylation readers in PHF20. We show that the plant homeodomain (PHD) finger of PHF20 recognizes dimethylated lysine 4 of histone H3 (H3K4me2) and represents an example of a native reader that selects for this modification. Biochemical and structural analyses help to explain this selectivity and the preference of Tudor2, another reader in PHF20, for dimethylated p53. Binding of the PHD finger to H3K4me2 is required for histone acetylation, accumulation of PHF20 at target genes, and transcriptional activation. Together, our findings establish a unique PHF20-mediated link between MOF histone acetyltransferase (HAT), p53, and H3K4me2, and suggest a model for rapid spreading of H4K16ac-enriched open chromatin.